Rhizoctonia Bataticola Lectin ( RBL ) Induces Apoptosis in Human Ovarian Cancer PA-1 Cells and Suppresses Tumor Growth In Vivo

Background: A lectin from the plant pathogenic fungus Rhizoctonia bataticola (RBL) has shown unique sugarbinding specificity towards the complex N-glycans, which are known to be expressed in many cancers, including ovarian cancer. Previous studies have demonstrated that RBL exerts cytotoxic effects on human ovarian cancer PA-1 cells. In the present study, we determined the signaling mechanism of RBL-induced cytotoxicity in PA-1 cells upon binding to cell surface receptors and also assessed the anti-tumor activity of RBL in NOD-SCID mice bearing ovarian xenografts. Methods: Cytotoxicity was determined by MTT assay, Cell cycle analysis, Annexin V staining, and expression of caspase-3 was determined by flow cytometry using specific fluorescent detection reagents. Western blotting was carried out to observe the intermediates of apoptotic pathway. Results: Treatment of PA-1 cells with RBL decreased their viability in a doseand timedependent manner. Cell cycle analysis revealed an increase in the hypo diploid population in response to lectin treatment. RBL-treated cells stained with Annexin V and PI showed an increase in both the early and late apoptotic populations. The mechanism of RBL-induced apoptosis in PA-1 cells was assessed by the observed decrease in MMP (ΔΨm), release of cytochrome-c, activation of caspase-9 and -3, and cleavage of PARP. Specific pharmacological inhibitors revealed the involvement of caspase-9 and -3 but not caspase-8. RBL at a subtoxic concentration (50 μg/mouse) suppressed tumor growth in NOD-SCID mice bearing PA-1 xenografts. Conclusions: The present study demonstrates that, RBL induced cytotoxicity in PA-1 cells is mediated by intrinsic apoptotic pathway. The present study strengthens the merit of RBL for its potential application in ovarian cancer research. *Corresponding author: Dr. Shashikala R Inamdar, Professor, Department of Biochemistry, Karnataka University, Dharwad -580003 India, Tel: 0091-8362771973, Fax: 0091-836-2747884; E-mail: pinkisri2001@yahoo.co.in Received February 11, 2013; Accepted March 14, 2013; Published March 21, 2013 Citation: Eligar SM, Pujari R, Savanur MA, Nagre NN, Barkeer S, et al. (2013) Rhizoctonia Bataticola Lectin (RBL) Induces Apoptosis in Human Ovarian Cancer PA-1 Cells and Suppresses Tumor Growth In Vivo. J Glycobiol S1: 001. doi:10.4172/2168-958X.S1-001 Copyright: © 2013 Eligar SM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The incidence of ovarian cancer, one of the leading causes of cancer death among women worldwide, is increasing every year [1].Most ovarian cancer cases are diagnosed at advanced stages when the cancer has already developed into a wide-spread disease [2].Epithelial ovarian cancer makes up 80-90% of all ovarian malignancies [3].Although there are many chemotherapeutic drugs that have shown high patient response rates after surgery, patients have a poor overall survival rate (25%); the survival rate decreases to 5% if patients are diagnosed at stage III or IV [4].There is a great need for nontoxic, antitumor molecules in the treatment of many cancers, including ovarian cancer.
Lectins are a well-known group of multivalent carbohydratebinding proteins that recognize specific glycans of cell surface receptors and mediate a variety of biological functions [5].Recent studies on fungal lectins have attracted the attention of researchers due to their interesting biological properties and sugar specificities [6].Various applications in cancer research are being developed for lectins that have specific affinity towards cancer-associated antigens and exhibit antitumor activity.As a result, some of these lectins are already currently used in clinical studies [7,8].
Recently, we reported the isolation of a mitogenic and immuno stimulatory lectin from the phyto pathogenic fungus Rizhoctonia bataticola (RBL) that exhibited complex sugar specificity.Glycan array analysis of RBL revealed its exclusive specificity towards N-glycans, primarily high mannose, tri-and tetra-antennary complex N-glycans, and tandem repeats of sialyl Lewis antigens.The various interactions of RBL have been studied using human ovarian cancer cells PA-1, which are known to express these glycans.The results showed that RBL has cytotoxic effects on PA-1 cells, which can be effectively blocked by the addition of competing glycoproteins [9][10][11].
Malignant transformation is associated with a variety of glycosylation changes and is known to be involved in important events such as transformation, invasion, and metastasis [12].One of the common glycosylation changes in cancer is the increased branching of N-glycans that express tri-and tetra-antennary oligosaccharides with terminal sialylation.The expression of sialyl Lewis a , sialyl Lewis x and their isomers on the N-and O-linked oligosaccharides is observed in various human malignancies that are involved in cancer progression.The expression of these altered glycans in breast, bladder, lung, ovary, and colon cancer correlates with poor prognosis [13].Many protein tumor markers on epithelial ovarian cancer have been studied and are known to express altered cancer-associated glycans [14].The ability of RBL to specifically recognize these altered glycans prompted us to study their interaction in ovarian cancer cells.
In the present study, we report the signaling mechanism involved in RBL-induced cell death in PA-1 cells in vitro and the anti-tumor effects of RBL in NOD-SCID mice with PA-1 xenografts in vivo.
The purification of RBL and the preparation of the FITC-labeled RBL were conducted according to methods described earlier [9].

Cell culture
The human ovarian cancer cell line PA-1 was procured from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in MEM (Gibco-Invitrogen, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 mM glutamine, 1 mM sodium pyruvate, 100 mg/ml streptomycin, and 100 units/ml penicillin at 37 °C in 5% CO 2 and 95% humidified air.Exponentially growing PA-1 cells (80-85% confluent) were used for all the said studies.

Analysis of surface binding by confocal microscopy
PA-1 cells were grown on cover slips at 2×10 5 cells/ml for 24 h before staining with FITC-RBL (2 µg/ml) for 1h at 4 °C.Unbound lectin was washed with PBS, and cells were fixed with 2% para-formaldehyde and visualized using a confocal laser scanning microscope (Zeiss, Germany).DAPI was used as a nuclear stain.

Growth inhibitory studies
PA-1 cells at 5000 cells/well were grown in 96 well plates for 24 h.The dose-and time-dependent effects of RBL on PA-1 cells were determined by treating the cells with different concentrations (1.25 to 10 µg/ml) of RBL for 10 h or different time points (6 to 48 h) using 5 µg/ml of RBL.After each time point, cell viability was measured by the MTT assay as described earlier [9].The percentage of viable cells was calculated compared to control treatments, which were considered 100% viable.

RBL-induced apoptosis in PA-1 cells
RBL-induced apoptosis was determined by cell cycle analysis.PA-1 cells at 2×10 5 cells/well in 6 well plate were grown for 24 h, then cells treated with RBL (5 µg/ml) for 3 and 6 h.Cells were harvested by gentle trypsinization and fixed in 70% chilled ethanol for 30 min at 4 °C.Cells were treated with 50 μl Ribonuclease A (5 mg/ml) and stained with propidium iodide (50 μg/ml).The DNA content was analyzed by flow cytometry (FACS Calibur, BD, San Jose, CA, USA) to observe the distribution of cells in different phases of the cell cycle.
The induction of apoptosis by RBL was confirmed by dual staining with FITC-labeled Annexin V and propidium iodide.Briefly, PA-1 cells at 2×10 5 cells/well in 6 well plate were grown for 24 h and treated with RBL for 3 and 6 h.Cells were harvested by gentle trypsinization and resuspended in binding buffer at a concentration of 1×10 6 cells/ ml, and stained with FITC-Annexin V and PI.The percentage of cells positive for Annexin V, PI, or both was determined by flow cytometry analysis.

Activation of caspase-3
PA-1 cells at 2×10 5 cells/well in 6 well plates were grown for 24 h and treated with RBL for 3 and 6 h.Cells were harvested by gentle trypsinization, and active caspase-3 was determined by using the caspase-3 detection kit (Calbiochem) according to the manufacturer's instructions.The FITC-labeled caspase-3 inhibitor (FITC-DEVD-fmk) was used to detect active caspase -3 by flow cytometry analysis.Cells that were not treated with RBL were used as controls.

Determining Mitochondrial Membrane Potential (ΔΨm)
PA-1 cells were grown on cover slips, treated with RBL (5 µg/ml) for 3 and 6 h, and stained with the mitochondrial-specific dye TMRE (200 nM) for 30 min.Cells were washed, fixed with 2% para-formaldehyde for 10 min, and visualized using a confocal laser scanning microscope.DAPI was used as a nuclear stain.

Western blot analysis
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 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).In another experiment, cells were pre-incubated with pan caspase inhibitors (40 µM) for 1 h, treated with RBL (5 µg/ml) for 12 h, lysed, and prepared as mentioned above.Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane.After blocking with 5% BSA, the blots were probed with primary antibodies for Caspase-8, -9, active caspase-3, and PARP followed by species-specific HRPlabeled secondary antibodies with appropriate dilutions as per the manufacturer's instructions.The bands were visualized by enhanced chemiluminescence (Pierce, USA).Actin was used as a loading control.
Cytosolic proteins from RBL-treated and untreated cells were isolated by methods described by McCarthy et al. [15].Cytosolic proteins were transblotted onto a PVDF membrane, probed with antibodies against cytochrome-c, and processed as mentioned above.

In vivo antitumor activity in NOD-SCID mice
Inbred NOD-SCID mice were procured, maintained at the Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), India on a synthetic diet, and used for the toxicity and anticancer studies with clearance from the ethics committee (IRB approval No.11/2009).
PA-1 cells (1×10 7 ) were injected subcutaneously into two NOD-SCID mice (donor mice).After 4 weeks, the tumors were excised from the donor mice and chopped into 2-3 mm sizes.Tumor pieces were transplanted subcutaneously into 18 NOD-SCID mice and maintained for 12 days to allow the tumor to grow to 5-6 mm.The mice were then divided randomly into three groups, with six animals in each group.Animals in Groups II & III were given intraperitoneal injections of RBL (25 and 50 µg/mice) every other day for 9 days.The animals in Group I were similarly injected with PBS as a negative control for 9 days.The animals were sacrificed after the 14 th day and photographed.Tumor volume and weight were measured after excising the tumor, and mean tumor weight and volume were considered.Different parameters of toxicity, including total blood cell count, hemoglobin content, and serum liver enzyme levels (aspartate transaminase and alanine transaminase) were measured.

Statistical analysis
Each experiment was performed at least three times, each time in triplicate.The results were analyzed by one-way ANOVA followed by the 'Newman-Keuls' test for multiple comparisons using the 'StatsDirect' software.Data were considered significant when p<0.05.

Surface-binding and cytotoxicity of RBL in PA-1 cells
The localization of FITC-RBL on the cell surface was visualized by confocal microscopy.Uniform binding of FITC-RBL was observed on the surface of PA-1 cells confirming its binding to cell surface (Figure 1A).The dose response profile of RBL-treated PA-1 cells revealed no significant growth inhibition at 1.25 and 2.50 µg/ml; however, at 5 and 10 µg/ml, significant cell death was observed (53 ± 4.68% and 56 ± 7.62%, respectively) (Figure 1B).Time kinetic studies revealed that cell death (53%) was observed as early as 6 h, and maximum cell death (89 ± 0.68%) was observed at 48 h (Figure 1C).These results suggest that RBL exhibits strong cytotoxicity in PA-1 cells at a 5 μg/ml dose in a time-dependent manner.

Induction of apoptosis by RBL
Treatment of PA-1 cells with RBL resulted in an increase of hypodiploid cells by 8.82% and 22.25% at 3 and 6 h, respectively, compared to 5.38% in the control sample.RBL treatment also decreased the S and G2/M phase cell population to 14.73% and 10.09% after 6 h, respectively, compared to 25.55% and 24.15% in the control group.An increase in the percentage of the G0/G1 population in RBL-treated cells (51.84 %) compared to untreated control cells (43.28%) was also observed after 6 h (Figure 2A).RBL treatment of PA-1 cells resulted in a significant increase in the Annexin-V positive population (early apoptotic) by 4.54 and 23.30% after 3 and 6 h, respectively.The percentage of cells positive for both Annexin-V and PI (late apoptotic) increased to 46.71% compared to 0.42% in the control group, and a small population of cells (10%) positive for PI (necrosis) was also observed at the end of 6 h (Figure 2B).  to a 5.29% increase in untreated cells (Figure 3A).RBL-treated PA-1 cells showed a time-dependent increase in the activation of caspase-9 and -3 at 2, 4, and 8 h; in contrast, the expression of procaspase-8 remained constant at all time points, which suggests that caspase-8 is not involved in RBL-induced apoptosis.The increase in PARP cleavage was observed in time dependent manner (Figure 3B).

Activation of caspases during RBL-induced apoptosis
To demonstrate the involvement of caspases in RBL-induced apoptosis, cells were pre-incubated with pan-caspase inhibitors before receiving RBL treatment and PARP cleavage was determined by western blot analysis.In the presence of pan-caspase inhibitors, the cleavage of PARP was prevented, which suggests the involvement of caspases in RBL-induced apoptosis (Figure 3C).
The role of caspases in RBL-induced cell death was analyzed by using pharmacological inhibitors for specific caspases and a pancaspase inhibitor.RBL-induced cell death was suppressed by 50 and 45% in the presence of caspase-9 and pan-caspase inhibitors, respectively; furthermore, there was no protection from apoptosis in cells pre-incubated with caspase-8 inhibitors (Figure 4A).

Loss of mitochondrial membrane potential and release of cytochrome-c
As RBL-induced apoptosis involves the activation of caspase-9, it was essential to analyze the breakdown of ΔΨm.The time-dependent loss of ΔΨm in RBL-treated PA-1 cells was visualized by confocal microscope.Untreated cells showed intact mitochondria with intense red staining that was visualized around the nucleus; meanwhile, RBL-treated cells displayed a significant reduction in red staining and a diffused appearance of the mitochondria (Figure 4B).
The loss of ΔΨm typically involves the release of mitochondrial cytochrome-c to the cytosol, where it combines with apoptotic protease activating factor 1 (Apaf-1) and procaspase-9 to form the apoptosome, which results in the activation of caspase-9 and caspase-3.Consistent with these facts, a time-dependent increase in cytosolic cytochrome-c was observed (Figure 3B).Overall, these results suggest that RBL-induced apoptosis may be executed through the activation of mitochondrial-mediated intrinsic apoptotic pathway.

RBL induces antitumor effects in vivo
The growth inhibitory effect of RBL was also analyzed by in vivo studies using NOD-SCID mice with PA-1 xenografts.A significant reduction in tumor size was observed in RBL-injected mice compared to control groups (Figure 5A).A reduction in average tumor volume and weight was observed in RBL-treated mice (50 µg/mice) after 9 days (Figure 5B).Tumor weight and volume reduced to 0.100 ± 0.012 g (P=0.029) and 193.5 ± 52 mm 3 (P=0.0036) in RBL-treated mice in Group-III in contrast to control mice that had a tumor weight and volume of 0.319 ± 0.043 g and 618.0 ± 190 mm 3 , respectively.RBLtreated mice in Group-II also showed a partial reduction in tumor weight and volume (Figure 5C and D).Analysis of toxicity parameters in these mice showed no considerable difference in RBL-treated and untreated groups (Table 1).

Discussion
In the present study, we evaluated the effect of an N-glycan-specific   lectin from Rizhoctonia bataticola on human ovarian cancer PA-1 cells both in vitro and in vivo.The study demonstrated that RBL induced strong cytotoxicity in PA-1 cells and that observed cell death is due to the activation of the intrinsic apoptotic pathway.RBL treatment showed significant tumor regression in ovarian cancer xenografts.
Cell surface expression of branched N-glycans, high mannose tri-and tetra-antennary, and bisecting N-glycans on glycoprotein is observed in many cancers, including ovarian cancer [13,16,17].Some of these glycoproteins are considered to be cancer markers.Previously, we reported the cytotoxic effect of a mitogenic lectin RBL on PA-1 cells [9][10].In the present study, the mechanism of RBL-induced cytotoxicity was studied in detail.RBL-induced PA-1 cell death was observed in a dose-and time-dependent manner.RBL induced apoptosis in PA-1 cells as determined by cell cycle analysis.RBL affected the cells in all phases of the cell cycle; specifically, RBL treatment decreased S and G2/M phase cell number and increased the G0/G1 and hypodipliod population.These results suggest that RBL may arrest cell growth at the G0/G1 phase of the cell cycle followed by the induction of apoptosis.RBL treatment increased the number of Annexin V-positive cells (70%) along with an increase in the necrotic cell population (10%) after 6 h.This observation suggests that RBL is a potent inducer of apoptosis.Activation of caspase-9 and -3 was observed in a time-dependent manner, but there was no significant difference in the expression of procaspase-8 after RBL treatment.Further, caspase-9 and pan-caspase inhibitors, but not caspase-8 inhibitors, significantly prevented RBLinduced cell death, which suggests the lack of involvement of caspase-8 in RBL-induced apoptosis.A decrease in ΔΨm in RBL-treated PA-1 cells and the release of mitochondrial cytochrome-c were also observed.All these results support that RBL possibly induces apoptosis by activating the intrinsic apoptotic pathway.
Lectins from higher fungi have been shown to possess antiproliferative activity against different cancer cells in vitro and in vivo [8].For many fungal lectins, the mechanism behind these apoptosis-inducing properties has not been studied in great detail.A lectin from Sclerotium rolfsii (SRL) has been shown to inhibit the growth of human ovarian (PA-1) and colon (HT29) cancer cells by inducing apoptosis through the activation of both the intrinsic and extrinsic apoptotic pathway [18,19].Agrocybe aegerita lectin (AAL)   NOD-SCID mice were injected intraperitoneally with RBL (25, and 50 µg/mouse) and TBS on alternate days for 9 days containing 6 animals in each treatment.The Average body weight, total blood cell count, and biochemical parameters in serum were determined on 14 th day and represented.had demonstrated inhibition of cell growth in many cancer cells, and its apoptosis-inducing property has been studied in detail in HeLa cells [20].The majority of plant lectins are known to induce apoptosis by arresting cell growth at the G0/G1 phase and activating both the extrinsic and intrinsic apoptotic pathways [21].Unlike the previously reported fungal lectins, RBL is potent because of its ability to induce apoptosis at low concentrations and at early time points by activating the intrinsic apoptotic pathway.
RBL suppressed tumor size and volume up to 70% in ovarian cancer xenografts, and RBL-treated mice were healthy without any signs of toxicity.Mushroom lectins from Pleurotus citrinopileatus and Russula lepida have shown antitumor activity in vivo in mice bearing Sarcoma 180 (S-180) [22,23].A lectin from Pleurotus ostreatus has been shown to induce antitumor effects in mice bearing S-180 and mouse hepatoma H-22 cells [24].The antitumoral effect of RBL is observed at a much lower concentration and more rapid time frame than the earlier-reported fungal lectins.These preliminary in vivo studies with RBL need to be further investigated before it is exploited for clinical applications.
In conclusion RBL induced potent toxicity in ovarian cancer PA-1 cells in vitro.The inhibition of growth can be attributed to its induction of apoptosis involving the mitochondrial-mediated intrinsic apoptotic pathway.RBL also suppressed tumor growth in vivo in an ovarian cancer xenograft model.The present study demonstrates the possible potential use of RBL in ovarian cancer therapeutics and warrants further investigation.
RBL-treated PA-1 cells showed a 31.49and 44.47% increase in the cells positive for active caspase-3 at 3 and 6 h, respectively, compared /mI) Time inh * represents p < 0.05.

Figure 1 :
Figure 1: RBL binding and cytotoxic effects on PA-1 cells.(A) Human ovarian cancer PA-1 cells were stained with FITC-RBL and DAPI and then visualized by confocal microscopy.Images were captured at 63X magnification and show a uniform surface-binding of RBL.(B) PA-1 cells were incubated with RBL (1.25 to 10 µg/ ml) for 10 h, and cell viability was measured by the MTT assay.(C) 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 the MTT assay.The data are representative of three experiments performed in triplicate.

Figure 2 :Figure 3 :*
Figure 2: RBL induces apoptosis in PA-1 cells.(A) Human ovarian cancer 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 the FL2-A channel of the flow cytometer.M1, M2, M3, and M4 represent the subdiploid/apoptotic, G0/G1, S, and G2/M phases, respectively.(B) PA-1 cells were incubated with RBL as above, processed for Annexin V and PI staining, and analyzed by flow cytometry.The X-axis depicts Annexin V positive cells, and the Y axis depicts PI positive cells.The numbers indicate the number of viable, early apoptotic, late apoptotic, and necrotic cells that are present in the respective quadrants.

Figure 4 :
Figure 4: Involvement of caspases in RBL induced cell death and Loss of MMP.(A) Cells were pre-incubated with specific caspase inhibitors before they were treated with RBL (5 μg/ml) for 12 h, and viable cells were measured by the MTT assay.

Figure 5 :
Figure 5: RBL inhibits tumor growth in vivo.(A) NOD-SCID mice with PA-1 tumors were injected intraperitoneally with RBL (25, 50 µg/mice) or control PBS on alternate days for 9 days.Representative images of the animals after 14 days are shown.Tumors were excised (B), and tumor weight and volumes were compared between the groups (C and D).