Accepted Manuscript Maackia amurensis agglutinin enhances paclitaxel induced cytotoxicity in cultured non-small cell lung cancer cells Rakhee Chhetra Lalli, Kiranjeet Kaur, Shashank Dadsena, Anuradha Chakraborti, Radhika Srinivasan, Sujata Ghosh PII:

S0300-9084(15)00136-4

DOI:

10.1016/j.biochi.2015.05.002

Reference:

BIOCHI 4712

To appear in:

Biochimie

Received Date: 17 September 2014 Accepted Date: 4 May 2015

Please cite this article as: R.C. Lalli, K. Kaur, S. Dadsena, A. Chakraborti, R. Srinivasan, S. Ghosh, Maackia amurensis agglutinin enhances paclitaxel induced cytotoxicity in cultured non-small cell lung cancer cells, Biochimie (2015), doi: 10.1016/j.biochi.2015.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Maackia amurensis agglutinin enhances paclitaxel induced cytotoxicity in cultured nonsmall cell lung cancer cells Rakhee Chhetra Lallia, Kiranjeet Kaura, Shashank Dadsenaa, Anuradha Chakrabortia, Radhika Srinivasanb, Sujata Ghosha* a

Department of Experimental Medicine and Biotechnology, Post Graduate Institute of

b

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Medical Education and Research (PGIMER), Chandigarh, India, 160012

Department of Cytology and Gynaecological Pathology, Post Graduate Institute of Medical

Education and Research (PGIMER), Chandigarh, India, 160012

E-mail address: [email protected], [email protected], [email protected],

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[email protected], [email protected], [email protected]

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*Corresponding author E-mail: [email protected]

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Tel No. 0091-172-2755236; Fax No. 0091-172-2744401

ACCEPTED MANUSCRIPT Abstract Maackia amurensis agglutinin (MAA) is gaining recognition as the potential diagnostic agent for cancer. Previous studies from our laboratory have demonstrated that this lectin could interact specifically with the cells and biopsy samples of non-small cell lung cancer (NSCLC) origin but not with normal lung fibroblast cells. Moreover, this lectin was also found to

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induce apoptosis in NSCLC cells. Further, the biological activity of this lectin was shown to survive gastrointestinal proteolysis and inhibit malignant cell growth and tumorigenesis in mice model of melanoma thereby indicating the therapeutic potential of this lectin. Paclitaxel is one of the widely used traditional chemotherapeutic drugs for treatment of NSCLC but it

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exerts side-effects on normal healthy cells too. Studies have revealed that lectins have potential to act as an adjuvant chemotherapeutic agent in cancer of different origin. Thus, in

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the present study, an attempt was made to assess the chemo-adjuvant role of MAA in three types of NSCLC cell lines [adenocarcinoma cell line (A549), squamous cell carcinoma cell line (NCI-H520) and large cell carcinoma cell line (NCI-H460)]. We have observed that the non-cytotoxic concentration of this lectin was able to enhance the cytotoxic activity of Paclitaxel even at low dose by inducing apoptosis through intrinsic /mitochondrial pathway in all the three types of NSCLC cell lines, although the involvement of extrinsic pathway of

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apoptosis in case of NCI-H460 cell line could not be ruled out. Further, this lectin was also found to augment the chemo-preventive activity of this drug by arresting cells in G2-M phase of the cell cycle. Collectively, our results have suggested that Maackia amurensis agglutinin may have the potential to be used as adjuvant chemotherapeutic agent in case of NSCLC.

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Keywords: Non-small cell lung cancer, Maackia amurensis agglutinin, Paclitaxel,

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cytotoxicity, chemo-adjuvant

ACCEPTED MANUSCRIPT 1. Introduction Lung cancer is one of the most common fatal malignancies throughout the world with nonsmall cell lung cancer (NSCLC) accounting for ~86% of all lung cancer cases [1-3]. It is the major cause of cancer related deaths in developed countries and is also rising at an alarming rate in developing countries including India [2, 4-5]. NSCLC represents a heterogeneous sub-

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group in terms of both behavior and therapeutic response and is usually associated with metastasis and poor prognosis [6]. The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Adenocarcinomas are the most prevalent among the three sub-types of NSCLC [7-8].

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Currently, the accepted modalities for the treatment of lung cancer involve surgery, radiation therapy and chemotherapy, singly or in combination [9-10]. The taxane group of drugs

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including Paclitaxel and Docetaxel are the most effective chemotherapeutic agents for NSCLC [11]. Paclitaxel has been shown to exert its effect by arresting cells at the G2-M phase of the cell cycle through microtubule stabilization and inducing apoptosis [12]. However, the use of such drugs is plagued by serious side effects due to their non-specific action on the normal healthy cells too. Moreover, after first round of chemo- and radiotherapy, NSCLC cells acquire drug resistance, thereby posing a significant challenge for

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successful therapy [13, 10]. Thus, medical science is in need of prospective studies to develop newer alternative strategies for the treatment of NSCLC. Altered glycosylation especially aberrant sialylation in the cellular glycoconjugates is a prominent biomolecular alteration in the cancer cells [14]. Changes in sialylation pattern of

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the cellular glycoconjugates affect their property in various ways to modulate different cellular activities associated with tumor progression [15-17]. Such altered patterns of

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glycosylation are detected by lectins [18, 19]. Numerous studies have followed the lectin based diagnostic approach in the form of histochemical analysis of malignant tumor tissues [20-23]. The diagnostic potential of different lectins was also assessed in case of NSCLC [2426]. Lectins were also shown to have anti-carcinogenic activities in vitro and in vivo that could be beneficial for the treatment of cancer [27, 28]. Various studies have revealed that lectins could induce apoptosis in different cancer cell lines [29-35]. Studies have also revealed the chemo-adjuvant property of lectins in cancer of different origin [36-39]. Maackia amurensis agglutinin (MAA) is a NeuNAcα (2-3) Gal β(1-4)GlcNAc/Glc specific lectin present in the seeds of the Amur Maackia, which has been used as a medicinal plant for centuries to treat a variety of ailments including cancer in parts of Asia [40,41]. MAA was

ACCEPTED MANUSCRIPT shown to have diagnostic potential in cancer cells of different origin [42-47, 15]. In the previous study, we have observed that this lectin showed strong reactivity, specifically with the NSCLC cell lines (NCI-H520 & NCI-H460) as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients but not with the normal lung fibroblast cells. Further, this lectin was found to activate the intrinsic apoptotic signal

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transduction pathway in NSCLC cells leading to DNA fragmentation and ultimately apoptosis [48]. MAA also could inhibit in vitro and in vivo melanoma cell growth via interaction through podoplanin, thereby indicating the therapeutic potential of MAA in melanoma [49]. Considering the importance of MAA in cancer, in the present study an

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attempt was made to assess the role of this lectin on drug induced cytotoxicity in NSCLC cell lines.

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2. Materials and methods 2.1 Chemicals

Paclitaxel, Maackia amurensis agglutinin (MAA), Monosialoganglioside GM3 (canine blood), neuraminidase (Clostridium perfringens Type V), MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide], Propidium Iodide (PI) and Trizol were purchased from Sigma-Aldrich (USA). Jacalin was purchased from Bangalore Genei (India). DMSO was

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purchased from Merck (Germany). Paclitaxel was dissolved at a concentration of 1mg/ml in DMSO and MAA and Jacalin were dissolved in saline at a concentration of 1mg/ml and all the further dilutions of Paclitaxel, MAA and Jacalin were made in RPMI 1640 media.

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Ribonuclease (RNase)-H was procured from Bangalore Genei (India). SYBR green I master mix was purchased from Roche (Germany) and First strand c-DNA synthesis kit was purchased from Thermo Scientific (USA). JC-1(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-

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benzimidazoylcarbocyanine iodide) was purchased from Invitrogen (USA). Antibodies against Bax, Bid, Bcl-XL, caspases-3,8,9 and cytochrome-c as well as the secondary antibodies [horseradish peroxidise (HRP)-conjugated anti-rabbit IgG antibody and HRPconjugated anti-mouse IgG antibody] were procured from Santa Cruz Biotech (CA, USA). 2.2 Cell lines Three human non-small cell lung cancer (NSCLC) cell lines [A549 (adenocarcinoma cell line), NCI-H520 (squamous cell carcinoma cell line) and NCI-H460 (large cell carcinoma cell line)] used in this study were procured from National Centre for Cell Sciences (Pune, India). All the cell lines were cultured in RPMI-1640 media containing 2mM L-glutamine

ACCEPTED MANUSCRIPT (Gibco, USA) supplemented with 10mM HEPES, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 1mM sodium pyruvate, 50U/ml penicillin, 50µg/ml streptomycin (Hi-Media, India) and 10% FBS (Gibco, USA) and maintained at 37˚C in humidified atmosphere of 5% CO2-95% air in a CO2 incubator (Eppendorf, Germany). 2.3 Binding of lectins to NSCLC cells

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Binding of MAA to the NSCLC cell lines was assessed by Western immunoblotting. Briefly, the membrane proteins of NSCLC cells (A549, NCI-H520 & NCI-H460) were prepared by the method of Adamo et al. [50]. Following SDS-PAGE [51] of the membrane proteins, the proteins bands were electrophoretically transferred onto the Hybond ECL membrane (GE

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Healthcare, USA) according to the method of Towbin et al. [52]. The non-specific sites of the membrane strips were blocked overnight with 5% skimmed milk in PBS (SM-PBS) at 4ºC,

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washed and incubated for 1h at 25ºC with MAA(1µg/ml) pre-treated (3h, 25ºC) with or without GM3(2µg/ml; an inhibitor of MAA [47]. In another set of experiments, after blocking the membrane strips were incubated for 1h at 25ºC with MAA in presence and absence of Paclitaxel (0.1µg). After extensive washing, the strips were incubated with IgGMAA (1:2500) for 2h at 37ºC. This was followed by washing and incubation with the HRP-conjugated goat anti rabbit antibody (1:1500, Santa Cruz Biotechnology,USA) and finally detection with

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Pierce® ECL Western Blotting Substrate kit (Thermo Scientific, USA). Binding of Jacalin to the NSCLC cell lines was assessed by flow cytometry. Briefly, the labeling of Jacalin with FLUOS was done using Fluorescein Labelling kit (Roche

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biochemicals, Germany) as per the manufacturers’ instructions. The cells (105) were suspended in 100 µl RPMI 1640 medium and the non-specific binding sites were blocked by incubation of cells with 10% FBS (30min). After washing, the cells were incubated with

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FLUOS-Jacalin at 0°C for 1h. The cells were washed, suspended in PBS, analysed by flow cytometry using ‘FACS Diva’ software programme and the results were expressed in view of mean fluorescence intensity (MFI). 2.4 PI uptake assay

Chemosensitivity in view of % apoptotic cells was determined by the PI uptake assay. PI is a fluorescent dye that intercalates into the DNA strands and the extent of PI incorporation in the cells can be monitored by flow cytometry [53]. For this assay, the NSCLC cells (5x104 /500µl media /well) grown in monolayer overnight in 24-well cell castors (Greiner Bio-One, Germany) were cultured in absence and in presence of different doses of Paclitaxel (10-

ACCEPTED MANUSCRIPT 500nM in case of A549 and NCI-H460 cells and 10-1000nM in case of NCI-H520 cells) in serum free media for 24h. In another set of experiments, the cells (5x104 cells/ 500µl media/well) were cultured with different doses of drug (10-1000nM) in serum-free media for 48h at 37ºC to determine the IC50 (the dose of the drug at which 50% cell death occurs) of Paclitaxel. The cells were trypsinized, washed and fixed in 70% ethanol for 1h at 4˚C. After

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washing, the cells were permeabilized with the hypotonic buffer (0.1% sodium citrate/ 0.1% Triton X-100 in PBS) containing RNase-H (10µg/ml) for 15min and finally PI was added at a final concentration of 10µg/ml. Analysis of the apoptotic cells was done on BD FACSCanto II cell analyser (USA) using the ‘FACS Diva’ software programme (Becton Dickinson,

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USA). The results were expressed in view of the mean fluorescence intensity of the labelled cells in the hypodiploid region, which directly correlates to the percentage of apoptotic cells. PI uptake assay was also done to evaluate MAA induced apoptosis separately in the three

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types of NSCLC cell lines. Briefly, the cells (5x104 /500µl media /well) were cultured in 24well cell castors in absence and presence of different doses of MAA (0.005-1µg in case of A549 cells and 0.005-0.5µg in case of NCI-H520 cells and NCI-H460 cells) in serum-free media for 24h at 37ºC. Similar experiments were also conducted in NSCLC cell lines (A549, NCI-H520 and NCI-H460) using Jacalin as the control plant lectin. The cells were harvested,

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washed and subjected to PI uptake assay. The apoptotic cells were analysed on BD FACSCanto II cell analyser.

Further, the effect of MAA on Paclitaxel induced apoptosis in NSCLC cells was assessed by PI uptake assay. Briefly, the cells (5x104/500µl/well) were treated with the optimum dose of

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the lectin (the dose required to induce maximum apoptosis at 24h) in serum free medium followed by different doses of Paclitaxel (25-250nM in case of A549 cells, 10-500nM in case

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of NCI-H520 and 10-100nM in case of NCI-H460 cells) for 24h. Appropriate controls were run in parallel. The cells were trypsinized, washed, subjected to PI uptake assay and analyzed by flow cytometry. The dose of Paclitaxel, which alongwith the optimum dose of the lectin could induce maximum increase in the percentage of apoptotic cells, was taken as the optimum dose of the drug. In a separate set of experiments, cells were treated with different doses of the lectin (0.005-0.1 µg) in serum free media followed by the optimum dose of the drug (as obtained from the previous experiments) for 24h. Appropriate controls were run in parallel. The cells were harvested and PI uptake assay was performed. The relatively noncytotoxic dose of the lectin, which alongwith the optimum dose of the drug could induce maximum increase in the percentage of apoptotic cells, was selected as the dose of the lectin

ACCEPTED MANUSCRIPT to be used in the subsequent experiments. In another set of experiments, NSCLC cells were treated with the non-cytotoxic dose of Jacalin (same as that selected in case of MAA) and the optimum dose of the drug under the same conditions and PI uptake assay was performed. Further, to authenticate the synergistic effect of MAA on drug induced cytotoxicity, the NSCLC cell lines (A549, NCI-H520 and NCI-H460) were cultured in serum free medium in

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presence of the non-cytotoxic dose of MAA (selected from the previous experiments), preincubated in presence and in absence of GM3 for 3 h followed by the optimum dose of the drug under the same conditions. In the other set of experiments, NSCLC cells were pretreated with neuraminidase (0.2U/106 cells/200µl media, 30min, 37ºC) and cultured under the same

cells were harvested and subjected to PI uptake assay.

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2.5 Study groups

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conditions in presence of MAA and Paclitaxel. Appropriate controls were run in parallel. The

In the present study, all the parameters in the three NSCLC cell lines were studied in four groups. Group a consisted of cells only; Group b consisted of cells cultured in presence of selected dose of the lectin; Group c consisted of cells cultured in presence of selected dose of the drug; Group d consisted of cells cultured in presence of selected dose of the lectin and the

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drug. 2.6 Cell Death Detection ELISA

The extent of apoptosis in the cells (5×104/500µl/well) cultured in 24-well cell castors was analysed by the photometric enzyme immunoassay using Cell Death Detection ELISA kit

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according to the manufacturers’ instructions. This assay is based on the quantitative sandwich-enzyme immunoassay principle using respective mouse monoclonal antibodies

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against DNA and histones. The HRP-conjugated anti-DNA antibody and 2, 2’-azino-di-[3ethylbenzthiazoline sulfonate] (ABTS) containing H2O2 are used as the secondary antibody and the chromogenic substrate respectively. This allows the specific determination of monoand oligo-nucleosomes present in the cytoplasm of the apoptotic cells, which can be monitored at 405nm on ELISA reader. The extent of apoptosis in the treated cells was expressed relative to that of the untreated cells as the apoptotic index. 2.7 Measurement of mitochondrial membrane potential The cationic fluorochrome JC-1(5,5’,6,6’-tetrachloro-1,1,3,3’-tetraethylbenzimidazoylcarbocyanine iodide), a dual emission potential-sensitive probe was used to evaluate the decrease in mitochondrial transmembrane potential (∆ψm) that accompanies the efflux of pro-

ACCEPTED MANUSCRIPT apoptotic molecules from the mitochondria to the cytosol. JC-1 forms aggregates that fluoresces red in presence of high mitochondrial membrane potential as in normal cells. When mitochondrial membrane potential decreases, as occurs when mitochondrial pores are opened during apoptotic process, JC-1 becomes monomeric and fluoresces green. For this assay, cells (106 cells/ml /well) cultured under different conditions in 6 well cell castors were

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harvested, washed and incubated with JC-1 (2µM, 15min, 37°C) in a humidified atmosphere. After washing with PBS, the cells were analysed by flow cytometry using ‘FACS Diva’ software programme [54]. 2.8 Cell Cycle Analysis

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The DNA content of the cells at different phases of cell cycle was monitored by flowcytometric analysis using PI. Histograms of growing cell culture display the characteristic x-

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axis distribution according to the DNA content, the first peak corresponding to the diploid (2c) peak, i.e. cells in the G0-G1 phase and the second peak to cells with 4c content, i.e. cells in the G2-M phase. Cells with an intermediate DNA content are in the S phase. When DNA is fragmented, as in case of apoptosis, the affinity with the intercalating propidium iodide decreases and a hypodiploid peak (or area) appears to the left of the G0-G1 peak i.e. the subG0 peak. For cell cycle analysis, cells (5×104/500µlmedia/well) were cultured in 24-well cell

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castors under different conditions as mentioned before. After trypsinization, the cells from all the four groups of all the three cell lines were harvested, washed and fixed in 70% ethanol (1h, 4˚C). Subsequently, the cells were permeabilized with the hypotonic buffer for 15min and PI was added. The cells were analysed on BD FACS Calibur cell analyser using ‘Cell

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Quest’ software programme (Becton Dickinson, USA) and expressed as percentage of cells in

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different phases of the cell cycle.

2.9 Study on the expression of different parameters of extrinsic and intrinsic pathways of apoptosis

The level of expression of different parameters of the apoptotic pathways in the three NSCLC cell lines cultured under different conditions was assessed at the mRNA level by Quantitative Real time PCR as well as at the protein level by Western immunoblotting. Appropriate controls were run in parallel.

ACCEPTED MANUSCRIPT 2.9.1 Quantitative Real Time PCR Expression of Bcl-2 family regulatory proteins (Bax, Bid & Bcl-XL) was assessed in all the groups of the three NSCLC cell lines at the m-RNA level by quantitative real-time PCR (qPCR). For this, total cellular RNA was extracted from the cells (106/ml) using Trizol method [55] following manufacturers’ instructions. The purity and amount of RNA was

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measured spectrophotometrically (BioPhotometer, Eppendorf, Germany).Concentration of RNA was measured in µg/ml as A260 x 40 x dilution factor, where 1O.D. at A260 corresponds to 40µg/ml of RNA and the purity of RNA was indicated by the ratio of A260 to A280 (1.82.0). The c-DNA was synthesized from 2µg of purified total RNA using First Strand c-DNA

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synthesis kit according to manufacturers’ instructions. This kit relies on a genetically engineered version of the Moloney Murine Leukaemia Virus reverse transcriptase with low

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RNaseH activity. Briefly, 2 µg of total RNA of each sample was mixed with 1µl oligo (dT)18 primer and final volume was adjusted to 12µl with DEPC water and incubated at 70˚C for 5 min. The reaction mixture was chilled on ice. This was followed by sequential addition of 5X reaction buffer (4 µl; provided in the kit), 1 µl RNase inhibitor (20U/µl), 10 mM dNTP mix (2 µl) and 2µl of RevertAidTM M-MuLV Reverse Transcriptase (200 U/µl), mixing and incubation at 37˚C for 1h. The reaction was stopped by inactivating the reverse transcriptase

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at 70˚C for 10 min and prepared c-DNA was further used for qPCR. Each qPCR reaction mixture contained 5µl of SYBR green I master mix, 0.2µM of each primer, 1µl (40ng) of the cDNA in a final reaction volume of 10µl. No template control reaction mixture was used as the negative control. The q-PCR was carried out by LightCycler® 480 system (Roche,

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Germany) using the conditions as mentioned in Table-1. The expression of β-actin (reference gene) was assessed for normalization against variation in

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the cDNA concentration in each set of experiment. Relative fold change in mRNA level was determined by ∆∆CT method [56]. CT value was inversely proportion to the gene expression reflecting indirect relation with the relative level of the target gene. 2.9.2 Western immunoblotting Expression of Bax, Bid, Bcl-XL and caspases (-3,-8 &-9) was assessed at the protein level by Western immunoblotting of the lysates prepared from cells (106/ml) cultured under different conditions. Briefly, after trypsinization, the cells were washed with PBS (pH 7.4) and incubated in lysis buffer [10mM HEPES (pH 7.5) containing 150mM NaCl,10% glycerol,10mM NaVO4,0.6% Triton X-100 and cocktail protease inhibitors (1:10)] for 30min at 4ºC [57]. This was followed by centrifugation (10,000rpm, 10min) to remove the cell

ACCEPTED MANUSCRIPT debris. The protein content of the supernatant was estimated by Bicinchoninic acid assay [58]. Following SDS-PAGE (12%) [51] of the lysates, the proteins bands were electrophoretically transferred onto the Hybond ECL membrane (GE Healthcare, USA) according to the method of Towbin et al. [52]. The non-specific sites of the membrane strips were blocked overnight

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with SM-PBS at 4ºC. After washing with PBST, the strips were probed with antibodies against Bax, Bid, Bcl-XL, caspase-3, caspase-8 and caspase-9 (1:250 in each case) separately, washed and incubated with HRP-conjugated respective secondary antibody (HRP-conjugated anti-rabbit/anti-mouse IgG antibody; 1:1000) followed by detection with Pierce® ECL

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Western Blotting Substrate kit (Thermo Scientific, USA). To assess the expression of β-actin, the same membranes were stripped off by incubation in the stripping buffer [62.5mM

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Tris/HCl (pH 6.7) containing 2% SDS & 100mM β-mercaptoethanol] for 30 min at 60ºC. The membranes were washed thoroughly with PBST, blocked in 5%SM-PBS and reprobed with the antibody against β-actin (internal control). The expression of β-actin (internal control) in the lysates of each group was assessed to check the quality of lysate preparation and for normalization against variation in the protein concentration.

For assessment of cytochrome c, mitochondrial and the cytosolic fractions were isolated from

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the cells [29]. Briefly, after harvesting and washing, the cells (5x106) were suspended in the isolation buffer [20µM HEPES (pH 7.4) containing 10 µM KCl, 1.5µM MgCl2, 1µM disodium salt of EDTA, 1µM dithiothreitol & cocktail protease inhibitor (1:10)]. After chilling on ice for 5 min, the cells were sonicated. The lysates were centrifuged (2500g,

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10min, 4°C) to remove unbroken cells and nuclei. Subsequently, the mitochondria were pelleted by centrifugation (9000g, 30 min, 4°C). The resulting supernatant was centrifuged

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(100,000g, 1h, 4°C) to give cytosolic fraction and light membrane fractions. After estimation of the protein content, an equal amount (60µg) of mitochondrial and cytosolic proteins from each group was separately resolved on 15% SDS-PAGE under reducing conditions and transblotted [52]. After blocking in 5% SM-PBS, the membranes containing the protein bands were probed with antibody against cytochrome c (1:250). This was followed by washing, incubation with respective HRP-conjugated secondary antibody (1:1000) and development by ECL. The expression of β-actin was assessed as mentioned earlier.

ACCEPTED MANUSCRIPT 2.10 Densitometric Analysis The change in band intensity in case of each parameter in Western immunoblotting was quantified by scanning densitometry using Scion image 4.3software. The values of all the parameters in all the study groups were normalized with the value of β-actin in the respective group. Within each experiment, the level of expression of each parameter in the control (cells

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cultured without any treatment) was set as 1and the increase or decrease (fold) in the level of expression of the respective parameter in the cells cultured with different treatments was calculated. In case of cytochrome c, the densitometric values of both the mitochondrial and cytosolic fractions of cytochrome c in all the study groups were normalized with that of β-

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actin. Within each study group, the level of expression of mitochondrial cytochrome c was set as 1 and the increase (fold) in the level of expression of the cytosolic cytochrome c was

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calculated. 2.11 Statistical Analysis

The data was analyzed by applying various statistical tests e.g. paired‘t’ test and ANOVA as per requirement. The probability value of less than 0.05 was considered as significant. 3. Results

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3.1 MAA and Jacalin bind to NSCLC cell lines

The Fig. 1A reveals the binding of MAA to the membrane proteins of the three NSCLC cell lines. MAA was found to interact with a protein band of molecular weight ~66 kDa in case of

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all the three NSCLC cell lines (A549: lane 1, NCI-H520: lane 3, NCI-H460: lane 5), the intensity of which was found to be reduced in the presence of GM3 (A549: lane 2, NCI-H520: lane 4, NCI-H460: lane 6), an inhibitor of MAA [47]. However, no alteration in the intensity

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of ~66kDa band was noted in presence of Paclitaxel. The Fig. 1B shows the representative bar diagram showing the binding of Jacalin to the three NSCLC cell lines. It was observed that A549, NCI-H520 and NCI-H460 cells showed 5 fold, 4.3 fold and 5.75 fold binding to Jacalin as compared to untreated cells. 3.2 Paclitaxel & MAA induce apoptosis in NSCLC cell lines PI uptake assays were performed to assess Paclitaxel and MAA sensitivity of the three NSCLC cell lines. Dose dependent apoptosis with Paclitaxel was noted in all the cell lines at 24h/48h as assessed by PI uptake assay. Maximum percentage of apoptotic cells in A549, NCI-H520 and NCI-H460 cell lines were found at 250nM (29.5± 0.85%), 500nM (20.6±

ACCEPTED MANUSCRIPT 2.8%) and 50nM (23.4±1.9%) respectively at 24h (Fig. 2A), while at 500nM (54.5±0.5%), 750nM (53.3±1.5%) and 500nM (58.0±0.5%) respectively at 48h (Fig. 2B) The IC50 values of Paclitaxel were found to be 171.3nM, 529.7nM and 213.1nM in case of A549, NCI-H520 and NCI-H460 cells respectively. Maackia amurensis agglutinin (MAA) was also found to induce dose dependent apoptosis in

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all the three NSCLC cell lines at 24h with maximum percentage of apoptotic cells detected at 0.5µg dose of MAA in A549 cells (26.1 ± 0.8%), while at 0.25µg dose of the lectin in NCIH520 cells (40.2 ± 0.28%) and NCI-H460 cells (35.9 ± 0.42%) as depicted in Fig.2C. However, the percentage of apoptotic cells found with Jacalin at the same dose as that of

13.7± 0.83% respectively as depicted in Fig.2D.

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MAA in A549 cells, NCI-H520 cells and NCI-H460 cells was 20.1±0.42%, 16.3 ± 0.53% and

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3.3MAA enhances the Paclitaxel induced apoptosis in NSCLC cell lines

To determine the effect of MAA on the Paclitaxel induced apoptosis, in one set of experiments, optimum dose of the lectin was used in combination with different doses of the drug. Maximum increase in the % of apoptotic cells was noted in case of 50nM Paclitaxel treated A549 cells in presence of 0.5µg dose of MAA, while in case of 250nM and 50nM Paclitaxel treated NCI-H520 cells and NCI-H460 cells respectively in presence of 0.25µg

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dose of the lectin (Table 2). In the other set of experiments, when optimum dose of the drug (as obtained in the previous experiment) alongwith different doses of the lectin were used, the % of apoptotic cells increased maximum in the A549 cells (18.5%) treated with 50nM Paclitaxel in presence of 0.005µg dose of MAA followed by NCI-H460 cells (15.2%) and

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NCI-H520 cells (12.4%) treated with 250nM and 50nM Paclitaxel respectively in presence of 0.01µg dose of the lectin (Table 3A). From the above experiments the non-cytotoxic/less-

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cytotoxic dose of MAA and the moderate cytotoxic dose of Paclitaxel were selected to be used in combination for the subsequent experiments. Jacalin at the same dose as that of MAA under the same conditions could enhance the drug induced cytotoxicity in A549 cells by 8.5%, NCI-H520 cells by 7.4% only and no synergistic effect was noted in case of NCI-H460 cells (Table 3B).

The Table 4 clearly depicts that GM3 (0.014 ng) could inhibit the enhanced effect of MAA (0.005µg) on Paclitaxel induced cytotoxicity in A549 cells by 64% and completely inhibited the enhanced effect of MAA (0.01µg) on Paclitaxel induced cytotoxicity in NCI-H520 and NCI-H460 cells. Moreover, the effect of MAA on the drug induced cytotoxicity in NSCLC

ACCEPTED MANUSCRIPT cells pretreated with neuraminidase was also found to be inhibited by 33% in A549 cells, 50% in case of NCI-H520 cells and 61 % in case of NCI-H460 cells. The Fig. 3 depicts the extent of apoptosis induced at 24h in each study group of the three NSCLC cell lines as evaluated by cell death detection ELISA. The apoptotic index of A549, NCI-H520 and NCI-H460 cells treated with the combination of the lectin and the drug was

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found to be increased by 2.4 fold, 2 fold and 2.4 fold respectively as compared to the respective cells treated with the drug alone (1.7 fold, 1.5 fold and 1.8 fold respectively). The apoptotic index of the cells treated with the lectin only was comparable to the control cells in case of all the cell lines.

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3.4 MAA augments Paclitaxel induced decrease in mitochondrial membrane potential in NSCLC cell lines

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The Fig. 4A-C shows the change in mitochondrial membrane potential (∆ψm) in different study groups of NSCLC cells. As depicted in Table 5, the percentage of depolarized cells in the green fluorescence region (lower right quadrants, Q4) increased significantly when the cells were treated with the drug alone as compared to the respective control cells. The percentage of cells in the green fluorescence region was found to be increased by 18%, 13% and 15% in case of A549, NCI-H520 and NCI-H460 cells respectively, when the cells were

treated cells.

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treated with the combination of the lectin and the drug as compared to the respective drug

3.5 MAA synergizes Paclitaxel induced cell cycle arrest in NSCLC cell lines

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The representative histograms of cell cycle analysis of all the study groups of the three NSCLC cell lines are shown in Fig.5A-C. Redistribution of the cells was observed in

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different phases of the cell cycle. Paclitaxel is known to exert its action by causing G2-M phase (mitotic) arrest of the cell cycle, as was observed in case of all the cell lines and the percentage of cells in this phase was increased significantly when the cells were treated with the combination of the lectin and the drug. The sub-G0 population in the drug treated cells in absence and in presence of the lectin was significantly higher as compared to the respective control cells. Further, the number of cells in this phase was significantly reduced in case of the cells treated with the combination of the lectin and the drug in comparison to the respective drug treated cells. The G0-G1, G2-M and the sub-G0 population in case of cells treated with the lectin was comparable to the respective control cells, as also depicted in Table-6.

ACCEPTED MANUSCRIPT 3.6 Evaluation of the expression of regulatory and effector proteins of the apoptotic pathways in NSCLC cell lines treated with Paclitaxel in presence of MAA Quantitative Real-Time PCR and Western immunoblotting were performed to assess the level of expression of pro-apoptotic proteins (Bax & Bid) and anti-apoptotic protein (Bcl-XL) at m-RNA level and protein level respectively. The level of expression of Bax transcript was

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upregulated by 10 fold, 2.5 fold and 2 fold in the drug treated A549, NCI-H520 and NCIH460 cells respectively, which was increased to 12 fold, 4 fold and 3.0 fold in the respective cells treated with the combination of the lectin and the drug as compared to the respective control cells . An increase in the level of expression of Bid transcript was noted only in the

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case of NCI-H460 cells which was 5.5 fold in the drug treated cells and 6 fold in the cells treated with a combination of the lectin and the drug as compared to the respective control

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cells. However, the level of expression of Bcl-XL transcript was found to be unaltered in all the study groups of the three NSCLC cell lines. (Fig. 6A).

Cell lysates of all the study groups of the three NSCLC cell lines were analyzed by Western immunoblotting to assess the level of expression of Bax, Bid and Bcl-XL respectively at the protein level. Bax expression was enhanced by 2.0, 1.8 and 1.4 fold in the cells treated with the drug only and 2.7, 2.2 and 1.8 fold in the cells treated with the combination of the lectin

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and the drug in case of A549, NCI-H520 & NCI-H460 cells respectively as compared to the respective control cells. Expression of Bid was found to be increased by 1.2 fold and 1.6 fold in NCI-H460 cells treated with the drug in absence and in presence of the lectin respectively as compared to the respective control cells. Bcl-XL expression was unaltered in all the study

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groups of the three NSCLC cell lines (Fig. 6B). The expression of cytochrome c in the cytosolic fraction of A549, NCI-H460 and NCI-H520

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cells cultured with the drug only was found to be upregulated by 1.6 fold, 1.3 fold & 1.4 fold respectively, which was further enhanced to 1.9 fold, 1.8 fold & 1.8 fold in the respective cells treated with the combination of the lectin and the drug as compared to the respective mitochondrial fraction, while that in the control as well as lectin treated cells remained unaltered (Fig.7A). The level of expression of activated caspase-9 and caspase-3 in the cells treated with the drug in absence and presence of the lectin was found to be upregulated (Caspase-9: 1.7 and 2.1 fold respectively in A549 cells, 1.4 and 1.6 fold respectively in NCI-H520 cells and 1.4 and 1.8 fold respectively in NCI-H460 cells; Caspase-3: by 1.6 and 1.9 fold respectively in A549 cells, 1.5 and 1.7 fold respectively in NCI-H520 cells and 1.8 and 2.1 fold respectively in

ACCEPTED MANUSCRIPT NCI-H460 cells) as compared to the respective control cells. The expression of activated caspase-8 was found to be increased by 1.4 fold and 1.6 fold only in the NCI-H460 cells cultured in presence of the drug alone and the combination of the lectin and the drug respectively as compared to the control cells, while in other cell lines caspase-8 was nondetectable (Fig. 7B).

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4. Discussion

The tremendous side effect of the currently accepted modalities for NSCLC treatment necessitates the development of the newer targeted clinically useful strategies to combat the disease. In this context, lectins are the important biomolecules having diagnostic as well as

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apoptosis inducing potential in cancer cells of different origin [27, 59-61]. Maackia amurensis agglutinin (MAA) is gaining recognition due to it’s diagnostic potential in various

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cancers including gastric cancer [42], prostate cancer [44], carcinoma of papilla of vater [43], colorectal cancer [45], acute lymphoblastic leukaemia [47] and cervical cancer [15]. The findings of our previous study have suggested that MAA may have the potential to serve as a unique probe for the detection of NSCLC and also as a specific apoptosis inducing agent in these cells [48]. In the present study, we have found that the intensity of the MAA specific glycoprotein band of Mr ~66kDa was reduced in the presence of GM3 (an inhibitor of MAA)

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[47]. Further, it has been reported that this lectin could survive gastrointestinal proteolysis and target Podoplanin to inhibit melanoma cell growth, migration and tumorigenesis [49]. All these findings have increased the importance of MAA in cancer. In the present study, we have explored the chemo-adjuvant property of this lectin in three different types of NSCLC

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(adenocarcinoma, squamous cell carcinoma and large cell carcinoma) cell lines. Paclitaxel was the drug of our choice in this study, since it is usually used in the first-line of

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chemotherapy for NSCLC. A549 cells revealed maximum sensitivity to Paclitaxel followed by NCI-H460 cells and NCI-H520 cells. Our findings are in accordance with the report of Loprevite et al. [62] where lung adenocarcinoma cell lines were also found to be more sensitive to Paclitaxel as compared to the squamous cell carcinoma cell lines. However, drugs including Paclitaxel also affect the normal healthy cells non-specifically. Thus, the need of the hour is to search for the novel anti-cancer agents, which can increase the cytotoxic efficacy of the comparatively low dose of the drug on the cancer cells and thus having less damaging effect on the normal healthy cells. Various studies have reported that lectins could enhance the cytotoxic and apoptosis-inducing effects of anti-cancer drugs when given in combination [36-39]. In the present study, we have

ACCEPTED MANUSCRIPT found that optimum dose of MAA (the dose required to induce maximum apoptosis) in combination with the lower dose (in comparison to the optimum dose) of Paclitaxel especially in case of NCI-H520 cells (2 fold lower dose) and A549 cells (5 fold lower dose) could induce maximum increase in the percentage of apoptotic cells. In case of NCI-H460 cell line, the drug dose remained same as the optimum dose, which in presence of the

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optimum dose of the lectin was found to induce maximum increase in the percentage of apoptotic cells. However, at the optimum dose, MAA itself showed appreciable cytotoxicity in case of all the three NSCLC-cell lines. Thus, an attempt was made to assess the effect of the non-cytotoxic/less cytotoxic dose of the lectin on Paclitaxel induced apoptosis in these

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cell lines. Interestingly, we have found that MAA at non-cytotoxic dose (in case of NCIH460 and A549 cells respectively) and less cytotoxic dose (in case of NCI-H520 cells) could appreciably enhance the drug induced cytotoxicity. In this context, it should be mentioned

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that Jacalin (the control plant lectin), which could also bind to all the NSCLC cell lines, could not reveal such synergistic effect under the same conditions. This finding has strongly established the importance of MAA as chemoadjuvant over the other plant lectin (Jacalin). To investigate the correlation between the susceptibility to Paclitaxel and the presence of ~66kDa glycoprotein, which could bind to MAA in all the NSCLC cell lines, we assessed the

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binding of MAA in presence and absence of Paclitaxel. No significant alteration in the intensity of the band was noted, which clearly indicated that paclitaxel could not interfere with the MAA binding. In fact, it has been reported that Paclitaxel, the microtubule stabilizing agent binds to β -tubulin subunits of microtubules before being internalized to the

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lumenal site of the microtubule wall [63].The finding of our study suggested that binding of MAA to the cell surface sialoglycoprotein(s) could increase Paclitaxel uptake by the cells,

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which might be due to the modulation in the cellular architecture thereby increasing the susceptibility of the cells to Paclitaxel. Further, the effect of MAA on Paclitaxel induced cytotoxicity was found to be attenuated in presence of the inhibitor of MAA i.e. GM3 [47], thereby authenticating the specific role of MAA in enhancing the drug induced cytotoxicity in NSCLC cells. This observation was further substantiated by the abrogation of the synergistic effect of MAA on the drug induced cytotoxicity in NSCLC cells pretreated with neuraminidase. Our finding was further confirmed by the results of CDD-ELISA, which also revealed an augmentation of the apoptotic index in case of all the three types of the NSCLC cell lines treated with the combination of MAA and Paclitaxel as compared to the respective drug

ACCEPTED MANUSCRIPT treated cells. Mitochondria play an important role in controlling the process of apoptosis. During apoptosis, several key events occur in mitochondria including the loss of mitochondrial transmembrane potential (∆Ψm) and release of cytochrome c as the caspase activator [64]. In the present scenario, a significant reduction in ∆Ψm was noted in all the three NSCLC cell

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lines treated with Paclitaxel in presence of MAA as compared to the respective drug treated cells. The decrease in ∆Ψm was maximum in case of adenocarcinoma cell line which is in accordance to our findings of PI uptake assay and CDD-ELISA. In this context, it should be mentioned that the apoptotic index as well as ∆Ψm in case of NCI-H520 cells treated with

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0.01µg dose of MAA was found to be comparable to that of the untreated cells and thus this dose of the lectin was considered as the non-cytotoxic dose for this cell line.

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The intrinsic/mitochondrial pathway of apoptosis is known to be controlled by the pro- and anti-apoptotic members of the Bcl-2 family proteins [65, 66]. The pro-apoptotic proteins including Bax constitute a requisite gateway to the intrinsic pathway of apoptosis [64]. Down-regulation of Bax has been associated with advanced stages of cancer [67]. An increased expression of Bax was observed in gastric cancer cell line (SGC-7901) treated with Paclitaxel [68]. In the present study, Paclitaxel induced upregulation of Bax was noticed in

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case of all the three types of NSCLC cell lines and the expression was further upregulated in the cells treated with the combination of MAA and Paclitaxel. Bid is another pro-apoptotic member of Bcl-2 family, which is cleaved at the N-terminus by the activated caspase-8 to truncated Bid during extrinsic apoptotic signaling. The truncated bid triggers Bax

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oligomerisation and thus serves to engage a mitochondrial amplification loop in certain cell types [69]. The level of expression of Bid was found to be unaltered in NCI-H520 and A549

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cells under all the conditions. However, in case of NCI-H460 cells, the expression of Bid was appreciable in the drug treated cells and the expression was increased in the cells treated with the combination of the lectin and the drug. Bcl-2 and Bcl-XL are the anti-apoptotic members of the Bcl-2 family which confers the survival advantage to the cancer cells. Over-expression of these molecules has been associated with the progression of various types of cancers [70].The level of expression of Bcl-XL was found to be enhanced in paclitaxel treated SNU-398, human hepatocellular carcinoma cell line [71]. Further, the findings of our previous study have suggested that Bcl-XL rather than Bcl-2 may be an important regulator of MAA mediated apoptosis in NSCLC cell lines [48]. Thus, in the present investigation, the level of expression of Bcl-XL

ACCEPTED MANUSCRIPT was assessed in the three types of NSCLC cell lines treated with Paclitaxel only and also in combination with non-cytotoxic concentration of MAA and it was found to be unaltered in all the groups. The level of expression of Bax in all the three NSCLC cell lines and Bid in NCI-H460 cells treated with the drug in absence and in presence of the lectin at the protein level was not as

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much as that of mRNA level. This might be due to post transcriptional events like alternative splicing, nuclear degradation, processing and nuclear export, thereby affecting the stability of Bax and Bid transcripts.

An early event in the process of cell death is the redistribution of cytochrome c into the

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cytosol. It is the balance between pro- and anti-apoptotic members of Bcl-2 family that influences the cytochrome c release from the mitochondria into the cytosol [72]. Once

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released into the cytosol, cytochrome c binds to an adapter protein Apaf-1(apoptosis protease activating factor-1), which in the presence of dATP/ATP, self-oligomerizes to form an Apaf-1 multimer [73]. The Apaf-1 complex then recruits procaspase-9 (45kDa) and promotes its proteolytic processing into activated p35 subunit [74]. Active caspase-9 further activates procaspase-3(32kDa) by proteolytic processing into activated p17 and p20 subunits, which are then released into the cytosol to affect the proteolytic degradation of its target substrates

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leading to cell death [75]. Activation of pro-caspase-8 (55kDa) requires proteolytic processing of the inactive zymogen into activated p18 and p10 subunits leading to the formation of truncated Bid, which is also involved for the release of cytochrome c from mitochondria. In the present study, the level of cytosolic cytochrome c in MAA (non-

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cytotoxic dose) treated NSCLC cell lines was comparable to that of the respective control cells, which was appreciably less as compared to Paclitaxel treated cells. However, the lectin

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at the non-cytotoxic dose could augment Paclitaxel induced release of cytochrome c from mitochondria into the cytosol as well as activation of procaspase-9 and procaspase-3 in all the three types of NSCLC cell lines. It was reported that Paclitaxel induced apoptosis in osteosarcoma cells (Saos-2) was also accompanied by an increased level of cytosolic cytochrome c and activation of procaspase-3 [76]. Caspase-8 was found to be activated only in NCI-H460 cells treated with the drug and the expression of the activated caspase -8 was upregulated in the presence of non-cytotoxic dose of MAA. This can be correlated with the increased level of Bid under the same conditions in this cell line indicating the activation of extrinsic pathway of apoptosis.

ACCEPTED MANUSCRIPT Phosphorylation of Bcl-XL at Ser62 has been detected at G2 checkpoint, most often in the cells treated with microtubule poisons, including Paclitaxel. The phospho-Bcl-XL (Ser-62) stabilizes G2 arrest by trapping of cyclin-dependent kinase Cdk1 (cdc2) in nucleolar structures thus slowing the mitotic entry of these cells [77]. In this study, we have found a significant redistribution of the cells in the three phases (sub-G0, G0-G1 and G2-M) of the cell

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cycle in case of the three NSCLC cell lines treated with the drug alone indicating the presence of apoptotic cells as well as cell cycle arrest at G2-M phase. The population in the G2-M phase was increased when all the cell lines were treated with Paclitaxel in presence of the non-cytotoxic dose of MAA, indicating the role of MAA in enhancing the

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chemopreventive effect of Paclitaxel in these cells. 5. Conclusions

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Our data clearly reveal that MAA even at non-cytotoxic dose could augment the apoptosis inducing activity of the low dose of Paclitaxel in all the three types of NSCLC cell lines (A549, NCI-H520 & NCI-H460) through the intrinsic/ mitochondrial apoptotic pathway, although the additional role of extrinsic apoptotic pathway could not be ruled out in case of NCI-H460 cells. Further, the non-cytotoxic dose of MAA was also found to enhance the chemopreventive effect of the low dose of Paclitaxel in all the three types of NSCLC cell

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lines by arresting cells in the G2-M phase of the cell cycle. It is possible that MAA even at the non-cytotoxic dose could modulate the cell surface architecture probably through the interaction with its target molecule(s), which might be the sialylated glycoprotein(s) containing the NeuNAcα (2-3) Gal unit [since the intensity of the MAA specific glycoprotein

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band of ̴ 66kDa was reduced in the presence of GM3 (an inhibitor of MAA) having the same disaccharide unit to which MAA could bind], rendering the cells more accessible to

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Paclitaxel and thereby increasing the efficacy of this drug even at a lower dose. Our findings regarding the chemo-adjuvant activity of MAA in NSCLC cells may have implications in the development of novel strategies for the treatment of NSCLC. Conflict of Interest

The authors declare that they have no conflict of interest associated with this work. Disclosure Dr. Sujata Ghosh helped in conception and study design of the research work as well as in the final layout of the manuscript. Dr. Anuradha Chakraborti and Dr. Radhika Srinivasan gave constructive and valuable suggestions throughout the research work. Rakhee Chhetra Lalli and Shashank Dadsena conducted experiments and analyzed data. Kiranjeet Kaur helped in

ACCEPTED MANUSCRIPT conducting the experiments required for the revision of the manuscript. Rakhee Chhetra Lalli wrote the manuscript. All authors have read and approved the manuscript. Acknowledgement

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This work was supported by grant from Indian Council of Medical Research, New Delhi, India.

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Fig. 1A. Western blot of membrane proteins of A549 cells, NCI-H520 cells & NCI-H460 cells interacting with MAA in absence (Lanes1, 3 & 5 respectively) and in presence of GM3 (Lanes 2, 4 & 6 respectively). Fig. 2B. Binding of Jacalin to the NSCLC cell lines. Bar diagram indicating the binding of Jacalin to A549 cells, NCI-H520 cells & NCI-H460 cells

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in view of Mean Fluorescence Intensity. Each bar represents mean ±S.D. values done in triplicates.***p