Mitomycin C induces bystander killing in homogeneous and heterogeneous hepatoma cellular models

Abstract

Background: 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).

Results: 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.

Conclusion: 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.

Figure 1

Cell growth and cytotoxicity assay in HepG2 and HepG2-EGFP cell lines. (A) Growth kinetics of HepG2 and HepG2-EGFP cells shows no significant alteration in growth properties. HepG2 and HepG2-EGFP cells (2 × 10³) were seeded in a 96 well plate and subsequently cultured for 24 h, 48 h, 72 h and 96 h, and MTT assay was performed. Absorbance was measured at 570 nm using 630 nm as reference filter. Optical density of 0.2 corresponds to 2 × 10³ cells. (B) MMC is equally cytotoxic to HepG2 and HepG2-EGFP cells. HepG2 and HepG2-EGFP cells (1 × 10⁴) were seeded in a 96 well plate and kept overnight for adherence. MMC treatment with indicated concentrations was given for 48 h. Cells were grown in a drug free medium for 24 h before performing the MTT assay. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d.

Figure 2

Cell growth and cytotoxicity assay for HeLa/HeLa-EGFP cells and for SiHa/SiHa-EGFP cells. Cells (2 × 10³) were seeded in a 96 well plate and subsequently cultured for 24 h, 48 h, 72 h and 96 h and MTT assay was performed. Absorbance was measured at 570 nm using 630 nm as reference filter. Optical density of 0.2 corresponds to 2 × 10³ cells. (A) Growth kinetics of HeLa and HeLa-EGFP cells. (B) Growth kinetics of SiHa and SiHa-EGFP cells. (C) MMC cytotoxicity on HeLa and HeLa-EGFP cells. (D) MMC cytotoxicity on SiHa and SiHa-EGFP cells. Cytotoxicity assay for HeLa, HeLa-EGFP, SiHa and SiHa-EGFP was performed as done for HepG2 and HepG2-EGFP. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d.

Figure 3

Co-culture experiments to evaluate MMC induced bystander killing. (A) Bystander effect mediated by effector HepG2 cells treated with MMC (0.3 nM, 3 nM, 30 nM and 150 nM) for 24 h, towards target HepG2-EGFP cells. Effector cells were plated in a 12-well culture plate at a density of 5 × 10⁴ cells per well and kept for adherence at 37°C for 24 h. MMC treatment was given for 24 h, after that medium was decanted and cells were washed twice with medium. 5 × 10⁴ target cells were co-plated and the co-cultures were allowed to grow at 37°C for 72 h. Subsequently cells were trypsinized and total live fluorescence was quantified. In order to avoid any possibility of cell death due to residual drug in the well, we used an empty well containing no effector cells but only medium containing 150 nM MMC and subsequently this well was processed similarly before plating the target HepG2-EGFP cells. (B) Bystander effect mediated by effector Hep3B cells treated with MMC (30 nM and 150 nM) towards target HepG2-EGFP cells. (C) Bystander effect mediated by effector Hep3B cells towards target Hep3B cells. MTT assay was performed to evaluate bystander cytotoxicity in Hep3B cells. Cells were plated and treated with MMC (30 nM, 150 nM and 200 nM) for 24 h and target Hep3B cells were co-plated as described in materials and methods. After 72 h medium was decanted and 50 μl MTT (1 mg/ml) was added in each well and incubated for 4 h at 37°C. Subsequently, formazan crystals were solubilized in 50 μl of iso-propanol by incubating with shaking for 10 min, at room temperature. Absorbance was measured at 570 nm using 630 nm as reference filter. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. * and ** significantly differs from there respective controls.

Figure 4

MMC fails to induce bystander killing in cervical cancer cell. (A) Effector HeLa and SiHa cells were plated and treated with MMC (200 nM and 500 nM respectively) for 24 h. EGFP expressing target HeLa and SiHa cells were co-plated and the co-culture were allowed to grow for 72 h after that cells were trypsinized and total live fluorescence was quantified. Data presented are representative of three independent experiments and are expressed as mean+s.d. of triplicate samples. (B) Fluorescent photomicrographs of co-cultured cells to evaluate MMC induced bystander killing: (a) untreated HeLa cells co-plated with HeLa-EGFP, (b) MMC treated HeLa cells (200 nM) co-plated with HeLa-EGFP, (c) untreated SiHa cells co-plated SiHa-EGFP, (e) MMC treated SiHa cells (500 nM) co-plated with SiHa-EGFP.

Figure 5

Medium transfer experiments to detect the presence of soluble cytotoxic effector molecules. (A) Time-course release of cytotoxic factor from MMC treated effector HepG2 cells. The effector cells were treated with MMC for 24 h and fresh medium was added as described in materials and methods. At the time indicated, corresponding supernatants were collected and added to the untreated target HepG2-EGFP cells with or without neutralizing anti-FasL antibody. Target cells were incubated with the supernatants for 48 h before quantitating fluorescence. The supernatants were supplemented with 0.2% FBS to avoid cell death due to growth factor depletion. The culture supernatant from untreated HepG2 cells was used as control and fluorescent intensity was calculated with respect to identical time point controls. (B) The target cell death increases with the increase in number of effector cells. Effector cells were treated with MMC for 24 h and fresh medium was added as described in materials and methods. The supernatants were collected after 48 h and used to culture target HepG2-EGFP cells for 48 h before quantitating fluorescence. (C) Involvement of death ligands in bystander killing of hepatoma cells. Bystander killing of HepG2-EGFP cells is mediated by FasL. Effector HepG2 and Hep3B cells were treated with MMC (150 nM) and target HepG2-EGFP cells were co-plated with or without neutralizing antibody against FasL (1 μg/ml) and TRAIL (2 μg/ml). (D) Bystander killing of target Hep3B cells by effector Hep3B cells treated with MMC (150 nM) is mediated by TRAIL. Trypan blue assay to detect the percentage cell viability in co-cultures of effector and target Hep3B cells in the presence of neutralizing anti-TRAIL antibody. After 72 h of growth in co-culture cells were harvested by trypsinization and stained with 0.005% trypan blue for 5 min at RT. Unstained as well as stained cells were counted in haemocytometer and percentage cell viability was calculated. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. *, ** and *** differs significantly at p < 0.05.

Figure 6

Sandwich ELISA to detect secreted form of death ligands in conditioned medium obtained from MMC treated hepatoma cells. (A) Sandwich ELISA for quantification of secreted FasL from untreated and treated HepG2 and Hep3B cells at different time points. (B) Sandwich ELISA for quantification of secreted TRAIL from untreated and treated HepG2 and Hep3B cells at different time points. Data presented are representative of three independent experiments performed in triplicates expressed as mean ± s.d. *** differs significantly from the respective control. (C) Fluorescent photomicrographs of co-cultured cells to evaluate MMC induced bystander killing of HepG2-EGFP cells: (a) untreated effector HepG2 cells, (b) effector cells treated with MMC (150 nM), (c) neutralizing anti-FasL antibody (1 μg/ml), (d) neutralizing anti-TRAIL antibody (2 μg/ml). (D) Photomicrographs of co-culture of effector and target Hep3B cells: (a) untreated effector Hep3B cells with target Hep3B cells, (b) MMC (150 nM) treated effector Hep3B cells with target Hep3B cells, (c) effector Hep3B cells treated with MMC and target Hep3B cells co-plated in the presence of neutralizing anti-TRAIL antibody (2 μg/ml), (d) effector Hep3B cells pretreated with EDTA (2 μM) followed by MMC treatment and then target Hep3B cells were co-plated.

Figure 7

Expression of death receptors increases after MMC treatment in hepatoma cells. HepG2 and Hep3B cells were treated with MMC for 24 h. Cells were washed twice with medium, fresh medium without MMC was added and cells were incubated again for 24 h at 37°C. After post-treatment growth in MMC free medium whole cell lysate was prepared and western blot was performed. (A) Western blot analysis: lane (1) control HepG2 cells, lane (2) HepG2 treated for 24 h with MMC and lane (3) HepG2 treated for 24 h with MMC followed by post treatment growth in drug free medium for 24 h. β-actin was detected as a loading control. Where ever required, blots were stripped by incubating the membranes at 50°C for 30 min in stripping buffer (62.5 mM Tris-Cl pH 6.7, 100 mM mercaptoethanol, 2% SDS) with intermittent shaking. Membranes were washed thoroughly with TBS and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting. (B) Immunofluorescence staining of HepG2 cells. Cells were treated with MMC for 24 h, washed twice with medium and fresh medium without MMC was added and cultured for 24 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X- 100 and blocked with 5% FBS. Cells were then incubated with anti-FasL, anti-Fas, anti-TRAIL and anti-DR4 primary antibodies for 2 h and subsequently stained with FITC conjugated secondary antibody for 1 h.

Figure 8

Secreted form of FasL involved in mediating bystander cytotoxicity and apoptosis in HepG2 cells. (A) Effector HepG2 cells were pretreated with EDTA (2 μM) for 1 h and MMC was added in the same medium containing EDTA. Subsequently, this medium was used to culture target HepG2-EGFP cells and fluorescent intensity was measured after 48 h. (B) Fluorescent photomicrograph: (a) untreated HepG2 effector cells co-plated with target HepG2-EGFP cells and (b) effector HepG2 cells pretreated with 2 μM EDTA followed by MMC treatment for 24 h. (C) Sandwich ELISA to detect FasL in CM from MMC treated and cells pretreated with EDTA followed by MMC treatment for 24 h. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. ** differs significantly at p < 0.005. (D) Effector HepG2 cells treated with MMC for 24 h as well as from effector HepG2 cells pretreated with EDTA for 1 h followed by MMC treatment for 24 h. Cells were then washed twice with medium, target HepG2-EGFP cells were co-plated and the co-culture were grown for 72 h, and whole cell lysates were made from the co-culture to perform western blot analysis. The levels of PARP p116 proform and its cleavage product p85 were detected. β-actin was detected as a loading control. Lane (1) untreated effector HepG2 cells. (2) MMC treated effector cell and harvested after 24 h. (3) MMC treated effector cells and harvested after 48 h. (4) EDTA (2 μM) pre-treatment followed by MMC treatment of effector cells and harvested after 24 h. (5) EDTA (2 μM) pre-treatment followed by MMC treatment of effector cells and harvested after 48 h.

Figure 9

MMC induced apoptotic factors. (A) HepG2 and Hep3B cells treated with MMC were harvested and cell lysates were prepared. The expression of apoptosis related proteins were dectected by western blot analysis. The protein levels of PARP p116 proform and its p85 cleavage product, pro caspase-3 p32 and its cleavage product p11, and BID p21 proform were detected. Lane (1) untreated cells, (2) cells treated with MMC for 24 h and (3) cells treated with MMC for 24 h followed by post treatment growth in MMC free medium for 24 h. β-actin was detected as a as loading control. Where ever possible blots were stripped and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting. (B) TUNEL assay for effector and target cells. For effector cells, HepG2 and Hep3B cells were treated with MMC (150 nM) for 24 h. Then the cells were washed with medium and cultured for additional 72 h before performing TUNEL assay. Similarly, for target cells, medium form untreated as well as from treated HepG2 or Hep3B cells were used to culture target HepG2 and Hep3B cells respectively for 48 h before performing TUNEL assay. The medium was supplemented with 0.2% FBS to avoid growth factor depletion.

Figure 10

Soft-agarose colony formation assays to evaluate the role of secreted FasL in mediating cytotoxic effect. Base agarose layer was made in DMEM without phenol red and top agarose layer containing 4 × 10³ HepG2 cells was poured over the base layer. After 4 weeks of growth in semisolid medium untreated and treated effector cells (5 × 10⁵) were added in the center well of the plates. These plates were incubated at 37°C for 4 weeks. (A) Number of colonies decreased in the presence of effector cells treated with MMC as compared to control effector cells. The number of colonies decreased drastically in the presence of recombinant FasL (6 μg) which served as a positive control. (B) Photomicrographs of soft-agarose plate containing MMC treated effector HepG2 cells. The colony size diminished and very small colonies (<20 μm in mean diameter) were observed in the area surrounding center well containing treated effector cells. (C) Photomicrographs of soft-agarose colonies at 4× magnification and 10× magnification corresponding to different fields from the plate containing control effector cells and the plate containing MMC treated effector cells, in the center well.

Figure 11

Release of secreted cytotoxic factors diminishes considerably the size of soft-agarose colonies. (A) Pie chart showing percentage distribution of colonies of different mean diameters (colonies >20 μm in diameter were considered) in the soft-agaose plate containing untreated effector HepG2 cells and in the plate containing MMC treated effector HepG2 cells. (B) Photomicrographs of colonies at higher magnification showing diminished growth when MMC treated effector HepG2 cells were added in the center well of soft-agarose plate containing colonies of target cells.

Figure 12

Proposed model. The generation of MMC induced bystander signals deciphering function of membrane bound as well as secreted form of death ligands.