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3. INTRODUCTION

Many of the key biological processes such as cell adhesion, molecular

trafficking and clearance, receptor activation, signal transduction, and

endocytosis are mediated by the cell surface glycans. Changes in the

glycosylation on the cell surface express numerous biomarkers which are

correlated with cell-cell interaction, differentiation, and in many diseased

conditions. This further demonstrates the involvement of glycans in multiple

disciplines including ontogeny, immunology, neurobiology, metabolism,

hematology, and cancer biology [Ohtsubo and Marth 2006]. The detailed studies

on the involvement of these altered glycans in many pathophysiological

conditions have provided a means of developing the pharmaceutical agents to

target these molecules for the improved treatment of many diseases including

cancer. Discovering new cancer specific glycans/alterations and understanding

them in detail for their use in cancer therapy is now becoming one of the

important aspects of cancer glycobiology. Molecules such as antibodies or

lectins which can decipher the information encoded in these glycans are useful

tools in cancer research. Lectins due to their specific sugar recognition property

are gaining attention of researchers in many fields of Life science. Recent

studies on the fungal lectins, attracted wide attention of researchers due to their

interesting biological properties and sugar specificities [Konaska 2006]. Lectins

from plants and mushrooms, which have specific affinity towards cancer

associated antigens, and with antitumor activity are finding application in cancer

research and some of them are under clinical studies [Liu et al. 2010; Fu et al.

2011; Khan and Khan 2011; Singh et al. 2010].

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The incidence of ovarian cancer is increasing every year and becoming

one of the leading causes of cancer death among the gynecologic malignancies

worldwide [Ferlay et al. 2010]. Most of the ovarian cancers are diagnosed at

advanced stage as it is asymptotic at early stages and at the time of diagnosis

most of the patients have a wide spread disease [van Dalen A et al. 2000]. The

epithelial ovarian cancer constitutes 80-90 % of ovarian malignancies amongst

the other tissue origins [Riman et al. 1998]. Although there are many

chemotherapeutic drugs which have shown high response rate after surgery but

patients have poor survivalance (25 %), which further decrease to 5 % when

patients detected at stage III and IV [Gupta and Lis 2009]. This demands the

need for nontoxic antitumor molecules for the treatment of many cancers

including ovarian cancer.

A variety of glycosylation changes are known to be involved in

important events of malignancies like transformation, invasion and metastasis

[Rudd et al. 2001]. The increased branching in the N-glycans is one of the

common glycosylation changes associated with cancer, mainly the increase in β-

1-6 branching results in the expression tri- and tetra-antennary oligosaccharides

with terminal sialylation [Dennis 1992; Dennis et al. 1987]. Expression of sialyl

Lewisa, sialyl Lewisx, and their isomers on the N- and O-linked oligosaccharides

are observed in various human malignancies that are involved in cancer

progression. Expression of these altered glycans in breast, bladder, lung, ovary

and colon cancer is correlated with poor prognosis and these changes are

associated with advanced forms of malignancies [Gorelik et al. 2001;

Powlesland et al. 2009; Nakamori et al. 1993; Davidson et al. 2000].

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Many tumor markers on epithelial ovarian cancer have been studied

which are known to express altered cancer associated glycans. CA125 is one of

the well studied ovarian tumor marker used for routine clinical diagnosis [Meyer

and Rustin 2000]. Cell surface expression and release of proteolytic fragments of

CA125 is observed during conversion of benign to malignant ovarian tumors.

Glycan analysis of the CA125 has revealed the expression of high mannose and

complex bisecting N-glycans [Wong et al. 2003].

Recently isolation of a mitogenic and immunostimulatory lectin from a

phytopathogenic fungus Rizhoctonia bataticola (RBL) has been reported from

our laboratory which exhibited complex sugar specificity when analyzed by

hapten inhibition studies. Glycan array analysis of RBL revealed the exclusive

specificity towards N-glycans, primarily recognizing high mannose, tri- and

tetra- antennary complex N-glycans, and also to tandem repeats of sialyl Lewis

antigen. The interaction of RBL has been studied with human ovarian cancer

cells PA-1 cells which are known to express these altered N-glycans. The results

showed that, RBL has a cytotoxic effect on PA-1 cells which can be effectively

blocked by competing glycoproteins [Nagre et al. 2010a; Pujari et al 2010].

In the present chapter we report the detailed signaling mechanism

involved in RBL-induced cell death of PA-1 cells in vitro, which is

demonstrated to be due to the induction of apoptosis through the activation of

intrinsic pathway.

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3.1. Materials and Methods

Bovine serum albumin (BSA), bovine submaxillarymucin, fetuin, Asialofetuin,

phenyl methyl sulfonyl fluoride (PMSF), nonidet P-40 (NP-40),

ethylenediaminetetra-acetic acid (EDTA), 2-mercaptoethanol, Triton X-100,

trypan blue, DAPI, formaldehyde, propidum iodide, Tetramethylrhodamine ethyl

ester (TMRE), DTT, sodium azide, bromophenol blue, trypan blue, N-[2-

hydroxy ethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES), formaldehyde,

CM-cellulose, Acrylamide, bis-acrylamide, Tris, sodium dodecyl sulphate

(SDS), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide],

and glycine were obtained from Sigma chemicals Co., St. Louis, USA, Isopropyl

alcohol, methanol, ethanol were from Himedia, India. Hybond poly vinylene

diflurodine (PVDF) membrane were obtained from GE Life Sciences (USA).

Protease inhibitor cocktail was from Roche (Germany). Sepharose-4B was from

Pharmacia. Caspase inhibitors- Z-VAD-FMK a pan-caspase inhibitor, Z-IETD-

FMK caspase-8 inhibitor and Z-LEHD-FMK caspase-9 inhibitor, mounting

medium, Annexin-V detection kit, and Tissue culture grade plastic-ware were

procured from BD Biosciences (USA). Antibodies against caspase-8, and active

caspase-3, cytochrome-c were from Epitomics (USA), Caspase-9 and PARP

were from PIERCE (USA), β-actin was from MP Biomedicals, USA. Species

specific HRP-labeled secondary antibodies were procured from BioRad (USA).

Active caspse-3 detection kit was procured from Calbiochem, USA. All other

chemical used were of analytical grade.

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3.1.1. Activation of Sepharose 4B and coupling of asialofetuin

Asialofetuin coupled Sepharose-4B required for the RBL purification was

prepared by activating Sepharose-4B and coupling with asialofetuin as described

by March et al [1974]. Briefly, Sepharose-4B (20 ml) was suspended in 2 M

sodium carbonate (1:1) for the activation and 0.05 volume of CNBr in

acetonitrile was added to the Sepharose 4B with constant stirring at room

temperature (RT). Change in the color from white to pale yellow indicates the

activation. The slurry was extensively washed with 0.1 M carbonate buffer (pH-

9.5) and resuspended in the binding buffer (0.2M carbonate buffer, pH 9.5).

Asialofetuin (10 mg/ml) in binding buffer was added to the slurry at 10 mg/ml of

sepharose and kept for coupling at 4 °C for 20 h with constant shaking.

Unreacted groups were masked by incubating the slurry with 1M glycine for 4 h.

After coupling the slurry was extensively washed with 0.1 M acetate buffer (pH

4.5), 2 M urea, and 0.1 M carbonate buffer (pH-10) containing 0.5 M sodium

chloride sequentially. Finally asialofetuin coupled Sepharose 4B was

resuspended in PBS and stored at 4° C until further use. Coupled protein was

estimated by Direct dye binding quantitative assay for solid-phase immobilized

protein as described by Martin Bonde et al [1992]. Briefly, the Sepharose

coupled asialofetuin was diluted with PBS (1:1) and incubated with 800 µl of

Bradford reagent. Sample was centrifuged and the absorbance of the supernatant

was measured at 465 nm (unreacted coomassie). The values were extrapolated

using the standard graph of BSA.

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3.1.2. Purification of RBL

RBL was purified from the fungal mycelia as described by Nagre et al (2010).

The following steps were used to purify RBL to homogeneity.

a. Extraction

R. bataticola cultures were grown in Byrde’s liquid synthetic media [Byrde et al.

1956] for 11 days, the mycelia mat was harvested, washed with distilled water

and freeze-dried and powdered in a glass mortar. Mycelial powder (10 g) was

suspended in extraction buffer (50 mM sodium acetate buffer, pH 4.3) and

sonicated briefly at 4 °C. The sample was kept for extraction at 4°C for

overnight on a magnetic stirrer. The extract was centrifuged (9,500×g, 30 min, at

4 °C), and supernatant was filtered using membrane filter (0.45 μm) and used as

crude extract.

b. Ion-exchange chromatography on CM cellulose

The clear filtrate was passed through a CM-cellulose column (20×1.5 cm)

equilibrated with extraction buffer and unbound fraction were washed using

same buffer. Adsorbed proteins were eluted using 50 mM sodium acetate buffer

pH 4.3 containing 500 mM NaCl. The fractions with highest OD (at 280 nm) and

hemagglutinating activity were pooled and kept for dialysis against PBS.

c. Affinity chromatography on asialofetuin-Sepharose 4B

The lectin was further purified by affinity chromatography on an asialofetuin-

Sepharose 4B column (10×1.3 cm) at 4 °C. The lectin was applied to the affinity

column equilibrated with PBS, the column was washed with PBS, and the

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affinity bound lectin was eluted using glycine-HCl buffer (100 mM, pH 2.0)

containing 500 mM NaCl. Fractions containing lectin activity were pooled,

dialyzed against PBS and stored at −20 °C for further studies.

3.1.3. Conjugation of RBL with FITC

FITC conjugated RBL used for confocal microscopy was prepared as described

in previous chapter by the method Goldman et al [1968].

3.1.4. Cell culture

Human ovarian cancer cell line PA-1 was procured from American Type Culture

Collection (ATCC Rockville, USA) and maintained in MEM (Gibco, BRL)

supplemented with 10% heat inactivated Fetal calf serum (FCS), 1mM

glutamine, 1 mM sodium pyruvate, 100mg/ml streptomycin and 100 units/ml

penicillin at 37°C in 5% CO2 and 95% humidified air.

3.1.5. Surface binding

The binding of RBL to the PA-1 cells has been shown earlier by flow cytometry

which revealed binding of RBL to more than 90 % of tested cells. The

expression and distribution of the RBL binding receptors on the cell surface was

determined by confocal microscopy. PA-1 cells were grown on cover slips in six

well plate for 24 h and washed with ice cold PBS. Non specific binding was

blocked by incubating with 3 % BSA for 1 h at 4 °C. Cells were washed with ice

cold PBS and then incubated with FITC-RBL (4 µg/ml) for 1 h at 4 °C.

Unbound lectin was washed with PBS. The cells were fixed with freshly

prepared 2 % para-formaldehyde for 10 min. After washing with PBS, cells were

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counter stained with DAPI for 30 min. The cover slips were mounted on slides

using mounting media and visualized using Confocal Laser Scanning

Microscope (Zeiss LSM 510, Germany) equipped with 488 nm and 560 nm

Argon lasers.

3.1.6. Localization of FITC-RBL in PA-1 cells

Localization of RBL in PA-1 cells was monitored at different time intervals

following its incubation. PA-1 cells grown on cover slips in six well plates were

incubated with FITC-RBL (4 µg/ml) in MEM with 0.5 % FCS for different time

intervals (1, 2, 3, and 6 h) at 37 °C in a humidified atmosphere. To observe

surface binding cells were incubated at 4 °C for 1 h (to block the internalization

of lectin). Cells were washed with PBS after each time point to remove excess

lectin and fixed in 2 % para formaldehyde for 10 min. Cells were washed and

counter stained with DAPI for 30 min at RT in dark. Cover slips were mounted

using mounting media on slides and visualized using confocal microscope.

3.1.7. Growth inhibitory studies

RBL induced cytotoxic effect was monitored by MTT assay. PA-1 cells were

seeded in a 96 well plate at 5x104 cells/ml and grown for 24 h before the lectin

treatment to study the dose and time dependent effect. Cells were treated with

RBL at different concentrations (1.25 to 10 µg/ml) in MEM with 0.5 % FCS for

10 h. Time dependent effect was studied using 5 µg/ml dose, PA-1 cells were

treated with RBL (5 µg/ml) for different time points (6 to 48 h) in a humidified

atmosphere (37 °C, 5 % CO2). After each time point 10 µl of MTT (5 mg/ml)

was added and cells were lysed using lysis buffer (10 % SDS with 0.01 N HCl)

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for 5-6 h at 37 °C. The absorbance was measured at 570 nm with a background

deduction at 640 nm. The percent viable cell number was calculated with respect

to controls considered 100 % viable.

3.1.7. Cell cycle analysis

The distribution of cells in different phases of cell cycle in response to RBL

treatment was determined by PI staining and analysis by flow cytometry. PA-1

cells were seeded at 2x105 cells/ml in a 6 well plate and incubated for 24 h

before lectin treatment. Cell were treated with or without RBL (5 µg/ml) for 3

and 6 h and harvested by gentle trypsinization. Cells were washed with PBS

twice and fixed in 70 % chilled ethanol for 30 min at 4 °C. Cells were rehydrated

by washing with PBS. Cells were treated with 50 μl ribonuclease A (5 mg/ml in

PBS, DNase free) to remove the RNA from the cells for 10 min at RT. Cells

were stained with propidium iodide (50 μg/ml in PBS) for 2 h at RT in dark. The

DNA content was analyzed on the FL-2A channel of Flow Cytometer (FACS

Calibur, BD) equipped with a 488 nm argon laser at linear scale were X-axis

represents fluorescent intensity and Y-axis represents number of events for cell

cycle analysis. The data was analyzed by Cell Quest Pro software for the

distribution of cells in different phases of cell cycle. Markers were applied to

differentiate the different phases of cell cycle (hypodiploid, G0/G1, S, and

G2/M) and compared between control and treated cells.

3.1.8. Detection of apoptosis by Annexin V staining

RBL induced apoptosis was quantified by using FITC-Annexin V labeling kit

(BD, Bioscience) and experiment was performed as per the manufacturer’s

86

instruction. Briefly, PA-1 cells were grown in 6 well plates at 2x105 cells/ml for

24 h before treating with RBL. Cells were treated with RBL (5 µg/ml) for 3 and

6 h in MEM with 0.5 % FCS in a CO2 incubator. After each time point the cells

were collected by gentle trypsinization and resuspended in binding buffer at a

concentration of 1x106 cells/ml. Cell suspension (100 μl) was incubated with 5

μl of FITC Annexin V and 5 μl propidium iodide for 15 min at room temperature

in dark. After incubation 400 μl of binding buffer was added to each tube and

analyzed by flow cytometry. The following controls are used to set up

compensation and quadrants- unstained cells, cells stained with FITC-Annexin V

(no PI) and cells stained with PI (no FITC-Annexin V). The percentages of cells

positive for Annexin V, PI alone and both Annexin V and PI were calculated by

dot blot analysis using Cell Quest Pro software.

3.1.9. Activation of caspase-3 in RBL treated PA-1 cells

Caspase-3 is an important executioner caspase which gets activated by different

pathway during apoptosis and upon activation it further cleaves many important

cellular proteins. RBL induced activation of caspase-3 was determined by the

FITC labeled caspase-3 inhibitor (FITC-DEVD-FMK) kit (Calbiochem) which

specifically recognizes active caspase-3 and can be analyzed by flow cytometry.

PA-1 cells (2x105 cells/ml) were grown for 24 h in a 6 well plate before treating

with lectin. Cells were treated with RBL (5 µg/ml) in MEM with 0.5 % FCS for

3 and 6 h and harvested by gentle trypsinization. Cells were washed with

washing buffer and resuspended in 500 µl of binding buffer and incubated with 5

µl of FITC-DEVD-FMK for 30 min in dark at RT. The data was acquired for

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10,000 events on BD FACS Calibur cytometer (Becton Dickinson, San Jose,

CA) and analyzed using cell quest-pro software. The following controls were

used to compare the positive shift in the treated cells; untreated cells without

FITC-DEVD-FMK, untreated with caspase-3 inhibitor, treated without caspase-

3 inhibitor. The results were presented as histograms.

3.1.10. Role of caspases in RBL induced cell death

The activation of caspases-3 is initiated by two important pathways; receptor

mediated (extrinsic) caspase-8 activation followed by the activation of caspase-

3, and the release of cytochrome-c followed by the cleavage of caspase-9 which

further activates caspase-3 (intrinsic). RBL induced cell death was protected

using specific inhibitors for different caspases and viability was determined by

MTT assay. PA-1 cells were seeded in 96 well plate at 5000 cells/well grown for

24 h. Cells were preincubated with the specific inhibitors for caspase-8 (z-IETD-

FMK), caspase-9 (z-LEHD-FMK) or pan-caspase (z-VAD-FMK – inhibitor for

caspase-3/7) for 1 h followed by treatment of RBL (5 µg/ml) for 12 h. At the end

of 12 h cell viability was measured by MTT assay as described above. Cells

treated with only RBL and only caspase inhibitors were used as controls to

compare the results.

3.1.11. Determination of Mitochondrial Membrane Potential (MMP)

The loss of mitochondrial membrane potential in RBL treated cells was

determined by the uptake of tetra methyl rhodamine ethylester (TMRE) dye. The

activation of intrinsic apoptotic pathway initiates with the binding of pro-

apoptotic proteins like Bid to the mitochondrial membrane. These anti apoptotic

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proteins may alter the mitochondrial membrane potential by directly forming the

pores in membrane, through which proteins and other molecules present in the

interstitial space are released into the cytosol. These pores also allow the leakage

of cytochrome-c along with the other contents. This is one of the initial steps

observed during the activation of intrinsic apoptotic pathway. The decrease in

the MMP can be determined by mitochondrial specific dye TMRE. The cationic

dye TMRE distinguishes the healthy cells from the cell undergoing apoptosis.

The dye accumulates and aggregates in healthy cells giving off intense red

fluorescence where as in apoptotic cells the altered mitochondrial membrane

potential makes the dye to stay as monomer in the cytoplasm resulting in

reduced fluorescence. These fluorescence signals can be determined by the flow

cytometry or by confocal microscopy.

PA-1 cells were grown on cover slips for 24 h and treated with RBL (5

µg/ml) for 3 and 6 h in a CO2 incubator. After each time incubation the cells

were stained with 1 µl of TMRE (200 nM) for 30 min in a CO2 incubator. Cells

were washed with PBS and fixed with 2 % para-formaldehyde for 10 min. Cells

were counter stained with DAPI for 30 min and visualized under confocal

microscope.

3.1.12. Western blotting to monitor the expression of apoptotic proteins

The expression of different apoptotic proteins upon treatment with RBL in PA-1

cells was determined by western blotting studies. PA-1 cells were grown in 6

well plate for 24 h before treating with the RBL. PA-1 cells were treated with

RBL (5 µg/ml) for different time intervals up to 8 h. At specific time intervals

the cells were harvested by using cell scraper and washed using ice cold PBS.

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Cells were lysed using RIPA lysis buffer (120 mM NaCl, 1.0 % Triton X-100,

20 mM Tris–HCl, pH 7.5, 100 % glycerol, 2 mM EDTA, and protease inhibitor

cocktail). Total protein of each sample was analyzed by modified Lowry method

using DC protein assay kit (BioRad). Equal concentrations of protein was

resolved on SDS-PAGE and blotted on to PVDF membrane. Blots were

incubated with 5 % BSA to block the non specific binding and probed with

primary antibodies for caspase-8, -9, active caspase-3, and PARP for 1 h at room

temperature using the appropriate dilutions as mentioned by the manufacturer’s

for each antibody. Blots were washed thrice and incubated with species specific

secondary antibodies conjugated with horseradish peroxidase for 1 h. The blots

were washed thrice and the bands were visualized by chemiluminescence, using

Super signal west femto maximum sensitivity substrate (Pierce, USA) as per the

manufacturer’s instructions. The membranes were exposed to X-ray films, fixed

and developed. Blots were reprobed for β-actin upon stripping the blots using

stripping buffer (50 mM Tric-HCl, pH-6.7, 2 % SDS, 100 mM mercapto

ethanol) for 30 min at 60 °C and followed by incubating with actin antibody and

processed as mentioned above.

Cytochrome-c release into the cytosol was analyzed by isolating cytosolic

proteins separately using HEPES buffer containing digitonin. Digitonin

specifically binds to cholesterol and increases the porosity of the plasma

membrane, which further releases the cytosolic proteins to the supernatant. The

presence of cholesterol is observed more in the plasma membrane when

compared to organelle membranes. Hence the exclusive recovery of cytosolic

proteins is achieved by the use of digitonin. PA-1 cells were treated with RBL

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(5 µg/ml) for different time points and after each time point the cells were

harvested by gentle trypsinization. Cells were incubated with HEPES buffer (25

mM HEPES, 5 % sucrose, 2 mM EDTA, protease inhibitor cocktail, 100 mM

NaCl, pH-6.8) with 20 mM digitonin for 30 min with gentle shaking at 4 °C.

Cells were centrifuged at 450 g for 10 min at 4 °C and supernatant was collected

and stored as cytosolic proteins at -20 °C. The extraction was repeated for two to

three times for efficient recovery of cytosolic proteins. Protein sample was

precipitated using 5 % TCA and precipitated proteins were redissolved in PBS.

Total protein was estimated and transblotted on to PVDF membrane. Blots were

then processed as mentioned above for cytochrome-c antibody.

Inhibition of PARP cleavage induced by RBL was analysed by

preincubating the cells with pan caspase inhibitor followed by western blotting.

Cells grown for 24 h were preincubated with pan caspase inhibitor (100 nM) for

1 h at 37 °C and treated with RBL (5 µg/ml) for 12 h in a CO2 incubator. Cell

lysate was prepared and the total protein was transblotted to a membrane as

mentioned above. Blot was incubated with 5 % BSA to block the nonspecific

binding and probed with PARP antibody and processed in a similar way as

mentioned above.

3.1.13. Statistical analysis

Each experiment was performed at least three times, each time in triplicate.

Results were analyzed by one-way ANOVA followed by ‘Newman-Keuls’ test

for multiple comparisons using ‘StatsDirect’ software and data considered

significant when P <0.05.

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3.2. RESULTS

3.2.1. Surface binding

In our previous studies the binding of RBL to PA-1 cells and its inhibition in

presence of different haptens by flow cytometry has been reported [Nagre et al.

2010]. In the present study we studied the interaction of RBL with PA-1 cells

using FITC labeled RBL followed by the confocal microscopic observation.

Uniform binding of FITC-RBL was observed all over the cell surface of PA-1

cells as depicted by the intense fluorescence on the cell surface (Fig. 1).

3.2.2. Localization of FITC-RBL at different time intervals

RBL was localized at different time following its incubation with PA-1 cells.

Some of the lectins are known to induce their physiological response upon their

internalization. The results of the localization studies are presented in Fig. 2.

Incubation of FITC-RBL for different time intervals up to 6 h at 37 °C revealed

the complete internalization of lectin in to the cells. The complete RBL uptake

was observed as early as 1 h and the lectins gets localized and concentrate in the

cytosol without entering into the nucleus as revealed in Fig. 2.

Fig. 1

Fig. 1. Binding of RBL to PA-1 cells: PA-1 cells were stained with FITC-RBL and DAPI, and visualized by confocal microscope. Images were captured at 63X magnification which shows the surface binding of RBL.

DAPI FITC-RBL Merged

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3.2.3. Growth inhibitory effect of RBL on PA-1 cells

RBL showed strong binding to PA-1 cells, hence the physiological responses of

RBL on PA-1 cells was studied. Previous studies has shown strong cytotoxic

effect of RBL on PA-1 cells in a dose dependent manner at 6,25, 12.5, and 25

µg/ml after 12 h incubation. In the present study we have tested the effect of

RBL at lower concentration and early time points to observe the RBL induced

cell death by MTT assay. Results have shown that RBL induces cytotoxic effect

upon binding in a dose and time dependent manner. The dose response profile

showed 53±4.68 % and 56±7.62% cell death at 5 and 10 µg/ml respectively

1 h 2 h 3 h 6 h

Merged

DAPI

FITC-RBL

Fig. 2

Fig. 2. Localization of FITC-RBL: PA-1 cells were incubated with FITC-RBL (4 µg/ml) for different time intervals in a CO2 incubator. After specific time points cell were fixed and counter stained with the DAPI. Images were captured at 63X magnification in a confocal microscope.

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(n=9, P<0.05) (Fig. 3A) on the other hand, RBL at 2.5 µg/ml showed only 12 %

cell death after 10 h. The percentage inhibition and RBL concentrations are

compared in Table 1. Time kinetics of RBL-induced cytotoxicity was analysed

using 5 µg/ml concentration for different time intervals up to 48 h. RBL showed

a time dependent increase in the cell death with maximum cell death of 89±0.68

% at 48 h (n=9, P<0.05) (Fig. 3B). Cell death was initiated as early as 6 h and

showed 46.88 % cell death. RBL at 12, 24, and 36 h, induced 60, 66, 82 % cell

death respectively. Comparison of the time and percentage of growth inhibition

are summarized in Table 2. These preliminary data suggest that RBL exhibits

strong cytotoxicity in PA-1 cells at 5 μg/ml dose in a time dependent manner.

Hence the detailed signaling mechanism involved in RBL induced cell death in

PA-1 cell was studied at 5 µg/ml.

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Table 1 Table 2

RBL (µg/ml) % of Inhibition Time in hr % of Inhibition 1.25 0.71 6 46.88

2.5 12.80 12 60.30

5 58.14 24 66.80 10 62.01 36 82.65

48 89.00

3.2.4. RBL induced apoptosis in PA-1 cells by cell cycle analysis

The percentage of cells undergoing apoptosis and the distribution of cells in the

different phases of cell cycle was determined by exposing PA-1 cells to RBL (5

μg/ml) for 3 and 6 h followed by propidium iodide (PI) staining and flow

cytometry analysis. The cell death was observed as early as 6 h and hence we

selected 3 and 6 h time points for the cell cycle analysis. Treatment of PA-1 cells

with RBL resulted in 8.82 and 22.25 % of hypo-diploid population at 3 and 6 h

Fig. 3

Fig. 3. Dose and time dependent effect of RBL on PA-1cells. A. PA-1 cells were incubated with different concentration of RBL (1.25 to 10 µg/ml) for 10 h and cell viability was measured by MTT assay. B. PA-1 cells were incubated with RBL (5 µg/ml) for different time points (6 to 48 h) and cell viability was measured after each time point by MTT assay. Data represents the representative of three experiments done in triplicate. * represents the P<0.05. Comparison of the percentage inhibition at different doses and time are presented in Table 1 and 2 respectively.

95

as compared to 5.38 % in control respectively. RBL treatment for 3 h didn’t

reveal noticeable difference compared to untreated cells. However after 6 h RBL

decreased the S and G2/M phase cell population to 14.73 and 10.09 % as

compared to 25.55 and 24.15 % in untreated cells respectively. The increase in

G0/G1 phase cell population in RBL treated cells by 51.84 % as compared to

43.28 % in untreated cells was observed after 6 h (Fig. 4). The distribution of

cell in different phases of cell cycle in RBL treated and untreated cells are

compared and presented in Table 4.

Table 4

Cell cycle phases UNT 3 h 6 h

Hypo diploid 5.38 8.82 22.25

G0/G1 43.28 44.25 51.84

S 25.55 24.29 14.73

G2/M 24.15 20.54 10.09

Fig. 4

Fig. 4. Effect of RBL on different phases of cell cycle. PA-1 cells were incubated with or without RBL (5 µg/ml) for 3 and 6 h. Cells were stained with PI and acquired on FL2-A channel of flow cytometer equipped with 488 nm laser. The X-axis represents the DNA content of the cells and the Y-axis represents the cell number. M1, M2, M3, and M4 represent the subdiploid/apoptotic, G0/G1, S, and G2/M phase respectively. The percentage cells at different phases were compared with untreated cells and presented in Table 4.

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3.2.5. Quantification of RBL induced apoptosis in PA-1 by Annexin V and

PI staining

The apoptotic potential of RBL with PA-1 cells was quantified by Annexin V

and PI staining to distinguish viable, early and late apoptosis/necrotic cells.

Treatment with RBL resulted in significant increase of the early apoptotic cells

(only Annexin V positive) to 4.54 and 23.3% after 3 and 6 h respectively. The

cells positive for both Annexin V and PI (late apoptotic) was increased to 46.71

% when compared to 0.42 % in untreated cells at 6 h (Fig. 5A). These results

further support the efficacy of RBL in inducing apoptosis quantitatively at early

time points. A small percentage (10 %) of necrotic cells (only PI positive) was

also observed at the end of 6 h. Comparison of the cells positive for early/late

apoptosis and necrosis are presented in Fig. 5B and summarized in Table 5.

Fig. 5

Fig. 5.AnnexinV –PI staining. PA-1 cells were treated with RBL (5 µg/ml) for 3 and 6 h followed by Annexin- V-FITC and PI staining. The X-axis depicts Annexin- V positive cells and Y axis depicts PI positive cells.

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PA-1 Cells UNT 3h 6h

Viable 99.52 79.75 20.75 Early apoptosis 0.00 4.54 23.3

Late apoptosis 0.06 4.83 46.71

Necrosis 0.42 9.24 10.88

3.2.6. Activation of caspase-3 in RBL treated PA-1 cells

Activation of caspase-3 during apoptosis is an important event. Different

methods can be used for the determination of active caspase-3. FITC labeled

caspase-3 inhibitor (FITC-DVED-fmk) is used to determine the active caspase-3

in RBL treated PA-1 cells by flow cytometry. Treatment of PA-1 cells with RBL

for 3 and 6 h showed a time dependent positive shift as observed in the overlay

histograms (Fig. 7). A significant increase of active caspase-3 by 31.49 and

44.47 % was observed after 3 and 6 h respectively when compared to 5.29 % in

untreated cells (Table 6). This suggests the activation of caspase-3 as early as

3 h.

0

20

40

60

80

100

120

UNT 3 6

Cel

ls (%

)

Time in h

Viable Early apoptosisLate apoptosisNecrosis

Fig. 6

Table 5

Fig. 6. Comparison of the Annexin V and PI positive cells. The percentage of PA-1 cells positive for early/late apoptosis and necrosis were compared with normal cells at different time in a bar graph and values are presented in Table 5.

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Time (h) % positive cells

0 5.29

3 31.49

6 44.47

3.2.7. Inhibition of RBL induced cell death by caspase inhibitors

To understand the role of different caspases in RBL induced cell death, specific

pharmacological inhibitors for the caspases were used to protect the cell death.

Inhibitors for caspase-8, -9, and a pan caspase were preincubated with PA-1 cells

before treating with RBL for 12 h. The viable cells were measured by MTT

assay. RBL induced cell death was suppressed by 50 and 45 % in caspase-9 and

pan-caspase inhibitors respectively and there was no suppression of cell death in

caspase-8 inhibitor preincubated cells (Fig. 8). These results suggest the possible

involvement of caspase-9 mediated intrinsic pathway in RBL induced cell death.

Fig. 7

Fig. 7. Activation of caspase-3. PA-1 cells were treated with RBL (5 μg/ml) for 3 and 6 h and the activity of active caspase-3 was determined by Flow cytometry analysis using FITC tagged caspase–3 inhibitor (FITC-DEVD-FMK). Table 6 represents the comparison of percentage of cells showing active caspase-3 at different time intervals.

Table 6

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3.2.8. Loss of MMP in RBL treated PA-1 cells

The loss of membrane potential across mitochondria attributes to the formation

of large pores across inner and outer mitochondrial membrane due to the signals

received within the cells such as DNA damage, excessive ROS, changes in the

calcium levels within the cells. Preincubation of PA-1 cells with caspase-9

inhibitor has shown the protection of RBL induced cell death. Hence it was

essential to analyze whether this activation is due to the loss of mitochondrial

membrane potential (MMP), which was visualized by confocal microscopy

using a mitochondrial specific fluorescent dye TMRE. PA-1 cells treated with

RBL for 3 and 6 h showed a time dependent decrease in the uptake of TMRE as

0123456789

Cel

l Vi

abili

ty (f

old

incr

ease

)

Fig. 8

Fig. 8. Inhibition of RBL induced cell death by caspase inhibitors. PA-1 Cells with or without pretreatment with 60 μM of caspase inhibitors (zVAD-FMK, zIETD-FMK, or zLEHD-FMK) for 2 h were incubated with 5 μg/ml of RBL for 12 h. Viable cells were measured after 12 h by MTT assay. The graph represents the mean ± SD values from three independent experiments. * represents p < 0.05

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observed by confocal microscope. Intact mitochondria were stained red and

observed around the nucleus in the control cells, whereas, RBL treated cells

showed diffused mitochondria in the cytosol and also weak fluorescent signal as

the time increased (Fig. 9).

3.2.9. Expression of different intermediates of apoptosis in RBL treated

PA-1 cells by Western blotting

The involvement of caspases in RBL induced cell death has been shown by

using specific pharmacological inhibitors. Activation of different caspases,

release of cytochrome-c, and cleavage of PARP was observed by Western

Fig. 9

Fig. 9. Loss of MMP in PA-1 cells. The loss of MMP was visualized in PA-1 cells treated with or without RBL (5 μg/ml) for 3 and 6 h by confocal microscopy. Cells were stained with TMRE after each time point and counter stained with DAPI. Images were captured at 63X magnification.

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blotting using specific antibodies to further substantiate our earlier observations.

Expression of different proteins in RBL treated PA-1 cells were monitored at

different time points (2, 4, and 8 h) and the results are presented in Fig. 10A.

Time dependent increase in active caspase-3 expression was observed, which

was initiated as early as 2 h and reached maximum by 8 h. The decrease in the

expression procaspase-9 was observed in a time dependent manner, whereas

there was no change observed in the expression procaspase-8.

The release of cytochrome-c from mitochondria to the cytosol is an

important step in the intrinsic apoptotic pathway. Cytosolic proteins from RBL

treated PA-1 cells were isolated and probed with cytochrome-c antibody. The

observed time dependent increase in the expression cytochrome-c falls in line

with the activation of caspase-9 and supports the possible involvement of

intrinsic apoptotic pathway.

Poly (ADP-ribose) polymerase (PARP) is a 116 kDa protein involved in

the DNA repair mechanism and other cellular events. PAPR gets cleaved by the

members of caspase family to 89 and 24 kDa fragments during apoptosis.

Detection of these cleaved fragments of PARP is a hallmark of the apoptosis.

RBL induced PARP cleavage was observed in a time dependent manner using

specific antibody. Native PARP band (116 kDa) decreased as the time increased

whereas the cleaved fragment (89 kDa) increased with increase in time. Caspase

dependent PARP cleavage was also confirmed by pre incubating the PA-1 cells

with pan caspase inhibitor for 1 h before treating the cells for 12 h with RBL.

The result shows inhibition of cleavage of native PARP band (116 kDa) in the

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pan caspase pre incubated cells, whereas, cells treated with only RBL showed

complete decrease of native PARP band (Fig. 10B).

PARP native

β-actin

116

44

UNT RBL

RBL +

Z-VAD Z-VAD

Active caspase-3

PARP

Pro caspase-8

Pro caspase-9

Cytochrome-c (Cytosolic)

β-actin

0 2 4 8 h kDa

17

116 89

55

46

14

44

Fig. 10 A

B

Fig. 10. Western blotting. A. PA-1 cells were treated with RBL (5 μg/ml) for 2, 4, and 8 h, and whole cell lysates were transblotted onto a membrane. The membrane was probed with antibodies for caspase-8, -9, active caspase-3, PARP and cytochrome-c. Actin was used as a loading control. B. Cells were pre-incubated with pan caspase inhibitors before they were treated with RBL (5 μg/ml) for 12 h, the cell lysate was transblotted similarly and probed for PARP antibody.

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3.3. DISCUSSION

The present chapter reports the cytotoxic effect of an N-glycan specific lectin

from Rizhoctonia bataticola on human ovarian cancer PA-1 cells is due to

induction of apoptosis involving mitochondrial mediated intrinsic pathway.

Alterations in N-glycosylation during transformation have been well

studied in epithelial cancers that are associated with carcinogenesis, invasion and

metastasis [Varki 1999; Dwek 1996]. Cell surface expression of branched N-

glycans and high mannose tri- and tetra-antennary bisecting N-glycans on

glycoproteins are observed in many cancers including ovarian cancer [Dennis et

al. 1987; Machado et al. 2011; Abbott et al. 2008]. Some of these glycoproteins

are considered as cancer markers. Tumor markers of ovarian cancer have been

studied in detail, and CA-125 is one of the best studied ovarian tumor marker,

which is known to express complex N-glycans. The unique sugar specificity of

RBL towards these cancer specific N-glycans prompted us to study its

interaction with ovarian cancer PA-1 cells. The cytotoxic effect of RBL on

human ovarian cancer PA-1 cells has been reported earlier [Nagre et al. 2010a].

In the present study the mechanism of RBL induced cytotoxicity is studied in

detail which shows that, RBL induces apoptosis in PA-1 cells. RBL shows high

toxicity in a dose and time dependent manner and cell death is initiated as early

as 6 h at 5 µg/ml. Cell cycle analysis reveals that RBL affects the cells in all the

phases of cell cycle. The decrease in the cell population of S and G2/M phase,

increase in the G0/G1 and sub-G0 apoptotic population was observed in a time

dependent manner. These results suggested that RBL may arrests the cells

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growth at G0/G1 phase of cell cycle followed by induction of apoptosis. The loss

of membrane asymmetry during apoptosis exposes phosphatidylserine towards

the outer leaflet of plasma membrane which can be determined by Annexin V

staining. PA-1 cells treated with RBL for 6 h showed 70 % Annexin V positive

cells, suggesting the potential of RBL in inducing apoptosis. There was also an

increase in small necrotic cell population (10 %) after 6 h treatment.

It is known that mitochondrial membrane gets depolarized during

apoptosis to activate intrinsic apoptotic pathway. RBL induced the decrease in

mitochondrial membrane potential in PA-1 cells in a time dependent manner.

The decrease in membrane potential results in release of cytochrome-c to the

cytosol which then activates caspase-9 by forming apoptosome. The treatment of

PA-1 cells with RBL induced the release of the cytochrome-c to cytosol and

increased the activation of caspase-9 as observed by western blotting studies.

The activation of caspase-9 further activates the executioner caspase-3 to

degrade the functional proteins including PARP. RBL induced cell death can be

inhibited by the presence of specific inhibitors for capsase-9 and pan caspase but

not with caspase-8 inhibitor. These results suggest the possible involvement of

intrinsic pathway in RBL induced apoptosis.

Lectins from higher fungi have been shown to possess antiproliferative

activity in different cancer cells in vitro and in vivo [Khan and Khan 2011]. The

detailed mechanism of the antiproliferative activity and induction of apoptosis

by fungal lectins is not been studied in many cases. However, there few reports

like lectin from Agrocybe aegerita (AAL) shows inhibition of cell growth in

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many cancer cells and apoptosis inducing property has been studied in HeLa

cells. AAL induces the increase of subG0/G1 cells to 31 % and Annexin V

positive cells to 14.15 % in HeLa cells after 36 h [Zhao at al. 2003; Liang et al.

2009]. AAL induces apoptosis by binding to nuclear ligand mortality factor 4-

like protein 15 (MRG15) [Liang et al. 2010], whereas AAL2 another lectin

isolated from the same edible mushroom with differing sugar specificity,

recognizing terminal GlcNAc has been cloned and is known to induce increase

of Annexin V positive cells to 10 and 36 % in H22 and Huh7 hepatoma cells at

2.4 µM respectively after 24 h for the induction of apoptosis [Jiang et al. 2012].

Plant lectins that are reported to induce apoptosis are usually known to arrest the

cell growth at G0/G1 phase and activate both extrinsic and intrinsic apoptotic

pathways [Lam and Ng 2011a]. Wheat germ agglutinin (WGA), an N-glycan

specific lectin induces the cell cycle arrest at G2/M phase and increases

apoptotic population at 0.5 µM after 24 h in mouse L929 Fibroblasts. WGA

induced apoptosis in L929 cells is known to involve caspase-3 and Bax [Liu et

al. 2004] whereas, in Jurkat cells it induced an increase of annexin V positive

cell to 52 % at 10 µg/ml involving both caspase-8 and -9 [Gastman et al. 2004].

Concanavalin A (ConA), one of most extensively studied N-glycan specific

lectin induces anti-cancer effect in many cancer cells both in vitro and in vivo

through mitochondrial mediated P73-Foxo1a-Bim signaling pathway for

apoptosis and BNIP3-mediated mitochondrial autophagy. Apart from these

effects ConA is also known to activate lymphocytes [Li et al. 2011]. Lectins

from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) also

106

inhibit the growth of human leukemic Molt-4 and HL-60 cells with IC50 values

of 3 and 20 μg/ml respectively. However, the lectins were not cytotoxic to

normal human peripheral blood lymphocytes even at 200 μg/ml. Both ConA and

ConBr induce the cell cycle arrest at Sub-G1 phase and decrease MMP to induce

the apoptosis in Molt-4 and HL60 cells [Glaucia et al. 2012]. Hemagglutinin

(HA) from dried Phaseolus vulgaris cultivar ‘French bean number 35’ inhibits

the proliferation of breast cancer MCF-7 cells with an IC50 of 2 μM. HA

induces the cell cycle arrest at G0/G1 and G2/M phases, and induces the increase

of early apoptotic cells by 45 % at 45 μM. HA induced apoptosis is mediated

through the death receptor, FasL followed by the activation of caspase-8 [Lam

and Ng 2011b]. A mannose binding lectin from Polygonatum cyrtonema induces

both apoptosis and autophagy in human melanoma A375 cells through

mitochondrial mediated ROS–p38–p53 pathway [Liu et al. 2009] whereas

Polygonatum odoratum another mannose biding lectin induced apoptosis involve

both caspase-8 and -9 [Yang et al. 2011].

There are reports on lectins from higher fungi with growth inhibitory

effect on different cancer cells. For example; lectin from dried Lactarius

flavidulus fruiting bodies (LFL) suppressed the proliferation of hepatoma

(HepG2) and leukemic (L1210) cells with an IC50 of 8.90 and 6.81 µM

respectively [Wu et al. 2011]. A lectin from toxic mushroom Inocybe umbrinella

inhibits the proliferation HepG2 and MCF7 cells with an IC50 of 3.5 mM and

7.4 mM respectively [Zhao et al. 2010] whereas, another lectin from fresh

fruiting bodies of the edible mushroom Lyophyllum shimeiji shows the similar

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antiproliferative activity towards the same cells with an IC50 of 10 and 6.2 µM

respectively [Zhang et al. 2010]. In a similar way the lectins isolated dried

fruiting bodies of monkey head mushroom Hericium erinaceum (HEL) [Li et al.

2010], medicinal mushroom Pholiota adipose (PAL) [Zhang et al. 2009], and

dried fruiting bodies of Agaricus arvensis [Zhao et al. 2011] have also shown

growth inhibitory effects on HepG2 and MCF-7 cells. However detailed

signaling mechanism involved in the growth inhibitory effect of these lectins is

not known. RBL appears to be more potent because of its ability to induce

apoptosis at low concentration and early time point in comparison with the

reported fungal lectins.

Aberrant glycosylation in ovarian cancer exposes many tumor markers

that are potential diagnostic targets. The expression of high mannose and

complex bisecting N-glycans on CA-125, an important ovarian tumor marker

under clinical use, has been observed [Gadducci et al. 2004; Ayhan et al. 2007].

Apart from these glycosylation changes the expression of sialyl-Lewis antigen

and derivatives of Lewis antigens on N- and O-glycans are also observed on the

primary as well as metastatic ovarian carcinoma [Davidson et al. 2000]. Specific

recognition of these glycans by RBL as revealed by glycan array analysis

suggests the possible involvement of CA-125 and other glycoproteins expressed

on ovarian cancer cells for its interaction. These observations further support the

possible potential of RBL in ovarian cancer research both in diagnostics and

therapeutics.

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In conclusion RBL induces potent toxicity in PA-1 ovarian cancer cells in

vitro. The inhibition of growth can be attributed to its induction of apoptosis

involving mitochondrial mediated intrinsic pathway. The present work opens

new perspectives to further explore the potential of RBL for its clinical

implications.