Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment

Abstract

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.

Figure 1

MMC induces expression of death ligands in cervical cancer cells. (a) Bystander killing in CM transfer experiment. The effector cells (HeLa and SiHa) were treated with indicated concentrations of MMC for 24 h, and then CM medium was collected after 48 h as described in Materials and Methods. Target HeLa (i) and SiHa (ii) cells were incubated with the respective CM for 24 h. Cell survival was evaluated by MTT assay. (b) Semi-quantitative RT-PCR for FasL mRNA. HeLa and SiHa cells were treated with indicated concentrations of MMC for 24 h, and were processed for RT-PCR. β-Actin was used as a loading control. Data are mean±S.D., and are representative of three independent experiments. (c) Western blot analysis of FasL. HeLa and SiHa cells were treated with indicated concentrations of MMC for 24 h, and cell lysates were subjected to SDS-PAGE and probed for protein levels of FasL. (d) Flow cytometric analysis of FasL expression. HeLa (i) and SiHa (ii) cells were treated with MMC as described above. Untreated or MMC-treated cells were probed with FasL primary antibody or IgG control (1 : 100), and further with PE-conjugated secondary antibody (1 : 200). Cells were then washed with PBS, and FasL expression was analyzed by flow cytometry. (e) Sandwich ELISA for quantification of sFasL from MMC-treated HeLa (i) and SiHa (ii) cells at the indicated time points. Data are mean±S.D., and are representative of three independent experiments (**P<0.01 when compared with their respective controls)

Figure 2

MMC induces death ligand expression via PPARγ and proteasomal inhibition increases level of death receptors in cervical cancer cells. (a) Analysis of expression of FasL in MMC-treated THP-1 MΦ. (i) Semi-quantitative RT-PCR for FasL mRNA. THP-1 MΦ were treated with indicated concentrations of MMC, and processed for RT-PCR. β-Actin was used as a loading control. Data are mean±S.D., and are representative of three independent experiments. (ii) THP-1 MΦ were treated with indicated concentrations of MMC, and whole-cell lysate were subjected to western blotting for FasL. (iii) Flow cytometric analysis of FasL expression. THP-1 MΦ were treated with MMC as described above. Untreated or MMC-treated cells were probed with FasL primary antibody or IgG control (1 : 100), and further with PE-conjugated secondary antibody (1 : 200). Cells were then washed with PBS, and FasL expression was analyzed by flow cytometry. (iv) Sandwich ELISA for quantification of sFasL from untreated and MMC-treated THP-1 MΦ at indicated time points. (b) Analysis of involvement of PPARγ in the regulation of FasL expression. (i) Western blot analysis of PPARγ in MMC-treated cells. HeLa, SiHa and THP-1 MΦ were plated in 35 mm culture dishes. After 24 h, MMC treatment was given at indicated concentrations, and cells were further incubated for 24 h. Cell lysates were then subjected to SDS-PAGE and western blotting for PPARγ. (ii) Effect of PPARγ inhibition on FasL expression. HeLa, SiHa and THP-1 MΦ were plated in 35 mm culture dishes. After 24 h, cells were pretreated with GW9662 (10 μM) for 2 h. Thereafter, MMC (500 nM) treatment was given and cells were incubated for 22 h. Whole-cell lysates were subjected to western blotting for FasL. (c) Effect of knockdown of PPARγ on MMC-induced expression of FasL. HeLa, SiHa and THP-1 MΦ were transfected with control siRNA or PPARγ siRNA for 15 h, and allowed to grow for a further 15 h. Control siRNA and PPARγ siRNA-transfected cells were exposed to MMC for 24 h, and cells were collected for western blot analysis of PPARγ and FasL. (d) MG132-induced expression of Fas. HeLa and SiHa cells treated with MMC and/or MG132 for 24 h. Western blot analysis of whole-cell lysates subjected to SDS-PAGE and probed for Fas. (e) Analysis of expression and localization of Fas in MG132-treated cervical cancer cells. (i) Immunofluorescence staining of HeLa and SiHa cells. Cells were treated with MMC and/or MG132 for 24 h, washed twice, then fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100 respectively, and blocked with 5% FBS. Cells were further incubated with anti-Fas primary antibodies (1 : 100) for 2 h and subsequently stained with FITC-conjugated secondary antibodies (1 : 200) for 1 h. (ii) Flow cytometric analysis of Fas expression in cervical cancer cells. HeLa and SiHa cells were treated with MG132 for 24 h. Untreated or MG132-treated cells were probed with primary antibody against Fas or IgG control (1 : 100) for 1 h, and further with PE-conjugated secondary antibody (1 : 200) for 30 min. Cells were then washed with PBS, and Fas expression was analyzed by flow cytometry. (f) Semi-quantitative RT-PCR for Fas mRNA in MMC-treated cervical cancer cells. HeLa and SiHa cells were treated with indicated concentrations of MMC for 24 h, and were processed for RT-PCR. β-Actin was used as a loading control

Figure 3

MG132 sensitizes cervical cancer cells to FasL-mediated cell death. (ai and bi) MTT assay in MG132 and MMC-treated HeLa and SiHa cells. HeLa (ai) and SiHa (bi) cells (7 × 10³/well) were seeded in 96-well plates. After 24 h, treatment of 5 μM MG132 was given for 2 h. Thereafter, 200 or 500 nM MMC was added for next 34 h, and MTT assay was performed. (aii and bii) Annexin V-FITC staining in MG132 and MMC-treated cervical cancer cells. HeLa (aii) and SiHa (bii) cells (1 × 10⁵) were seeded in 35 mm plate. After 24 h, treatment of 5 μM MG132 was given for 2 h. Thereafter, 500 nM MMC was added for further 34 h. Next, cell were stained with annexin V-FITC, and analyzed by flow cytometry. Data are mean±S.D., and are representative of three independent experiments (*P<0.05, **P<0.01 when compared with their respective controls). (c and d) Involvement of sFasL in mediating bystander cytotoxicity. The effector cells (HeLa and SiHa) were treated with 500 nM MMC for 24 h and then CM medium was collected after 48 h as described in Materials and Methods. Target HeLa (c) and SiHa (d) cells were incubated with the respective CM in the presence or absence of MG132 and/or anti-FasL antibody or IgG control antibody for 36 h. CM was supplemented with 0.2% FBS to avoid cell death owing to growth factor depletion. Cell survival was evaluated by MTT assay. Data are mean±S.D., and are representative of three independent experiments (*P<0.05, **P<0.01, when compared with their respective controls)

Figure 4

Co-plating experiments to evaluate contact-dependent bystander killing in homogeneous system. Analysis of apoptotic cell death in target EGFP-expressing cervical cancer cells. (ai) Histograms for effector (HeLa) and target cell (HeLa-EGFP) populations alone or in co-culture are shown after annexin V-PE staining as described in Materials and Methods. 1: Untreated HeLa cells; 2: Untreated HeLa-EGFP cells; 3: Untreated effector cells were co-plated with target EGFP cells; 4: Effector cells were treated with MMC for 24 h, washed with medium and target EGFP cells were co-plated; 5: Untreated effector cells were co-plated with target EGFP cells and treated with MG132 for 24 h; 6: Effector cells were treated with MMC for 24 h, washed with medium, and target EGFP cells were co-plated and then treated with MG132 for 24 h; 7: Effector cells were treated with GW9662 for 2 h followed by the addition of MMC, for 24 h, and then washed, co-plated with target EGFP cells along with treatment of MG132 and GW9662; 8: Effector cells were treated with MMC for 24 h, washed and co-plated with target EGFP cells, and further incubated in the presence of MG132 and anti-TRAIL antibodies; are shown with annexin V-PE positive counts and are indicated as percentages of apoptotic cells. Upper right quadrant represents proportion of apoptotic target EGFP cells. (aii) The bar graph shows annexin V-PE positive cells in the same experiment. Data are mean±S.D., and are representative of three independent experiments (**P<0.01, when compared with their respective controls). (bi) Similar experiment was performed in SiHa cells (effector SiHa and target SiHa-EGFP). (bii) The bar graph shows annexin V-PE positive cells in the same experiment. Data are mean±S.D., and are representative of three independent experiments (**P<0.01, when compared with their respective controls)

Figure 5

CM transfer and co-plating experiments to evaluate bystander killing in heterogeneous system. Involvement of sFasL in mediating bystander cytotoxicity. The effector cells (THP-1 MΦ) were treated with 500 nM MMC for 24 h, and then CM was collected after 48 h as described in Materials and Methods. Target HeLa (a) and SiHa (b) cells were incubated with the CM in the presence or absence of MG132 and/or with anti-FasL antibody for 36 h. CM was supplemented with 0.2% FBS to avoid cell death owing to growth factor depletion. Cell survival was further evaluated by MTT assay. Data are mean±S.D., and are representative of three independent experiments performed in triplicates (**P<0.01 when compared with their respective controls). (ci) Apoptotic cell death in target HeLa-EGFP cells in heterogeneous system. Histograms for effector and target cell populations alone or in co-culture are shown by annexin V-PE staining using flow cytometry. 1:Untreated THP-1 MΦ 2: Untreated HeLa-EGFP cells; 3: Untreated effector cells (THP-1 MΦ) were co-plated with target EGFP cells; 4: Effector cells (THP-1 MΦ) were treated with MMC for 24 h, washed with medium and target EGFP cells were co-plated; 5: Untreated effector cells (THP-1 MΦ) were co-plated with target EGFP cells and treated with MG132 for 24 h; 6: Effector cells (THP-1 MΦ) were treated with MMC for 24 h, washed with medium, and target EGFP cells were co-plated and then treated with MG132 for 24 h; 7: Effector cells (THP-1 MΦ) were treated with GW9662 for 2 h followed by addition of MMC for 24 h, and then washed, co-plated with target EGFP cells along with treatment of MG132 and GW9662; 8: Effector cells (THP-1 MΦ) were treated with MMC for 24 h, washed and co-plated with target EGFP cells, and further incubated in the presence of MG132 and anti-TRAIL antibodies; are shown with annexin V-PE positive counts and are indicated as percentages of apoptotic cells; are shown with annexin V-PE positive counts indicated as percentages of apoptotic cells. Upper right quadrant represents proportion of apoptotic bystander target EGFP cells. Effector cells were washed with medium three times before co-plating with the target cells. (cii) The bar graph shows annexin V-PE positive cells in the same experiment. Data are mean±S.D., and are representative of three independent experiments (**P<0.01 when compared with their respective controls). (d) Western blot analysis for PARP cleavage. Target HeLa cells were pretreated with 5 μM MG132 for 2 h. Thereafter, CM collected from MMC-treated effector cells (HeLa and THP-1 MΦ) was added in the presence or absence of MG132 for an additional 24 h. Whole-cell lysates were prepared to perform western blot analysis. The levels of PARP (p116) and its cleaved product (p85) were detected. β-Actin was used as a loading control

Figure 6

PPARγ knockdown abrogates MMC-induced bystander killing. (a and b) The effector cells (HeLa, SiHa and THP-1 MΦ) were transfected with control siRNA or PPARγ siRNA for 15 h, fresh medium was added and allowed to grow for the next 15 h. Effector cells (transfected or non-transfected) were treated with 500 nM MMC for 24 h, and then CM medium was collected after 48 h as described in Materials and Methods. Target HeLa (a) and SiHa (b) cells were incubated with the respective CM in the presence or absence of MG132 for 36 h. CM was supplemented with 0.2% FBS to avoid cell death due to growth factor depletion. (c and d) Similar experiments were performed in heterogeneous system by incubating target HeLa (c) and SiHa (d) cells in CM collected from THP-1 MΦ. Cell survival was evaluated by MTT assay. Data are mean±S.D., and representative of experiments performed in triplicates (*P<0.05, **P<0.01, ***P<0.001 when compared with their respective controls)

Figure 7

The combination treatment of MMC and proteasomal inhibitor reduces tumor progression in HeLa cells xenografted mouse tumor model. (a) Experimental layout of in vivo study. HeLa cells (1 × 10⁶ in 100 μl PBS) were injected on the right flank of the mice to form tumors. Tumor-bearing mice were treated with combination of MMC (1 mg/kg/every third day) and MG132 (10 μM/kg/day) as described in Materials and Methods. Control mice were administered with equal volume of vehicle on the same treatment day. (b) Tumor progression after drug administration in control and treated mice. (c and d) Bar graph showing tumor volume and tumor weight in mice at the end of the experiment. (e) Changes in body weight in mice during the course of the experiment. (f) Histopathological analysis of major vital organs collected from experimental mice. Kidney, heart, liver and lungs were fixed in 4% formaldehyde. The tissues sections were stained with hematoxylin and eosin (H&E; magnification, × 400). (g) TAMs were isolated from tumor as described in Materials and Methods. Cells were analyzed for FasL expression by flow cytometry. Cells were dually stained with CD11b (1 : 100) and FasL (1 : 100), and CD11b-positive cells were gated to analyze FasL expression. (h) Representative images of immunostained section analysis of Fas (i) TUNEL assay (ii) in tumor tissues of different treatment groups (magnification, × 40 with inset at × 400)

Figure 8

Proposed model for bystander effect. MMC induces expression of membrane bound and secretory forms of death ligands (FasL) in cancer cells as well as macrophages. Restoration of Fas by inhibiting proteasomal degradation facilitates bystander killing of tumor cells and, thus effectively retarding the tumor progression