Mitomycin C induces bystander killing in homogeneous and heterogeneous hepatoma cellular models
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Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide that is particularly refractory to chemotherapy. Several studies have proposed combination chemotherapy regimen for HCC treatment. However, these therapies are not effective in regressing tumor and prolonging survival of patient's suffering from HCC. Therefore, the development of more effective therapeutic tools and new strategies for the treatment of HCC are urgently needed. Over the last decade much attention has been focused on "bystander effect" as a possible therapeutic strategy for the treatment of certain human tumors. Interest in this therapeutic approach originated from numerous reports describing the radiation induced bystander effect. However, the knowledge about chemotherapy induced bystander effect is still limited. Hence, chemotherapy induced bystander phenomenon in hepatoma cells was explored by utilizing Mitomycin C (MMC).
MMC induced bystander killing was observed only in hepatoma cells and it did not occur in cervical cancer cells. MMC induced bystander killing was transferable via medium. It occurred in co-cultured cells indicating the involvement of secreted as well as membrane bound factors. FasL and TRAIL were detected in the conditioned medium from treated cells. In medium transfer experiment, pre-treatment with EDTA (a broad range protease inhibitor) diminished MMC induced bystander killing. Following drug exposure, expression of Fas and TRAIL receptors increased and treatment with neutralizing antibodies against FasL and TRAIL inhibited bystander killing.
Our results highlight the therapeutic importance of MMC in the treatment of HCC and implicate role of membrane bound and secreted forms of FasL and TRAIL in MMC induced bystander killing.
KeywordsConditioned Medium HepG2 Cell Enhance Green Fluorescent Protein Bystander Effect Hep3B Cell
Methyle Thiazole Tetrazolium
Ethylene Diaminetetra acetic acid.
For solid tumors chemotherapy is characteristically the choice of treatment other than surgical resection. However, there are inherent limitations associated with chemotherapy of solid tumors. These include toxicity to normal cells, development of resistant cells within a tumor mass and inadequate delivery of cytotoxic drugs due to inaccessibility to certain cells within the same tumor mass. Moreover, the effectiveness of a chemotherapeutic drug is a combined characteristic of the drug, tumor and patient. The universal adoption of cancer chemotherapy is limited by treatment complexities which make it mandatory to explore drugs that, instead of assuring delivery to 100% of tumor cells, utilize the ability of exposed cells to kill neighboring unexposed tumor cells. Therefore, the concept of "Bystander Effect" comes into the picture. The name was initially borrowed from the field of gene therapy, where it usually referred to the killing of several types of tumor cells by targeting only one type of cell within a mixed population . Radiation induced bystander effect has been extensively studied [2, 3, 4]. The available data concerning this effect falls into two categories: (i) confluent cultures where physical contacts between irradiated and non-irradiated cells are made and where intercellular communication is essential for the process and (ii) sparsely populated cultures where the bystander effect may be mediated by damage signals released into the culture medium by irradiated cells' [4, 5, 6, 7].
Hepatocellular carcinoma (HCC) is one of the most common solid tumors that are particularly refractory to the available chemotherapeutic drugs administered either alone or in combination . During the last few decades many drugs have become available for treatment of a wide variety of neoplastic diseases. Mitomycin C (MMC) is one such drug used for treating solid tumors including hepatocellular carcinoma and cervical cancer [9, 10, 11, 12]. It is already known that chemotherapeutic DNA-alkylating agents can induce in vivo and in vitro paracrine factors that cause growth inhibition. In most cases there is production of soluble factors which have a distal effect on untreated cells . Moreover, single acute exposure of MMC has been implicated in the changes in genomic stability of exposed cells that signals the unexposed bystander cells to respond similarly . Various molecules that are involved in the genesis of bystander effect remain unidentified. There are reports demonstrating involvement of reactive oxygen species, nitric oxide and cytokines such as TGF β that promote bystander cell killing in sparsely populated cell cultures [15, 16].
HCCs are heterogeneous tumors. They commonly emerge on the histological background of chronic liver disease. Heterogeneity of a tumor occurs due to clonal variations within a tumor mass during its development. However, in spite of heterogeneity, there are certain common molecular alterations in all sub-clones within a tumor that correlates with the monoclonal origin or homogeneity of a tumor. These molecular alterations and signaling pathways play a crucial role in development of HCC and also can be explored to develop an effective therapeutic tool for treatment of HCC .
Our study reveals the mechanism of bystander killing by MMC in hepatoma cells. We have earlier reported that 5-fluorouracil induces bystander killing in mixed co-cultures of breast cancer cells, a heterogeneous cell model . In the present work we addressed the ability of MMC to specifically induce bystander killing of hepatoma cells in both homogeneous and heterogeneous cell models. We investigated the involvement of death ligands in the bystander killing of hepatoma cells. Interestingly, MMC induced bystander killing was transferable through medium and it occurred also in co-cultured cells, implicating the involvement of soluble as well as membrane bound death effector molecules.
Cell lines and reagents
Hepatoma cell lines HepG2, Hep3B and cervical cancer cell lines HeLa, SiHa were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in our in-house National Cell Repository. Cells were routinely cultured in minimum essential medium (MEM) supplemented with 10% heat inactivated fetal bovine serum (Sigma, MO, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Invitrogen Corporation, CA, USA) at 37°C with 5% CO2. Chemotherapeutic drug Mitomycin C (MMC), purchased from Sigma was dissolved in methanol to make a stock solution of 5 mM. Methylethiazole tetrazolium (MTT) (USB, OH, USA) was reconstituted in DMEM without phenol red to make 1 mg/ml working solution. Antibodies against Fas, FasL, TRAIL, DR4, caspase-3, BID, PARP, β-actin, recombinant human FasL, HRP conjugated and FITC-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Neutralizing antibodies against FasL and TRAIL (BD Biosciences, CA, USA) were reconstituted in sterile PBS to make 1 mg/ml solution.
Development of EGFP expressing cell lines
The cell lines, expressing enhanced green fluorescent protein (EGFP) were established by transfecting HepG2, HeLa and SiHa cells with pEGFPN1 plasmid (Clontech, CA, USA) by Lipofectamine 2000 (Invitrogen Corporation, CA, USA) as described in the manufacturer's protocol and selected on G418 (USB, OH, USA) 800 μg/ml (for HepG2) and 400 μg/ml (for HeLa and SiHa cells). These cells were subsequently maintained in medium containing G418 (100 μg/ml).
Cell growth and cytotoxicity assay for HepG2 and HepG2-EGFP cell lines
Cells were seeded in a 96 well plate and the growth kinetics was performed as described previously . To assay cytotoxicity of drug, cells seeded in a 96 well plate were treated with indicated concentration of MMC for 48 h and then cultured for additional 24 h in drug free medium. Subsequently, MTT assay was performed as described previously .
In vitro co-culture experiments were designed to evaluate whether the cytotoxicity induced by MMC in HepG2, Hep3B, HeLa and SiHa cells (MMC treated cells were termed as "Effector Cells") causes death in co-plated bystander HepG2-EGFP, HeLa-EGFP and SiHa-EGFP cells (EGFP expressing bystander cells which were not treated with MMC were termed as "Target Cells"). Death of target cells was evaluated by quantification of total live GFP fluorescence by exciting at 485 nm and measuring emission at 510 nm (Fluoroskan Ascent FL, Labsystems, Finland). Briefly, effector cells were plated and treated with MMC. Target cells were co-plated and bystander cytotoxicity was measured as described previously . Wherever medium transfer experiments were performed, the medium was supplemented with 0.2% FBS to avoid cell death due to growth factor depletion.
MTT assay to evaluate bystander cytotoxicity in co-cultures containing effector and target Hep3B cells
Effector Hep3B cells were plated (3.5 × 103 and 7.5 × 103 cells per well) in a 96 well plate and allowed to adhere for 24 h at 37°C. Cells were treated with MMC for 24 h. Subsequently, effector cells were washed twice with medium and target Hep3B cells (7.5 × 103 cells per well) were co-plated. After 72 h medium was decanted and MTT assay was performed.
Trypan blue dye exclusion assay to detect involvement of TRAIL in mediating MMC induced bystander effect in co-cultures containing effector and target Hep3B cells
Hep3B cells (2 × 105 cells per well) were plated in a six well plate and allowed to adhere for 24 h at 37°C. Cells were treated with MMC for 24 h. Thereafter, effector cells were washed twice with medium and target Hep3B cells (2 × 105 cells per well) were co-plated with or without neutralizing anti-TRAIL antibody. After 72 h of growth in co-culture, trypan blue dye exclusion assay was performed as described previously .
Conditioned medium collection and sandwich ELISA for detection of secreted FasL and TRAIL from effector cells
Secreted FasL and TRAIL were detected in conditioned medium (CM) by sandwich ELISA. To obtain CM, 7 × 105 effector cells were plated in 60 mm tissue culture dishes and kept overnight for adherence. Cells were treated with MMC (150 nM) for 24 h. Wherever EDTA was used, cells were pre-treated with 2 μM EDTA for 1 h and MMC treatment was given in the presence of EDTA for 24 h. Subsequently, medium was decanted, cells were washed twice with medium and phenol-red free DMEM without FBS was added to the cells. Cells were cultured for additional 24 h, 48 h, 72 h and 96 h. After each time period CM was collected, centrifuged at 50,000 × g for 30 min and supernatants were concentrated to half of the original volume by SpeedVac (Thermo Savant, USA). For ELISA, each well of micro-titre plate was coated with 50 μl of anti-FasL Mab and/or anti-TRAIL Mab (250 ng/50 μl) for 3 h at 37°C, followed by blocking of free sites with 100 μl of blocking reagent (5% BSA in PBS) for 2 h at 37°C. Test samples (50 μl) were added and incubated for 3 h at 37°C followed by incubation with 50 μl of second anti-FasL and/or anti-TRAIL polyclonal antibody, for 3 h at 37°C. After that, secondary HRP conjugated antibody was added in each well. Finally, ABTS in 0.1 M Na-citrate buffer (pH 4.0) was added to each well and incubated for 10 min at room temperature before taking absorbance at 405 nm. Five washes with PBS containing 0.05% Tween-20 were performed between each step and plates were pat dried.
Western blot analysis and immunostaining
Whole cell lysates were made and western blotting was performed as described previously . For immunostaining, cells were fixed, permeabilized and immunofluorescence was performed as described earlier .
Bystander cell killing in soft-agarose colony formation assay
Base agarose layer (1%) was made in a tissue culture plate and a top agarose layer (0.8%) containing HepG2 cell was added over the base layer. Consequently, plates were kept in incubator and cells were allowed to grow inside the semisolid agarose containing culture medium. After 4 weeks when sizable colonies appeared, a center well was made in plates with the help of micropipette tip in which untreated cells were added in control plate and treated cells were added in the experimental plate. These wells were covered with 0.8% agarose. The plates were incubated for additional 4-5 weeks. After that colonies in both plates were counted manually under microscope and pictures were taken. The mean diameter of colonies was calculated using ImagePro Plus 5.0 software (MediaCybernetics, Silver Spring, MD, USA). Data thus obtained on number of colonies and size of colonies was depicted by graph.
Detection of apoptosis in effector and target cells by TUNEL assay
TdT mediated dUTP Nick End Labeling (TUNEL) assay was performed by using APO-DIRECT (BD Biosciences, CA, USA) and/or by In-situ cell death detection kit (Roche Applied Science, PA, USA) following the manufacturer's protocol. Briefly, HepG2 and Hep3B cells (7 × 103) were seeded in multi-well slides (MP Biomedicals, OH, USA). For effector cells, MMC treatment was given for 24 h with the indicated concentration. Thereafter, drug containing medium was decanted and fresh medium without MMC was added to each well. Cells were cultured in drug free medium for additional 72 h. In case of target cells medium transfer experiment was performed. Effector cells (5 × 104) were plated in a 35 mm tissue culture dish and treated with indicated concentration of MMC for 24 h. Following treatment the effector cells were washed twice with medium and fresh medium without drug was added to the cells and incubated for additional 48 h. The medium thus obtained was utilized to culture target cells for 48 h before performing TUNEL assay. The medium was supplemented with 0.2% FBS to avoid growth factor depletion. For TUNEL assay cells were washed with PBS and fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100 and reaction mixture containing TdT enzyme, FITC labeled dUTP was added to cells. The reaction was performed at 37°C for 1 h. Cells were then washed with rinsing buffer, PI solution was added to each well and kept at 37°C for 30 min. After washing cells thoroughly they were overlaid with mounting medium containing antifade (Santa Cruz Biotechnology, CA, USA). The slides were subjected to confocal microscopy (Zeiss LSM510, Heidelberg, Germany). Images were processed by Adobe Photoshop software. Wherever In-situ cell death detection kit was used to detect apoptosis, PI (50 μg/ml) was added to the cells and incubated at 37°C for 30 min before they were overlaid with mounting medium.
The data were calculated with sigma plot 10 and values are presented as arithmetic mean ± standard deviation (mean ± s.d). Unpaired student's t-test was used to determine the statistical significance of differences between samples and p-value < 0.05 were considered significant. * is for p-value < 0.05, ** is for p-value < 0.005 and *** is for p-value < 0.0005.
Growth pattern and chemosensitivity of EGFP expressing cells was similar to their corresponding parental cells
MMC induced bystander cytotoxicity in hepatoma cells is proportional to drug dosage
Bystander cytotoxicity is not observed in co-cultured cervical cancer cells treated with MMC
MMC induced bystander effect is transferable through medium and extent of killing depends upon the number of effector cells
Both membrane bound and secreted form of death ligands are involved in MMC induced bystander effect
Expression of death receptors increases following MMC treatment
Treatment with EDTA decreases bystander cytotoxicity
Both effector and target cells undergo apoptosis following MMC treatment
Death factors released by MMC treated cells significantly reduce the size of colonies in soft-agarose assay
In the present study we addressed the efficacy of MMC in hepatoma cells by exploiting its ability to promote bystander killing in addition to its direct toxicity. Our results from the co-culture experiments indicated that the bystander signal initiated after 24 h of treatment, even at a low dose of 0.3 nM MMC. Significant increase in bystander killing was observed up to 72 h after drug treatment which was dependant upon the number of effector cells treated and on the MMC dosage used. MMC induced bystander killing was cell type specific with no bystander effect occurring in cervical cancer cells, although the drug did induce direct cytotoxicity in these cells per se.
Chemotherapeutic drugs induce expression of death receptors [21, 27, 28], and their role in drug induced bystander effect is still not clear. MMC treatment increased Fas expression in HepG2 cells and these cells were sensitive to Fas/FasL mediated apoptosis [29, 30]. Whereas, Hep3B cells are reported to be resistant to Fas mediated apoptosis [21, 31]. Our data clearly demonstrated that Hep3B cells express Fas, though reduced percentage of cells displayed the expression and this observation was in agreement with the report of Lamboley et al . Moreover, in MMC treated Hep3B cells, no significant increase in the levels of Fas was detected. Therefore, it is unlikely that the Fas-FasL pathway was involved in MMC induced bystander effect in co-cultures of effector Hep3B and target Hep3B cells. Concurrently, MMC treatment caused a significant increase in TRAIL receptor DR4 in Hep3B cells. In the presence of neutralizing anti-TRAIL antibody, the MMC induced bystander killing of Hep3B diminished, indicating involvement of the TRAIL pathway in mediating a bystander effect. Interestingly, the presence of neutralizing anti-FasL antibody prevented bystander killing in HepG2 cells, but cell death was not prevented in the presence of neutralizing anti-TRAIL antibody. In mixed co-cultures of effector Hep3B cells and target HepG2-EGFP cells, there was also a decrease in bystander killing in the presence of neutralizing anti-FasL antibody, suggesting involvement of the Fas-FasL pathway. Additionally, the significant increase in FasL and TRAIL in CM of MMC treated effector cells was suggestive of involvement of secreted form of these ligands being responsible for transferring a bystander signal through the medium. The involvement of these ligands was reconfirmed by culturing MMC treated effector cells with/without neutralizing anti-FasL and/or anti-TRAIL antibody in target HepG2 and Hep3B cells, respectively (Additional file 4). The pro-apoptotic role of secreted forms of FasL and TRAIL in hepatocellular carcinoma has been reported [32, 33, 34], though there are contradictory reports suggesting an anti-apoptotic role of secreted FasL [35, 36, 37]. It has been reported that the cleaved product of human FasL has cytotoxic activity , but it is relatively weaker than the membrane associated FasL . Also, the cytotoxic activity is enhanced if the death ligands are present in cross linked or aggregated form [32, 33, 34, 35]. Thus, the detection of the high molecular weight form of FasL (≈ 48 kDa) in CM from treated HepG2 cells suggested its involvement in bystander killing (Additional file 1). The significance of secreted death ligands in bystander effect was further confirmed by treatment with EDTA. As shown in Fig. 8A, in the presence of EDTA, the bystander killing decreased in medium transfer, as well as under co-culturing conditions (Fig. 8B). Treatment with EDTA led to a decreased abundance of FasL in CM that correlated well with diminished PARP cleavage in HepG2 cells co-cultures (Fig. 8C and Fig. 8D) Although, HepG2 effector cells also released TRAIL, the amount was insignificant in comparison to the amount of FasL released in the medium.
For a chemotherapeutic drug to be effective in eliminating tumor completely, the drug or its effect should reach not only the cells in periphery, but also the inner cellular mass of the tumor. One of the possibilities to achieve this was to explore the feasibility of diffusion of released cytotoxic effector molecules from the tumor cells exposed to the drug directly. Therefore, to verify that indeed released cytotoxic effector molecules from the treated cells were able to diffuse through semisolid medium thereby mimicking in-vivo conditions, we performed soft-agarose colony formation assays. Because MMC treated effector HepG2 secret FasL, we hypothesized that this ligand should diffuse through semisolid medium. Interestingly, we found that in the plate in which treated HepG2 effector cells were added in the centre well, colony size was smaller and they were of distinct shapes, suggestive of diminished cell growth (Fig. 11B). The decrease in size and alterations in shape of colonies was prevented when the treated effector cells were added in the centre well along with neutralizing anti-FasL antibody (Additional file 2d).
We thank Dr. G.C. Mishra, Director, NCCS for being very supportive and giving all the encouragement to carry out this work. We also thank Department of Biotechnology, Government of India for providing financial support. RK thank UGC, AS thanks CSIR and AKA thanks ICMR for providing fellowships.
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