review article indirubin and indirubin derivatives for...

Review ArticleIndirubin and Indirubin Derivatives for CounteractingProliferative Diseases

Tina BlaDeviT1 Elke H Heiss1 Atanas G Atanasov1 Johannes M Breuss2

Verena M Dirsch1 and Pavel Uhrin2

1Department of Pharmacognosy University of Vienna 1090 Vienna Austria2Department of Vascular Biology andThrombosis Research Center for Physiology and Pharmacology Medical University of Vienna1090 Vienna Austria

Correspondence should be addressed to Pavel Uhrin paveluhrinmeduniwienacat

Received 3 June 2015 Revised 23 August 2015 Accepted 24 August 2015

Academic Editor Isabel Andujar

Copyright copy 2015 Tina Blazevic et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Indirubin is the active component of Danggui LonghuiWan a traditional Chinese medicine formulationThe encouraging clinicalresults from the 1980s obtained in chronic myelocytic leukemia patients treated with indirubin stimulated numerous studies onthis compound These investigations explored the use of indirubin in different types of cancer and reported the synthesis of novelderivatives with improved chemical and pharmacokinetic properties In this paper we review the impressive progress that has beenmade in elucidating themechanistic understanding of how indirubin and its derivatives affect physiological and pathophysiologicalprocesses mainly by inhibition of cell proliferation and induction of cell death Furthermore we survey the therapeutic use of thesecompounds in combating proliferative diseases such as cancer restenosis and psoriasis

1 Introduction

Historically natural products have been successfully usedin the management of a large number of human diseases[1] Natural products may also serve as a basis for syn-thesis of derivatives aiming to reduce toxic side effects toimprove their pharmacokinetic properties and to increasetheir efficacy [1ndash3]Themolecular targets andmechanisms ofthese substances in physiology or pathophysiology have beenelucidated only in the last decades

Indirubin is the active ingredient of Danggui LonghuiWan a traditional Chinese medicine containing plants suchas Indigofera tinctoria L and Isatis tinctoria L The interestin clinical use of indirubin was evoked in the 1980s inChina when medical doctors together with scientists startedtesting its clinical use for treatment of chronic myelocyticleukemia (CML) a slowly progressive disease characterizedby the overproduction of granulocytes [4ndash7] Over 50of the treated CML patients exhibited partial or completeremission [6 8 9] similar to the standard treatment using thecytostatic agent busulfan [8] Indirubin toxicity was low andthe side effects experienced by about half of the participants

comprised mild abdominal pain diarrhea and nausea [7] Inthree cases reversible pulmonary arterial hypertension andcardiac insufficiency were reported [10]

These encouraging results with CML stimulatedresearchers to explore the use of indirubin and its novelderivatives in other types of cancer as well as other diseases[11ndash14] Here we survey the outcome of these studies incombating proliferative diseases such as cancer pathologicalangiogenesis restenosis and psoriasis and discuss theunderliningmechanisms regarding how indirubin influencescellular signaling In the text we refer to natural indirubin aswell as its chemical derivatives as indirubins

2 Synthetic Indirubin Derivatives

Indirubin a stable red isomer of the blue indigo is chem-ically a 321015840-bisindole In many laboratories the structureof indirubin was employed as a skeleton for the synthesisof new derivatives with improved chemical and pharma-cological properties such as solubility and absorption Theantiproliferative effects of indirubins were mostly attributed

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 654098 12 pageshttpdxdoiorg1011552015654098

2 Evidence-Based Complementary and Alternative Medicine

to the inhibition of cell cycle-related kinases like cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3120573(GSK-3120573) and later on to other target proteins as describedfurther in the text New indirubin derivatives in additionto increased antiproliferative properties served as importanttools in revealing specific targets that mediate their cellulareffects

The first classes of synthesized indirubin-basedcompounds included alkylated halogenated and N- andO-substituted derivatives Among these N-ethyl-indir-ubin 5-halogen-indirubins andN-methylisoindigotin (meis-oindigo) demonstrated much higher antitumor potency inanimal models than natural indirubin [15ndash17] Indirubin-31015840-monoxime (a substance with numerous synonyms suchas indirubin-31015840-oxime indirubin-3-oxime indirubin-3-monoxime indirubinoxime and indirubin-31015840-monoxime)was synthesized in 1996 by Li and colleagues [18] and waslater found to inhibit CDKs amajor target of indirubins withhigher potency when compared to the parent compound[9] To date indirubin-31015840-monoxime has become one of themost studied synthetic indirubin derivatives This substancesimilar to some other newly synthesized indirubins (5-iodoindirubin-31015840-monoxime indirubin-5-sulfonic acidindirubin-5-sulfonamide and 5-halogenoindirubins) inhib-its efficiently at nanomolar range another indirubintarget the GSK-3120573 [19] Next using cocrystal structuresand modeling approaches novel and more potent CDKand GSK-3120573 inhibitors such as 6-bromoindirubin werediscovered [33] Thereafter 6-bromoindirubin-31015840-oximehaving higher potency towards GSK-3120573 was developed [20]Additionally novel 31015840-substituted 7-halogenoindirubinslacking the inhibitory effects towards CDKs and GSK-3120573 were generated which nevertheless still induced celldeath in a diversity of human tumor cell lines [21] Thisallowed researchers to point to the importance of othermechanisms mediating indirubin effects [21] The synthesisand characterization of several other derivatives such asE564 E728 and E804 demonstrated the inhibitory effect ofthese compounds towards signal transducers and activatorsof transcription 3 (STAT3) signaling thereby contributingto the induction of apoptosis in human cancer cells [22]STATs are transcription factors that transmit signals frommembrane receptors to the nucleus where they promote thetranscription of their target genes or play an important rolein regulating cell cycle progression and in apoptosis [34 35]

The study on the novel indirubin derivatives 5-nitro-indirubinoxime 5-fluoro-indirubinoxime and 5-trimeth-ylacetamino-indirubinoxime (originally [23] denoted incor-rectly as substituted at the five-prime position on theindirubin molecule) demonstrated their antitumor activityin vitro and in several animal models for cancer [23ndash27] while 5-nitro-indirubinoxime additionally exhibitedanti-inflammatory properties in human vascular endothe-lial cells [28] In comparison with other known indiru-bins 7-azaindirubin bearing a heterocyclic nitrogen atomat position C7 as well as its 31015840-oxime derivative showsreduced kinase inhibitory activity but nevertheless demon-strates high antiproliferative activity [29] Newly synthesized(di)azaindirubins exhibit a highly selective inhibitory activity

against casein kinase 2 as revealed by testing of a panelof more than 200 protein kinases [36] PHII-7 becamea promising potent derivative of indirubin with cytotoxiceffects in multidrug resistant cells which represent a majorobstacle for chemotherapy in many human malignancies[37] Indirubins carrying a 51015840-carboxylate moiety (such ascompound 7-bromo-51015840-carboxyindirubin-31015840-oxime) yieldeda novel inverse binding mode with improved selectivity forDYRK kinases that are implicated in alternative pre-mRNAsplicing and in pathologies associated with neurons includ-ing Alzheimerrsquos disease and Down syndrome [30] Finally5-diphenylacetamido-indirubin-31015840-oxime was described as anovel mitochondria-targeting agent with antileukemic activ-ities [31]

Due to the planarity of structure the developed hydrogenbonds and hydrophobic 120587-interactions and the rigid crystalstructure indirubins generally suffer from low water solu-bility [38] Achieving an increased though not excessivewater solubility without loss of bioactivity represents amajor goal in the synthesis of new indirubins [20] Tothis end attempts to intervene in the scaffold of indirubin(eg the conversion of 31015840-carboxyl group to an oxime)and to destroy the planarity of indirubins have been made[39] Alternatively chemical modifications by adding polarhydrophilic groups to the scaffold were accomplished asin the case of novel analogues of 6-bromo-indirubin-31015840-oxime [40] The latter substances were then successfullyapplied to alter circadian rhythm in cell cultures [40]Lately indirubin-5-carboxamides a novel class of indiru-bins carrying amide substituents with basic centers weregenerated Here quaternization or protonation of the alky-lamino substituents improved their solubility also withoutloss of bioactivity [41] The recently developed 51015840-OH-5-nitro-indirubin oxime (AGM130) had improved solubilitycompared to indirubin and was effectively applied to induceapoptosis of CML cells that were resistant to imatinib ablocker of the pathological BCRABL fusion protein [32]Additionally extensive series of more soluble halogenatedanalogues have been generated as reviewed elsewhere [1342] Also the structure of meisoindigo has beenmodified andthe derivatives have been tested in a panel of cancer cells [43]The substance (E)-1-(2-(4-methylpiperazin-1-yl)ethyl)-[331015840-biindolinylidene]-221015840-dione (5-4) was at least 40 times bettersoluble than meisoindigo and exhibited little or no tendencyto aggregate in solution and was capable of significantlyextending the lifespans of animals in aK562 tumor xenograftsmodel [43]

Besides the attempts to increase solubility of the indiru-bins studies aiming to increase their in vivo bioavail-ability have been performed These include for examplethe establishment of supersaturatable self-microemulsifyingdrug delivery system (S-SMEDDS) that improved indirubinrsquosrelease in vitro and its bioavailability in rats when orallyapplied [44] In another study use of a self-nanoemulsifyingdrug delivery system (SNEDDS) increased oral bioavailabil-ity of E804 by sim980 when compared with the aqueoussuspension [45]

Typical features of some of these above-mentionedindirubins as well as their structures are depicted in Table 1

Evidence-Based Complementary and Alternative Medicine 3

Table1Th

ecores

tructure

ofindirubin(R5=O

)with

listedstr

ucturesa

ndactio

nsof

selected

synthetic

indirubinderiv

ativesX

deno

tesh

alogen

atom

s(BrIC

landF)

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

Cores

tructure

ofindirubins

N HN

O

12

34

56

78

93998400

1998400

R6 5998400

R54998400

R2

3998400

9998400

6998400

7998400

8998400

2998400

R3

R4

R1

Indirubin

-H-H

-H-H

=O-H

Antitu

mor

effect

[4ndash10]

N-Ethyl-in

dirubin

5-halogeno

indirubins

N-M

ethylisoind

igotin

(meisoindigo)

-CH2CH3

-H-H

-H=O

-H

Highera

ntitu

mor

potencies

comparedto

indirubin

[15ndash17]

-H-X

-H-H

=O-H

NO

N HO

CH3

Indirubin-31015840-m

onoxim

e-H

-H-H

-H=N

OH

-HInhibitio

nof

CDKs

with

high

potency

[918]

5-Iodo

indirubin-31015840-

mon

oxim

e-H

-I-H

-H=N

OH

-H

Inhibitio

nof

GSK

-3120573

[19]

Indirubin-5-sulfo

nica

cid

-H-SO3H

-H-H

=O-H

Indirubin-5-sulfo

namide

-H-SO2NH2

-H-H

=O-H

5-Halogenoind

irubins

-H-X

-H-H

=O-H

C6andC5

C6halogen

substitofind

irubin

-H-H

or-X

-X-H

=O-H

Morep

otentC

DKandGSK

-3120573

inhibitors

[20]


Table1Con

tinued

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

31015840-Sub

stituted

7-halogeno

indirubins

-Hor

-CH3

-H-H

-X=N

OH=

NOCH3=

NOCO

CH3

andothers

-HLackingtheinh

ibito

ryeffects

towards

CDKs

andGSK

-3120573bu

tstillindu

cing

celldeath

[21]

E564

-H-H

-H-H

=NOCH2CH2OCH2CH2OH

-HInhibitory

effecttow

ards

STAT

3sig

nalin

gcontrib

utingto

apop

tosis

inhu

man

cancer

cells

[22]

E728

-H-O

CH3

-H-H

=NOH

-HE8

04-H

-H-H

-H=N

OCH2CH2CH

(OH)C

H2OH

-H5-Fluo

ro-in

dirubino

xime

-H-F

-H-H

=NOH

-HAntitu

mor

activ

ityin

vitro

andin

severalanimalmod

els

[23ndash27]

5-Trim

ethylacetamino-

indirubino

xime

-H-N

HCO

t Bu

-H-H

=NOH

-H

5-Nitro-indirubino

xime

-H-N

O2

-H-H

=NOH

-HAd

ditio

nalanti-infl

ammatory

prop

ertie

sinHUVEC

s[28]

7-Azaindirubin-31015840-oxime

N HN

H

N

O

N

HO

Potent

antip

roliferativep

roperties

incancer

celllin

esandinhibitio

nof

aserieso

fkinases

[29]

7-Brom

o-51015840-

carboxyind

irubin-31015840-oxime

-H-H

-H-Br

=NOH

-COOH

Novelinverseb

inding

mod

ewith

improved

selectivity

forD

YRK

kinases

[30]

5-Diphenylacetamido-

indirubin-31015840-oxime

-H-N

HCO

CH(C6H5) 2

-H-H

=NOH

-HNovelmito

chon

dria-ta

rgeting

agentw

ithantileukemicactiv

ity[31]

51015840-O

H-5-nitro-indirubin

oxim

e(AG

M130)

-H-N

O2

-H-H

=NOH

-OH

Improved

solubilitycomparedto

indirubinandeffectiv

eind

uctio

nof

apop

tosis

ofim

atinib-resistant

CMLcells

[32]


3 Proteins and Signaling PathwaysTargeted by Indirubins

The intensive research during the several last decades onindirubins revealed that these substances interact with differ-ent proteins and influence a number of signaling pathwaysThe main effects of indirubins include the inhibition of cellproliferation as well as induction of cell differentiation andcell death as surveyed below

31 Indirubin-Mediated Inhibition of Cell Proliferation andInduction of Differentiation The suppression of malignanttransformation in CML patients by indirubin reported in the1980s was originally attributed to DNA intercalation [17 46]as well as interference with microtubule polymerization [17]Only later the potent inhibition of CDKs and of GSK-3120573 was found to contribute mainly to the antiproliferativeeffects of indirubins [9 19] Besides these effects indirubinfrom Realgar-Indigo naturalis formula used for APL patienttreatment caused intensified ubiquitinationdegradation ofthe promyelocytic leukemia- (PML-) retinoic acid receptoralpha (RARalpha) oncoprotein [47]

CDKs are very important regulators of the eukaryoticcell cycle [48] Identification of CDKs as specific moleculartargets of indirubins was reported by the group of Meijer [9]Their work discovered the cocrystal structure of a complex ofCDK2 and indirubin-31015840-monoxime thereby demonstratingindirubin-31015840-monoxime interaction with the ATP-bindingsite of CDK2 In a later study indirubin-31015840-monoximepotently inhibited cell proliferation of amammary carcinomacell line (MCF-7) by arresting the cells in the G2M phase ofthe cell cycle possibly by inhibiting CDK1 [49] Furthermoreindirubin-31015840-oxime suppresses in a CDK2-dependent man-ner abnormal centriole duplication induced by the humanpapillomavirus in a human osteosarcoma cell line [50] Thecompound also increases the level of the CDK inhibitorp27Kip1 in a human neuroblastoma cell line (LA-N-1) andreduced the expression of CDK2 and cyclin E leading to cellcycle arrest at the G0G1 phase [51]

Cell cycle-inhibitory (cytostatic) activity of indirubinswas additionally attributed to the inhibition of GSK-3120573 [19]a regulatory serinethreonine kinase related to CDKs GSK-3120573 exhibits a plethora of cellular effects and is a therapeutictarget for the treatment of cancer chronic inflammationtype II diabetes and other diseases [52 53] Such inhibitionby 6-bromoindirubin acetoxime was important not only forsuppression of migration of adult gliomas [54] but alsofor inhibiting migration of pediatric glioma by instigatingcytoskeletal rearrangement [55]

Another mechanism for how indirubins contribute totheir cytostatic effects is the interaction with the aryl hydro-carbon receptor (AhR) [56] AhR plays an important rolein the cellular metabolism of xenobiotics and AhR signal-ing is considered as a promising drug target particularlyfor cancer inflammation and autoimmune diseases [57]Indirubin and indigo bind to AhR with high affinities [58]These compounds are present in normal human urine at02 nM concentration and at such physiological levels they

may activate AhR-mediated signaling mechanisms in vivo[59] The kinase-independent cytostatic effect of indiru-bins was demonstrated using 1-methyl-indirubins a sub-family of kinase-inactive indirubins and involved the AhR-mediated expression of a potent CDK2 inhibitor p27Kip1[56] In another study overexpression of the AhR in theabsence of exogenous ligands was found to rapidly disruptcentriole duplication control in breast cancer cells [60]Nonetheless the AhR agonist indirubin-31015840-oxime and akinase-inactive inhibitor 1-methyl-indirubin-31015840-oxime stillsignificantly reduce centriole overduplication stimulated byectopic AhR expression [60] The role of indirubins in AhRsignaling seems to be complex as indirubin activated AhRin human HaCaT and HepG2 cells with significantly higheralthough still transient potency as compared with dioxin theprototypical AhR ligand [61]

Finally indirubins may induce cell differentiation orinhibit cellular proliferation by suppression of protoonco-genes or by interference with growth factor signaling Forexample the indirubin derivative meisoindigo was shownin the 1990s to induce differentiation of ML-1 humanmyeloblastic leukemia cells and to downregulate c-mybgene expression [62] Indirubin-31015840-monoxime suppresses theautophosphorylation of fibroblast growth factor receptor 1(FGFR1) and by blocking receptor-mediated cell signal-ing also inhibits proliferation of NIH3T3 fibroblasts [63]Indirubin-31015840-monoxime further potently suppresses Notch1signaling in a manner that is dependent on GSK-3120573 but inde-pendent of proteosomal degradation [64] The compound 5-nitro-indirubinoxime (denoted incorrectly as substituted atthe five-prime position on the indirubin molecule) inhibitsthe beta1 integrinfocal adhesion kinaseAkt pathway andthereby decreases the metastatic ability of human head andneck cancer cells [26] Indirubin-31015840-monoxime in humanleukemic KBM-5 cells suppresses tumor necrosis factor-(TNF-) 120572-induced activation of NF-120581B a central transcrip-tion factor involved in the regulation of various inflammatorygenes as well as antiapoptotic genes [65 66] Although theroles of NF-120581B in different types of cancer are complex [67]these data indicate that the antiproliferative and the anti-inflammatory activity previously assigned to indirubin [28]may be mediated in part through the suppression of the NF-120581B pathway

32 Indirubin-Mediated Induction of Cell Death Additionalimportant hallmarks of the indirubin-related effects whichinclude induction of proapoptotic genes interference withmitochondrial function and STAT signaling leading to celldeath are surveyed below

Indirubin-5-nitro-31015840-monoxime in human lung cancercells induces apoptosis via p53- andmitochondria-dependentpathways [68] The compound additionally stimulates cellcycle arrest as well as apoptosis as shown with carci-noma cells This process was instigated after the release ofcytochrome c and the activation of caspases Also indirubin-31015840-monoxime causes apoptosis in human cervical hepatomaand colon cancer cells by inducing proapoptotic Bcl-2 fam-ily members Bid and Bax [69] as depicted in Figure 1


CDKsAhR GP Src p53

AhR

Cell cycleprogression

STAT

Cell survival

GlycogenolysisBid Bax

Cytochrome cCaspasecascade

Indirubins

Indirubins Indirubins

Nucleus

Nucleus

Cytosol

Cytosol

Membrane Membrane

Mitochondria

GSK-3120573

Mcl-1 survivin

p27kip1p27kip1

Figure 1 Cellular effects mediated by indirubins By direct inhibition of CDKs and GSK-3120573 and by the activation of AhR-mediatedupregulation of cell cycle inhibitor p27Kip1 indirubins inhibit cell cycle progression They also suppress cell survival by inducing caspase-mediated apoptosis upon interfering with mitochondrial function and affecting Src and STAT-mediated expression of cell survival proteinsFurthermore by inhibiting GP indirubins suppress glycogenolysis thus hampering the glucose supply of cancer cells These effects lead toinhibition of cell cycle progression and induction of cell death (rarr activationrelease perp inhibition)

Indirubin-31015840-oxime (synonym of indirubin-31015840-monoxime)further alters mitochondrial function in human neuroblas-toma cells by curtailing the mitochondrial membrane poten-tial and elevating the levels of reactive oxygen species [51]Moreover this substance induces apoptotic and autophagicdeath in acute lymphoblastic as well as chronic myelogenoushuman leukemia cells by a mechanism possibly involvingcaspase-3 [70]

An additional indirubin derivative 51015840-methoxyindirubininduces apoptosis in human neuroblastoma cells [71] Treat-ment of melanoma cells with 8-Rha-120573-indirubin enhancestheir sensitivity for death ligands overcoming their resis-tance to two important factors namely TNF-120572-relatedapoptosis-inducing ligand (TRAIL) and CD95 agonists[72] A similar drug-potentiation effect was observed forthe novel indirubin derivative PHII-7 that increased adri-amycin cytotoxicity (via inhibiting P-glycoprotein expres-sion) and cell apoptosis in MCF-7ADR human breast can-cer cells [37] Application of 6-bromo-indirubin-31015840-oximeeffectively abrogates cellular growth and induces apop-tosis in both breast cancer and bladder carcinoma celllines and prevents tumor cell resistance towards TRAIL[73] Finally 5-diphenylacetamido-indirubin-31015840-oxime wasrecently described as a novel mitochondria-targeting agentwith antileukemic activities that is able to increase theopening of mitochondrial permeability transition pores[31]

Inmany reported cases indirubin-induced apoptosis wascaused by an interference with STAT signaling For exampleindirubin derivatives E564 E728 and E804 potently blockconstitutive STAT3 signaling in human breast and prostatecancer cells [22] The compound E804 in addition directlyinhibits Src a kinase acting upstream STAT proteins andsuppresses the expression of antiapoptotic proteins Mcl-1and survivin [22] Importantly a subtoxic concentration of

E804 resensitized cancer treatment-resistant cells (MV4-11-R) by inhibiting STAT1 STAT3 and STAT5 signaling and byabolishing survivin expression [74] In human K562 chronicmyelogenous leukemia cells E804 potently suppresses STAT5signaling and induces apoptosis [75] E738 works as a noveldual inhibitor of STAT-activating kinases including Januskinases (JAKs) and Src family kinases (SFKs) thereby induc-ing apoptosis in humanpancreatic cancer cells [76] In humanmelanoma cells 6-bromoindirubin-31015840-oxime inhibits JAKand STAT3 signaling [77] and 7-bromoindirubin (designatedas MLS-2438) inhibits STAT3 and Akt signaling with bothderivatives inducing apoptosis [78]

Besides the ability to induce apoptosis indirubins alsoinduce necrosis Specifically indirubin-31015840-monoxime causesnecrosis in human breast carcinoma cell line [79] and 7-bromoindirubin-31015840-oxime instigates necrosis in humanTHP-1 macrophages and in a human mesothelioma cell line [80]

Finally indirubin-5-sulphonate allosterically inhibitsglycogen phosphorylase (GP) [81] This effect might beimportant for the depletion of glucose in cancer cells therebyhampering their survival

The main pathways of indirubin-mediated effects onproliferation and cell death are outlined in Figure 1

4 Indirubins CounteractingProliferative Diseases

In addition to the in vitro effects outlined above indirubinswere tested and their potency was shown in many preclinicalmodels These studies clearly showed the potential therapeu-tic use of indirubins for counteracting proliferative diseasessuch as cancer pathological angiogenesis restenosis andpsoriasis as reviewed in the following sections


5 Indirubins Thwart Cancer andPathological Angiogenesis

One of the first animal studies pointing to an efficacy ofindirubins in the treatment of cancer involved the applica-tion of various indirubins to skin xenografts Specificallydirect injection of indirubins into xenografts of RK3E-ras rat kidney epithelial cells harboring the k-ras genewas shown to reduce cancer in male Sprague-Dawley rats[23] In the aforementioned study several indirubins suchas 5-nitro-indirubinoxime 5-fluoro-indirubinoxime and5-trimethylacetamino-indirubinoxime were injected everyother day for 10 days and this significantly inhibited tumorgrowth and induced apoptosis [23] Indirubin-3-monoximewas applied to a murine benzo(120572)pyrene [B(120572)P]-inducedlung cancer model at a dose of 10mgkg for 5 d per weekand this resulted in a reduced adenocarcinoma growth due toapoptosis [82] Clinically treatment of a CML patient usingimatinib in combination with indirubin and meisoindigoresulted in complete cytogenetic response and the longestrecord of survival among CML patients in the literature[83]The isoindigo derivative natura-alpha (N-methyl-Δ331015840-dihydroindole-221015840-diketone) demonstrated both in vitro andin vivo effects In case of the former growth of both androgen-dependent (LNCaP) and androgen-independent (LNCaP-AI PC-3 and DU145) prostate cancer cells was inhibitedwhereas the compound inhibited androgen receptor signal-ing upon coinjection with human prostate cell lines in axenograft model using nude mice [84] Importantly natura-alpha also reduced tumor volume in a patient with hormone-refractory metastatic prostate cancer [84] Indirubin-31015840-monoxime also induced apoptosis in oral cancer cell linesin vitro through activation of cytochrome c and inhibitionof a number of factors including focal adhesion kinaseurokinase-type plasminogen inhibitor and matrix metallo-proteinaseWhen applied topically tomice in an adhesive gelindirubin-31015840-monoxime suppressed oral cancer through thedownregulation of survivin [85]

In addition to their ability to suppress primary tumourgrowth indirubins also suppress lung tumour metastasesas demonstrated by administration of 6-bromo-indirubin-31015840-oxime in a 4T1mousemodel for aggressive breast cancer [86]These findings were fully consistent with in vitro data show-ing that 6-bromo-indirubin-31015840-oxime inhibited adhesionmigration and invasion of a variety of metastatic cell typesThis was achieved by influencing the JAKSTAT3 signalingpathway and inhibiting several kinase cascades involved inthe metastasizing of cancer cells Such competence of indiru-bins to influence multilaterally several cellular signalingnetworks and metabolism strongly suggests that indirubinscould be used as drugs either alone or in combination withconventional antineoplastic agents to overcome multidrugresistance which represents a major impediment to theeffective therapy of many human malignancies [87]

The experimental evidence for the importance of induc-ing and sustaining angiogenesis the formation of new bloodvessels from existing ones for tumor growth is compelling[88 89] Pathological angiogenesis is a hallmark of cancer aswell as various ischaemic and inflammatory diseases [90ndash92]

Therefore the ability of indirubins to inhibit angiogenesis asoutlined below may significantly contribute to the inhibitionof tumor growth and metastasis

The antiangiogenic potential of indirubin-31015840-monoximewas discovered in vivo in an automated quantitative screeningassay using transgenic zebrafish embryos and confirmedin vitro by applying tube formation and proliferation inhuman umbilical vein endothelial cells HUVECs [93] Ina subsequent study indirubin-31015840-monoxime was appliedto endothelial cells which not only influenced prolifera-tion cell cycle apoptosis migration and tube formationbut also acted on AKT p38 ERK12 c-Src and GSK-3120573 kinases Indirubinrsquos scaffold was therefore suggested tobe promising for the development of novel antiangiogenicdrugs [94] Additionally the inhibitory effect of the par-ent compound indirubin on angiogenesis was visualized intransgenic zebrafish embryos and confirmed in endothelialcell cultures [95] 5-Nitro-indirubinoxime reduced angio-genesis primarily by inhibiting the expression of vascularendothelial growth factor VEGF [26] This was shown invivo using the chorioallantoic membrane (CAM) assay infertilized chicken eggs and in the cornea of mice [26]In ex vivo assays indirubin-31015840-monoxime was also ableto inhibit the number of microvessels growing from theaortic rings whereas in other in vivo tests using micethe compound induced neovascularization in matrigelplugs The molecular mechanisms underlying the antian-giogenic effects of indirubin-31015840-monoxime in endothelialcells depended at least partly on the downregulation of theactivation of VEGFR2 (vascular endothelial growth factorreceptor 2) [96 97] The in vivo inhibitory effect of E804on angiogenesis and tumor growth was shown in syngenicBalbc mice that were transplanted subcutaneously withcolon cancer cells Subsequently the intratumor injections ofE804 inhibited vascular growth of the inoculated allograftsas demonstrated by a decrease in both themicrovessel densitymarker CD31 and the proliferative indicator Ki-67 but also byan increase in the apoptosis index [98] The antiangiogeniceffect of E804 was further corroborated in vitro by inhibitionof proliferation migration and tube formation of HUVECsand in vivo by monitoring growth factor-induced neovesselformation in the matrigel plug model in mice [99] Thesegrowth factors included VEGF and basic fibroblast growthfactor (bFGF)Altogether these data clearly show the potencyof indirubins to suppress cancer growth in part by inhibitingpathological angiogenesis

6 Indirubins Counteracting Restenosis

Restenosis refers to the thickening of the arterial wall andis characterized not only by increased platelet and immunecell infiltration but also by increased migration and pro-liferation of vascular smooth muscle cells (VSMCs) intothe intimal layer Restenosis represents a serious clinicalproblem in patients undergoing balloon angioplasty andstenting and may result in life-threatening vessel occlu-sions [100 101] Studies on VSMC proliferation in vitroand experimentally induced neointima formation in vivo


revealed the inhibitory action of indirubin-31015840-monoxime onrestenosis In our previous work indirubin-31015840-monoximeinhibited VSMC proliferation induced by the BB isoform ofplatelet-derived growth factor (PDGF-BB) by arresting cellsin the G0G1 phase of the cell cycle Moreover the indiru-bin derivative specifically blocked the phosphorylation ofSTAT3 upon PDGF interferon-120574 and thrombin stimulation[102] In a murine femoral artery cuff model indirubin-31015840-monoxime prevented neointima formation while reducingboth STAT3 phosphorylation and the amount of proliferatingKi67-positive cells In addition this compound increasedendothelial nitric oxide production thereby potentially ame-liorating endothelial dysfunction [102] A further study onindirubin-31015840-monoxime demonstrated that it inhibits themigration of vascular smooth muscle cells after stimulationwith leukotriene or platelet-derived growth factor More-over the compound suppresses leukotriene biosynthesisin monocytes by direct inhibition of 5-lipoxygenase [103]Oral administration of indirubin-31015840-monoxime significantlyretarded occlusion in a rat carotid artery-injury model aswell as ADP- and collagen-induced platelet aggregationThis effect was due to the suppression of glycoprotein VI-mediated signaling pathways in platelets through blocking ofphospholipase C1205742 phosphorylation [104] A further com-pound 5-nitro-indirubinoxime (incorrectly designated asmodified at the five-prime position) was applied toHUVECsand this inhibited the TNF-120572-induced anti-inflammatoryresponse and the adhesion of U937 cells by decreasingthe expression of the cell adhesion molecules ICAM-1 andVCAM-1 [28] The above described inhibitory effects ofindirubins on VSMC proliferation platelet aggregation andactivation of endothelial cells showcase the potential use ofindirubins especially that of indirubin-31015840-monoxime as leadcompounds for preventing restenosis

7 Indirubins for Treating Psoriasis

Psoriasis is a common inflammatory skin disease affectingabout 2of the general population in thewesternworld [105]One of the hallmarks of this disease is an uncontrolled prolif-eration of keratinocytes which is accompanied by abnormaldifferentiation of cells [106 107]Mechanistic studies revealedthat in psoriatic skin lesions indirubin treatment interfereswith the proliferation and differentiation of keratinocytesunderscored by a decrease in proliferationmarker PCNA andan increase in the expression of protective involucrin [108]Indirubin additionally inhibits the activation of epidermalgrowth factor receptor (EGFR) as well as CDC25B geneexpression induced by epidermal growth factor (EGF) [109]In cultured human keratinocytes and in psoriatic lesionsindirubin also causes upregulation of claudin-1 which func-tions as major constituent of tight junction complexes thatregulate the permeability of epithelia [110] Application of 5-nitro-indirubinoxime (incorrectly [111] designated as mod-ified at the five-prime position) to mouse skin epidermalcells results in the inhibition of neoplastic cell transformationinduced by EGF or 12-O-tetradecanoylphorbol-13-acetate(TPA) Such treatment additionally inhibits a number of

activities induced by EGF or TPA including those of Raf-1MEK12 ERK12 JNK and c-Jun Also in a mouse modelof skin psoriasis local application of indirubin and of indigonaturalis was demonstrated to be effective and safe [112] Ina recent study indigo naturalis treatment was additionallyshown to be effective in treating nail psoriasis in humans acondition that is notoriously difficult to treat and which lacksstandardized therapeutic regimens [113]

8 Conclusions

The use of indirubin and its derivatives in several clinicalsettings has been patented as recently surveyed in anotherreview [38] and holds promise for future therapeutic appli-cations In addition to the traditional use of indirubin in thetreatment of CML indirubin and its numerous derivativeswere shown to be effective in counteracting many prolifer-ative diseases as demonstrated by cell culture studies andpreclinical models This review highlights the contemporaryprogress in elucidating the mechanisms of how indirubinsinfluence multiple molecular pathways leading to cell cycleinhibition and apoptosis Although the most pronouncedeffects of indirubin and its derivatives were seen in cancertreatment of psoriasis and restenosis may also benefit fromthis group of compounds

The Key Abbreviations Used

AhR Aryl hydrocarbon receptorbFGF Basic fibroblast growth factorCDK Cyclin-dependent kinaseCML Chronic myelocytic leukemiaeNO Endothelial nitric oxideEGF Epidermal growth factorEGFR Epidermal growth factor receptor (EGFR)FGFR1 Fibroblast growth factor receptor 1GP Glycogen phosphorylaseGSK-3120573 Glycogen synthase kinase-3120573HUVECs Human umbilical vein endothelial cellsICAM-1 Intercellular adhesion molecule 1PDGF Platelet-derived growth factorSTAT Signal transducers and activators of

transcriptionTNF-120572 Tumor necrosis factor-120572TPA 12-O-Tetradecanoylphorbol-13-acetateTRAIL TNF-120572-related apoptosis-inducing factor

ligandVCAM-1 Vascular cell adhesion molecule 1VEGF Vascular endothelial growth factorVEGFR2 Vascular endothelial growth factor

receptor 2VSMC Vascular smooth muscle cell

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgment

The authors thank Aner Gurvitz for his careful reading andediting of this paper

References

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[2] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011

[3] A G Atanasov B Waltenberger E M Pferschy-Wenzig etal ldquoDiscovery and resupply of pharmacologically active plant-derived natural products a reviewrdquo Biotechnology Advances2015

[4] ldquoClinical and experimental studies in the treatment of chronicgranulocytic leukemia with indirubinrdquo Zhonghua Nei Ke ZaZhi vol 18 no 2 pp 83ndash88 1979

[5] GYWuandFD Fang ldquoStudies on themechanismof indirubinaction in the treatment of chronic granulocytic leukemia IIEffects of indirubin on nucleic acid and protein synthesis inanimal transplantable tumor cells and normal proliferating cellsin vitro (authorrsquos transl)rdquo Zhongguo Yi Xue Ke Xue Yuan XueBao vol 2 no 2 pp 83ndash87 1980

[6] M Z Ma and B Y Yao ldquoProgress in indirubin treatment ofchronic myelocytic leukemiardquo Journal of Traditional ChineseMedicine vol 3 no 3 pp 245ndash248 1983

[7] Z Xiao Y Hao B Liu and L Qian ldquoIndirubin andmeisoindigoin the treatment of chronic myelogenous leukemia in ChinardquoLeukemia amp Lymphoma vol 43 no 9 pp 1763ndash1768 2002

[8] Z N Zhang E K Liu T L Zheng and D G Li ldquoTreatmentof chronic myelocytic leukemia (CML) by traditional Chinesemedicine and Western medicine alternatelyrdquo Journal of Tradi-tional Chinese Medicine vol 5 no 4 pp 246ndash248 1985

[9] R Hoessel S Leclerc J A Endicott et al ldquoIndirubin theactive constituent of aChinese antileukaemiamedicine inhibitscyclin-dependent kinasesrdquo Nature Cell Biology vol 1 no 1 pp60ndash67 1999

[10] S Jiang G Yu and J Cao ldquoAdverse effect of indirubin on thecardiovascular system a report of 3 casesrdquo Chinical Journal ofHematology vol 7 no 1 p 30 1986

[11] G Eisenbrand F Hippe S Jakobs and S Muehlbeyer ldquoMolec-ular mechanisms of indirubin and its derivatives novel anti-cancer molecules with their origin in traditional Chinese phy-tomedicinerdquo Journal of Cancer Research and Clinical Oncologyvol 130 no 11 pp 627ndash635 2004

[12] G Karapetyan K Chakrabarty M Hein and P Langer ldquoSyn-thesis and bioactivity of carbohydrate derivatives of indigo itsisomers and heteroanaloguesrdquo ChemMedChem vol 6 no 1 pp25ndash37 2011

[13] K Vougogiannopoulou and A L Skaltsounis ldquoFrom tyrianpurple to kinase modulators naturally halogenated indirubinsand synthetic analoguesrdquoPlantaMedica vol 78 no 14 pp 1515ndash1528 2012

[14] Y Wang P M Hoi J Y W Chan and S M Y Lee ldquoNewperspective on the dual functions of indirubins in cancertherapy and neuroprotectionrdquo Anti-Cancer Agents in MedicinalChemistry vol 14 no 9 pp 1213ndash1219 2014

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[20] P Polychronopoulos P Magiatis A L Skaltsounis et alldquoStructural basis for the synthesis of indirubins as potent andselective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinasesrdquo Journal of Medicinal Chemistry vol 47 no4 pp 935ndash946 2004

[21] Y Ferandin K Bettayeb M Kritsanida et al ldquo31015840-substituted 7-halogenoindirubins a new class of cell death inducing agentsrdquoJournal of Medicinal Chemistry vol 49 no 15 pp 4638ndash46492006

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[24] S A Kim S W Kim S Chang J H Yoon and S G Ahnldquo51015840-nitro-indirubinoxime induces G2M cell cycle arrest andapoptosis in human KB oral carcinoma cellsrdquo Cancer Lettersvol 274 no 1 pp 72ndash77 2009

[25] J H Yoon S A Kim S M Kwon et al ldquo51015840-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis insalivary gland adenocarcinoma cells through the inhibitionof Notch-1 signalingrdquo Biochimica et Biophysica ActamdashGeneralSubjects vol 1800 no 3 pp 352ndash358 2010

[26] S A Kim S M Kwon J A Kim K W Kang J H Yoon andS G Ahn ldquo51015840-Nitro-indirubinoxime an indirubin derivativesuppresses metastatic ability of human head and neck cancercells through the inhibition of Integrin 1205731FAKAkt signalingrdquoCancer Letters vol 306 no 2 pp 197ndash204 2011

[27] H E Yoon S A Kim H S Choi M Y Ahn J H Yoon and SGAhn ldquoInhibition of Plk1 andPin1 by 51015840-nitro-indirubinoximesuppresses human lung cancer cellsrdquoCancer Letters vol 316 no1 pp 97ndash104 2012

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[29] M Kritsanida P Magiatis A L Skaltsounis Y Peng P Li andL P Wennogle ldquoSynthesis and antiproliferative activity of 7-azaindirubin-31015840-oxime a 7-aza isostere of the natural indirubin


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[32] W S KimM J Lee D H Kim et al ldquo51015840-OH-5-nitro-Indirubinoxime (AGM130) an Indirubin derivative induces apoptosisof Imatinib-resistant chronicmyeloid leukemia cellsrdquo LeukemiaResearch vol 37 no 4 pp 427ndash433 2013

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[36] X Cheng K H Merz S Vatter J Christ S Wolfl and GEisenbrand ldquo771015840-diazaindirubinmdasha smallmolecule inhibitor ofcasein kinase 2 in vitro and in cellsrdquo Bioorganic and MedicinalChemistry vol 22 no 1 pp 247ndash255 2014

[37] R Shi W Li X Zhang et al ldquoA novel indirubin derivativePHII-7 potentiates adriamycin cytotoxicity via inhibiting P-glycoprotein expression in human breast cancer MCF-7ADRcellsrdquo European Journal of Pharmacology vol 669 no 1ndash3 pp38ndash44 2011

[38] N Gaboriaud-Kolar K Vougogiannopoulou and A Skaltsou-nis ldquoIndirubin derivatives a patent review (2010mdashpresent)rdquoExpert Opinion on Therapeutic Patents vol 25 no 5 pp 583ndash593 2015

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[41] X Cheng P Rasque S Vatter K H Merz and G Eisen-brand ldquoSynthesis and cytotoxicity of novel indirubin-5-carboxamidesrdquo Bioorganic amp Medicinal Chemistry vol 18 no12 pp 4509ndash4515 2010

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[63] Y Zhen V Soslashrensen Y Jin Z Suo and A WiedłochaldquoIndirubin-31015840-monoxime inhibits autophosphorylation ofFGFR1 and stimulates ERK12 activity via p38 MAPKrdquoOncogene vol 26 no 44 pp 6372ndash6385 2007

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[71] H Saito K Tabata S Hanada Y Kanda T Suzuki and SMiyairi ldquoSynthesis of methoxy- and bromo-substituted indiru-bins and their activities on apoptosis induction in human neu-roblastoma cellsrdquo Bioorganic and Medicinal Chemistry Lettersvol 21 no 18 pp 5370ndash5373 2011

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constitutively-activated Stat3 signaling associated with apopto-sis of human pancreatic cancer cellsrdquo Molecular Oncology vol7 no 3 pp 369ndash378 2013

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[79] E Damiens B Baratte D Marie G Eisenbrand and LMeijer ldquoAnti-mitotic properties of indirubin-31015840-monoxime aCDKGSK-3 inhibitor induction of endoreplication followingprophase arrestrdquoOncogene vol 20 no 29 pp 3786ndash3797 2001

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[81] M N Kosmopoulou D D Leonidas E D Chrysina etal ldquoBinding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogenphosphorylase b comparison with ligand binding to pCDK2-cyclin a complexrdquo European Journal of Biochemistry vol 271no 11 pp 2280ndash2290 2004

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[90] P Carmeliet and R K Jain ldquoAngiogenesis in cancer and otherdiseasesrdquo Nature vol 407 no 6801 pp 249ndash257 2000

[91] J Folkman ldquoRole of angiogenesis in tumor growth and metas-tasisrdquo Seminars in Oncology vol 29 supplement 16 no 6 pp15ndash18 2002

[92] DHanahan andRAWeinberg ldquoThehallmarks of cancerrdquoCellvol 100 no 1 pp 57ndash70 2000


[93] T C Tran B Sneed J Haider et al ldquoAutomated quantitativescreening assay for antiangiogenic compounds using transgeniczebrafishrdquoCancer Research vol 67 no 23 pp 11386ndash11392 2007

[94] S Zahler J Liebl R Furst and AM Vollmar ldquoAnti-angiogenicpotential of small molecular inhibitors of cyclin dependentkinases in vitrordquo Angiogenesis vol 13 no 3 pp 239ndash249 2010

[95] D Alex I K Lam Z Lin and S M Y Lee ldquoIndirubin showsanti-angiogenic activity in an in vivo zebrafish model and an invitro HUVEC modelrdquo Journal of Ethnopharmacology vol 131no 2 pp 242ndash247 2010

[96] X Zhang Y Song Y Wu et al ldquoIndirubin inhibits tumorgrowth by antitumor angiogenesis via blocking VEGFR2-mediated JAKSTAT3 signaling in endothelial cellrdquo Interna-tional Journal of Cancer vol 129 no 10 pp 2502ndash2511 2011

[97] J K Kim E K Shin Y H Kang and J H Y Park ldquoIndirubin-31015840-monoxime a derivative of a chinese antileukemia medicineinhibits angiogenesisrdquo Journal of Cellular Biochemistry vol 112no 5 pp 1384ndash1391 2011

[98] E K Shin and J K Kim ldquoIndirubin derivative E804 inhibitsangiogenesisrdquo BMC Cancer vol 12 article 164 2012

[99] Y K Chan H H Kwok L S Chan et al ldquoAn indirubinderivative E804 exhibits potent angiosuppressive activityrdquoBiochemical Pharmacology vol 83 no 5 pp 598ndash607 2012

[100] R Hoffmann and G S Mintz ldquoCoronary in-stent restenosismdashpredictors treatment and preventionrdquo European Heart Journalvol 21 no 21 pp 1739ndash1749 2000

[101] V Farooq B D Gogas and P W Serruys ldquoRestenosis delin-eating the numerous causes of drug-eluting stent restenosisrdquoCirculation Cardiovascular Interventions vol 4 no 2 pp 195ndash205 2011

[102] A V Schwaiberger E H Heiss M Cabaravdic et alldquoIndirubin-31015840-monoxime blocks vascular smooth muscle cellproliferation by inhibition of signal transducer and activator oftranscription 3 signaling and reduces neointima formation invivordquoArteriosclerosisThrombosis and Vascular Biology vol 30no 12 pp 2475ndash2481 2010

[103] T Blazevic A M Schaible K Weinhaupl et al ldquoIndirubin-31015840-monoxime exerts a dual mode of inhibition towardsleukotriene-mediated vascular smooth muscle cell migrationrdquoCardiovascular Research vol 101 no 3 pp 522ndash532 2014

[104] J J Lee J H Han S H Jung et al ldquoAntiplatelet action ofindirubin-31015840-monoxime through suppression of glycoproteinVI-mediated signal transduction a possible role for ERKsignaling in plateletsrdquoVascular Pharmacology vol 63 no 3 pp182ndash192 2014

[105] E Christophers ldquoPsoriasismdashepidemiology and clinical spec-trumrdquoClinical and Experimental Dermatology vol 26 no 4 pp314ndash320 2001

[106] M A Lowes A M Bowcock and J G Krueger ldquoPathogenesisand therapy of psoriasisrdquoNature vol 445 no 7130 pp 866ndash8732007

[107] F O Nestle D H Kaplan and J Barker ldquoPsoriasisrdquo The NewEngland Journal of Medicine vol 361 no 5 pp 444ndash509 2009

[108] Y K Lin Y L Leu S H Yang H W Chen C T Wang andJ H S Pang ldquoAnti-psoriatic effects of indigo naturalis on theproliferation and differentiation of keratinocytes with indirubinas the active componentrdquo Journal of Dermatological Science vol54 no 3 pp 168ndash174 2009

[109] W L Hsieh Y K Lin C N Tsai T M Wang T Y Chenand J H S Pang ldquoIndirubin an acting component of indigonaturalis inhibits EGFR activation and EGF-induced CDC25B

gene expression in epidermal keratinocytesrdquo Journal of Derma-tological Science vol 67 no 2 pp 140ndash146 2012

[110] Y K Lin H W Chen Y L Leu Y L Yang Y Fang andT L Hwang ldquoIndigo naturalis upregulates claudin-1 expres-sion in human keratinocytes and psoriatic lesionsrdquo Journal ofEthnopharmacology vol 145 no 2 pp 614ndash620 2013

[111] P Khanal H K Choi G M Namgoong et al ldquo51015840-nitro-indirubinoxime inhibits epidermal growth factor- and phorbolester-induced AP-1 activity and cell transformation throughinhibition of phosphorylation of Pin1rdquo Molecular Carcinogen-esis vol 50 no 12 pp 961ndash971 2011

[112] Y K Lin L C See Y H Huang et al ldquoComparison of refinedand crude indigo naturalis ointment in treating psoriasis ran-domized observer-blind controlled intrapatient trialrdquoArchivesof Dermatology vol 148 no 3 pp 397ndash400 2012

[113] Y K Lin L C See Y H Huang et al ldquoEfficacy and safety ofIndigo naturalis extract in oil (Lindioil) in treating nail pso-riasis a randomized observer-blind vehicle-controlled trialrdquoPhytomedicine vol 21 no 7 pp 1015ndash1020 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


to the inhibition of cell cycle-related kinases like cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3120573(GSK-3120573) and later on to other target proteins as describedfurther in the text New indirubin derivatives in additionto increased antiproliferative properties served as importanttools in revealing specific targets that mediate their cellulareffects

The first classes of synthesized indirubin-basedcompounds included alkylated halogenated and N- andO-substituted derivatives Among these N-ethyl-indir-ubin 5-halogen-indirubins andN-methylisoindigotin (meis-oindigo) demonstrated much higher antitumor potency inanimal models than natural indirubin [15ndash17] Indirubin-31015840-monoxime (a substance with numerous synonyms suchas indirubin-31015840-oxime indirubin-3-oxime indirubin-3-monoxime indirubinoxime and indirubin-31015840-monoxime)was synthesized in 1996 by Li and colleagues [18] and waslater found to inhibit CDKs amajor target of indirubins withhigher potency when compared to the parent compound[9] To date indirubin-31015840-monoxime has become one of themost studied synthetic indirubin derivatives This substancesimilar to some other newly synthesized indirubins (5-iodoindirubin-31015840-monoxime indirubin-5-sulfonic acidindirubin-5-sulfonamide and 5-halogenoindirubins) inhib-its efficiently at nanomolar range another indirubintarget the GSK-3120573 [19] Next using cocrystal structuresand modeling approaches novel and more potent CDKand GSK-3120573 inhibitors such as 6-bromoindirubin werediscovered [33] Thereafter 6-bromoindirubin-31015840-oximehaving higher potency towards GSK-3120573 was developed [20]Additionally novel 31015840-substituted 7-halogenoindirubinslacking the inhibitory effects towards CDKs and GSK-3120573 were generated which nevertheless still induced celldeath in a diversity of human tumor cell lines [21] Thisallowed researchers to point to the importance of othermechanisms mediating indirubin effects [21] The synthesisand characterization of several other derivatives such asE564 E728 and E804 demonstrated the inhibitory effect ofthese compounds towards signal transducers and activatorsof transcription 3 (STAT3) signaling thereby contributingto the induction of apoptosis in human cancer cells [22]STATs are transcription factors that transmit signals frommembrane receptors to the nucleus where they promote thetranscription of their target genes or play an important rolein regulating cell cycle progression and in apoptosis [34 35]

The study on the novel indirubin derivatives 5-nitro-indirubinoxime 5-fluoro-indirubinoxime and 5-trimeth-ylacetamino-indirubinoxime (originally [23] denoted incor-rectly as substituted at the five-prime position on theindirubin molecule) demonstrated their antitumor activityin vitro and in several animal models for cancer [23ndash27] while 5-nitro-indirubinoxime additionally exhibitedanti-inflammatory properties in human vascular endothe-lial cells [28] In comparison with other known indiru-bins 7-azaindirubin bearing a heterocyclic nitrogen atomat position C7 as well as its 31015840-oxime derivative showsreduced kinase inhibitory activity but nevertheless demon-strates high antiproliferative activity [29] Newly synthesized(di)azaindirubins exhibit a highly selective inhibitory activity

against casein kinase 2 as revealed by testing of a panelof more than 200 protein kinases [36] PHII-7 becamea promising potent derivative of indirubin with cytotoxiceffects in multidrug resistant cells which represent a majorobstacle for chemotherapy in many human malignancies[37] Indirubins carrying a 51015840-carboxylate moiety (such ascompound 7-bromo-51015840-carboxyindirubin-31015840-oxime) yieldeda novel inverse binding mode with improved selectivity forDYRK kinases that are implicated in alternative pre-mRNAsplicing and in pathologies associated with neurons includ-ing Alzheimerrsquos disease and Down syndrome [30] Finally5-diphenylacetamido-indirubin-31015840-oxime was described as anovel mitochondria-targeting agent with antileukemic activ-ities [31]

Due to the planarity of structure the developed hydrogenbonds and hydrophobic 120587-interactions and the rigid crystalstructure indirubins generally suffer from low water solu-bility [38] Achieving an increased though not excessivewater solubility without loss of bioactivity represents amajor goal in the synthesis of new indirubins [20] Tothis end attempts to intervene in the scaffold of indirubin(eg the conversion of 31015840-carboxyl group to an oxime)and to destroy the planarity of indirubins have been made[39] Alternatively chemical modifications by adding polarhydrophilic groups to the scaffold were accomplished asin the case of novel analogues of 6-bromo-indirubin-31015840-oxime [40] The latter substances were then successfullyapplied to alter circadian rhythm in cell cultures [40]Lately indirubin-5-carboxamides a novel class of indiru-bins carrying amide substituents with basic centers weregenerated Here quaternization or protonation of the alky-lamino substituents improved their solubility also withoutloss of bioactivity [41] The recently developed 51015840-OH-5-nitro-indirubin oxime (AGM130) had improved solubilitycompared to indirubin and was effectively applied to induceapoptosis of CML cells that were resistant to imatinib ablocker of the pathological BCRABL fusion protein [32]Additionally extensive series of more soluble halogenatedanalogues have been generated as reviewed elsewhere [1342] Also the structure of meisoindigo has beenmodified andthe derivatives have been tested in a panel of cancer cells [43]The substance (E)-1-(2-(4-methylpiperazin-1-yl)ethyl)-[331015840-biindolinylidene]-221015840-dione (5-4) was at least 40 times bettersoluble than meisoindigo and exhibited little or no tendencyto aggregate in solution and was capable of significantlyextending the lifespans of animals in aK562 tumor xenograftsmodel [43]

Besides the attempts to increase solubility of the indiru-bins studies aiming to increase their in vivo bioavail-ability have been performed These include for examplethe establishment of supersaturatable self-microemulsifyingdrug delivery system (S-SMEDDS) that improved indirubinrsquosrelease in vitro and its bioavailability in rats when orallyapplied [44] In another study use of a self-nanoemulsifyingdrug delivery system (SNEDDS) increased oral bioavailabil-ity of E804 by sim980 when compared with the aqueoussuspension [45]

Typical features of some of these above-mentionedindirubins as well as their structures are depicted in Table 1


Table1Th

ecores

tructure

ofindirubin(R5=O

)with

listedstr

ucturesa

ndactio

nsof

selected

synthetic

indirubinderiv

ativesX

deno

tesh

alogen

atom

s(BrIC

landF)

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

Cores

tructure

ofindirubins

N HN

O

12

34

56

78

93998400

1998400

R6 5998400

R54998400

R2

3998400

9998400

6998400

7998400

8998400

2998400

R3

R4

R1

Indirubin

-H-H

-H-H

=O-H

Antitu

mor

effect

[4ndash10]

N-Ethyl-in

dirubin

5-halogeno

indirubins

N-M

ethylisoind

igotin

(meisoindigo)

-CH2CH3

-H-H

-H=O

-H

Highera

ntitu

mor

potencies

comparedto

indirubin

[15ndash17]

-H-X

-H-H

=O-H

NO

N HO

CH3


onoxim

e-H

-H-H

-H=N

OH

-HInhibitio

nof

CDKs

with

high

potency

[918]

5-Iodo

indirubin-31015840-

mon

oxim

e-H

-I-H

-H=N

OH

-H

Inhibitio

nof

GSK

-3120573

[19]

Indirubin-5-sulfo

nica

cid

-H-SO3H

-H-H

=O-H

Indirubin-5-sulfo

namide

-H-SO2NH2

-H-H

=O-H

5-Halogenoind

irubins

-H-X

-H-H

=O-H

C6andC5

C6halogen

substitofind

irubin

-H-H

or-X

-X-H

=O-H

Morep

otentC

DKandGSK

-3120573

inhibitors

[20]


Table1Con

tinued

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

31015840-Sub

stituted

7-halogeno

indirubins

-Hor

-CH3

-H-H

-X=N

OH=

NOCH3=

NOCO

CH3

andothers

-HLackingtheinh

ibito

ryeffects

towards

CDKs

andGSK

-3120573bu

tstillindu

cing

celldeath

[21]

E564

-H-H

-H-H

=NOCH2CH2OCH2CH2OH

-HInhibitory

effecttow

ards

STAT

3sig

nalin

gcontrib

utingto

apop

tosis

inhu

man

cancer

cells

[22]

E728

-H-O

CH3

-H-H

=NOH

-HE8

04-H

-H-H

-H=N

OCH2CH2CH

(OH)C

H2OH

-H5-Fluo

ro-in

dirubino

xime

-H-F

-H-H

=NOH

-HAntitu

mor

activ

ityin

vitro

andin

severalanimalmod

els

[23ndash27]

5-Trim

ethylacetamino-

indirubino

xime

-H-N

HCO

t Bu

-H-H

=NOH

-H

5-Nitro-indirubino

xime

-H-N

O2

-H-H

=NOH

-HAd

ditio

nalanti-infl

ammatory

prop

ertie

sinHUVEC

s[28]


N HN

H

N

O

N

HO

Potent

antip

roliferativep

roperties

incancer

celllin

esandinhibitio

nof

aserieso

fkinases

[29]

7-Brom

o-51015840-

carboxyind


-H-H

-H-Br

=NOH

-COOH

Novelinverseb

inding

mod

ewith

improved

selectivity

forD

YRK

kinases

[30]



-H-N

HCO

CH(C6H5) 2

-H-H

=NOH

-HNovelmito

chon

dria-ta

rgeting

agentw


ity[31]

51015840-O

H-5-nitro-indirubin

oxim

e(AG

M130)

-H-N

O2

-H-H

=NOH

-OH

Improved



eind

uctio

nof

apop

tosis

ofim

atinib-resistant

CMLcells

[32]













CDKsAhR GP Src p53

AhR


STAT

Cell survival



Indirubins


Nucleus

Nucleus

Cytosol

Cytosol

Membrane Membrane

Mitochondria

GSK-3120573

Mcl-1 survivin

p27kip1p27kip1

























8 Conclusions










Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Th

ecores

tructure

ofindirubin(R5=O

)with

listedstr

ucturesa

ndactio

nsof

selected

synthetic

indirubinderiv

ativesX

deno

tesh

alogen

atom

s(BrIC

landF)

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

Cores

tructure

ofindirubins

N HN

O

12

34

56

78

93998400

1998400

R6 5998400

R54998400

R2

3998400

9998400

6998400

7998400

8998400

2998400

R3

R4

R1

Indirubin

-H-H

-H-H

=O-H

Antitu

mor

effect

[4ndash10]

N-Ethyl-in

dirubin

5-halogeno

indirubins

N-M

ethylisoind

igotin

(meisoindigo)

-CH2CH3

-H-H

-H=O

-H

Highera

ntitu

mor

potencies

comparedto

indirubin

[15ndash17]

-H-X

-H-H

=O-H

NO

N HO

CH3


onoxim

e-H

-H-H

-H=N

OH

-HInhibitio

nof

CDKs

with

high

potency

[918]

5-Iodo

indirubin-31015840-

mon

oxim

e-H

-I-H

-H=N

OH

-H

Inhibitio

nof

GSK

-3120573

[19]

Indirubin-5-sulfo

nica

cid

-H-SO3H

-H-H

=O-H

Indirubin-5-sulfo

namide

-H-SO2NH2

-H-H

=O-H

5-Halogenoind

irubins

-H-X

-H-H

=O-H

C6andC5

C6halogen

substitofind

irubin

-H-H

or-X

-X-H

=O-H

Morep

otentC

DKandGSK

-3120573

inhibitors

[20]


Table1Con

tinued

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

31015840-Sub

stituted

7-halogeno

indirubins

-Hor

-CH3

-H-H

-X=N

OH=

NOCH3=

NOCO

CH3

andothers

-HLackingtheinh

ibito

ryeffects

towards

CDKs

andGSK

-3120573bu

tstillindu

cing

celldeath

[21]

E564

-H-H

-H-H

=NOCH2CH2OCH2CH2OH

-HInhibitory

effecttow

ards

STAT

3sig

nalin

gcontrib

utingto

apop

tosis

inhu

man

cancer

cells

[22]

E728

-H-O

CH3

-H-H

=NOH

-HE8

04-H

-H-H

-H=N

OCH2CH2CH

(OH)C

H2OH

-H5-Fluo

ro-in

dirubino

xime

-H-F

-H-H

=NOH

-HAntitu

mor

activ

ityin

vitro

andin

severalanimalmod

els

[23ndash27]

5-Trim

ethylacetamino-

indirubino

xime

-H-N

HCO

t Bu

-H-H

=NOH

-H

5-Nitro-indirubino

xime

-H-N

O2

-H-H

=NOH

-HAd

ditio

nalanti-infl

ammatory

prop

ertie

sinHUVEC

s[28]


N HN

H

N

O

N

HO

Potent

antip

roliferativep

roperties

incancer

celllin

esandinhibitio

nof

aserieso

fkinases

[29]

7-Brom

o-51015840-

carboxyind


-H-H

-H-Br

=NOH

-COOH

Novelinverseb

inding

mod

ewith

improved

selectivity

forD

YRK

kinases

[30]



-H-N

HCO

CH(C6H5) 2

-H-H

=NOH

-HNovelmito

chon

dria-ta

rgeting

agentw


ity[31]

51015840-O

H-5-nitro-indirubin

oxim

e(AG

M130)

-H-N

O2

-H-H

=NOH

-OH

Improved



eind

uctio

nof

apop

tosis

ofim

atinib-resistant

CMLcells

[32]













CDKsAhR GP Src p53

AhR


STAT

Cell survival



Indirubins


Nucleus

Nucleus

Cytosol

Cytosol

Membrane Membrane

Mitochondria

GSK-3120573

Mcl-1 survivin

p27kip1p27kip1

























8 Conclusions










Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Con

tinued

Indirubinderiv

atives

R1R2

R3R4

R5R6

Maineffects

prop

ertie

sRe

ference

31015840-Sub

stituted

7-halogeno

indirubins

-Hor

-CH3

-H-H

-X=N

OH=

NOCH3=

NOCO

CH3

andothers

-HLackingtheinh

ibito

ryeffects

towards

CDKs

andGSK

-3120573bu

tstillindu

cing

celldeath

[21]

E564

-H-H

-H-H

=NOCH2CH2OCH2CH2OH

-HInhibitory

effecttow

ards

STAT

3sig

nalin

gcontrib

utingto

apop

tosis

inhu

man

cancer

cells

[22]

E728

-H-O

CH3

-H-H

=NOH

-HE8

04-H

-H-H

-H=N

OCH2CH2CH

(OH)C

H2OH

-H5-Fluo

ro-in

dirubino

xime

-H-F

-H-H

=NOH

-HAntitu

mor

activ

ityin

vitro

andin

severalanimalmod

els

[23ndash27]

5-Trim

ethylacetamino-

indirubino

xime

-H-N

HCO

t Bu

-H-H

=NOH

-H

5-Nitro-indirubino

xime

-H-N

O2

-H-H

=NOH

-HAd

ditio

nalanti-infl

ammatory

prop

ertie

sinHUVEC

s[28]


N HN

H

N

O

N

HO

Potent

antip

roliferativep

roperties

incancer

celllin

esandinhibitio

nof

aserieso

fkinases

[29]

7-Brom

o-51015840-

carboxyind


-H-H

-H-Br

=NOH

-COOH

Novelinverseb

inding

mod

ewith

improved

selectivity

forD

YRK

kinases

[30]



-H-N

HCO

CH(C6H5) 2

-H-H

=NOH

-HNovelmito

chon

dria-ta

rgeting

agentw


ity[31]

51015840-O

H-5-nitro-indirubin

oxim

e(AG

M130)

-H-N

O2

-H-H

=NOH

-OH

Improved



eind

uctio

nof

apop

tosis

ofim

atinib-resistant

CMLcells

[32]













CDKsAhR GP Src p53

AhR


STAT

Cell survival



Indirubins


Nucleus

Nucleus

Cytosol

Cytosol

Membrane Membrane

Mitochondria

GSK-3120573

Mcl-1 survivin

p27kip1p27kip1

























8 Conclusions










Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























CDKsAhR GP Src p53

AhR


STAT

Cell survival



Indirubins


Nucleus

Nucleus

Cytosol

Cytosol

Membrane Membrane

Mitochondria

GSK-3120573

Mcl-1 survivin

p27kip1p27kip1

























8 Conclusions










Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























8 Conclusions










Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgment


References






























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article indirubin and indirubin derivatives for...

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