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Abrus precatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells

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Birendra Behera, Debasish Mishra 1, Bibhas Roy, K. Sanjana P. Devi, Rajan Narayan, Joyjyoti Das, Sudip K. Ghosh, Tapas K. Maiti ⇑ Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India

a r t i c l e

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Article history: Received 29 March 2014 Received in revised form 17 July 2014 Accepted 28 August 2014 Available online xxxx Keywords: Abrus agglutinin derived peptides ROS p38 JNK Apoptosis and autophagy Hollow fiber assay

a b s t r a c t 10kDAGP, a tryptic digest of Abrus precatorius lectin ‘Agglutinin’ is known to induce apoptosis by mitochondria-dependent pathways in human cervical cancer (HeLa) cells. The present study was focused on deciphering the detailed molecular mechanism of apoptosis induction in vitro by 10kDAGP and also its in vivo therapeutic efficacy. For in vivo model, HeLa cell encapsulated hollow fiber was implanted in Swiss Albino mice and treated with 10kDAGP. Our results showed that 10kDAGP was able to enter the cell within a span of 20 min and co-localized with mitochondria after 90 min. of incubation. A drastic loss of mitochondrial membrane potential was noted within 6 h of 10kDAGP administration along with an increase in ROS generation. ROS further led to symptoms of early apoptosis by deregulating Akt (Protein Kinase B) and activating c-Jun N-terminal Kinase (JNK), p38 Mitogen Activated Protein Kinase (MAPK), p53, and autophagy starting from 8 h of incubation. Besides in vitro conditions, 10kDAGP activated JNK to mediate cancer cell killing in vivo. Therefore, 10kDAGP can be an excellent therapeutic agent as it can act through different ways in the cellular system. Future studies are directed to screen out active peptides from the pool of peptides and to study whether the mode of action is in synergistic way or in individual forms. Ó 2014 Published by Elsevier Ireland Ltd.

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1. Introduction

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Apoptosis is a highly essential process for development and maintenance of homeostasis in multicellular organisms. It is a key feature of cancer cells that they avoid growth inhibitory signals and develop resistance to apoptosis by regulating expression of proapoptotic and antiapoptotic proteins. Due to the apoptosis avoiding nature, most of the anticancer drugs target apoptosis via activating or deactivating different pathways and signaling molecules. Among the drugs that target apoptosis in cancer, peptides or moreover hydrolysate of proteins are of greater interest as they have less side effects than traditional chemotherapies [19,27,7,40]. For instance, anticancer peptide Lunasin from soy protein hydrolysate induced apoptosis that was mediated by upregulating tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activities [29].

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⇑ Corresponding author. Tel.: +91 3222 283766; fax: +91 3222 278707. E-mail address: [email protected] (T.K. Maiti). Present address: School of Biological Science and Technology, Vellore Institute of Technology, Tamil Nadu 632014, India. 1

Rapeseed peptide isolated from rapeseed protein hydrolysate induced apoptosis by cell cycle arrest at S phase [41]. Similarly, >30 kDa fraction of Sargassum coreanum hydrolysates induced apoptosis in HL-60 cancer cell line by increasing expression of proapoptotic Bax, activating caspase-3 and cleavage of PARP [poly(ADP-ribose) polymerase] [21]. Although many lectins had been shown to have anticancer effects [10,24], they still suffer from large size and immunization problems. Hydrolyzing lectins to get smaller, non-immunogenic anticancer peptides can be a novel strategy to obtain efficient anticancer peptides. Bhutia et al. [4] had demonstrated that 10 kDa permeate tryptic hydrolysate (10kDAGP) of agglutinin lectin from seeds of Abrus precatorius had potent anticancer and immunostimulatory effect both in vitro and in vivo. Moreover, it also induced apoptosis in HeLa through increase in Reactive Oxygen Species (ROS), decrease in mitochondrial potential, increase in Bax/Bcl2 ratio and DNA fragmentation. However, detailed molecular pathways associated with apoptosis and the time-scale of events involved in this process have not been completely elucidated. Hence, in this study we deciphered the in vitro molecular mechanisms of mitochondrial apoptosis by 10kDAGP in HeLa cells. Besides, we also attempted to

http://dx.doi.org/10.1016/j.cbi.2014.08.017 0009-2797/Ó 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: B. Behera et al., Abrus precatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.08.017

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validate the applicability of 10kDAGP in the in vivo system. For in vivo work we have employed a model called ‘Modified Hollow Fiber Assay’ as described by Sharma et al. [32]. In this assay, HeLa cells were encapsulated within hollow fiber (20 mm length  1 mm ID) and implanted in Swiss Albino mice in both subcutaneous and intraperitoneal compartments followed by 10kDAGP treatment. After treatment, fibers were retrieved and cellular conditions were analyzed. It is a time saving and less expensive model started by National Cancer Institute (NCI) to screen huge number of compounds before the clinical trials.

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2. Materials and methods

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2.1. Chemicals and reagents

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MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Dimethyl Sulfoxide (DMSO), Propidium Iodide (PI), Q2 RNase A, Trypsin for protein digestion, MEM non-essential amino acids, Sodium Pyruvate, N-acetylcysteine (NAC), 3-methyle adenine (3MA), and different inhibitors of signaling molecules (SPF600125, LY294002, SB202190) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Amicon Ultra (10 kDa) was purchased from Millipore, India. Fetal bovine serum (FBS) South American origin, DMEM, MEM and RPMI-1640 media, were from Invitrogen, India. Primary antibodies for p-JNK-1 p-p53, JNK1, and p53 were obtained from Santa Cruz Biotechnologies, USA. Primary antibodies for LC3, p-Akt, p-p38, Akt, p38, active Caspase3 and all secondary antibodies were obtained from Cell Signaling Technologies, USA.

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2.2. Isolation of agglutinin derived peptides

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Peptides from agglutinin were isolated by proteolysis according to methods described in earlier reports [4] with slight modifications. Briefly, Abrus agglutinin was digested by trypsin (10  103 BAEE unit/mg of protein) in a buffer system of 10 mM Tris Buffer (pH 8.0), in 1:50 ratio (w/w) and the proteolytic digestion was carried out at 37 °C overnight (18 h). After incubation, the mixture was passed through Amicon Ultra 10 kDa cut-off membrane and the membrane permeate was taken as the 10kDAGP. The 10kDAGP was lyophilized and kept at 20 °C until further use. Quantification of peptides was done using Lowry method whenever necessary.

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2.3. Cell lines HeLa cell line was obtained from The National Centre for Cell Sciences (NCCS), Pune. It was maintained in Minimum Essential Medium (MEM) supplemented with 1 mM Sodium pyruvate, 100 unit Penicillin, 0.1 mg Streptomycin and 10% Fetal Bovine Serum (FBS).

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2.4. Cytotoxicity test in HeLa cells

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Cytotoxic activity of 10kDAGP was evaluated in HeLa cell as described by Mosmann et al. [28]. Briefly, logarithmically growing cells were trypsinized and seeded in 96 well tissue culture plates at 104 cells/ml concentrations. After overnight incubation of the plates inside humidified 5% CO2 incubator, 10kDAGP was treated with or without pretreatment of 10 mM NAC, JNK inhibitor (25 lM, SP600125), p38 inhibitor (20 lM, SB202190), autophagy inhibitor (10 mM 3MA) and Akt inhibitor (50 lM, LY294002). After 72 h treatment, viability of cells was checked by MTT assay.

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2.5. FITC labeling of peptides and cell internalization studies

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Cellular internalization studies of 10kDAGP were done by labeling the peptide fractions with Fluorescein Isothiocyanate (FITC). FITC (dissolved in DMSO) and 10kDAGP were taken in 1:1 ratio (w/w) in 100 mM NaHCO3 and kept for 4 h in dark at room temperature. Thereafter, 1 M ethanolamine was added to block the unbound FITC. Free FITC was removed from FITC-10kDAGP peptide fraction by acetone precipitation as described earlier by Vive’s and Lebleu [38]. HeLa cells were treated with FITC-10kDAGP and Mitotracker Red dye /Lysotracker Red dye simultaneously for different time intervals. After the treatment, cells were washed and examined under Confocal microscope (Olympus IX81, FV1000) for internalization and co-localization of FITC-10kDAGP. To study membrane co-localization, cells were prestained with 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethyl-indocarbocyanine perchlorate (DiI) before cell seeding. The percentage of peptides present in membrane and in cytoplasm was calculated by ImageJ software.

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2.6. Reactive Oxygen Species (ROS) measurement

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For ROS measurement, HeLa cells were seeded in 12-well tissue culture plates. Then cells were pretreated with Dichlorofluorescein diacetate (DCFDA) for 30 min. before treatment with 10kDAGP for different time intervals. Non-fluorescent DCFDA gets cleaved inside cell to form fluorescent dichlorofluorescein (DCF) which fluoresce according to ROS levels. So, after completion of treatment, cells were trypsinzed and fluorescence was measured by flow cytometry using FACS Calibur and Cell quest pro software (BD Biosciences, CA).

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2.7. Mitochondrial membrane potential measurement

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Logarithmically growing cells were trypsinized and seeded in 12-well tissue culture plates. Cells were treated with 10kDAGP for different time intervals and stained with Rhodamine 123 at the end of treatment. Mitochondrial membrane potential was measured by examining fluorescence of above stained cells through flow cytometry using FACS Calibur and Cell quest Pro software.

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2.8. Annexin V FITC-PI assay

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For apoptotic studies, HeLa cells were seeded in 6-well tissue culture plates. Cells were treated with 10kDAGP with or without 10 mM NAC for different time intervals and stained with Annexin-V FITC-PI Kit (BD Biosciences, CA) according to the company’s protocol. Cells were then observed in flow cytometry using FACS Calibur and Cell Quest-pro software.

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2.9. Western blotting

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Western blotting was done according to the method described by Mallick et al. [26]. Briefly, HeLa cells were treated with 10kDAGP for 8 or 10 h with or without pretreatment of 10 mM NAC, JNK inhibitor (25 lM, SP600125), p38 inhibitor (20 lM, SB202190), and Akt inhibitor (50 lM, LY294002). Cells were then lysed using Cell Lytic™ (Sigma–Aldrich, USA) supplemented with protease and phosphatase inhibitor. Protein was loaded on SDS– PAGE and proteins were blotted on to a PVDF membrane after gel electrophoresis. Expressions of p-JNK, p-p53, p-38, LC3, Akt, and active caspase-3 were evaluated using respective antibodies and b-actin was taken as equal loading control. HRP- conjugated secondary antibodies and ECL substrates (Sigma–Aldrich, USA) were used for detection and band development in photographic films. Band intensity was measured using ImageJ software [22,11].

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2.10. Hollow fiber encapsulation of HeLa cells

2.14. Statistical analysis

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All data were represented as mean plus standard deviation of three independent experiments. Statistical significance was calculated using two-tailed student T-test and data with P < 0.05 were considered as significant.

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3. Results

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3.1. 10kDAGP showed time and dose dependent toxicity in HeLa cells

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2.11. Mice maintenance

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Swiss Albino mice were obtained from National Centre for Laboratory Animal Sciences (NCLAS) and maintained in Animal House facility available in the institute. Mice were kept inside isolated cages connected to ventilation regulator. Mice were provided with pellet food and water ad libitum. Mice were kept at 23–25 °C with 12–14 h dark and light cycles. All experiments were performed with intensive care and following permission and instructions of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and Institute Animal Ethics Committee (Reg. No. 764/03/ac/CPCSEA).

Bhutia et al. [5] had reported that IC50 value of 10kDAGP for HeLa cell (for 72 h) was 2.4 ± 0.3 lg/ml whereas IC50 value for normal cells like 3T3 or Hacat cell was more than 100 lg/ml. This shows 10kDAGP is more specific towards cancer cells than normal cells and it makes 10kDAGP an interesting drug candidate for further study. Although toxicity of 10kDAGP in HeLa cells had been previously described by Bhutia et al. [5], time dependent toxicity was not revealed indicating that mechanistic and sequential events occurring inside the cellular compartment still need to be investigated. Hence, in the present study, toxicity of 10kDAGP was studied taking different time intervals (24, 48, and 72 h) and different doses (25, 12.5, 6.25, and 3.12 lg/ml). Fig. 1 described the% proliferation index of HeLa cells obtained for different times and doses of treatment. As the proliferation index reached near IC50 within 24 h at 25 lg/ml, this dose was selected for further time dependent mechanism study.

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Hollow fiber assay to evaluate anti-cancer effect of 10kDAGP was done according to method described by Hall et al. [15] with some modifications. Dry hollow fibers (Spectrum Lab, USA) of 1 mm i.d. and 2 cm length were taken and activated by keeping them in 100% ethanol for 30 min. Fibers were then washed and immersed in distilled water and autoclaved. Sterile fibers were then kept in complete media for 24 h inside 37 °C humidified 5% CO2 incubator. Logarithmically growing HeLa cells were trypsinized and 5  104 cells were injected inside each fiber using syringe. Both ends of the fibers were heat-sealed by smooth ended needle holders. Hollow fiber encapsulated cells were kept inside incubator for 24 h before in vivo implantation.

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2.12. Hollow fiber assay

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3.2. Uptake and co-localization of FITC-10kDAGP with cell membrane and mitochondria

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As intra-cellular targets may decipher further activity of drug molecule, co-localization studies were done taking FITC dyeconjugated 10kDAGP. Fig. 2 describes co-localization of peptides with cell membrane (a), lysosome (b) and mitochondria (c). It is evident from the above figures that some part of 10kDAGP got incorporated into the membrane within 20 min and a fraction of it moved inside (50% of total fluorescence was from membrane region). After 150 min of incubation, maximum part of the peptides co-localized with mitochondria in a time dependent manner. It was also found that the peptides did not co-localize with lysosome within the time of study. It would be worthwhile to mention that a part of the peptides were still located in the cytoplasm suggesting cytoplasmic localization of peptides.

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For implantation of hollow fiber encapsulated HeLa cells in the in vivo system, nine Swiss Albino mice (22 ± 2 g wt. and 6–8 week old) were kept in overnight starvation and anesthetized using mixture of Ketamine/Xylazine (5 mg/kg body weight). Dorsal surface below neck and left dorso-lateral portion of the mice were shaved with shaving razor. A small incision on dorsal surface below neck was made to push one cell-loaded hollow fiber in head-to-tail direction inside the subcutaneous cavity. The incision was sealed by suturing with silk thread. Similarly, 0.2 mm cut was made on left dorso-lateral side to expose peritoneal cavity (P.C.); one hollow fiber with cells was inserted inside the PC and sutured. Sealed portions were applied topically with Betadine and Neosporin for preventing infection. Mice were then treated with warm PBS (S.C.) and kept at 37 °C warm condition for recovery from anesthesia. All the operations were done in aseptic conditions with temperature regulators. Mice were treated with TAXIM antibiotic (100 lg/kg body wt., i.p.) for five consecutive days for wound healing as post-operative care. Mice were then divided into three groups (n = 3). One group served as control and other two groups received 1 and 2 mg/kg body wt. of 10kDAGP treatment (i.p. route, daily for 6 days). After treatment mice were sacrificed by over-anesthesia and fibers were isolated under aseptic condition. Fibers were then washed in PBS, cleaned for removing external tissue and immersed in serum-free media containing 0.01 mg/ml Resazurin for alamar blue assay. After 6 h of incubation, fibers were taken out from media, washed in PBS and fixed with Bouin’s fluid for histological studies.

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2.13. Immunohistochemistry

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Fixed cell containing hollow fibers were embedded in paraffin and sections of 15 lm thickness were made using microtome. Sections were fixed on APTES (3-Aminopropyl) triethoxysilane)derivatized glass slides and taken for H&E staining. Parts of the sections were also subjected to immunohistochemistry for expression of p-JNK [36,6].

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Fig. 1. Time and dose dependent toxicity of 10kDAGP on HeLa cells. HeLa cells were treated with 10kDAGP for 24, 36 or 72 h time intervals at concentrations of 6.25, 12.5 or 25 lg/ml and% proliferation index was calculated. IC50 was obtained to be 25 lg/ml for 24 h treatment and was selected for further mechanism study (⁄P < 0.05, ⁄⁄P < 0.01).

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Fig. 2. Time dependent uptake and co-localization of 10kDAGP with cell membrane, lysosome and mitochondria. For (a) membrane co-localization study, HeLa cells were pre-stained with 4 lg/ml 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI) and then treated with 25 lg/ml FITC labeled 10kDAGP(or 0.75 lg per 3  104 cells). For (b) lysosome and (c) mitochondrial co-localization study HeLa cells were first treated with 1 lM Lysotracker Red dye and 1 lM Mitotracker Red dye respectively and then treated with FITC-10kDAGP. Pearson’s Co-efficient (PC) was calculated for five regions per picture and results are expressed as Mean ± SD. (Bar represents 10 lm.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3. Time dependent decrease in mitochondrial potential

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Mitochondrial membrane permeabilization and loss of membrane potential is the near-universal hallmark and a critical step for subsequent cell death [35]. Fig. 3(a) describes that after 10kDAGP treatment, mitochondrial membrane potential (MMP) decreased with time, starting from 6 h of treatment. There were no significant changes in MMP during the initial time periods which indicate that localization of peptides within mitochondria was the first step in induction of death signals.

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3.4. Time dependent increase in ROS generation in HeLa cells and role of ROS in cell death As ROS generation is an after-effect of decrease in MMP [35,12,43], change in ROS was measured by DCFDA staining. Fig. 3(b) shows that ROS generation was enhanced with treatment of 10kDAGP in time dependent manner and ROS production initiated from 8 h of treatment. No significant change in ROS level was noted up to 8 h of treatment. As the decrease in MMP starts from 6 h of treatment, it confirms that enhanced ROS level may be due to the loss of MMP. In addition, pretreatment with known ROS scavenger, NAC (N-acetylcysteine) increases% proliferation index (Fig. 3c). This suggests that the ROS generated by loss of MMP was a key player in cancer cell death promotion. Time dependent induction of apoptosis was studied using Annexin-V FITC and PI staining. From Fig. 3(d) it may be inferred that apoptosis

commenced from 8 h of treatment and increased with time. With NAC treatment, apoptotic population decreased partly as compared to the only treated group. This further suggests that ROS is a key player in triggering apoptosis.

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3.5. 10kDAGP-induced apoptosis was regulated by ROS dependent signaling pathway

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As ROS is a key player of apoptosis, it is necessary to know which signaling pathway is activated by ROS to stimulate death in HeLa cells. It is known that ROS dependent cell death can take place through deregulation of AKT [16] or up-regulation of c-Jun N-terminal Kinase or JNK [44], p38 MAPK [8];), p53 [18], and autophagy [14]. Hence, Western blotting analysis was done to evaluate expressional level of all above signaling molecules. Results shown in Fig. 4(a) demonstrated that phospho-JNK, phospho-p38 MAPK, autophagic marker protein LC3 II and phosphop53 increased while phospho-Akt decreased with treatment of 10kDAGP after 8 h of incubation. The above effects were reversed with pretreatment of NAC, which suggested that ROS was the upstream key player of all the above events. As indicated from Fig. 4(b), pretreatment with inhibitors of JNK (SP600125), p38 (SB202190) and autophagy (3MA) increased proliferation index of HeLa cells whereas Akt inhibitor (LY294002) severely decreased the% proliferation index with respect to only 10kDAGP treatment. In addition, when pretreatment with inhibitors of different signaling molecules was given before addition of 10kDAGP, it was found

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Fig. 3. Time dependent change in Mitochondrial Membrane Potential (MMP), ROS, and early apoptosis and effect of NAC on proliferation. (a) HeLa cells were treated with 25 lg/ml 10kDAGP for indicated time intervals and after that cells were stained with 5 lg/ml Rhodamine 123 to measure MMP by flow cytometry. (b) Cells were stained with 5 lM DCFDA and then treated with 10kDAGP for different time intervals. ROS was measured by flow cytometry thereafter. (c) HeLa cells were pretreated for 1 h with 10 mM N-acetylcysteine (NAC) before treatment with 10kDAGP and proliferation index was calculated. (d) Cells were treated with 10kDAGP with or without NAC pretreatment for different time interval and then stained with Annexin V-FITC and PI for evaluation of apoptotic population by flow cytometry. (⁄P < 0.05, ⁄⁄P < 0.01.)

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that active caspase-3 (an apoptosis marker) was decreased (Fig. 4c). More precisely, there was about 4- and 2-fold decrease in active caspase-3 with pretreatment of JNK and p38 inhibitors respectively. Active-caspase-3 expression also increased with pretreatment of Akt inhibitor. Again, from Fig. 4(d), it is evident that with JNK inhibition, p-38, and p-p53 increases; with p-p38 inhibition, p-JNK increases but p-p53 decreases; and with Akt inhibition, levels of p-p38, p-p53, and p-JNK increase. Taken together, Fig. 3(a–e) suggest that treatment of 10kDAGP caused death by (i) inducing ROS production which resulted in activation of multiple stress-activated pathways like JNK, p38 and autophagy (ii) activation of p38; (iii) 10kDAGP took alternative pathways (JNK or p38) if either of these was inhibited; and (iv) 10kDAGP not only activated death-promoting molecules but also suppressed survival-promoting factors like Akt.

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3.6. Growth inhibition of HeLa in hollow fiber assay

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Application of drug in the in vivo system is necessary to stage it as a preferable therapeutic molecule. Cancer models like the

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modified hollow fiber assay provides a suitable platform to test efficacy of drug molecule on transgenic cancer cells which otherwise will not form tumor in vivo. In addition, it also allows interaction of chemical change instead of cellular interactions which prevents immune rejection in case of transgenic tumor implantation. Thus, here HeLa cells were encapsulated inside hollow fibers and implanted in intraperitoneal and subcutaneous compartment of Swiss Albino mice. The doses of 10kDAGP were decided based on the toxicity level (5 mg/kg body weight) as reported earlier by Bhutia et al. [4]. In addition to this, no significant difference in control and treated mice was observed during gross examination of body weight, fur conditions, activeness and weight of vital organs such as liver, spleen, and kidney. This ensured that there was no general toxicity of the Hollow Fiber in the systemic level. Alamar blue assay of hollow fiber encapsulated HeLa cells (isolated from both subcutaneous and intra-peritoneal compartment of control and treated group mice) revealed that tumor cell inhibition reached 50% with treatment of 10kDAGP at a dose of 2 mg/kg body weight (Fig. 5a–d). From the results, it may be inferred that 10kDAGP has anticancer property against human cervical cancer

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Fig. 4. Change in level of different signaling molecules and effect of pharmacological inhibitors as analyzed by Western blotting and MTT methods. Cells were treated with 10kDAGP for 8 and 10 h with or without pretreatment of NAC and then whole cell lysates were subjected to Western blotting. Protein levels were detected by chemiluminescence and recorded using X-ray films. Band intensity was calculated by ImageJ analysis and ratio of band intensity of protein of interest to that of b-actin (taken as loading control) was taken for evaluation of change in protein level. (a) Changes in expression of phosphorylated and non phosphorylated form of JNK1, Akt, p38, p53, and LC3. (b) Changes in proliferation index upon pretreatment with JNK inhibitor (SP600125), p38 inhibitor (SB202190), autophagy inhibitor (3MA) and Akt inhibitor (LY294002) (⁄P < 0.05). (c) Changes in expression of active caspase-3 upon pretreatment with JNK, p38 and Akt inhibitors. (d) Changes in expression of p-JNK, p-p38, and p-p53 upon pretreatment with JNK, p38 and Akt inhibitors.

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cells in vivo in addition to various other murine tumor models like Dalton’s Lymphoma [4], Ehrlich’s ascites, and B16 melanoma (data unpublished). 3.7. H&E staining and immunohistochemistry of hollow fiber isolated from mice

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After alamar blue viability assay, the hollow fibers were subjected to histological sectioning, H&E staining to examine morphology and immunostaining for partial analysis of mode of action of 10kDAGP in mice system implanted with hollow fibers. H&E staining (Fig. 5e) demonstrated that cells were more densely packed and with prominent nuclear stain in control group whereas cells were sparse and with light or no nuclear stain in case of treated group. This was indicative of the fact that cells undergo nuclear degradation due to 10kDAGP treatment. Moreover, decrease in expression levels of JNK phosphorylation in treated group compared to control was observed (as obtained by immunostaining for p-JNK, Fig. 5f and g) suggesting 10kDAGP mediated cell death via stress-activated JNK pathway both in vitro and in vivo.

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4. Discussions

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Our previous study showed that 10kDAGP is a peptide pool containing peptides in the range of 918–1976 Da. Two of the peptides from the 10kDAGP peptide pool have been successfully identified

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so far [2]. These peptides were found to contain amino acid sequences similar to preproagglutinins SGASDDEEIAR (PI – 3.92, 1149.1 Da) and ICSSHYEPTVRIGGR (PI – 8.23, 1674.9 Da) along with other peptides that are still under identification. According to the present results obtained, it may be inferred that some fraction of 10kDAGP (50% of total fluorescence comes from cell membrane) binds to membrane while some enters the cytosol within 20 min of incubation. On internalization, part of 10kDAGP co-localizes with mitochondria and some part stays in the cytoplasm. This may be a critical step for the decrease in mitochondrial membrane potential (MMP) and ROS production after 6 and 8 h of incubation respectively. Loss of Mitochondrial Membrane Potential (MMP) can occur due to various reasons including (i) matrix remodeling (ii) mitochondrial swelling (ii) inhibition of synthesis and transport of ATP (iii) opening of permeability transition pore (PTP) (iv) increase of pro-apoptotic proteins (Bax, Bak, and Bad) or decrease of anti-apoptotic proteins (Bcl-xl, Bcl-2, Mcl-1) (v) increase of Reactive Oxygen Species (ROS) and (vi) release of Cytochrome-c. All above events are neither mutually independent events nor always a first step in process of MMP loss and they may occur as an after effect of MMP loss. In fact, the mechanism of MMP loss is not universal and it depends on the cell type, the treatment type, and with the fluorochrome used for detection [25]. There are peptides such as BM-1197 which bind to anti-apoptotic proteins Bcl-2/ Bcl-xl in both Small cell lung cancer cells and Mcl/ Mouse Embryonic Fibroblast and induce Bax/Bak dependent opening of PTP and cause disruption of MMP [1]. There are other compounds

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Fig. 5. In vivo anticancer activity of 10kDAGP in hollow fiber encapsulated HeLa cells that were implanted in peritoneal and subcutaneous cavity of Swiss Albino mice. Hollow fiber encapsulated HeLa cells were retrieved from (a) peritoneal and (b) subcutaneous cavity of mice. (c) Recovered hollow fibers were subjected to (d) alamar blue viability assay, (e) Hematoxylin and Eosin (H&E) staining and (f, g) immunohistochemistry (f: nuclear staining, DAPI, g: p-JNK, Alexa F565). (⁄P < 0.05, bar represents 20.2 lm.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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like staurosporine which inhibit ATP transport in Jurkat cells, exhibits initial increase in MMP, but later induce mitochondrial swelling and MMP loss [31]. As Bhutia et al. [5] had shown that 10kDAGP increases Bax/Bcl-2 ratio in HeLa cells, it can be proposed that localization of 10kDAGP to mitochondria inhibits Bcl-2 and activates Bax which in turn open up the PTP and induce MMP loss. However, the exact time dependent changes in events like Bcl-2 inhibition, Bax activation, mitochondrial swelling and change in ATP/ADP ratio are yet to be studied for clarifying the detailed mechanism of MMP loss by 10kDAGP. ROS is necessary for tumor cell proliferation, secretion, differentiation and defense but at the same time high ROS generation also paves the path of tumor cell apoptosis and senescence [3]. Increase in survivability and decrease in apoptotic population of HeLa cells with NAC (known ROS inhibitor) pretreatment confirms that ROS may be the key death initiator in 10kDAGP-mediated cell death. However, as there is only 30% recovery from cell death, it indicates that there is also a direct role of peptides in killing of cells apart from killing through ROS generation. This may be further due to synergistic effect of two or more peptides. In addition, studies regarding involvement of signaling molecules reveals that 10kDAGP treatment increased levels of active JNK, p38, p53 and LC3 II whereas it decreased level of Akt after 8 h of treatment. No increment in JNK expression was observed up to 8 h of treatment which might be due to no significant change in ROS level. It is known that JNK and p38 are members of Mitogen Activated Protein Kinase (MAPK) family which

respond to different stress conditions like oxidative stress, UV radiation, hyperosmosis, and inflammatory cytokines [9]. In nonstressed conditions, JNK and p38 are deregulated by binding with other proteins [37] or dephosphorylation by MAPK phosphatases or deactivation of common upstream kinases. But when high ROS is produced, JNK and p38 become free from other proteins and MAPK phosphatases are inactivated probably by oxidation of critical cysteine residues [20]. As ROS is a common upstream player, it may be the plausible reason for activation of both JNK and p38 with 10kDAGP treatment. Activation of both JNK and p38 was also observed in case of few other anticancer agents like Aplidin™ [8], and adaphostin [42] but the cross talk between JNK and p38 was not studied by them. Attempts to check the cross talk between JNK and p38 (Fig. 4d) revealed that when one of them was inhibited the other one got upregulated. This explains that JNK and p38 act independently and it also confirms the presence of ROScontrolled common upstream activator of both JNK and p38. Studies regarding the pro-apoptotic tumor suppressor protein p53 suggested that it was activated with treatment of 10kDAGP and decreased when either ROS or p38 was inhibited (Fig. 4a and d). It suggests that 10kDAGP activated ROS which in turn activated p38 and p53 was further activated by p38. It is to be noted that JNK and p53 can act for cell survival in high ROS conditions ([33,8] but as there was sustained increase in both JNK and p53 (from 8 to 10 h, Fig. 4a) this may have been the cause of death triggered by 10kDAGP.

Please cite this article in press as: B. Behera et al., Abrus precatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.08.017

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Fig. 6. Schematic representation of signaling molecules involved in HeLa cell apoptosis induced by 10kDAGP. ‘?’ represents upregulation and ‘–’ represents down regulation ‘"’ represents further increment and ‘;’ represents further decrement starting from the time of observation. Dotted lines or arrows represent probable pathways reported earlier.

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In contrast to the stress (ROS) activated apoptotic pathways, activated PI3K/Akt is one of the key players that help in tumor cell survival and proliferation. Therefore, effect of 10kDAGP on p-Akt was evaluated. Like the compounds adaphostin [42] and diclofenac [17], 10kDAGP also deregulates PI3K/Akt in a ROS-dependent manner. Furthermore, when cells were treated with both Akt inhibitor LY294002 and 10kDAGP, there was increased activation of active caspase-3, p53, p38 and JNK which led to enhanced cell death (Fig. 4b–d). It is worthwhile to note that high ROS increases Akt activation and cell survival [34]. However, as p53 is activated by 10kDAGP treatment, this may be another reason behind Akt downregulation as explained by Gottlieb et al. [13]. Besides apoptosis, another pathway that plays crucial role in cancers is ‘autophagy’. From the results it can be inferred that 10kDAGP also increased autophagy like other lectins and its parent protein [10,24,30]. According to earlier reports, autophagy can lead to either survivability or to apoptosis in oxidative (ROS) stress conditions. As autophagy inhibitor 3-MA increased the proliferation index (Fig. 4b), it tells that autophagy led to cell death in our case. All these results support the earlier findings that down regulation of Akt [30], increase of ROS [23] and activation of p38 MAPK [39] can lead to autophagy but which pathway is more responsible in case of 10kDAGP needs to be ascertained through further investigation. Apart from in vitro studies, in vivo validation is an important pre-requisite of any therapeutic molecule. Hence, Bhutia et al. [4] had demonstrated in vivo anticancer activity of 10kDAGP in Dalton’s Lymphoma Ascites mice tumor model. To further verify it is antitumor property in human tumor models like HeLa cells, one will need immune-deficient nude mice system which is expensive and time taking. For quick evaluation, here NCI approved hollow fiber assay had been followed. However, instead of nude mice in hollow fiber assay, as described by Hall et al. [15], in-bred Swiss Albino mice was taken for our experiments according to a protocol reported earlier by Sharma et al. [32]. Interestingly, 10kDAGP treatment showed 50% survivability of HeLa cells in both subcutaneous and intraperitoneal compartments at a dose of 2 mg/kg

body weight. Furthermore, immuno-histochemical study of Hollow Fiber encapsulated HeLa cells (isolated from mice) showed higher expression of p-JNK which is in line with the obtained in vitro results. All together, above results indicate that 10kDAGP acts against cancer cells through multiple pathways, which are summarized in Fig. 6. It is thus clear that10kDAGP is anticancerous not Q3 only in vitro but also in vivo as analyzed using hollow fiber systems.

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The 10kDAGP obtained from tryptic digests of A. precatorius lectin ‘agglutinin’ has been found to induce apoptosis by modulating different pro-apoptotic mechanisms including decrease in mitochondrial potential; induction of ROS; activation of JNK, p38 MAPK, p53, and deregulation of AKT. It not only activates apoptosis but also triggers autophagy which also contributes to total death induction. In addition to in vitro conditions, it is also capable of inhibiting cancer cells in the in vivo system using hollow fiber model. In conclusion, 10kDAGP can be a better potential anticancer therapeutic agent as it can act through multiple pathways and is associated with less side-effect. However, as it is a pool of peptides, the current experiments are directed towards fractionation, identification, synthesis, and activity study of each fraction of 10kDAGP. This will help to decipher whether one or more peptides are responsible for activation of multiple pathways and/or whether there is any synergistic effect among them.

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Conflict of Interest

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The authors declare no conflict of interest.

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Acknowledgements

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We would like to acknowledge UGC, Government of India for giving fellowship to B. Behera; DBT, Government of India for giving

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Please cite this article in press as: B. Behera et al., Abrus precatorius agglutinin-derived peptides induce ROS-dependent mitochondrial apoptosis through JNK and Akt/P38/P53 pathways in HeLa cells, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.08.017

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fellowship to K.S.P. Devi; and CSIR, Government of India for giving fellowship to D. Mishra, B. Roy and R. Narayan. We also acknowledge CSIR, Government of India for funding the project work (Scheme number: 27(0208)/09/EMR-II).

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