the effects of indirubin derivitives on gene expression

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THE EFFECTS OF INDIRUBIN DERIVITIVESON GENE EXPRESSION AND CELLULARFUNCTIONS IN THE MURINEMACROPHAGEAbigail BabcockClemson University, [email protected]

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THE EFFECTS OF INDIRUBIN DERIVATIVES ON GENE EXPRESSION AND

CELLULAR FUNCTIONS IN THE MURINE CELL LINE, RAW 264.7

A Dissertation Presented to

the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy Biological Sciences

by Abigail Sue Babcock

August 2008

Accepted by: Dr. Charles D. Rice, Committee Chair

Dr. Lyndon L. Larcom Dr. Thomas R. Scott Dr. Alfred P. Wheeler

ii

ABSTRACT

Indirubin is the active ingredient in the ancient herbal remedy, Danggui

Longhui Wan, which is used as an anti-leukemic, anti-inflammatory, and

detoxification treatment. Indirubin-3’-monoxime (IO) is the most widely studied of

the indirubin derivatives. Most have been shown to inhibit cyclin-dependent

kinases and glycogen synthase kinases, triggering cell cycle arrest and apoptosis

with different levels of activity depending on the particular structure. These

kinases inhibited by indirubin(s) play key roles in cellular proliferation and

immune functions, therefore synthetic indirubins may have significant

applications as therapeutic agents for cancer, inflammation, and neurological

diseases.

Indirubin is also a potent aryl hydrocarbon receptor (AhR) agonist. The

AhR is a cytosolic protein, which upon ligand binding is translocated to the

nucleus and acts as a transcription factor for genes involved in drug metabolism,

oxidative stress, and inhibitors of the cell cycle. Known anthropogenic ligands

(PCBs, dioxins, PAHs etc.) are mutagenic, carcinogenic, and immunotoxic. In

this study, I examined the effects of select indirubins on gene/protein expression

profiles, AhR activation, and cellular functions in the mouse macrophage cell line

RAW 264.7. Structure activity relationships (SAR) show that different derivatives

of indirubin may differentially affect AhR activation, kinase inhibition, and/or other

cellular functions, including intracellular killing of bacteria. The ultimate goal was

to identify a synthetic indirubin derivative that would be beneficial to cancer and

inflammation research without being overtly toxic.

iii

From this research, Indirubin-3’-(2,3 dihydroxypropyl)-oximether) (E804),

and 6-bromoindirubin-3-monoxime (BIO) were shown to be very potent AhR

ligands with strong anti-inflammatory properties. This study also shows that E804

has more clinical promise than its more famous counterpart indirubin-3’-

monoxime.

iv

DEDICATION

I dedicate this dissertation to my parents Joe and Sue Babcock. I could

not have gotten through these last 4 years without your love, support, and

unwavering belief in me. I feel very blessed to have the best parents in the world,

who instilled in me the confidence, morality, and perseverance I needed to

succeed. I further dedicate this work to my brothers, Taylor and Joey, my

grandparents, family and friends. You have been a great support system for

which I am grateful.

v

ACKNOWLEDGEMENTS

I would like to first acknowledge my advisor and mentor, Dr. Charlie Rice,

who set the bar high and had the confidence I would reach it. Your love of

science is contagious, your knowledge impressive, and your humor hilarious. I

will take with me all your stories and “advice”. I have the deepest respect and

appreciation for you and my time here. I would also like to thank my graduate

committee members, Lyn Larcom, Tom Scott, and Hap Wheeler for your

expertise, guidance, and belief in me. Thank you for the knowledge I received

from your classes and the example you set as great scientists.

I would like to thank my former labmates, Marlee Beckham Marsh, Laura

Hunt, Shannon Billings, Moss Matsebatlela, Palllavi Vedantam, and Josephine

Woydyl. I am so grateful for your knowledge, patience, and hard work over the

last four years; you all have truly made my time here wonderful.

Tim, I could not have made it through this time without your love, support,

and understanding. I want to especially thank you for all the slushes that kept me

going. You are my best friend and I feel very blessed to have such a great man in

my life. To my ALIVE crew, thank you for all the prayers, laughs, and shoulders

to cry on. Your friendships are invaluable to me. Finally, thank you to Sara and

Jason for spoiling me when I needed it the most.

vi

TABLE OF CONTENTS

TITLE PAGE ...................................................................................................... i ABSTRACT....................................................................................................... ii DEDICATION................................................................................................... iv ACKNOWLEDGEMENTS .................................................................................v LIST OF TABLES............................................................................................ vii LIST OF FIGURES ......................................................................................... iX LITERATURE REVIEW ....................................................................................1 INTRODUCTION ............................................................................................21 MATERIALS AND METHODS ........................................................................31 RESULTS .......................................................................................................42 DISCUSSION..................................................................................................80 APPENDIX......................................................................................................89 REFERENCES CITED..................................................................................124

vii

LIST OF TABLES Table Page 1 Functional gene groupings of the Mouse Stress and

Toxicity Pathway Finder™ RT2 Profiler™ PCR Array ...............................................................................51

2 Altered inflammatory gene expression in RAW 264.7

macrophages treated for 24 hours with LPS, Io, E804, or PCB-126........................................................52

3 Altered inflammatory gene expression in RAW 264.7

macrophages treated simultaneously with LPS and Io, E804, or PCB-126 for 24 hours..............................53

4 Altered proliferation and carcinogenesis gene expression

in RAW 264.7 macrophages treated for 24 hours with LPS, Io, E804, or PCB-126 ................................................54

5 Altered proliferation and carcinogenesis gene expression in

RAW 264.7 macrophages treated simultaneously with LPS and Io, E804, or PCB-126 for 24 hours ......................55

6 Altered growth arrest and senescence gene expression

in RAW 264.7 macrophages treated for 24 hours with LPS, Io, E804, or PCB-126 ................................................56

7 Altered growth arrest and senescence gene expression in

RAW 264.7 macrophages treated simultaneously with LPS and Io, E804, or PCB-126 for 24 hours ......................57

viii

List of Tables (Continued) Page 8 Altered apoptosis gene expression in RAW 264.7

macrophages treated for 24 hours with LPS, Io, E804, or PCB-126 ................................................58

9 Altered apoptosis gene expression in RAW 264.7

macrophages treated simultaneously with LPS and Io, E804, or PCB-126 for 24 hours ....................................................................................59

10 Altered stress related gene expression in RAW 264.7

macrophages treated for 24 hours with LPS, Io, E804, or PCB-126 ................................................60

11 Altered stress related gene expression in RAW 264.7

macrophages treated simultaneously with LPS and Io, E804, or PCB-126 for 24 hours ....................................................................................61

LIST OF FIGURES

Figure Page 1 Structure of indirubin derivatives..........................................................13 2 Dual mechanisms in which indirubins my induce cell cycle arrest .........................................................................29

3 Comparison of indirubins and TCDD in their interactions

in two AhR reporter systems and two kinase assays............................................................................30

4 Cytotoxicity of compounds to RAW 246.7 cells....................................47 5 Cytotoxicity of compounds to rat glioma D74 cells...............................48 6 Effects of 1µM of compounds on apoptosis in

RAW 246.7 macrophages .........................................................49 7 Effects of 1µM of compounds with LPS on apoptosis

in RAW 246.7 macrophages......................................................50 8 Nuclear translocation of AhR in RAW 246.7 cells

treated with media control for 4 hours .......................................62 9 Nuclear translocation of AhR in RAW 246.7 cells

treated with 1µM Io for 4 hours..................................................63 10 Nuclear translocation of AhR in RAW 246.7 cells

treated with 1µM BIO for 4 hours...............................................64

x

List of Figures (Continued) Nuclear translocation of AhR in RAW 246.7 cells

treated with 1µM E804 for 4 hours ............................................65 12 Nuclear translocation of AhR in RAW 246.7 cells

treated with 1µM PCB-126 for 4 hours ......................................66 13 Nuclear translocation of AhR in RAW 246.7 cells

treated with 1µM PCB-104 for 4 hours ......................................67 14 Nitric oxide stimulation in RAW 264.7 cells treated for 24

hours with indirubins or PCB-126 ..............................................68 15 Nitric oxide stimulation in RAW 264.7 cells treated for 48

hours with indirubins or PCB-126 ..............................................69 16 Immunoblot for 24 hour exposure to compound iNOS (a), ACTIN (b), 48 hour exposure to compound iNOS (c), and ACTIN (d) ...........................................................70 17 Effects of 24 hour exposure of indirubins and PCB-126

on the production of interleukin-6 in RAW 264.7 macrophages .........................................................71

18 Effects of 48 hour exposure of indirubins and PCB-126

on the production of interleukin-6 in RAW 264.7 macrophages .........................................................72

xi

List of Figures (Continued) Page 19 Effects of 24 hour exposure of indirubins and PCB-126

on ROI production in RAW 246.7 macrophages........................73 20 Effects of 48 hour exposure of indirubins and PCB-126

on ROI production in RAW 246.7 macrophages........................74 21 Effects of 24 hour regime exposure of compounds on

lysozyme activity in RAW 246.7 macrophages.........................75 22 Effects of 48 hour regime exposure of compounds on

lysozyme activity in RAW 246.7 macrophages.........................76 23 RAW 264.7macrophage phagocytosis under 24 hour treatment regime...............................................................77 24 RAW 264.7macrophage phagocytosis under 48 hour treatment regime...............................................................78 25 Intracellular bacterial killing by RAW 264.7 macrophages under both 24 hour and 48 hour treatment regime....................79

1

LITERATURE REVIEW

The Institute of Hematology, Chinese Academy of Sciences (1976) treated

16 chronic myelogenous leukemia (CML) patients with ingredients from an

ancient medicinal recipe called Danggui Longhui Wan (DLW), and reported that 4

patients showed complete remission and 5 partial remission. The ancient remedy

is made from extracts of several indigo-producing plants including Polygonum

tinctorium, Isatis indigotica and Isatis tinctoria. According to the Chinese

Academy of Medical Sciences (1975), I. tinctoria extracts significantly prolonged

the life span of rats with Walker carcinosarcoma 256 tumors, whereas other plant

extracts from DLW did not. The anti-leukemic properties from DLW were found to

come from the ingredient Qing Dai which is made up of indirubin, an isomer of

indigo. Indirubin is a red colored bis-indole metabolite of tryptophan found in

indigo-producing plants (Wu et al., 1979). In a later clinical trial, 253 patients with

CML were given 150-200mg/day of indirubin synthesized from indigo blue. Over

60% of the patients showed at least partial remission (Reviewed in Xiao et al.,

2002). Although these findings were exciting, due to indirubin’s poor solubility

patients had severe gastrointestinal side-effects and were forced to stop

treatment (Zheng et al., 1979). Recently, several derivatives have been

synthesized to increase the efficacy of the compound and decrease the toxicity

side effects of indirubin.

The mechanisms of action which indirubin and its derivatives possess are

not well known. The 11 herb remedy, DLW has a broad list of pharmacologically

2

relevant effects that could be exploited. Consumers also use the ancient herbal

medicine for its detoxification and anti-inflammatory properties in treating chronic

disease (Kunikata et al 2000). It is prudent to examine nutraceutical remedies not

only for safety and efficacy but for future drug therapies. One critical aspect for

this investigation is the effect indirubin has on gene expression, cellular signaling,

protein function, and inflammation in macrophages.

Activated macrophages display several important physiological functions

such as phagocytosis, antigen presentation, and release of a diverse arsenal of

biological mediators, for example, reactive oxygen intermediates and reactive

nitrogen species, cytokines, and hydrolytic enzymes (Reviewed in Billack 2006).

These actions make macrophages vital cells in the immune system, particularly

in their role in innate immunity. The broad and essential roles these cells play

make them attractive targets for therapeutic drug treatments.

A major aspect of innate immunity is the inflammatory response which

can be stimulated by diverse chemical, infectious, or mitogenic stimulants.

Mediators of the immune system include, chemokines, enzymes that promote

vascular permeability and migration of immunomodulators, chemoattractant

molecules responsible for the recruitment of neutrophils and monocytes, and the

inflammatory family of cytokines which include interleukin 1 (IL-1), IL-6, IL-18,

tumor necrosis factor α (TNF-α), colony stimulating factors (CSF) and many

others that regulate the duration and potency of the inflammatory response

(Reviewed in O’Shea et al., 2008; Jones et al., 2000). The innate immune system

is in close association with the more specific adaptive immune response. IL-18 is

3

one of the cytokines produced during the inflammatory response involved in

crucial crosstalk in promoting T helper cell 1 response in the adaptive immune

response (Wuttge et al., 2003).

Hematopoietic stem cells develop into monocytes in the presence of

macrophage colony stimulating factor and granulocyte-macrophage colony

stimulating factor within the bone marrow. Monocytes enter the blood stream and

migrate to tissues where they differentiate into macrophages. Macrophages are

activated by the recognition of pathogen associated molecular patterns (PAMPs),

which are conserved sequences on microbial non-self molecules, by pattern

recognition receptors like toll like receptors (TLRs). To date there are at least 13

different TLRs in mammals (Roach et al., 2005) that bind to PAMPs and self-

altered molecules (Akira et al., 2004). Toll-like receptor 4 (TLR4) is a

transmembrane receptor that recognizes several ligands notably

lipopolysaccharide (LPS). Ligand (LPS) binding initiates the recruitment of

various adaptor proteins to the cytosolic Toll-IL-1 receptor (TIR) domain which

activates a signaling pathway leading to the activation and subsequent nuclear

translocation of nuclear factor kappa B (NF-кB). The induction of inflammatory

genes as well as genes that regulate cell cycle progression, apoptosis and cell

differentiation are expressed depending on the nature and specific manner in

which ligand binds (Yan et al., 2007). Other toll-like receptors also mediate

inflammation, for example, TLR1 and TLR2 ligand binding also initiates the

translocation of NF-κB promoting the gene expression of IL-6 (Krishnan et al.,

2007).

4

The role cytokines play in the immune response has been an area vastly

studied particularly in inflammation. Interleukins, interferons, and colony-

stimulating factors bind to type I and II cytokine receptors (Boulay et al., 2003)

that commonly have a cytoplasmic Janus kinase (Jak) family associated with it.

Specific receptor/cytokine binding activates Jak kinases to phosphorylate

tyrosine residues in the receptor cytoplasmic domain creating a docking site for

Src homology 2 domain proteins, like Stat transcription factors (signal

transducers and activators of transcription). This cascade of cytokine signaling is

known as the JAK-STAT pathway (Kisseleva, et al., 2002). There are seven

members of the Stat family of DNA-binding proteins, Stat1, Stat2, Stat3, Stat4,

Stat5a, Stat5b, and Stat6, which regulate gene expression by the specific

cytokine bound. A hallmark of acute inflammation is a change in plasma proteins

called acute phase proteins; IL-6 is the principle cytokine responsible for the

concentration level change of these proteins (Gauldie et al., 1987). Agents that

inhibit IL-6 production show promise in clinical trials of patients with arthritis and

chronic inflammatory diseases (Crocker et al., 2003).

In addition to cytokines macrophages produce enzymes that are key

components in innate immunity that result in the production of nitric oxide, a

potent player in inflammation. There are three types of nitric oxide synthases,

NOS1 and NOS3 also known as neuronal NOS (nNOS) and endothelial NOS

(eNOS) are named for cells they were characterized in. NOS2 better known as

inducible nitric oxide synthase (iNOS) is not constitutively expressed but

synthesized upon activation in cells. iNOS acts on the amino acid L-arginine to

5

convert it to L-citrulline and nitric oxide (NO) and released in what is called the L-

arginine-NO pathway (Moncada et al., 1989; Moncada et al., 2002). Regulation

of iNOS is achieved by cytokines (Liew et al., 1997) and mediating the stability

and activation of mRNA. Several transcription factors regulate the iNOS gene

promoter including NF-кB and signal transducer and activator of transcription 1

(STAT1) (Reviewed in Tripathi et al., 2007). LPS alone is capable of inducing

Nos2 gene in the mouse macrophage cell line RAW 264.7; however, in most

other cell lines cytokines and other stimulating factors are needed (MacMicking

et al., 1997). Knockout mice deficient of the iNOS gene show greater

susceptibility to infection and an increase in fatality when they encounter bacteria

compared to their wild-type controls (MacMicking et al., 1997; MacMicking et al.,

1995).

Nitric oxide is a toxic free radical gas that is cell permeable and can react

with molecular oxygen to form reactive nitrogenous intermediates (Eiserich et al.,

1998). These highly reactive species are capable of killing or dramatically

reducing the replication of microbial agents including viruses, bacteria, and fungi.

On the contrary, however, the overproduction of NO can lead to necrosis of

healthy tissue and host toxicity (Laskin et al., 1996). The role of the small NO

molecule has drastic effects on virtually ever level of the inflammatory response

including vascular permeability, vasodilatation, leukocyte migration and

adhesion, and microbe clearance (Guzik et al., 2003).

Danggui Longhui Wan has been used for centuries in the treatment of

chronic inflammatory disease, although the means of action remain elusive, it is

6

reasonable to look at the NF-кB signaling pathway, also known as the central

mediator of the immune response. It is a key transcription factor that when

stimulated expresses several inflammatory genes. NF-кB is a member of the Rel

domain-containing proteins family, consisting of a heterodimer of p50 and p65. It

is found in the cytoplasm of cells in association with IкB which holds it in its

inactive form. It has been reported that there are over 150 extracellular means of

activation of this pathway including numerous pathogens, cytokines, and stress

stimuli. Only when activated does IкB kinase (IKK) phosphorylate IкB, signaling

its degradation to the proteosome, thereby disassociating it from its inhibitory

hold on the heterodimer. This unmasks the translocation factor enabling the p65

subunit to be phosphorylated this leads to the translocation of NF-kB to the

nucleus. NF-kB is responsible for the regulation and activation of many diverse

genes and pathways, including the expression of many immune modulators,

inflammatory cytokines, stress responses, and cell proliferation and

differentiation molecules (Li et al 2002). The transcription factor regulates

antiapoptotic, inflammatory and stress related genes including SURVIVN, Bcl,

Cox-2, and cell cycle genes cyclin D1 and c-MYC (Sethi et al, 2006).

Following earlier observations the effects of indirubin-3’monoxime and

other indirubin derivatives on the function of RAW 264.7 macrophages, and

consequently the inflammatory response were investigated; focusing on several

key modulators of the inflammatory response and report the effect that indirubin

and its soluble derivatives have on this important aspect of immunity.

7

In 1999, Hoessel et al. reported on a finding that would give great insight,

interest, and understanding into indirubin and its derivatives. They showed the

co-crystallization structure of indirubins and cyclin-dependent kinases (CDK1 and

CDk2), key factors in the progression of the cell cycle. This revealed a

competitive inhibition of the indole compounds with ATP for the catalytic binding

site of the kinase. Cyclin-dependent kinase inhibitors halt the cell cycle, therefore

inducing apoptosis, making them excellent candidates for cancer therapy.

The mammalian cell cycle is composed of four stages; two gap stages G1

and G2, a DNA synthesis stage S, and mitosis, M. G1 is a critical checkpoint in

the progression of a cell from the resting G0 phase through to S phase. The cycle

is under tight control through the degradation and production of regulatory

molecules creating an oscillating concentration of protein subunits called cyclins

which function to activate cyclin dependent kinases (CDKs). The heterodimer of

cyclin/CDK then phosphorylates key proteins, involved in the progression of the

cycle, activating or inactivating them in a series of orchestrated regulated events

through the stages of the cell cycle. The Gap1 cyclin, cyclin D, binds to CDK4

and CDK6 which are involved in the checkpoint regulation of G1 progression into

the S phase. CDK4 (6)/cyclin D phosphorylates many substrates including the

retinoblastoma protein (Rb). The phosphorylation of Rb (pRb) releases its

repression leading to the activation of the E2F family of transcription factors

which are responsible for the expression of various genes that are involved in cell

cycle progression (Reviewed in Johnson et al., 1999). The over-expression of

cyclin D mRNA and/or protein levels is seen in over 50% of breast cancers (Roy

8

et al., 2006). Therefore, finding a high affinity inhibitor of this cyclin subunit could

be pivotal in the future of breast cancer treatment, as well as other cancers.

Hoessel et al., (1999) also reported on the kinase selectivity of various

derivatives acting on several CDK/cyclin complexes. Derivatives have

preferential binding affinities for specific CDKs or other structurally related

kinases based on the chemical makeup and confirmation of the derivative. Indigo

did not show an inhibition for the enzymes tested, furthering the evidence that the

active anti-leukemic component of Danggui Longhui Wan is indirubin and not

indigo. Polychronopoulos et al., (2003) tested numerous indirubin derivatives

against CDK1/cylin B, CDK5/p25, and GSK-3β, showing that the difference in the

chemical substitutions of indirubin have different affinities to kinases. The

selectivity is based on the substituted group that is in contact with the CDK-ATP

catalytic binding pocket.

Leclrec et al., (2000) also reported that indirubin and some of its soluble

derivatives are potent inhibitors of glycogen synthase kinase-3β (GSK-3 β) and

CDK5 by competing with ATP in the active site of the kinase, similar to its

inhibition on CDK1 and CDK2 as reported by Hoessel et al., (1999). This finding

is of much interest due to GSK-3 and CDK5’s role in neurological disorders like

Alzheimer’s disease and bipolar disease. GSKs are responsible for numerous

distinct physiological pathways including cell cycle progression (Diehl et al.,

1998), WNT signaling pathway (Willert et al., 1998), and inflammation (Woodgett

et al., 2005). Imahori et al (1997) reported that the two kinases responsible for

the formation of neurofibrillary tangles (NFT) are GSK-3B and CDK5, which is

9

one of the hallmarks of Alzheimer’s disease. The neurological disease is

comprised of paired helical filaments from the abnormal hyperphosphorylation of

Tau protein by the glycogen synthase and cyclin-dependent-5 kinases (Ferrer et

al., 2005). The ability of a compound to inhibit such phosphorylation could lead to

promising agents in the fight against Alzheimer’s and other such diseases.

Another key turning point in the study of indirubin and its derivatives came

in 2001 (Adachi et al.) from a finding that indirubin purified from human urine has

the ability to bind to the cytosolic aryl hydrocarbon receptor (AhR). Polygonum

tinctorium extracts in addition to alleviating inflammation and cancer are also

used for enhancing detoxification processes, suggesting that this plant contains

compounds that induce the family of cytochrome P450 enzymes through the aryl

hydrocarbon receptor AhR. Adachi et al (2001) showed that indirubin is the most

likely candidate because it is an AhR ligand capable of inducing CYP1A, a phase

I drug metabolizing enzyme of the P450 family (Kawajiri et al., 2007). Kawanishi

et al., (2003) showed that using a reporter yeast construct that indirubin is a

potent AhR ligand. The CYP1A1 gene expression is induced over twelve fold

compared to DMSO (carrier control) in human U937 macrophages upon

treatment with indirubin-3’-monoxime (Springs et al., 2006).

The immunotoxic, homeostatic, and biologic consequences of many

halogenated aromatic hydrocarbons (HAH), polychlorinated biphenyls (PCB),

and other xenobiotic pollutants are a direct effect of their interaction with the AhR

(Hahn, 1998). The AhR is in the basic helix-loop-helix-PER-ARNT-SIM (bHLH-

PAS) superfamily of transcription factors (reviewed in Schmidt et al., 1996). The

10

bHLH family members have two distinct highly conserved domains, the basic-

region involved in DNA binding and the helix loop helix region involved in protein

interactions. There are two PAS domains that help facilitate the interaction

between the receptor and Ah receptor nuclear translocator (ARNT) and ligand

binding (Emma et al., 1992; Coumailleau et al., 1995). The AhR is also known as

the dioxin receptor for its high affinity to the ligand 2,3,7,8-tetrachlorodibenzo-p-

dioxin (dioxin or TCDD) which achieved notoriety in the 1970's when it was found

to be a contaminant in the herbicide Agent Orange. TCDD was also used as

chemical warfare agent during the Vietnam War (Reviewed in Frumkin, 2003).

In the inactivated state, the AhR is a cytosolic ligand-activated

transcription factor found within most tissues and cells. It is associated with heat

shock protein 90 (HSP-90) homodimer (Chen et al., 1994), a p23 chaperone

(Cox et al., 2004), and an immunophilin-related protein XAP-2 (Meyer et al.,

1998). Upon ligand binding, the coupled proteins are disassociated and the AhR-

ligand complex is translocated to the nucleus where it binds with AhR nuclear

translocator (ARNT). The ligand/receptor/ARNT complex binds to xenobiotic

response element (XRE), a cassette of specific DNA sequences upstream of

various genes such as drug metabolizing enzymes CYP1A1, glutathione S-

transferase (GST) (Rushmore et al., 1990) and UDP-glucoronsyltransferase

(UGT) (Iyanagi et al., 1986) increases the transcription rate of these genes.

(Reviewed in Shimba et al., 2001; Nohara et al., 2005; Nguyen, et al., 2007;

Marlowe et al., 2005). These phase II enzyme genes associate with the Nrf2

11

gene battery which binds to antioxidant response elements (AREs) encoding

several enzymes related to oxidative stress (Kohle et al., 2007).

The aryl hydrocarbon receptor is classified as an orphan receptor, in that

a known endogenous ligand has not yet been found. However, recently it was

discovered that indirubin, found naturally in human urine, binds the AhR and thus

may be an endogenous ligand for the receptor (Adachi et al., 2001; Spink et al.,

2003, Schnitzer et al., 2005). Knockaert et al., (2004) showed that Io and various

other derivatives, including methylated indirubins, rapidly translocate the

cytosolic AhR to the nucleus. This was an interesting finding because the

substituted methyl group interferes with the molecules ability to bind to the cyclin-

dependent kinase catalytic site, and therefore is not an inhibitor of the kinase.

This suggests that some of the effects on cytotoxicity and cell cycle arrest are

independent of CDK inhibition and could be AhR-dependent.

This is alarming because most anthropogenic compounds that bind to the

AhR, for example TCDD and PCB-126 are highly mutagenic, immunotoxic, and

carcinogenic (Hankinson, 1995 and Andersson et al 2002). Aryl hydrocarbon

receptor ligands can have both antagonistic and agonistic effects on the AhR

signaling pathway (Nishiumi et al, 2008). Some ligands that bind to the receptor

could induce apoptosis and therefore cause growth inhibition in some tumors and

cancer cells while other ligands promote tumorogenesis (Bradshaw et al 2002,

Damiens et al 2001, and Koliopanos et al 2002). The alteration of this pathway in

a non-toxic manner could lead to another mechanism in which indirubins could

arrest the cell cycle and be beneficial pharmacological agents.

12

Although the anti-cancer properties of indirubins, via CDK-inhibition, are

the subject of increased area of studies, the effects on inflammation and

detoxification mechanisms are not well established. This dissertation investigates

the effects of indirubin derivatives, in comparison with PCB-126 a known AhR

ligand, on gene/protein expression, AhR activation, and cellular function using a

murine macrophage cell line RAW 264.7.

13

Indirubin (IR) Indirubin-3’-monoxime (IO)

6-Bromoindirubin-3’-monoxime Indirubin-3’-(2, 3 dihydroxypropyl)- (BIO) oximether (E804) Figure 1. The structure of indirubin and indirubin derivatives.

14

LITERATURE CITED

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21

INTRODUCTION

The multibillion dollar Nutraceuticals industry is growing rapidly, especially

in the use and production of ancient herbal remedies. Some of the most well

known of these products are oriental supplements and teas which are readily

consumed by the American public on the marketing promise of anti-cancer, anti-

inflammation, and anti-disease that leads to longevity and health. The public

lacks an appropriate knowledge of the ingredients, effects, or modes of action of

these holistic medicines. Many herbal remedies consist of several ingredients

leading to the activation of different cellular pathways and “healing” capacities.

Recently there has been a push to isolate the key active compounds and test

their efficacy and safety. This study presented herein describes the effects of one

such natural components, on gene expression, cellular signaling and finally on

select immunologic cellular functions.

The Chinese Academy of Medicine identified Qing Dai (indigo naturalis) to

be the active component of Danggui Longhui Wan, an ancient Chinese herbal

tea used to treat several conditions, including neoplasia, detoxification, and

chronic inflammation. The ingredients are derived, in part, from the plants

Polygonum tinctorium (Polygonaceae) and Istasis indigotica (Brassicaceae) (Wu

et al., 1979). The minor constituent indirubin (IR), an indigo blue isomer, was

purified from the active Qing Dai substance, and found to be the compound

responsible for the anti-leukemic properties that the Chinese tea seemed to

possess. Indirubin, a metabolite of the amino acid tryptophan, has a colorful red

appearance formed by the spontaneous dimerization of its colorless precursors

22

indoxyl and isatin (reviewed in Hoessel et al., 1999). In some rare cases, patients

with catheters get purple urine bag syndrome (PUBS), and will produce purple

colored urine in the urinary bag, this was found to consist of indirubin and indigo

(Dealler et al., 1988). In patients with PUBS tryptophan is metabolized by graham

negative bacteria that act to degrade indican into indigo and indirubin producing

the purple coloration (Vallejo-Manzur, 2005). The exact mechanism in which

indirubin is formed from tryptophan varies among its natural sources which

include; indigo producing plants, Tyrian purple-producing mollusks, wild-type and

recombinant bacterial strains and mammalian urine including human and bovine

(Maugard et al., 2001; reviewed in Gillam et al., 2000; Meijer et al., Bhushan et

al., 2000; 2003; Adachi et al., 2001).

Over the past two decades indirubin has sparked much interest as a

pharmacological agent, especially in the use in cancer treatment with the finding

that indirubin arrests the cell cycle by binding to and inhibiting cyclin-dependent

kinases (Hoessel et a., 1999). Early clinical trials showed great promise in

treating chronic myelogenous leukemia (CML) as well as granulocytic leukemia.

In the patients with CML who received indirubin, 26% showed complete

remission and 33% partial remission (Gan et al., 1985; reviewed in Hoessel et

al., 1999). However, the compound is poorly soluble and can cause gastro-

intestinal and toxicity problems. In an effort to minimize side effects and increase

efficacy of the compound, soluble derivatives have been synthesized, most

notable indirubin-3’-monoxime (IO). IO has been used in many laboratories to

study the compounds modes of action and potential for various therapeutic

23

medicines in cancer, inflammation, and neurological disorders. Some of the

newly synthesized derivatives have similar effects in the body without the

insolubility side effects associated with indirubin (Marko et al., 2001; Guengerich

et al., 2004).

Recently, it was reported that indirubin, found naturally in human urine,

binds with high affinity to the aryl hydrocarbon receptor (AhR) (Adachi et al.,

2001; Spink et al., 2003, Schnitzer et al., 2005). The fact that indirubin was found

naturally in mammalian urine gives intrigue to the possibility of an endogenous

ligand for the AhR. To date the AhR is considered an orphan receptor, in that a

known endogenous ligand has not been found. Several halogenated aromatic

hydrocarbon (HAH) and polycyclic aromatic hydrocarbons (PAH) that are

considered environmental pollutants bind to the AhR. The most potent and

famous of which is 2,3,7,8-tetrachlorodibenzene-p-dioxin (TCDD or dioxin).

Binding of aromatic hydrocarbons to the receptor initiates the AhR signaling

pathway leading to a wide range of toxic and carcinogenic responses (Marlowe

et al., 2005).

The AhR is a member of the PER ARNT SIM (PAS) superfamily of

proteins (Gu et al., 2000). It is found in the cytoplasm of most cells in an

inactivated state held by chaperone p23 and heat shock proteins (Chen et al.,

1994; Cox et al., 2004). Ligand binding dissociates these proteins from the

receptor freeing it to translocate the nucleus. Once in the nucleus the ligand/AhR

forms a heterodimer with aryl hydrocarbon receptor nuclear translocator (ARNT).

These complexes can then bind to and manipulate the xenobiotic response

24

element of genes, including drug-metabolizing enzymes such as the cytochrome

P450 family including CYP genes (Hahn, 1998). Knockaert et al., (2004) showed

that IO and various other derivatives translocate the cytosolic AhR to the

nucleus. Further, IO has been found to increase the gene expression of CYP1A1

in U937 human macrophage cell line (Springs et al., 2006)

Immune suppression, reproductive, and developmental disorders are

associated with anthropogenic AhR ligand exposure (Holsapple et al., 1996).

Therefore, it is alarming that these natural compounds could bind and trigger

such toxic results. However, in recent years it has become evident that ligands

of the AhR not only trigger toxicant enzyme activities but independent of and in

combination with these activation pathways trigger other signal transduction

pathways as well (Puga et al., 2002). AhR signaling has been shown to regulate

developmental cues, cell cycle progression, and homeostasis along with

chemical toxicity (Reviewed in Nguyen et al., 2008). Several studies have shown

that the AhR has a role in cell cycle regulation, although the mechanisms are not

well known. Ge et al. (1998) reported on a direct protein-protein interaction

between the AhR and the retinoblastoma protein (Rb), a critical molecule in the

cell cycle. In response to ligand binding there is an increase in gene expression

of p27, a CDK2 inhibitor, halting the cell cycle in G1. The dual mechanism of

indirubins is shown in Figure 1 (Knockaert et al., 2003). Dependent on the

chemical makeup and confirmation of the synthesized derivative (Figure 3.) the

affinity may be greater for AhR binding, cyclin-dependent kinase inhibition, or

other cellular pathways. A key question for the future of this area of research is to

25

find a molecule with the beneficial pharmacology effects for medicinal drug

therapy, without the traditional AhR immunotoxicity and mutagenesis.

Although the adverse effects of anthropogenic compounds have been

studied, very little is known about the effects endogenous ligands have on the

function of macrophages or the immune system. Previous work has shown that

macrophages are susceptible to AhR activation in terms of phagocytic activity

(Tweari et al., 1979; van Grevenynghe et al., 2003), intracellular killing, ability to

present antigen, and produce cytokines (van Grevenynghe et al., 2003). This

study will describe the effects of indirubin derivatives on gene expression, AhR

signaling pathway, and on select immunologically relevant cellular functions in

RAW 264.7 macrophages.

A key turning point in the story of indirubin came in 1999 when Hoessel et

al. established that indirubin and some of its derivatives (indirubins) can

competitively bind to the ATP-binding pocket of cyclin-dependent kinases (CDK1

and CDK2) by means of van der Waals interactions and hydrogen bonding,

thereby inhibiting their role in cell cycle progression. This finding is the probable

mechanism through which the anti-leukemic properties of the herbal medicine

are seen. Although the exact pathway and mechanism of action is not clear, it is

becoming evident that indirubin(s) act to arrest cells and induce apoptosis by

means of CDK inhibition, making them a prospective anti-cancer compound.

The discovery of the cyclin-dependent kinase inhibitory activity of indirubin

peaked interest in the use and identification of indirubins that have high affinity

binding to CDKs, therefore becoming potential anti-cancer targets. Indirubin(s)

26

have been found to inhibit proliferation in many human cancer cell lines (Libnow

et al., 2008), including human tumor cells, through the arrest in G2/M phase by

blocking the action of CDK2, CDK1 and cyclin B kinase (Marko et al., 2000).

Recently, Kameswaran et al., (2008) showed that IO increased cell death and

apoptosis in human laryngeal carcinoma cells by inducing p21, a CDK inhibitor

protein and further by the inhibition of cyclin D1. Springs et al., (2006) showed

that Io reduced the p21 gene expression in human U937 cell line. The sudden

influx of the synthesis of various indirubin derivatives has led to numerous

reports of selective and potent inhibition of specific cancers. For example,

meisoindigo has been reported to inhibit microtubule assemblage and DNA

synthesis in tumor cells (Ji et al 1991) and decrease c-myb gene expression

thereby inducing leukemic cell differentiation (Liu et al., 1996). Indirubin-N’-

glycosides (Libnow et al., 2008) have showed great promise in arresting several

cell lines notably the breast cancer cell line MCF-7. Kim et al., (2007) reported

antitumor activity with 5’-nitro-indirubinozime, 5’-fluoro-indirubinoxime, and 5’

trimethylacetamino-indirubinoxime in a Sprague-Dawley rat tumor model.

Knockaert et al., (2004) showed that IO and various other derivatives,

including methylated indirubins, rapidly translocate the cytosolic AhR to the

nucleus. This was an interesting finding because the substituted methyl group

interferes with the molecules ability to bind to the cyclin-dependent kinase

catalytic site, and therefore is not an inhibitor of the kinase (Figure 3). This

suggests that some of the effects on cytotoxicity and cell cycle arrest are

independent of CDK inhibition and could be AhR-dependent. This is alarming

27

because most compounds that bind to the AhR, for example TCDD and PCB-126

are highly immunotoxic and carcinogenic (Hankinson, 1995 and Andersson et al

2002). However, it has been reported that aryl hydrocarbon receptor ligands can

have both antagonistic and agonistic effects on the AhR signaling pathway

(Nishiumi et al, 2008). Some ligands that bind to the receptor could induce

apoptosis and therefore cause growth inhibition in some tumors and cancer cells

(Bradshaw et al 2002, Damiens et al 2001, and Koliopanos et al 2002). The

alteration of this pathway in this manner could lead to another mechanism in

which indirubins could arrest the cell cycle and be beneficial pharmacological

agents.

In addition to IO’s role in halting the cell cycle and inducing apoptosis,

indirubins have been reported to be an anti-inflammatory agent. Hamburger et al

(2002) reported that leaf extracts from I. tinctoria, an indigo producing plant,

significantly inhibited cyclooxygenase-2 (COX-2) and iNOS in mast cells.

Recently a study using extracts as a topical cream on induced contact dermatitis

in humans showed significantly smaller increase of visual inflammation

(Heinemann et al., 2004). Kunikata et al (2000) showed a suppression of

interleukin 6 and interferon gamma inflammatory cytokines in mouse splenocytes

and human myelomonocytic HBL-28 cells, respectively. They also reported on

the inhibition of indirubin on induced delayed-type hypersensitivity as

demonstrated by 2,4,6-trinitro-1-chlorobenzene ear swelling in mice. These

studies suggest indirubin derivatives are potent anti-inflammatory compounds.

Sethi et al., (2006) showed that the inflammatory signaling pathway nuclear

28

factor kappa B (NF-kB) is inhibited with the treatment of indirubin through the

inhibition of IκB kinase and other phosphporylating events. A similar molecule

Indol-3-carbinol, a byproduct in plants and cruciferous vegetables, suppresses

NF-kB and IkB kinase activation, leading to the suppression of several gene

products including cyclin D1, COX-2, and anti-apoptotic factors (Takada et al.,

2005).

For the first time the effects indirubins have on gene expression, protein

expression, and the function of key components of the inflammatory response at

a cellular level are presented using a relevant RAW 246.7 macrophages in the

presence and absence of LPS.

29

Figure 2. The dual mechanisms in which indirubins may induce cell cycle arrest in dividing cells. (Knockaert et al., 2004)

30

Figure 3. Study by Knockaert et al. (2004) on indirubins in comparison with

TCDD on the interactions in two AhR reporter systems and two kinase assays. EC50 and IC 50 values were calculated from dose-response curves in µM amounts. * represents compounds inactive at the highest indicated concentration tested.

31

MATERIALS AND METHODS Cells and cell culture

The murine macrophage cell line RAW 264.7 was obtained from the

American Type Culture Collection (Manassas, VA) and maintained in Dulbecco’s

modified Eagle’s medium (DMEM; Cellgro) supplemented with 10% fetal bovine

serum (Atlas; Fort Collins CO), 10mM HEPES solution, 100mM L-glutamine

penicillin streptomycin solution, 3 g/L glucose, and 1.5 g/L of sodium bicarbonate,

each from Sigma (Sigma Aldrich, St. Louis MO, USA). Cells were grown at 37°C

with 5% CO2 in Corning 75cm2 culture flasks. Unless otherwise noted, each

experiment had two regimes. A 24 hour regime consisted of cells treated with

compound alone or simultaneously with compound and ultra pure

lipopolysaccharide (LPS) (Alexis Biochemical, San Diego CA, USA). A 48 hour

regime entailed treatment of cells with compound for 48 hours or with compound

and LPS for the last 24 hour of incubation.

Chemicals Specific indirubins and PCB congeners were selected based on the literature

and predicted structure-activity-relationships (SARs) relevant to AhR binding.

Compounds were first solubilized in DMSO (Sigma Aldrich) to a stock

concentration of 10-2 M and diluted to a final working concentration using

unsupplemented media.

IR: Indirubin (synthetic) (Alexis Biochemical, San Diego, CA)

IO: Indirubin-3’-monoxime (A.G. Scientific Inc, San Diego, CA)

32

E804: Indirubin-3’-(2,3 dihydroxypropyl)oximether (Alexis Biochemical,

San Diego, CA)

5Iodo: 5-Iodoindirubin-3’-monoxime (Alexis Biochemical, San Diego, CA)

PCB-126: 3,3',4,4',5-pentachlorobiphenyl (Ultra Scientific, North Kingston

RI, USA)

BIO: 6-Bromoindirubin-3’-oxime (GSK-1 Inhibitor X) (EMD Biosciences

Inc, San Diego, CA)

5-Iodo: Indirubin-5-sulphonate. Na (Alexis Biochemical, San Diego, CA)

PCB-104: 2,4,6,2',6'-pentachlorobiphenyl (Ultra Scientific, North Kingston,

RI)

DMSO: Dimethyl sulphoxide Hybri-Max® (Sigma Aldrich)

LPS: Lipopolysaccharide from E. coli, Serotype R515 (Re) (ultra pure,

TLR4grade™) (Alexis Biochemical, San Diego, CA)

PMA: Phorbol 12-myristate 13-acetate (A.G. Scientific, San Diego, CA)

Experiment: 1. Effective dosage and cell viability

Cytotoxicity – MTT assay

Cellular respiration as an indication of viability was measured by MTT (3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. RAW 264.7

macrophages and D74 rat glioma cells from the American Type Culture

Collection (Manassas, VA) were plated into 96 well Costar plates at 1x105 cells

per well in 100µl of DMEM media. After 4 hr incubation at 37°C for adherence,

compounds and DMSO carrier control were added in triplicate at a concentration

33

at serial dilutions of from 10-5 M/well. The cells incubated with compound for

either 24 or 48 hr. Four hours before termination of experiment each well

received 20µl of MTT at 5mg/ml in unsupplemented media. After centrifugation,

the supernatant for each well was discarded and the formazan solubilized with

100µl of acidified isopropanol. Following a brief period of shaking, the optical

density for each well was read at wavelength of 550λ on an uQuant microplate

reader (BioTek Instruments Inc). Data were expressed as percentage of the

control O.D. values for each experiment.

ApoScreen™ Annexin V Apoptosis Assay

Since the MTT assay quantify only cellular respiration and viability, and

not cell death due to apoptosis, apoptosis was quantified by measurement of

externalized phosphatidylserine residues as detected using an ApoScreen™

Annexin V Apoptosis Kit (Southern Biotech, AL, USA). Raw 264.7 murine

macrophages were seeded at 2.5 x 106 cell/well in 24 well plates for 3 hr for

adherence, and treated with 1µM indirubin-3’-monoxime, E804, or PCB-126

alone, or compound and LPS (1µg/ml) for 24 hr. Other compounds were not

further tested based on the MTT results. After treatment cells were washed with

ice-cold PBS and 500µl of Annexin V binding buffer. Binding buffer (100µl) was

added to all wells, along with 5µl of Annexin V-FITC was added, and the mixture

incubated for 15 min on ice in the dark. Without washing, 380µL of cold 1X

binding buffer and 10uL of PI were added to each. After washing the samples,

34

aliquots were processed by flow cytometer using FACScan™ (BD Biosciences,

San Jose, CA) and 10 000 cells were scored in each event.

Experiement: 2. Gene expression

rRT PCR Arrays for Stress & Toxicity

Murine macrophage RAW 264.7 cells were seeded at 5 X 106 cells/ml in

75 cm2 culture flasks (Corning Inc., NY) and allowed to adhere to flask surfaces

for 3 hours. Cells were then treated for 24 hr with 1µM of indirubin-3’-monoxime,

E804, and PCB-126 or compound and LPS. The adhered macrophages were

scraped from the flask surface using BD FalconTM cell scraper (BD Biosciences,

Bedford, MA) and harvested by centrifugation. TRI-reagent® was added (1ml) to

all cell pellets and the mixture was homogenized by gentle pippeting several

times. The homogenate was incubated at room temperature for 5 min and 0.2 ml

chloroform added. After incubating the mixture for 15 min, the samples were

centrifuged at 12,000 g for 15 min at 4°C. The aqueous phase was transferred to

a new 1.5 ml microcentrifuge tube precipitated with 0.5 ml isopropyl over a 10

min period, then centrifuged again at 12,000 rpm for 15 min and redissolved in

DEPC-treated water. After collecting 1mg RNA from each tube, Genomic DNA

contamination was eliminated by using Genomic DNA elimination mixture

(SuperArray Bioscience Corporation, Frederick, MD) and cDNA synthesis was

carried using the RT cocktail (SuperArray Bioscience Corporation, MD). Mouse

Oxidative Stress RT2 Profiler PCR Array and RT2 Real-Timer SyBR Green/qPCR

Master Mix were purchased from SuperArray Bioscience Corporation (Frederick,

35

MD). PCR was performed on BioRad iQ5 real-time PCR detection system

(Applied Biosystems). For data analysis the ∆∆Ct method was used; for each

gene fold-changes were calculated as difference in gene expression between

untreated controls and treated cell cultures. A positive value indicates gene up-

regulation and a negative value indicates gene down-regulation.

Experiment: 3 AhR activation

Translocation of AhR

Activation of the aryl hydrocarbon receptor (AhR) was determined by

fluorescent microscopy (Knockaert, et al., 2004). RAW 264.7 cells were grown in

minimum essential medium (Cellgro) without phenol red supplemented with 10%

FBS (Atlas; Fort Collins CO), 10mM HEPES solution, 100mM L-glutamine

penicillin streptomycin solution, 3 g/L glucose and 1.5 g/L of sodium bicarbonate

each from Sigma (Sigma Aldrich, St. Louis MO, USA). 100µl of macrophages

were seeded at 1 x 106 cells/ml in each well of an 8-chambered glass slide

(Fisher) and were allowed to adhere for 4 hours. Cells were treated for 4 hours

with 1µM of IO, E804, PCB126, and PCB104. Because the platform design could

accommodate an additional compound, cells were also treated with 1µM BIO.

The macrophages were fixed with 3% methanol free paraformaldehyde in HBSS

for 30 min followed by a brief 5 min treatment with 0.1% sodium borohydride to

quench auto-fluorescence. Cells were permeablized with 0.1% Triton X (Sigma)

in HBSS for 30min, then blocked with 3% Fraction V bovine serum albumin for 2

hours at room temperature in a humidified chamber. Polyclonal anti-AhR primary

36

antibody (Biomol Research Laboratories, Plymoth PA, USA 1:100) was

incubated overnight at 4°C rocking slowly. FITC conjugated secondary antibody

was added followed by DAPI labeling. Cells were mounted in 50% glycerol and

observed on a microscope equipped with a Canon camera on a Zeiss Axiovert

135 fluorescent microscope (Zeiss Gruppe, Germany).

Experiment: 4 Cellular functions

Nitric Oxide production

RAW 264.7 murine macrophages were seeded in 96 well COSTAR plates

at 1x105 cells per well in Minimum Essential Medium-alpha (MEM) without phenol

red (Gibco-Invitrogen), supplemented with 4µM L-Arginine. Cells were treated

with 1µM IO, BIO, E804, PCB-126 or compound and LPS. 100µl aliquots were

removed from the conditioned medium and incubated in 96 well plates at room

temperature for 15 min with an equal volume of Griess reagent (equal parts

Reagent 1; N-(1-naphthyl)ethylenediamine dihydrochloride (NED) and Reagent

2; Sulfanilamide) (Ding et al, 1988). The absorbance (OD) was measured using

an uQuant microplate reader (BioTek Instruments Inc) at 550nm. A standard

curve of known concentration of nitric oxide was made using sodium nitrite. The

values were averaged and then plotted as a stimulation index against the control

(Burleson et al 1995).

37

iNOS Western Blot

Western blot analysis for inducible nitric oxide synthase (iNOS), the

enzyme responsible for the production of NO, was determined from RAW

264.7cell lysates harvested after treatment with IO, BIO, E804, PCB-126 and

compound with LPS treated cells after 24 hr and 48 hr. The cells were lysed in

ice cold lysis buffer (50mM Tris, 150 mM NaCl, 1% IGEPAL, 0.1% SDS, pH8)

supplemented with HALT protease inhibitors (Pierce). The samples were

centrifuged at 15,000 for 10 min and the supernatant collected. Total protein was

measured using BCA protein reagent assay (Pierce, Rockford IL). Equal

concentration (30µl) of protein for each treatment were loaded on lanes of a 4-

20% SDS-PAGE criterion gel (Biorad) at 200 volts then transferred to a

nitrocellulose membrane (Millipore Corp, Billerica MA). After blocking with BSA

containing 0.01% gelatin, the membranes were probed with antibody specific for

iNOS (rabbit anti-iNOS, Signal Transduction Labs, Lexington KY: 1:500) followed

by goat anti rabbit secondary antibody (1:1000). The blots were then developed

with NitroBT/BCIP substrate solution.

Interleukin-6 ELISA

Interleukin-6 (IL-6) production by RAW 264.7 cell supernatant followed by

treatment was determined by a mouse READY-SET-GO IL-6 ELISA system

(eBioscience). Briefly, cells were seeded onto a 96 well plate and incubated with

compound in the presence or absence of LPS for 24hr or 48hr. One hundred µl

of supernatant were added to a precoated capture antibody Costar maxisorb

38

plate and incubated overnight at 4°C. Detection antibody was added followed by

treatment of avidin-HRP and a colorimetric substrate solution. A phosphoric acid

stop solution was added, and the plate read at 450nm on an uQuant microplate

reader (BioTek Instruments Inc). Concentrations of IL-6 were plotted against the

standard curve of known concentrations of mouse IL-6 supplied by the

manufacturer.

Measurement of ROS

The ability of indirubin(s) to either enhance total reactive oxygen species

(ROS) production or to modify PMA induced ROS was determined by

florescence resulting from the oxidation of dichlorodihydrofluorescein diacetate,

H2DCFDA (Molecular Probes, Eugene, OR) as described by Ding et al. (1988).

Briefly, cells were seeded onto black 96 well plates at 1 x 105 cells/well using

MEM without phenol red (Invitrogen). After treatment with compounds or

compounds with LPS for 24h or 48h, 22µl of 10-4 H2DCFDA was added to all

wells for 30 min. PMA (10-6M) was added to appropriate wells and incubated for

an additional 30 min before fluorescent 2’7’-dichlorofluorescin was measured on

a FLX-800 microplate fluorescence reader (BioTek Instruments, Inc) at excitation

485nm and emission 528nm (Mattson et al., 1995, revised by Henning et al.,

2002). Data was presented as RFU compared to their controls.

39

Phagocytosis

Phagocytosis was measured by a modification of previously described

methods (Bozzaro et al., 1987; Peracino et al., 1998). RAW264.7 cells were

added to 96-well round bottom plates at 1 x 105 cells per well in 100µl of

complete media. Cells then received 100µl of media containing 1µM Io, E804,

BIO, PCB-126, PCB-104 or carrier control in the presence or absence of LPS.

After treatments for 24 hr or 48 hr, cells were washed with PBS. Washed

fluorebrite carboxylate YG, 1 micron, microspheres (Polyscience, Inc Warrington

PA, USA) diluted in 50mM Na2HP04, were added to RAW 264.7 cells in the same

round bottom 96 well plates at a ratio of 100:1 (1 x 107 beads to 1 x 105 cells per

well), and centrifuged for 5 min for maximal contact. The plates were incubated

at 37°C for 45min or 90min. Extracellular microspheres (those beads that were

not phagocytosized) were cleared through extensive washes in a series of

centrifugations. Phagocytosis was stopped by the addition of 100µl of ice cold

PBS with 1mM CaCl2 and MgCl2. Plates were centrifuged, the well contents

removed by aspiration, and replaced with a Ca++/Mg** – free HBSS containing

0.01% trypsin. Cells were then transferred to an all-black 96 well plate. Trypan

blue (2mg/ml) dissolved in 20mM citrate and 150mM NaCl, pH 4.5, was added

for 5 minutes to quench extracellular fluorescence. Plates were then read on a

FLx-800 fluorescent spectrophotometer at an excitation wavelength of 485nm

and emission wavelength 528nm. Data were recorded as RFUs relative to the

control.

40

Lysozyme assay

Lysozyme activity was measured in RAW 264.7 cell lysates based on

procedures of Parry et al., (1965) and modified by Burton et al., (2002). Cells

were seeded onto COSTAR 6 well plates at 2 x 106 per well in DMEM complete

media and treated with 1µM IO, E804, PCB-126, PCB-104, or carrier control in

the presence or absence of LPS for 24 and 48 hr. Cells were then lysed and the

protein concentration normalized to 1mg/ml. One hundred µl of cell lysate, in

triplicate, were added onto 96 well plates followed by the addition of 200 µl of

2mg/ml of heat killed Micrococcus lysodietikus (MP Biomedicals, Solon, Ohio

USA) in 0.01 sodium phosphate buffer, pH 4.0. The plates were read at

wavelength 450nm every 5 min for 30minutes, then again at 10 minute intervals

for an additional 30 minutes. One unit of lysozyme activity was defined as a

decrease in absorbance of 0.001 min-1.

Bactericidal Assay

Intracellular killing of XL-1 Blue E.Coli (Stratagene, La Jolla, CA, USA) by

RAW 264.7 cells was measured using a novel approach based on Boleza et al.,

(2001) and communication with Stratagene. RAW 264.7 cells were seeded onto

96 well plates at a concentration of 1 x 105 cells/well under both the 24 hr and 48

hr treatment regimes with 1µM Io, BIO, E804, PCB-126 or carrier control (with

and without 1µM PMA). Bacteria were grown overnight in LB broth without

antibiotic, centrifuged and resuspended in sucrose-magnesium buffer (SM buffer)

to an O.D. of 0.5 at 600nm, which is equivalent to 4 x 108 cells (technical

41

sources from Stratagene). Bacteria were added to the pretreated cells at a ratio

of 25:1 and incubated for 180 minutes at 37°C. Extracellular bacteria were

washed away extensively with PBS, and residual bacteria killed by the addition of

20µl of 10000U Penicillin and 10g/mL steptomyocin solution (Sigma Aldrich) for

10 min. Cells were then permeablized with 0.1% Tween-20 in PBS for 15 min to

release the engulfed bacteria. Viable bacteria were grown up in LB broth for 12

hours at 37°C. The survival of bacterial was measured by a MTT assay. The

results are presented as the mean of percentage of control treated cells for three

experiments.

42

RESULTS

Cytotoxicity and effective dosage

In studies described herein, the anti- or pro-inflammatory effects of select

indirubin derivatives and reference PCBs were examined in the murine

macrophage cell line RAW 264.7. Cells were treated with compounds alone or

with LPS for 24 or 48 hours and endpoints relevant to gene expression,

transcription factor activation, and functional endpoints were measured. Since

the literature on indirubins suggests a pro-apoptotic effect in various cell lines,

cytotoxicity was first determined in an effort to find a suitable dose for

subsequent experiments. As indicated in Figure 4, RAW 264.7 cells are very

sensitive to indirubins, while the reference cell line D74, a rat glioma line, is

relatively resistant (Figure 5). Based on the MTT assay, exposure to levels less

than 1µM of tested compound were not cytotoxic in terms of affecting cellular

respiration. Therefore, 1µM was used for all subsequent experiments and

regimes to determine gene and protein expression, as well as functional cellular

assays.

To confirm that 1 uM was not only non-cytotoxic, but non-pro-apoptotic as

well, annexin-V binding was quantified. The flipping of phosatidylserine to the

outer cell membrane is indicative of early apoptosis and can be detected through

the binding and subsequent staining of annexin-V-FITC. As apoptosis continues

the cell membrane becomes degraded allowing molecules to enter the nuclear

membrane. In late apoptosis, cells that have lost membrane integrity can be

43

measured by propidium iodide staining of DNA. The results of annexin-V staining

coincide with the MTT assay results in that most of the cells after 24h exposure

to compounds (Figure 6) or compounds with LPS (Figure 7) are viable and not

labeling annexin-V.

QrRT PCR Arrays for Stress and Toxicity

RT2 Profiler™ PCR microarrays (SuperArray Bioscience Corporation,

Frederick, MD) for stress and toxicity profile the expression of 84 genes involved

in oxidative or metabolic stress, proliferation and carcinogenesis, growth arrest

and senescence, inflammation, and apoptosis. The Array was used to determine

the effect of indirubin-3’-monoxime, E804, and PCB-126 under the 24 hr regime

on gene expression in the absence or presence of LPS. The select functional

gene groupings are shown in Table 1. The corresponding tables (Table 2-11)

represent fold changes in gene expression compared to appropriate control

(cells alone, or cells treated with only LPS); a negative number indicates a down-

regulation, while a positive number is indicative of an induced gene.

A notable effect is seen in the expression of inflammatory genes. As

expected LPS activated macrophages show the increased expression of several

inflammatory genes (Table 2). Cells treated with indirubin-3’-monoxime alone

show a decrease in the expression of the NOS2, while E804 and PCB-126 both

down regulated chemokine ligand 10, TNFb, and Serpine1 compared to cells

treated without compound. In cells simultaneously activated with LPS (Table 3)

indirubin-3’monoxime and E804 shut down the expression of several

inflammatory genes, including IL-6, while PCB-126 did not.

44

Of interest in the proliferation and carcinogenesis gene groupings (Table

4) Indirubin-3’-monoxime and E804 decrease the expression of cyclin C and

cyclin D1, further E804 drastically inhibits the gene expression of E2F

transcription factor 1. The expression of proliferation and carcinogenesis genes

in macrophages activated with LPS consistently showed a decrease in most of

the genes tested (Table 5). The gene expression of growth arrest and

senescence, surprisingly, showed an overall decrease in regulation in both

compound alone and LPS activated cells, notably, p21 and p53, two important

genes in the regulation of progression of the cell cycle (Table 6 and Table 7).The

genes involved in apoptotic signaling varied in the expression results, all three

compounds showed an upregulation of caspase 1 and caspase 8 (Table 7) while

Bcl2-like 1 and NF-kB inhibitor were downregulated. The suppression of NF-kB

inhibitor, not surprisingly, due to its important role in many cellular functions,

Nfkb1 was induced in most treatment scenarios. LPS-activated macrophages

displayed an overall decrease in the expression of apoptotic genes, E804 and

PCB-126 showed a dramatic downregulation of Bcl211 (Table 8). However, E804

did induce Bax.

E804 was the only compound to show an increase in the expression of the

cytochrome P450 family of genes (Table 10) while heme oxygenase 1, an

inducible gene in response to oxidative stress, showed an increase in expression

in all compounds in all regimes (Table 11).

45

Translocation of AhR

To confirm the activation and subsequent translocation of the aryl

hydrocarbon receptor from the cytoplasm to the nucleus in RAW 246.7

macrophages, fluorescent microscopy was used. The derivatives IO (Figure 9),

BIO (Figure 10), and E804 (Figure 11) each translocated the AhR to the nucleus,

as well as the well documented AhR ligand PCB-126 (Figure 12). As expected,

the AhR in control treated cells (Figure 8) and PCB-104, a non-planar, non AhR-

binding PCB congener, (Figure 13) did not leave the cytoplasm and translocate

to the nucleus.

Measuring NO, IL-6, and ROS concentrations

To determine the effects of indirubins on key players of the inflammatory

response, several assays were employed. The measurement of nitrate, a stable

form of nitric oxide was achieved using Greiss reagent. E804 was the only

compound that significantly lowered the stimulation index for NO produced by

LPS-activated macrophages in both time regimes, (Figure 14 and Figure 15).

iNOS protein expression, as measured by SDS-PAGE and western blotting,

confirmed the NO production assay. iNOS protein concentrations were reduced

by exposure to E804 (Figure 16). E804 also significantly decreased the

expression of IL-6 (Figure 17 and Figure 18) in LPS activated cells. The 48 hr

regime showed a decrease in activity for all compounds exposed to activated

macrophages (Figure 18).

46

The measurement of intracellular ROS was determined by RAW 264.7

cells ability to cleave H2DCFDA. Most of the indole compounds showed an

increase in the production of ROS after 24h of exposure (Figure 19). However,

cells exposed to indirubin-3’-monoxime and BIO under the 48h regime showed a

decrease in ROS production (Figure 20).

Functional Immunological Endpoints

Three functional endpoints were selected due to their importance in

cellular immunocompetency. Cellular expression of lysozyme activity,

phagocytosis of fluorescent latex beads, and intracellular bactericidal assays

were selected and optimized. To determine lysozyme enzymatic activity the

ability for RAW 246.7 macrophage cell lysates to clear Micrococcus lysodietikus

was measured. In LPS-activated cells, E804 decreased the units of enzymatic

activity after the 24 hr of exposure (Figure 21). Further, after 48 hr (Figure 22)

cells treated with compound alone and with compound and LPS showed a

decrease in enzymatic activity.

The phagocytic ability of RAW264.7 macrophages to engulf 1 micron

latex beads after 45 minutes was hindered by all indole compounds tested

(Figure 23) but not as drastic after 90 minutes (Figure 24). These indirubins

inhibited intracellular killing of E. Coli in macrophages in the 24 hr regime not

activated by PMA (Figure 25). E804 was the only compound that showed

significant increase in the percentage of surviving bacteria (suppressed

intracellular killing) for the 48h regime.

47

Indirbubin-3-Monoxime

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

BIO

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

E804

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

5-Iodo

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

Indirubin-Sufonic Acid

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

PCB-126

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

PCB-104

0 2 4 6 8 100

20

40

60

80

100

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ival

DMSO

0 2 4 6 8 100

30

60

90

12024 HR

48 HR

Conc (uM)

Perc

en

t S

urv

ial

Figure 4. Cytotoxicity of indirubins, PCB-126, and PCB-104 to RAW 246.7 cells after treatment for 24 hours and 48 hours as determined by MTT assay. Data represents the percent survival means of three experiments +/- standard error. DMSO was included as carrier control.

48

Indirubin-3'-monoxime

0.0 2.5 5.0 7.5 10.0 12.50

25

50

75

100

125

Conc (uM)

Perc

ent S

urv

ival

BIO

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

E804

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

5-Iodo

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)P

erc

ent S

urv

ival

Indirubin-Sufonic Acid

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

PCB 126

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

PCB 104

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

TbT

0.0 2.5 5.0 7.5 10.0 12.50

20

40

60

80

100

120

Conc (uM)

Perc

ent S

urv

ival

Figure 5. Cytotoxicity of indirubins, PCB-126, and PCB-104 to D74 glioma cells after treatment for 24 hours as determined by MTT assay. Data represents the percent survival means of three experiments +/- standard error. Tributyltin chloride (TbT) was included as positive cellular toxicity control.

49

Figure 6. Effects of 1µM of compounds on apoptosis in RAW 246.7 macrophages as measured by flow cytometry with annexin V (x-axis) and propidium iodide (y-axis). I- Early apoptosis II-Late apoptosis III-Viable cells, not undergoing apoptotic events IV- Early apoptosis.

Control (Annexin V)

PCB-126 E804

Indirubin-3’-monoxime

I II

IV III

50

Figure 7. Effects of 1µM of compounds + LPS on apoptosis in RAW 246.7 macrophages as measured by flow cytometry with annexin V -labeling (x-axis) and propidium iodide-labeling (y-axis). I- Early apoptosis II-Late apoptosis III-

Viable cells, not undergoing apoptotic events IV- Early apoptosis.

LPS

E804 +LPS

Indirubin-3’-monoxime + LPS

PCB-126 + LPS

I II

IV III

51

Table 1. Functional Gene Groupings of the Mouse Stress and Toxicity Pathway Finder™ RT2 Profiler™ PCR Array.

Inflammation: Ccl21b, Ccl3 (MIP-1a), Ccl4 (MIP-1b), Csf2 (GM-CSF), Cxcl10 (Scyb10), Il1a, Il1b, Il6, Il18, Lta (TNF b), Mif, Nfkb1, Nos2 (iNOS), Serpine1 (PAI-1). Proliferation and Carcinogenesis: Ccnc (cyclin C), Ccnd1 (cyclin D1), Ccng1 (cyclin G), E2f1, Egr1, Pcna.

Growth Arrest and Senescence: Cdkn1a (p21Waf1/p21Cip1), Ddit3 (GADD153/CHOP), Gadd45a, Igfbp6, Mdm2, Trp53 (p53). Apoptosis: Anxa5 (annexin v), Bax, Bcl2l1 (bcl-x), Casp1 (Caspase1/ICE), Casp8 (caspase8/FLICE), Fasl (Tnfsf6), Nfkbia (ikBa/Mad3), Tnfrsf1a (TNFR1), Tnfsf10 (TRAIL), Tradd

Oxidative or Metabolic Stress: Cryab (a-Crystallin B), cytochrome p450 family, Ephx2, Fmo1, Fmo4, Fmo5, Gpx1 (glutathione peroxidase), Gpx2, Gsr (glutathione reductase), Gstm1 (glutathione S-transferase mu1), Gstm3, Hmox1, Hmox2, Mt2, Polr2k (Mt1a), Por, Sod1, Sod2.

52

Table 2. Altered inflammatory gene expression in RAW 264.7 macrophages treated for 24h with 1µM LPS, indirubin-3’-monoxime, E804, or PCB-126 as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and control treated cells.

GENE LPS IO E804 PCB126

Chemokine ligand 21b

1.03 -1.06 2.68 1.23

Chemokine ligand 3

7.17 1.01 4.81 -1.01

Chemokine ligand 4

190362 25710 633278.7 19791

Colony-Stimulating

factor 2

27.28 -1.06 2.68 1.23

Chemokine ligand 10

3.34 1.14 -72.65 -158.9

Interleukin 18 -1.4 -1.19 -1.16 -1.06

Interleukin 1 alpha

169.9 9.32 2.68 9.44

Interleukin 1 beta

35.45 -1.21 -2.29 1.05

Interleukin 6 67.89 -1.2 2.38 1.29

Tumor necrosis factor

beta

1.53 -1.35 -4.10 -2.2

Macrophage migration

inhibitory factor

350089 167864 4862.56 331157

Nuclear factor kappa B (p105)

16920 9244 6500 7706

Inducible nitric oxide synthase

10.19 -239.23 2.36 -1.55

Serpine 1 3.46 1.65 3.31 -2.35

53

Table 3. Altered inflammatory gene expression in RAW 264.7 macrophages treated simultaneously with 1µM LPS and indirubin-3’-monoxime, E804, or PCB-126 for 24h as determined by PCR array analysis. Data represents fold change difference in RFU between compound + LPS and LPS treated cells.

GENE LPS IO+LPS E804+LPS PCB126+LPS

Chemokine ligand 21b

1.03 -1.89 -1.47 1.13

Chemokine ligand 3

7.17 -1.67 -1.7 1.01

Chemokine ligand 4

190362.2 -1.31 -38.6 -1.21

Colony-Stimulating

factor 2

27.29 -2.83 -1.64 1.28

Chemokine ligand 10

3.34 -2.17 -928.16 1.16

Interleukin 18 -1.4 -2.06 -1669.83 1.17

Interleukin 1 alpha

169.9 -1.28 -14.75 -146.55

Interleukin 1 beta

35.45 -1.43 -1.89 1.5

Interleukin 6 67.89 -140.87 -1.94 1.46

Tumor necrosis factor

beta

1.53 -2.94 1.27 -1.47

Macrophage migration

inhibitory factor

350089.6 -3.47 -1.39 1.06

Nuclear factor kappa B (p105)

16920.76 -1.63 -1.48 -2

Inducible nitric oxide synthase

10.19 -1.27 -1.35 -1.16

Serpine 1 3.46 -1.51 -1.01 -1.34

54

Table 4. Altered proliferation and carcinogenesis gene expression in RAW 264.7 macrophages treated for 24h with 1µM LPS, indirubin-3’-monoxime, E804, or PCB-126 as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and control treated cells.


Cyclin C -1.21 -1509 -335.31 1.01

Cyclin D1 -1.51 -2366 -3.564.05 -1.06

Cyclin G1 -1.07 -15.34 7.2 1.4

E2f transcription factor 1

-1.18 1.09 -968.65 -1.62

Early growth response 1

8989 1208 2.68 3734

Proliferating cell nuclear antigen

15.98 4.91 -5 -1967

55

Table 5. Altered proliferation and carcinogenesis gene expression in RAW 264.7 macrophages treated simultaneously with1µM LPS and indirubin-3’-monoxime, E804, or PCB-126 for 24h as determined by PCR array analysis. Data represents the fold change difference in RFU between compound + LPS and LPS treated cells.


Cyclin C -1.21 -1385.62 -1072.65 -649.7

Cyclin D1 -1.51 -7792.47 1.02 -1.13

Cyclin G1 -1.07 -26.45 -11.9 -4197.59

E2f transcription

factor 1

-1.18 -1.55 -3201.7 -1.08

Early growth response 1

8989 -1.83 -1.81 1.26

Proliferating cell nuclear antigen

15.98 -8.46 -1.27 -1.06

56

Table 6. Altered growth arrest and senescence gene expression in RAW 264.7 macrophages treated for 24h with 1µM LPS, indirubin-3’-monoxime, E804, or PCB-126 as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and control treated cells.


Cyclin-dependent

kinase inhibitor 1a (p21)

-1.06 -4.26 -3.92 1.12

DNA-damage inducible

transcript 3 (Ddit3)

2.09 -56.39 -7.77 -1.2

Growth arrest and DNA-damage-

inducible 45 alpha

2.75 -22.54 -5.34 -1.2

Insulin-like growth factor

binding protein 6

1.01 -1.68 2.68 1.33

Transformed mouse 3T3 cell double minute 2

25458 10705 2.68 14380

p53 1.15 -5.93 -6387 -1.05

57

Table 7. Altered growth arrest and senescence gene expression in RAW 264.7 macrophages treated simultaneously with1µM LPS and indirubin-3’-monoxime, E804, or PCB-126 for 24h as determined by PCR array analysis. Data represents the fold change difference in RFU between compound + LPS and LPS treated cells.


Cyclin-dependent

kinase inhibitor 1a (p21)

-1.06 -3.34 1.13 1.09

DNA-damage inducible

transcript 3 (Ddit3)

2.09 -20.46 -2.3 1.19

Growth arrest and DNA-damage-

inducible 45 alpha

2.75 -9.07 -1.64 1.44

Insulin-like growth factor

binding protein 6

1.01 -1.89 -1.46 1.13

Transformed mouse 3T3 cell double minute 2

25458 -1.67 -1.95 -17.66

p53 1.15 -8.69 -1.35 -1.12

58

Table 8. Altered apoptosis gene expression in RAW 264.7 macrophages treated for 24h with1µM LPS, indirubin-3’-monoxime, E804, or PCB-126 as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and control treated cells.


Annexin A5 91288 94751 1733843 98147

Bcl2-associated X protein

-1.05 -1.22 3.95 -11926

Bcl2-like 1 2.55 -1364 -302.91 -1.55

Caspase 1 6462 2076 446703 1.33

Caspase 8 418.16 399 265.75 420

Fas ligand 1.01 -1.68 2.68 1.33

Nuclear factor kappa B inhibitor

2.88 -6.07 -13.53 1.3

Tumor necrosis factor receptor

1a

1.2 -1.32 -756 -2.3


10

2.13 2.61 2.68 4.6

TNFRSF1A-associated

death domain

-1.0 -1.1 5.74 1.38

59

Table 9. Altered apoptosis gene expression in RAW 264.7 macrophages treated simultaneously with 1µM LPS and indirubin-3’-monoxime, E804, or PCB-126 for 24h as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and LPS treated cells.


Annexin A5 91288 -1.12 -1.09 1.19

Bcl2-associated X protein

-1.05 1.04 -21897.7 -13263.5

Bcl2-like 1 2.55 -3879.33 -1.35 -1818.97

Caspase 1 6462 -3.25 -1.39 1.26

Caspase 8 418.16 -1.68 -1.37 1.06

Fas ligand 1.01 -1.89 -1.46 1.13

Nuclear factor kappa B inhibitor

2.88 -6.27 1.01 1.15


1a

1.2 -1.72 1.1 -2129.69


10

2.13 -1.88 -3.08 1.02

TNFRSF1A-associated

death domain

-1.0 -1.64 -1.5 1.13

60

Table 10. Altered oxidative and metabolic gene expression in RAW 264.7 macrophages treated for 24h with 1µM LPS, indirubin-3’-monoxime, E804, or PCB-126 as determined by PCR array analysis. Data represents the fold change difference in RFU between compound and control treated cells.

GENE LPS IO E804 PCB-126 Crystallin B

1.01 -1.68

2.68

1.33

Cytochrome P450 family

1.01 -1.68

2.68 1.33

Epoxide hydrolase 2

1.01 -1.68

2.68 1.33

Flavin containing monoxygenase 1

1.01 -1.68

2.68 1.33


5.23 -1.68

2.68 1.33


-1.75 -243.35

-54.06

1.38

Glutathione peroxidase 1

-1.37 -2.41

-32.58

1.09


-2.21 1.3 -3.42

-1.01

Glutathione reductase

-1.05 -23.22

2.25 -1.74

Glutathione S-transferase mu1

-1.15 -2281

-507

-1.51


1.01 -1.68

2.68 1.33

Heme oxygenase 1

1.01 2729

1582.53

1.33

Heme oxygenase 2

1.07 -2.28

1.10 1.33

Metallothionein 2

1.52 -1.27 -4.96 -1.02

Polymerase II polypeptide K

-1.21 -237

8.9

1.62

P450 oxidoreductase

1.29 -1.68

1.81

-1578

Superoxide dismutase 1

66465 312

323782

99398


2.05 -7.26

-457.67

-922

61

Table 11. Altered oxidative and metabolic stress gene expression in RAW 264.7 macrophages treated simultaneously with1µM LPS and indirubin-3’-monoxime, E804, or PCB-126 for 24h as determined by PCR array analysis. Data represents the fold change difference in RFU between compound + LPS and LPS treated cells.

GENE LPS IO + LPS E804 + LPS PCB-126 + LPS

Crystallin B

1.01 -1.89

-1.46

1.13

Cytochrome P450 family

1.01 -1.89

-1.46

1.13

Epoxide hydrolase 2

1.01 -1.89

-1.46

1.13


1.01 -1.89

-1.46

1.13


5.23 -9.79

-7.58

-1.19


-1.75 -155.48

-1.21

1.86


-1.37 -3.67

-1.48

-1.47


-2.21 1.49

-2.52

1.41

Glutathione reductase

-1.05 -3.51

1.84

1.84


-1.15 -2.17

-1.36

-1.18


1.01 -1.89

-1.46

1.13

Heme oxygenase 1

1.01 6529.36

6448.8

6798.29

Heme oxygenase 2

1.07 -2.51

-1.15

1.07

Metallothionein 2

1.52 -1.6

-1.55

225352

Polymerase II polypeptide K

-1.21 -326.06

-1.06

1.2

P450 oxidoreductase

1.29 -1.32

1.1

1.02


66465 -147.65

-1.49

-1.03


2.05 -6.77

-1.69

-2189.75

62

Figure 8. Nuclear translocation of AhR in RAW246.7 cells treated with media control for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light). Sale bar, 10µm.

A B

D C

10µm

63

Figure 9. Nuclear translocation of AhR in RAW246.7 cells treated with 1µM indirubin-3’-monoxime for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light).

A B

D C

64

Figure 10. Nuclear translocation of AhR in RAW246.7 cells treated with 1µM BIO for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light).

C D

B A

65

Figure 11. Nuclear translocation of AhR in RAW246.7 cells treated with 1µM E804 for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light).

A

D C

B

66

Figure 12. Nuclear translocation of AhR in RAW246.7 cells treated with 1µM PCB126, a positive AhR ligand control, for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light).

C D

B A

67

Figure 13. Nuclear translocation of AhR in RAW246.7 cells treated with 1µM PCB104, a non planar AhR ligand negative control, for 4 hours. DAPI was used to stain nuclear DNA. (A- AhR FITC staining, B- DNA DAPI staining, C- Merged AhR+DAPI images, D- white light).

C

B A

D

68

Indirubin-3'monoxime

Co 0.1µµµµM 1µµµµM LPS 0.1µµµµM 1µµµµM0

3

6

9

12S

tim

ula

tio

n U

nit

BIO

Co 0.1 µµµµM 1µµµµM LPS 0.1 µµµµM 1µµµµM0

3

6

9

12

Sti

mu

lati

on

Un

it

E804


3

6

9

12

**

Sti

mu

lati

on

Un

it

PCB-126


3

6

9

12

Sti

mu

lati

on

Un

it

Figure 14. Nitric oxide stimulation in RAW 264.7 cells treated for 24h with indirubins or PCB-126. White bars indicate the effect of compound alone on the macrophage model, while black bars indicate simultaneous treatment of compound and LPS. ** represents p ≤0.01.

69



5

10

15

20S

tim

ula

tio

n U

nit

BIO


5

10

15

20

Sti

mu

lati

on

Un

it

E804


5

10

15

20

*

Sti

mu

lati

on

Un

it

PCB-126


5

10

15

20

Sti

mu

lati

on

Un

it

Figure 15. Nitric oxide stimulation in RAW 264.7 cells treated for 48h with indirubins or PCB-126. White bars indicate the effect of compound alone on the macrophage model, while black bars indicate simultaneous treatment of compound and LPS. * represents p ≤0.05.

70

1 2 3 4 5 6 7 8 9 10

a 1 2 3 4 5 6 7 8 9 10

b

1 2 3 4 5 6 7 8 9 10

c

1 2 3 4 5 6 7 8 9 10

d Figure 16. iNOS protein expression in RAW 264.7 cells following exposure to 24h regime treatment (a) and 48h regime treatment (c). Cells were treated, lysed, and subjected to SDS-PAGE and probed for iNOS protein (a,c) or actin protein (b,d). Lane 1, control; lane 2, LPS control; lane 3, Io; lane 4, Io+ LPS; lane 5, BIO; lane 6, BIO + LPS; lane7, E804; lane 8, E804 +LPS, lane 9, PCB126; lane 10 PCB126 + LPS.

131kDa

42kDa

131kDa

42kDa

71



10

20

30** **

IL-6

mg

/ml

BIO


10

20

30 * *

IL-6

mg

/ml

E804


10

20

30

*

IL-6

mg

/ml

PCB-126


10

20

30IL

-6 m

g/m

l

Figure 17. The effects of indirubins and PCB-126 on the production of the inflammatory cytokine IL-6 on RAW 264.7 macrophages (white bars) and LPS-stimulated macrophages (black bars) treated with the 24h regime. Data represents the mean +/- the standard error. ** represents p ≤0.01, * represents p ≤0.05 compared to appropriate control.

72

Indirubin 3'-monoxime

Co 0.1 µµµµM 1µµµµM LPS 0.1 µµµµM 1µµµµM-10

0

10

20

30

40

50

60

** **

mg

/mL

BIO


0

10

20

30

40

50

60

** **

mg

/mL

E804


0

10

20

30

40

50

60

**

*

mg

/mL

PCB-126


0

10

20

30

40

50

60

**

mg

/mL

Figure 18. The effects of indirubins and PCB-126 on the production of the inflammatory cytokine IL-6 on RAW 264.7 macrophages (White bars) and LPS-stimulated macrophages (Black bars) treated with the 24h regime. Data represents the mean +/- the standard error. ** represents p ≤0.01, * represents p ≤0.05 compared to appropriate control.

73


CO .01µµµµM .1µµµµM 1µµµµM PMA .01µµµµM .1µµµµM 1µµµµM0

20

40

60

80

100

120 * *%

Co

ntr

ol

BIO


20

40

60

80

100

120

% C

on

tro

l

E804


20

40

60

80

100

120**

% C

on

tro

l

PCB-126


20

40

60

80

100

120

% C

on

tro

l

Figure 19. The effects of indirubins and PCB-126 on reactive oxygen intermediate production in RAW 246.7 macrophages (black bars) and PMA-stimulated macrophages (white bars) treated with the 24h regime with three different concentrations of compound. Data represents the mean +/- the standard error of the percent control cells treated without compound. ** represents p ≤0.01, * represents p ≤0.05 compared to appropriate control.

74

Indirubin-3'monoxime


20

40

60

80

100

120

* * *%

Co

ntr

ol

BIO


20

40

60

80

100

120

* * *

% C

on

tro

l

E804


20

40

60

80

100

120

% C

on

tro

l

PCB-126


20

40

60

80

100

120

% C

on

tro

l

Figure 20. The effects of indirubins and PCB-126 on reactive oxygen intermediate production in RAW 246.7 macrophages (white bars) and PMA-stimulated macrophages (black bars) treated with the 48h regime using three different concentrations of compound. Data represents the mean +/- the standard error of the percent control cells treated without compound. * represents p ≤0.05 compared to appropriate control.

75

Co Io BIO E804 1260

25

50

75

100

125

** ** **

45 min

1µµµµM Compound

Perc

en

t C

on

tro

l

45 min

LPS Io BIO E804 1260

25

50

75

100

125

** ** **

1µµµµM Compound + 1µµµµM LPS

Perc

en

t C

on

tro

l

90 min

Co Io BIO E804 1260

25

50

75

100

125

**

1µµµµM Compound

Perc

en

t C

on

tro

l

90 min


25

50

75

100

125

*

1µµµµM Compound + 1µΜµΜµΜµΜ LPS

Perc

en

t C

on

tro

l

Figure 21. The effects of 1µM indirubins and PCB-126 on phagocytosis incubated for 45min or 90min with FITC-labeled latex beads; treated under the 24h regime. Data represents the percent control mean from three experiments +/- the standard error. ** represents p ≤0.01, * represents p ≤0.05 as compared to appropriate control.

76

45min

Co Io BIO E804 1260

50

100

150

1µµµµM Compound

Perc

en

t C

on

tro

l

45min


50

100

150

1µµµµM Compound + 1 µµµµM LPS

Perc

en

t C

on

tro

l

90min

Co Io BIO E804 1260

50

100

150

*

1µµµµM Compound

Perc

en

t C

on

tro

l

90min


50

100

150

1µµµµM Compound + 1 µµµµM LPS

Perc

en

t C

on

tro

l

Figure 22. The effects of 1µM indirubins and PCB-126 on phagocytosis incubated for 45min or 90min with FITC-labeled latex beads; treated under the 48h regime. Data represents the percent control mean from three experiments +/- the standard error. * represents p ≤0.05 as compared to appropriate control.

77

Co LPS IO IO E804 E804 126 126 104 1040

20

40

60

80

** ** **

Lyti

c U

nit

s

Figure 23. The effects of 1µM indirubin-3’monoxime and E804 on lysozyme activity in RAW 246.7 macrophages (white bars) and LPS-activated macrophages (black bars) treated with the 24h regime. Data represents the means +/- standard error. ** represents p ≤ 0.01.

78

Co LPS IO IO E804 E804 126 126 104 1040

25

50

75

100

125

150

** **

**

Lyti

c U

nit

s

Figure 24. The effects of 1µM indirubin-3’monoxime and E804 on lysozyme activity in RAW 246.7 macrophages (white bars) and LPS-activated macrophages (black bars) treated with the 48h regime. Data represents the means +/- standard error. ** represents p ≤ 0.01, * represents p ≤ 0.05.

79

24h

Ecoli Co Io BIO E804 1260

100

200

300

* *

1µµµµM Compound

Perc

en

t C

on

tro

l

24h

Ecoli PMA Io BIO E804 1260

100

200

300*

1µµµµM Compound + 1µµµµM PMA

Perc

en

t C

on

tro

l

48h

Ecoli Co Io BIO E804 1260

100

200

300

1µµµµM Compound

Perc

en

t C

on

tro

l

48h

Ecoli PMA Io BIO E804 1260

100

200

300

*

1µµµµM Compound + 1µµµµM PMA

Perc

en

t C

on

tro

l

Figure 25. The effects of 1µM indirubins and PCB-126 on intracellular bacterial killing in RAW 264.7 macrophages under both 24h and 48h regimes. Data represents the mean of three experiments +/- standard error. * represents p ≤0.05 as compared to appropriate control.

80

DISCUSSION

Based on a large literature base that supports the utility of the murine

macrophage cell line, RAW 264.7, in the field of immunopharmcology, this cell

line was used to examine the effects of IO and E804, as well as BIO in some

cases, on macrophage inflammation-like functions. Because many of the

reported affects of these compounds on the biology of cells have been linked to

the AhR, this study also used the coplanar halogenated hydrocarbon PCB-126,

which is a potent AhR agonist. Moreover, when appropriate, the non-coplanar

congener PCB-104 was used as a negative control for PCB-126. This work

bridges the existing gap between the effects of treatments on alteration of gene

expression and cellular function in RAW 264.7 cells by indirubins.

As with previous work in this lab (Springs and Rice, 2006), concentrations

of 1 µM for each compound were not cytotoxic, even for up to 48 hr of treatment.

This turns out to be fortuitous because these compounds can be compared on a

molecule-to-molecule level, based on Avagadro’s number. One of the most

interesting early observations was that RAW 264.7 cells are far more sensitive to

indirubins than D74 glioma cells, suggesting that macrophages are more

sensitive than undifferentiated and anaplastic cell lines. Earlier work in this lab

used U937 cells, which are human histiocytes that can be differentiated into

macrophage-like cells via exposure to PMA. As with the RAW 264.7 cell line,

U937 cells seem to be more sensitive to indirubin toxicity as they become more

differentiated. Flow cytometry results also indicated that after 24h exposure to IO,

81

E804, PCB-126 and LPS most of the cells are not undergoing apoptosis and are

viable.

It has been well established that planar hydrocarbons are ligands for the

aryl hydrocarbon receptor (AhR). The potency of AhR signaling is dependent on

the affinity of the compound for the receptor. As a case in point, TCDD has the

highest known affinity, PCB-126 and other coplanar halogenated hydrocarbons

have a potency (affinity for the AhR) ranging from 1/10th to 1/1000th of TCDD

(Kafafi, et al., 1993). Most of the known and well characterized high affinity

ligands for the AhR are anthropogenic, but the affinity of natural (endogenous?)

compounds to the AhR is still uncertain. However, recently a reporter yeast

system showed that indirubin, a planar hydrocarbon, found naturally in plants,

mollusks, bacteria, and human urine binds to the AhR with an affinity higher than

TCDD (Adachi, et al., 2001). However, in whole-cell systems the potency of

indirubin is lower than what is seen in reporter systems.

As previously reported, indirubin-3’-monoxime and other derivatives are

able to activate the aryl hydrocarbon receptor, causing it to translocate to the

nucleus (Adachi et al., 2001; Spink et al., 2003; Knockaert et al., 2004; Springs

and Rice 2006) and promote the transcription of genes associated with the

xenobiotic response element. To date, all ligands that bind to the AhR induce the

drug metabolizing CYP1A-family of enzymes. Although gene expression did not

show an increase for the cytochrome P450 family of enzymes in cells exposed to

IO or PCB-126, there was a 2.68 fold increase when treated with E804 alone for

24 hours. This is not surprising because Springs et al. (2006) reported maximum

82

expression of CYP1A1 in undifferentiated U937 cells compared with low levels of

expression in fully differentiated cells also treated with LPS. Though much of the

historical attention has focused on indirubin, and it’s synthetic derivative IO, it is

clear from this study that E804 is extremely potent in terms of it’s affinity for the

AhR.

The induction of CYP genes by high affinity ligands to the AhR creates

oxidative stress within the cell due to leakage of oxygen radicals as the

monooxygenases consume oxygen (Whitlock 1993). In general, one of the most

severe toxic endpoints of strong AhR ligands is uncontrolled oxidative stress

(Radjendirane et al., 1999). Recently it was reported that AhR ligands interact

with the Nrf2 gene battery which encodes enzymes responsible for oxidative

stress (Kohle et al., 2007). Studies suggest that Nrf2 is a direct target gene for

the AhR (down-stream of the response element), and thus is transcribed along

with other members of the “gene cassette” that includes CYP1a1, Cyp1a2, and

Cyp1b1. Therefore, it is not surprising to see an increase in the expression of

heme oxygenase 1 (Hmox1) and superoxide dismutase 1 (Sod1) and other

stress related genes after exposure to IO, E804, and in PCB-126.

Both IO and E804 increased ROS production in RAW 264.7 cells after 24

hours, which is in line with Green et al. (2008) who, observed that the dioxin-like

PCBs induce Cyp1a1 and Cyp1b1 genes and intracellular production of ROS.

However, ROS production in IO and E804 treated cells was reduced after 48 hr,

indicating that several radical scavenging mechanisms were involved. Length of

time may be a factor, as activation of AhR also triggers the expression of aryl

83

hydrocarbon receptor repressor, AhRR. The repressor acts as a negative

feedback loop blocking the transcription of XRE genes by competitively inhibiting

the interaction with ARNT (Evans et al., 2008). Alternatively, and more likely, the

radical scavenging system was able to abate oxidative stress. As part of the

experimental design, cells were also treated with PMA for 30 minutes just before

measuring ROS production. In primary macrophages, and especially

neutrophils, PMA acts as a diacyl-glycerol (DAG) mimic and activates protein

kinase C (PKC). PKC then activates the membrane-associated NADPH-oxidase

system, which in turn converts molecular O2 to ROS such as superoxide anion.

Superoxide anion is further reduced to hydrogen peroxide by superoxide

dismutase (SOD). Since RAW 264.7 cells are not primary macrophages, they

may not possess the ability to respond to PMA in the same fashion, therefore

explaining why PMA-treatment did not result in large amounts of ROS

generation.

Activation and translocation of the AhR to the nucleus was seen within

four hours of exposure to IO, BIO, E804, and PCB-126. PCB-104 was used as a

negative halogenated hydrocarbon control; the non-planar (globular)

configuration of the molecule inhibits its binding with the receptor. Using

antibody-labeling for the AhR has become a gold standard for showing activation

and translocation following treatment (Knockaert, et al., 2004); however, this

technique alone does not show transcription factor binding to its particular

response element. Future studies with E804 and other experimental AhR ligands

should (and will) include ChiP assays and possibly electrophoretic mobility shift

84

assays (EMSAs). Still, as a screening mechanism, cytosolic vs nuclear staining

of the AhR is a quick and efficient method for identifying AhR ligands.

AhR signaling is not only involved in toxicity and oxidative stress, it plays a

role in several different pathways including cell cycle regulation, development,

and immunity (Marlowe et al., 2005). TCDD has been shown to decrease cyclin

D1 levels and CDK-dependent kinase activities (Wang et al., 1998), as have

other AhR ligands. The end effect of decreased cyclin D1 and CDK-dependent

kinases is cell cycle arrest (Reiners et al., 1999).

Hoessel et al. (1999) reported that indirubin is a cyclin-dependent kinase

inhibitor, leading to the arrest of the cell cycle by competitively binding to the ATP

catalytic site of the kinase. The interaction and concentration levels of cyclins and

cyclin-dependent kinases regulate the cell cycle progression in all eukaryotic cell

studies to date. In this study, Cyclin C, Cyclin D1, and Cyclin G were all inhibited

by the treatment of IO and E804, but treatment with PCB-126 alone did not. This

finding suggests that the halting of the cell cycle, at least in part, is independent

of AhR signaling and could be due to the regulation of cyclins and inhibition of

kinases.

The E2F family regulates the progression of the cell cycle, and there are

two subgroups divided into activating and repressing transcription factors. E2F1

is a major component in progressing the cell during the G1/S phase. The

hyperphosphorylated retinoblastoma (Rb) factor remains bound to E2F until

cyclin D/CDK4 releases its inhibition. This allows E2F to function as a positive

transcription factor increasing several factors needed to progress into the S

85

phase. Studies have indicated that the activated AhR can bind to Rb and

suppress E2F activity (Puga et al., 2000). A decrease in this key factor would halt

the cell cycle in the G1/S phase. E804 dramatically decreased the expression of

E2F1 gene, indicating that E804 might be a promising anti-cancer drug with an

array of mechanisms for halting the cell cycle.

The herbal remedy in which indirubin is the active ingredient, not only

showed promising results in terms of cancer and detoxification but also chronic

inflammation. It has been previously reported that indirubin inhibits the gene

expression of inducible nitric oxide and cyclooxygenase in mast cells (Hamburger

et al., 2002). Studies have also shown that indirubin suppresses IL-6 and IFN-γ

production in mouse splenocytes and human myelomonoctic cell lines, as well as

an inhibition on induced-delayed-type hypersensitivity in mice (Kunikata et al.,

2000). The study herein showed that expression of several inflammatory genes

was decreased in cells treated with IO and E804. Notably, NOS2 and IL-6 genes

were down-regulated. This corresponds well with the functional protein assays as

seen in nitric oxide (NO) and IL-6. NO is a product of iNOS that has an important

role in killing of intracellular bacteria and other pathogens. IL-6 is a key

inflammatory cytokine that is released from macrophages in response to antigen

activating the Toll-like receptor singling pathway production of several

inflammatory mediators. The gene expression for IL-6 in E804 treated cells is

induced after 24h of treatment, however the functional assay showed an

inhibition of IL-6 activity. This finding shows the importance of researching not

only gene expression but protein function as well. E804 inhibits the cytokine

86

cytoplasmic Stat signaling cascade of IL-6 by inhibiting Src kinase activity (Nam

et al., 2005). Therefore, although the gene expression of the cytokine may not

be suppressed, the down-stream signaling activity of the cytokine is modified. A

novel finding from this study indicates that E804 is a more potent inhibitor of

inflammatory functions and gene expression than its more famous IO

counterpart.

The NF-κB pathway is known as the central mediator of the innate

immune system. Most carcinogens and inflammatory agents activate NF-κB

through the degradation of its inhibitor IκB by IκB-kinase (IKK). It is then

translocated to the nucleus where it is involved in the expression of several gene

products including anti-apoptotic genes such as SURVIVIN, TRAF, BCL2, and

BCLxL, several inflammatory cytokines, and cell cycle genes including cyclin D1.

Sethi et al. (2006) reported that indirubin inhibits IKK degradation as well as the

phosporylation of p65, which is required for the translocation of NF-κB. The

study herein showed that IO and E804 had a dramatic effect on the expression of

several genes that are regulated by NF- κB including the antiapoptotic gene

BCLx, inflammatory cytokines IL-6, IL-1, and IL-18, as well as cell cycle

regulators Cyclin C and Cyclin D1. This suggests that the inhibition of NF-κB

signaling cascade could be the major mechanism by which these compounds

exert anti-inflammatory function.

The ability for RAW 264.7 macrophages to phagocytosize fluorescent

latex beads over a 45 min period was inhibited by all the indirubins. However,

after 90 min only BIO showed inhibition. It is evident from several previous

87

reports that AhR ligands inhibit phagocytosis in macrophages. van Grevenynghe

et al. (2003) reported that PAHs inhibit the macrophage’s ability to differentiate

into an active phagocytic macrophage in the presence of colony stimulating

factors. Others have shown that IO (Springs and Rice, 2006) and 3’-

methylcholantherene (Tewari et al., 1979) (a potent AhR ligand) suppress

phagocytosis in murine macrophages. The mechanisms in which indirubins and

other AhR ligands affect phagocytosis needs to be further examined.

Although indirubins may exert their effects through several diverse

pathways, it is becoming easier to develop indirubin derivatives with specific

activity towards one particular receptor or target. For example, brominated

indirubins bind with high affinity to GSK-3 (Meijer et al., 2003; Polychronopoulos

et al., 2004), while other derivatives bind to CDKs with higher affinity (Hoessel et

al., 1999; Moon et al., 2006). Recent studies show that methylated indirubins

have reduced binding to kinases, however they are potent ligands of the AhR

(Knockaert, et al., 2004). From a pharmacological standpoint, indirubins have

great potential for therapeutic applications by exploiting their selectivity towards a

particular kinase, receptor, or target, without the activation of other toxic

pathways. To date, however, none of the derivatives of indirubin lack AhR

binding activity, suggesting that the AhR may be a critical target, and have a

critical role in future anti-cancer and anti-inflammatory drug design approaches.

This is the first study that simultaneously examined the effects of multiple

indirubins on gene expression and corresponding cellular function in

88

macrophages. Of the indirubin derivatives examined, E804 was the most potent

in terms of biological effects.

In conclusion:

� IO, E804, and BIO bind to, and activate the AhR leading to it’s

translocation to the nucleus.

� IO and E804, but not PCB-126 suppressed the expression of genes

associated with cell cycling. E804 exposure drastically suppressed

expression of the E2f1 gene, indicating at least one mechanism of effect

that is independent of the AhR.

� E804 is a more potent inhibitor of inflammatory genes, proteins, and

cellular functions than was has been reported for indirubin or indirubin-3’-

monoxime.

� Indirubins affect normal cellular functions in RAW 264.7 cells in terms of

phagocytosis and intracellular killing.

� This investigation revealed that E804 has more significant effects and

clinical promise than its more famous counterpart indirubin-3’-monoxime.

� From a pharmacological standpoint, indirubins show great promise as

anti-cancer, anti-inflammatory, and detoxification compounds.

89

APPENDIX

Scatter plots of altered gene expression in RAW 264.7 macrophages. Data represents RFU fold changes compared to control.

Figure 25. Scatter plot of the effects of LPS on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

Mif

Ccl4

Nfkb1

IL1a

IL6

Csf2

IL1b

Serpine1

Cxcl10

Nos2

Ccl3

Lta

Ccl12b

IL18

Hsp90

Actb

Gapdh

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

90

Figure 26. Scatter plot of the effects of 1µM Io on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between Io and control treated cells.

Mif

Ccl4

Nfkb1

Il1a

Nos2

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

91

Figure 27. Scatter plot of the effects of 1µM E804 on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between E804 and control treated cells.

HOUSEKEEPING GENES

SUPPRESSED GENES

INDUCED GENES

Ccl4

Mif

Nos2

Serpine1

Ccl3

IL6

Ccl21bCsf2 IL1a

Cxcl10 Lta IL1b

92

Figure 28. Scatter plot of the effects of 1µM PCB-126 on RAW 264.7 macrophage inflammatory related gene expression. Data represents the RFU fold change between PCB-126 and control treated cells.

Cxcl10

SUPPRESSED GENES

INDUCED GENES

HOUSEKEEPING GENES

Mif

Ccl4

Nfkb1

IL1a

93

Figure 29. Scatter plot of the effects of 1µM Io + LPS on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between Io + LPS and LPS treated cells.

IL-6

Mif

IL18

Cxcl10

Csf2

Lta

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

94

Figure 30. Scatter plot of the effects of 1µM E804 + LPS on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between E804 + LPS and LPS treated cells.

Ccl4

IL18 Cxcl10

IL1a

IL6 *fold change -1.95

SUPPRESSED GENES

INDUCED GENES

HOUSEKEEPING GENES

95

Figure 31. Scatter plot of the effects of 1µM PCB-126 + LPS on RAW 264.7 macrophage inflammatory related gene expression. Data represents the fold change in RFU between PCB-126 + LPS and LPS treated cells.

IL1a

Nfkb1

HOUSEKEEPING GENES INDUCED

GENES

SUPPRESSED GENES

96

Figure 32. Scatter plot of the effects of LPS on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Gadph

Actb

Hsp90

Egr1

Pcna

Ccnc

E2f1

Ccng1

Ccnd1

97

Figure 33. Scatter plot of the effects of 1µM Io on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between Io and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Pcna

Egr1

Ccng1

Ccnd1

Ccnc

98

Figure 34. Scatter plot of the effects of 1µM E804 on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between E804 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Pcna

Ccng1

Ccnd1 E2f1 Ccnc

Egr1

99

Figure 35. Scatter plot of the effects of 1µM PCB-126 on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between PCB-126 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Egr1

Pcna

100

Figure 37. Scatter plot of the effects of 1µM Io + LPS on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between Io + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Ccnc Ccnd1

Ccng1

Pcna

101

Figure 38. Scatter plot of the effects of 1µM E804 + LPS on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between E804 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Ccnc E2f1

Ccng1

102

Figure 39. Scatter plot of the effects of 1µM PCB-126 + LPS on RAW 264.7 macrophage proliferation and carcinogenesis related gene expression. Data represents the fold change in RFU between PCB-126 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Ccnc Ccng1

103

Figure 40. Scatter plot of the effects of LPS on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES Hsp90 Actb

Gapdh

Mdm2

Trp53

Igfbp6

Cdkn1a

Gadd45a

Ddit3

104

Figure 41. Scatter plot of the effects of 1µM Io on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Mdm2

Trp53

Ddit

Gadd45a

Cdkn1a

105

Figure 42. Scatter plot of the effects of 1µM E804 on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between E804 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Mdm2, Igfbp6

Trp53

Cdkn1a Gadd45a

Ddit

106

Figure 43. Scatter plot of the effects of 1µM PCB-126 on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between PCB-126 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Mdm2

107

Figure 44. Scatter plot of the effects of 1µM Io + LPS on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between Io + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Cdkn1a

Ddit2 Trp53

Gadd45a

108

Figure 45. Scatter plot of the effects of 1µM E804 + LPS on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between E804 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Ddit2

109

Figure 46. Scatter plot of the effects of 1µM PCB-126 + LPS on RAW 264.7 macrophage growth arrest and senescence related gene expression. Data represents the fold change in RFU between PCB-126 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Mdm2

110

Figure 47. Scatter plot of the effects of LPS on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Chek2

Fasl

Tnfsf10

Casp8

Casp1

Anxa5

Bcl2l1

Nfkbia

Hsp90

Actb

Gapdh

Bax Tradd

Tnfrsf1a

111

Figure 48. Scatter plot of the effects of 1µM Io on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between Io and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Tnfsf10

Casp8

Casp1

Anxa5

Bcl2l1

Nfkbia

112

Figure 49. Scatter plot of the effects of 1µM E804 on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between E804 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Fasl Tnfsf10

Casp1

Anxa5

Bcl2l1

Nfkbi

Bax

Tradd

Tnfsf1a

Casp8

113

Figure 50. Scatter plot of the effects of 1µM PCB-126 on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between PCB-126 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Tnfsf10

Casp8

Anxa5

Bax

Tnfsf1a

114

Figure 51. Scatter plot of the effects of 1µM Io + LPS on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between Io + LPS and LPS treated cells.

INDUCED GENES

HOUSEKEEPING GENES

SUPPRESSED GENES

Bcl2l1

Casp1

Nfkbia

115

Figure 52. Scatter plot of the effects of 1µM E804 + LPS on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between E804 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Bax Tnfsf10

116

Figure 53. Scatter plot of the effects of 1µM PCB-126 + LPS on RAW 264.7 macrophage apoptotic related gene expression. Data represents the fold change in RFU between PCB-126 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Bax Bcl121 Tnfrsf1a

Gapdh

Actb

Hsp90

117

Figure 54. Scatter plot of the effects of LPS on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between LPS and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES Sod1

Fmo4 Gpx2

Cryab, Cyp’s Ephx2,Fmo1 Gstm3, Hmox1

Fmo5

Gstm1 Gsr

Por Sod2

Polr2k

Gapdh

Gpx1

Actb

Mt2 Hsp90

118

Figure 55. Scatter plot of the effects of 1µM Io on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between Io and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Hmox1

Sod1

Hmox2

Gpx1

Polr2k

Sod2

Gsr

Gstm1 Fmo5

119

Figure 56. Scatter plot of the effects of 1µM E804 on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between E804 and control treated cells.

Cryab, Cyp’s, Ephx2, Fmo1, Fmo4, Gstm3

SUPPRESSED GENES

INDUCED GENES

HOUSEKEEPING GENES

Gpx2 Fmo5 Sod2, Gstm1

Gpx1

Mt2

Sod1

Hmox1

Gsr

Pol2k

120

Figure 57. Scatter plot of the effects of 1µM PCB-126 on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between PCB-126 and control treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Sod1

Por Sod2

121

Figure 58. Scatter plot of the effects of 1µM Io + LPS on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between Io + LPS and LPS treated cells.

Hmox1

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Fmo4 Fmo5

Polr2k

Sod1 Sod2

Gsr Gstm1

Hmox2

Gpx1

122

Figure 59. Scatter plot of the effects of 1µM E804 + LPS on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between E804 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Hmox1

Fmo4

123

Figure 60. Scatter plot of the effects of 1µM PCB-126 + LPS on RAW 264.7 macrophage stress related gene expression. Data represents the fold change in RFU between PCB-126 + LPS and LPS treated cells.

INDUCED GENES

SUPPRESSED GENES

HOUSEKEEPING GENES

Hmox

Sod2 Mt2

124

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the effects of indirubin derivitives on gene expression

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