Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors

Abstract

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.

Introduction

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is a potent cancer cell-specific apoptosis-inducing agent with little to no effect on normal tissues.^1,2 The TRAIL receptor (TRAIL-R) agonist antibodies, mapatumumab and lexatumumab, have advanced to clinical testing² and remain attractive candidate anti-cancer drugs.^3-6 However, acquired resistance of cancer cells to TRAIL is a roadblock, allowing them to evade the pro-apoptotic effects of TRAIL.^7,8 Mechanisms of resistance include overexpression of the inhibitor of caspase-8 activation c-FLIP, hypermethylation of caspase-8, reduced cell surface TRAIL-R expression, overexpression of anti-apoptotic Bcl-2 family members such as Bcl-XL or Mcl-1, loss of pro-apoptotic Bax and overexpression of the inhibitor of apoptosis (IAP) family members.^9-12 Therefore, the effectiveness of TRAIL and TRAIL-R agonistic antibodies as mono-therapies may be limited by resistance, as it is for other anti-cancer agents, and this may be addressed through appropriate combinations with other therapeutic agents.

Cancer chemotherapy drugs are commonly combined to augment treatment efficacy and suppress the emergence of resistant clones. Thus, agents that can modulate some of these mechanisms of resistance to TRAIL have a potential in improving the cytokine therapy. It has been reported that DNA-damaging agents and small-molecule inhibitors effectively sensitize cancer cells to TRAIL.^10,13,14

Mitomycin-C (MMC) is an antibiotic that has demonstrated antitumor activity in preclinical and clinical studies and is widely used to treat various cancers. MMC is known to act synergistically with capecitabine and irinotecan.^15,16 Some studies suggested that the combination of 5-FU plus MMC is more active in vitro than mono-therapy in colorectal cancer.¹⁷ The efficacy of the combination of MMC with other cytotoxic agents such as capecitabine and raltiterxed for colorectal cancer has been reported.^18,19

MMC has been reported to overcome acquired resistance to TRAIL in DLD1 colon cancer cells.²⁰ Treatment with the combination of TRAIL and MMC led to enhanced activation of caspase-3 and induced overexpression of the BAX gene, which was correlated with enhanced TRAIL-induced cell killing in DLD1 cells. Following MMC exposure, expression of DR4 was found to be increased in hepatocellular carcinoma (HCC) cells, then leading to the bystander killing in homogeneous and heterogeneous hepatoma cellular models.²¹

These factors led us to investigate whether MMC can modulate TRAIL-induced apoptosis in other human colon cancer cell lines HCT116 and HT-29 and, if so, through what mechanism. We found that MMC can indeed enhance TRAIL-induced apoptosis through the downregulation of various cell survival proteins, upregulation of various apoptotic proteins and via upregulation of TRAIL receptors. The upregulation of death receptors by MMC was mediated through expression of C-Jun N terminal kinase.

Results

MMC enhances TRAIL-induced apoptosis in HCT116 (p53−/−) cells

Because p53 mutations commonly arise in colorectal cancer cells,²² the use of DNA-damaging agents for TRAIL sensitization would likely be less effective in the absence of wild-type p53. We initially set out to identify therapeutic combinations of MMC and TRAIL in colon carcinoma HCT116 (p53−/−) cells. The HCT116 (p53−/−) cells were minimally sensitive to either MMC or TRAIL alone. However, surprisingly, combination treatment with MMC and TRAIL decreased cell viability significantly (Fig. 1A). We also examined the effect of MMC on TRAIL-induced suppression of cell proliferation using crystal violet staining. Although MMC and TRAIL alone were moderately effective, MMC substantially enhanced the effect of TRAIL on suppression of the cell proliferation (Fig. 1B). To confirm the effect of MMC on TRAIL-induced apoptosis, we measured apoptosis by FACS analysis of the sub-G₁ fraction. We found that MMC and TRAIL treatment alone induced 9.5% and 35.0% apoptosis, respectively. However, combination treatment with MMC and TRAIL enhanced apoptosis to 66.6% (Fig. 1C).

Figure 1. MMC potentiates TRAIL-induced apoptosis of HCT116 (p53−/−) cells. (A) Right: A representative bioluminescence image corresponding to cell viability is shown from HCT116 (p53−/−) cells that were pretreated with 5 µM MMC for 24 h and then co-treated with TRAIL (25 ng/mL) and MMC (5 µM) for 12 h. Left: Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05). (B) Cell viability was determined by crystal violet staining in HCT116 (p53−/−) cells treated with 5 µM MMC, 25 ng/mL TRAIL or the combination. (C) Cells were treated with 5 µM MMC for 24 h and then treated with TRAIL (25 ng/mL) for 12 h. Cells were stained with PI, then analyzed by FACS. (D) Western blot showing the processing of caspases and the caspase-3 substrate PARP in HCT116 (p53−/−) cells treated with MMC, TRAIL or the combination. Ran was used as a protein loading control. (E) To examine the effect of caspase inhibition, HCT116 (p53−/−) cells were pretreated with caspase-8 inhibitor (z-IETD-fmk) and canspase-9 inhibitor (z-LEHD-fmk) (20 µM), respectively, for 1 h and then treated with MMC, TRAIL or the combination. Cells were stained with PI then analyzed by FACS or subjected to western blot for PARP cleavage (F). (G) MMC increases colon cancer cell sensitivity to TRAIL-R–activating antibodies. Cell viability was measured in HCT116 (p53−/−) cells after treatment with 5 μM MMC for 24 h followed by mapatumumab (50 ng/ml), or lexatumumab (50 ng/ml) for an additional 12 h. A luciferase-based assay system was used to measure cell viability. Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05).

After pretreatment with MMC, TRAIL more efficiently initiated processing of caspase-8, -9 and -3, as well as cleavage of the caspase-3 substrate poly-ADP-ribose polymerase (PARP), further indicating that MMC enhances TRAIL-induced apoptosis (Fig. 1D). To further investigate whether the combined treatment of MMC plus TRAIL triggered cell death through caspases, we used a caspase-8 and -9 inhibitor, z-IETD-fmk and z-LEHD-fmk. Pretreatment with z-IETD-fmk and z-LEHD-fmk effectively blocked the apoptosis (Fig. 1E) and PARP cleavage (Fig. 1F) induced by the combined treatment. This indicates that MMC sensitizes HCT116 (p53−/−) cells to TRAIL-induced apoptosis in a caspase-dependent manner.

MMC sensitizes TRAIL-resistant cells

We next investigated whether MMC affects TRAIL-resistant cancer cells. HT-29 cells were minimally sensitive to either MMC or TRAIL alone. However, the combination of MMC and TRAIL significantly suppressed cell viability (Fig. 2A) and cell proliferation of HT-29 cells (Fig. 2B). FACS analysis of apoptosis also revealed that pretreatment with MMC potently and significantly enhanced TRAIL-induced apoptosis from 5.7% and 6% to 25.7% (Fig. 2C). Consistent with these results, following pretreatment with MMC, TRAIL more efficiently initiated processing of caspase-8, -9 and -3, as well as cleavage of PARP, as shown in Figure 2D. Together, our results indicate that MMC can enhance TRAIL-induced apoptosis in TRAIL-resistant HT-29 cells.

Figure 2. MMC potentiates TRAIL-resistant HT-29 cells to TRAIL. (A) Right: A representative bioluminescence image corresponding to cell viability is shown from HT-29 cells that were pretreated with 5 µM MMC for 12 h and then co-treated with TRAIL (25 ng/mL) for 12 h. Left: Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05). (B) Cell viability was determined by crystal violet staining in HT-29 cells treated with 5 µM MMC, 25 ng/mL TRAIL or the combination. (C) Cells were treated with 5 µM MMC for 12 h and then treated with TRAIL (25 ng/mL) for 12 h. Cells were stained with PI then analyzed by FACS. (D) Western blot showing the processing of caspases and the caspase-3 substrate PARP in HT-29 cells treated with MMC, TRAIL or the combination. Ran was used as a protein-loading control. (E) Cell viability was measured in HT-29 cells after treatment with 5 μM MMC for 12 h followed by mapatumumab (50 ng/ml), or lexatumumab (50 ng/ml) for an additional 12 h. A luciferase-based assay system was used to measure cell viability. Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05).

MMC potentiates the cytotoxicity of TRAIL-R agonistic antibodies

Mapatumamab and lexatumamab are two agonistic TRAIL-R antibodies that selectively target DR4 and DR5, respectively. These two antibodies induce apoptosis by the same mechanism as TRAIL. However, the half-life of mapatumumab is ~9 days compared with less than 30 min for TRAIL in vivo.^23,24 The stability of mapatumumab and lexatumumab makes them attractive agents for use in humans. Based on this, we next investigated whether MMC sensitizes colon cancer cells to mapatumumab and lexatumumab. The results showed that pretreatment with MMC further enhanced the sensitivity to lexatumumab and mapatumumab in HCT116 (p53−/−) cells (Fig. 1G) and HT-29 cells (Fig. 2E), respectively.

MMC induces the expression of TRAIL receptors DR4 and DR5

To identify the mechanism by which MMC sensitizes colon cancer cells to TRAIL and TRAIL agonistic antibodies, we quantified multiple extrinsic and intrinsic cell death pathway components that could be affected by MMC and lead to more efficient apoptotic signaling. We detected appreciable changes in anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL and Mcl-1 upon MMC treatment (Fig. 3A). MMC also downregulated caspase-inhibitor family members c-IAP-1 and XIAP (Fig. 3A). By contrast, MMC increased the expression of pro-apoptotic proteins such as Bax and Bim (Fig. 3A). Furthermore, death receptors DR4 and DR5 were increased by MMC in a dose-dependent manner (Fig. 3B). Changes of cell surface receptor density, as determined by FACS, corresponded to death receptor protein content (Fig. 3C). We investigated whether TRAIL receptors are induced by MMC at the transcriptional level by RT-PCR. We found that MMC substantially upregulates DR4 and DR5 mRNA expression in a dose-dependent manner (Fig. 3D). The results are consistent with the Western blot results. Collectively, we conclude that MMC can sensitize colon cancer cells to TRAIL-induced apoptosis through downregulation of anti-apoptotic proteins, and upregulation of cell survival proteins and TRAIL death receptors.

Figure 3A–C. MMC suppresses anti-apoptotic proteins and induces pro-apoptotic proteins and death receptor expression. (A) HCT116 (p53−/−) cells (left panel) and HT-29 cells (right panel) were pretreated with the indicated doses of MMC for 24 h. Whole-cell extracts were prepared and subjected to western blotting for Bcl-2 family members(Mcl-1, Bcl-2, Bcl-XL, Bax, Bim and Bid) and IAP family members (cIAP-1 and XIAP). The same blots were stripped and re-probed with Ran antibody to verify equal protein loading. (B) HCT116 (p53−/−) cells (left panel) and HT-29 cells (right panel) were treated with the indicated doses of MMC. Whole-cell extracts were then prepared and analyzed for DR4 and DR5 by western blot. (C) HCT116 (p53−/−) cells (left panel) and HT-29 cells (right panel) were treated with 5 µM MMC for 24 h and then harvested for analysis of cell surface DR4 and DR5 by immunofluorescent staining and subsequent flow cytometry.

Figure 3D–F. (D) HCT116 (p53−/−) cells were treated with indicated concentration of MMC for 24 h, and total RNA was extracted and examined for expression of DR4 and DR5 by RT-PCR. GAPDH was used as an internal control to show equal RNA loading. (E) Blockage of DRs-induction reverses the ability of MMC to augment TRAIL-induced apoptosis. HCT116 (p53−/−) cells were transfected with control shRNA, DR4 shRNA, DR5 shRNA alone or combined. After treatment with MMC for 24 h, whole-cell extracts were prepared and analyzed by western blotting. (G) Cells were exposed to indicated doses of MMC for 24 h and then treated with 25 ng/mL TRAIL. Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05).

We also investigated whether upregulation of DR4 and DR5 by MMC is specific to HCT116 and HT-29 cells. MMC induced DR4 and DR5 expression in breast cancer cells (BT549), liver cancer cells (HEP3B), ovarian cancer cells (SKOV3) and thyroid cancer cells (8505C) (Fig. S1). MMC induced only DR4 expression in prostate cancer cells (DU145) and lung cancer cells (H1299 and H460). These results indicate that upregulation of DR4 and DR5 expression by MMC is not cell type-specific.

Gene silencing of death receptors abolishes the effect of MMC on TRAIL-induced cytotoxicity

In order to determine the role of DR4 and DR5 modulation in mediating MMC/ TRAIL-induced apoptosis, we used cancer cells stably transfected with shRNA against DR4 (HCT116^shDR4), DR5 (HCT116^shDR5) or DR4 and DR5 (HCT116^shDR4/shDR5) and then examined the impact of the gene silencing on suppression of cell viability by the combination of MMC and TRAIL. As shown in Figure 3E, transfection of cells with shRNA of DR4, DR5 or DR4 and DR5, but not with the control shRNA, reduced MMC-induced DR4 or DR5 expression in the respective cells. We next examined whether the suppression of DR4 or DR5 could abolish the effects of MMC on TRAIL-induced cell viability suppression. We found that the silencing of DR5 or both receptors increased the cell viability from 52% to 79% or 74%, respectively, but silencing of DR4 had no significant effect on cell viability (Fig. 3F). These results suggest that DR5 plays an important role in the effect of MMC on TRAIL-induced cytotoxicity.

MMC-induced upregulation of TRAIL receptors is p53-, Bax- and Bim-independent

Given the observed induction of DR4 and DR5 by MMC and the important role of DR5 in the synergistic effects as demonstrated above, we were particularly interested in the mechanisms by which MMC induces expression of DR4 and DR5. MMC upregulated DR4 and DR5 in p53−/− HCT116 cells, demonstrating the p53-independent nature of this effect, which is consistent with prior work²⁵ (Fig. 4A). We further investigated whether MMC upregulates DRs through upregulation of Bax and Bim. For this, we used Bax-knockout HCT116 and Bim-knockdown HCT116 (p53−/−) colon cancer cells. MMC induced expression of DR5 and DR4 in Bax-knockout parental cells, HCT116 (p53−/−) and Bim-knockdown HCT116 (p53−/−) cells (Fig. 4A and C), indicating that induction of TRAIL receptors occurs independently of Bax and Bim expression.

Figure 4. MMC induced DRs expression is p53, Bax and Bim-independent. (A) HCT116 wild-type (left panel), p53-knockout HCT116 (middle panel) and Bax-knockout (right panel) cells were pretreated with the indicated doses of MMC for 24 h. Whole-cell extracts were prepared and subjected to western blotting with the relevant antibodies. (B) HCT116 wild-type, HCT116 (p53−/−) and HCT116 (Bax−/−) cells were pretreated with MMC for 24 h and co-treated with MMC and TRAIL for 12 h. Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05). (C) HCT116 (p53−/−) cells were transfected with Bim siRNA. After 24 h transfection, the cells were treated with MMC for 24 h. Whole-cell extracts were prepared and subjected to western blotting with the relevant antibodies.

MMC-induced upregulation of death receptors is mediated through the activation of JNK

We sought to further identify pathways and transcription factors involved in MMC-induced DR4 and DR5 upregulation. Activation of JNK/c-Jun/activating protein 1(AP1) pathway leads to death receptor upregulation and increased TRAIL sensitivity in cell lines from multiple cancer types.^26-28 We hypothesized that an effect of MMC activity could be to stimulate the JNK/c-Jun/AP-1 pathway leading to upregulation of pro-apoptotic TRAIL death receptors. Indeed, we found that MMC increased the levels of phosphorylation of JNK and its downstream substrate, phosphorylation of c-Jun (Fig. 5A), indicating that MMC activates the JNK pathway.

Figure 5. Effects of MMC on JNK kinase expression, which mediates DR4 and DR5 expression. (A) HCT116 (p53−/−) cells were pretreated with the indicated doses of MMC for 24 h or indicated time point. Whole-cell extracts were prepared and analyzed by western blotting using the specific antibodies to proteins. The same blots were stripped and re-probed with Ran antibody to verify equal protein loading. (B) HCT116 (p53−/−) cells were treated with JNK inhibitor (SP600125) for 1 h and then exposed to 5 μM MMC for 24 h. Whole-cell extracts were prepared and analyzed for the expression of DR4, DR5 and Ran using relevant antibodies. (C) HCT116 (p53−/−) cells were were transfected with JNK1 siRNA and JNK2 siRNA, after 24 h transfection, the cells were treated with MMC for 24 h. Whole-cell extracts were prepared and subjected to western blotting with the relevant antibodies. (D) Cells were pretreated with MMC for 24 h and co-treated with MMC and TRAIL for 12 h. Quantitative analysis of cell viability (means ± SD, n = 3) from three experiments. Statistical significance was determined with Student’s t-test (*p < 0.05).

We studied the effects of MMC on DR4 and DR5 upregulation in the presence of the JNK inhibitor SP600125. The JNK-specific inhibitor SP600125 abrogates upregulation of DR4 and DR5 (Fig. 5B), indicating that MMC induces DR4 and DR5 expression through a JNK-dependent mechanism.

To further demonstrate the role of JNK activation in mediating MMC-induced DR4 and DR5 expression, we transfected JNK-specific siRNA to inhibit JNK activation through gene-silencing of JNK expression, and then examined its impact on DR4 and DR5 expression. As presented in Figure 5C, transfection of JNK1 siRNA and JNK2 siRNA reduced basal levels of JNK1 and JNK2, respectively, indicating the successful knockdown of JNK. MMC induced DR4 expression in control and control siRNA-transfected cells, but not in JNK1 and JNK2 siRNA-transfected cells. MMC did not induce DR5 expression only in JNK2 siRNA-transfected cells.

Next we tested the combination effect of MMC and TRAIL on JNK1 and JNK2-knockdown cells. We found that the synergistic effect of MMC and TRAIL decreased in the JNK1-knockdown cells (Fig. 5D). Because JNK2 siRNA was cytotoxic to the cells, it was difficult to study the synergistic effect.

Collectively, we conclude that MMC induces DR4 and DR5 expression through a JNK-dependent mechanism.

Mitomycin C enhances TRAIL sensitivity in vivo

We next tested the therapeutic combination of MMC and TRAIL in vivo. Mice bearing xenografted HCT116 (p53−/−) colon tumors and HT-29 colon tumors were treated with an intra-peritoneal dose of MMC (1 mg/kg) and an intravenous dose of TRAIL (100 μg) every other day. Animals were treated with 10 consecutive cycles of the combination therapy regimen. The combination therapy suppressed tumor growth significantly (Fig. 6A and B) and did not impact the weight of the mice (Fig. 6C), indicating that the therapeutic combination of MMC and TRAIL is well-tolerated and has anti-tumor activity in vivo.

Figure 6. MMC enhances TRAIL sensitivity in vivo. (A) HCT116 (p53−/−) and HT-29 xenograft tumors were established in nude mice by subcutaneous injection of 1 × 10⁶ or 2 × 10⁶ cells, respectively. When tumors reached a diameter of 0.2 cm, mice were treated with MMC (1 mg/kg) by intraperitoneal injection plus one intravenous dose of purified rhTRAIL (100 ug) for three weeks. Animals were sacrificed, and the tumors were excised. (B) HCT116 (p53−/−) and HT-29 xenograft tumors were established in nude mice and treated as described in (A). Tumor volumes were determined weekly by caliper measurements and using the following formula for the volume of an ellipsoid [volume (mm³) = 0.52 × (width)² × (length)]. Quantitative analysis of tumor volume (means ± SD, n = 7–8). Statistical significance was determined with Student’s t-test (*p < 0.05). (B) Effect of combinatorial therapy on the body weight of the mice (n = 7–8/group ± SD). (C) Schematic representation of mechanism by which MMC potentiates TRAIL-induced apoptosis.

Discussion

Although both TRAIL and agonistic antibodies to TRAIL receptors are currently in clinical trials for treatment of cancer patients, resistance of tumor cells to apoptosis is one of the major hurdles in the usefulness of this cytokine. Thus, agents that can either potentiate the effect of TRAIL or overcome resistance are urgently needed. In this study, we demonstrated that the classical chemotherapeutic agent MMC, in combination with TRAIL exhibits, enhanced apoptosis-inducing activity in human colon cancer HCT116 (p53−/−) cells and TRAIL-resistant HT-29 cells. Our findings are in agreement with previous findings that combination MMC can overcome acquired resistance to TRAIL in DLD1 colon cancer cells.²⁰ The observed activation of caspase-3, -8 and -9 in our experiments suggests that MMC potentiates both the extrinsic and intrinsic pathways of apoptosis. Moreover, we have shown that the combination of MMC and TRAIL exhibits enhanced effects on inhibiting the growth of HCT116 (p53−/−) cells and HT-29 cells xenografts in vivo (Fig. 6). Thus, the current results warrant further evaluation of the MMC and TRAIL combination as a potential therapeutic regimen against human colon cancer.

We also identified several sensitization mechanisms acquired for the sensitization of MMC in TRAIL-induced apoptosis. One of the mechanisms of sensitization involves the regulation of anti-apoptotic proteins. MMC downregulates the expression of Bcl-2, Bcl-xL and Mcl-1, all of which have been linked to tumor cell resistance to TRAIL.^9,29,30 XIAP is a potent inhibitor of caspase-3 activity and has previously been shown to confer resistance to TRAIL.³¹ Our results showed that MMC decreases the expression of XIAP. The enhanced caspase-3 activation we observed in cells treated with MMC plus TRAIL could also be due to reduced levels of XIAP.

Mutational inactivation of Bax has been shown to induce resistance to TRAIL,³² suggesting that Bax plays a critical role in TRAIL-induced apoptosis. In our study, we found that MMC upregulates the expression of pro-apoptotic proteins such as Bax and Bim, which could be mechanisms of sensitization. We found that the MMC and TRAIL combination induces a moderate increase in cell death in the cells treated with Bax (Fig. 4B) and Bim siRNA (data not shown).

DR4 and DR5 expression is also critical for sensitization of cells to TRAIL, as MMC induces DR4 and DR5 expression in a dose-dependent manner, and gene silencing of DR5 or DR4 and DR5 abolishes TRAIL-induced apoptosis. Thus, DR5 plays an important role in the effect of MMC on TRAIL-induced cytotoxicity. Unlike the DR5, DR4 cell surface expression is minimally changed although the DR4 protein content is decreased in DR4 shRNA transfected HCT116 cells (data not shown). So further research is needed to confirm the role of DR4 in the synergistic effect of MMC and TRAIL. Furthermore, we found that the upregulation of DR4 and DR5 by MMC was not restricted to colon cancer cells, but also occurred in breast, liver, ovarian, thyroid, prostate and lung cancer cells (Fig. 4E). Thus, MMC is likely to potentiate the effect of TRAIL in a wide variety of cells. However, induction of TRAIL receptors in some cells is much more pronounced than in other cell types.

The expression of death receptors is known to be regulated by p53-dependent or p53-independent mechanisms.^25,33,34 MMC induces the expression of DR4 and DR5 in a colon cancer cell line, regardless of p53 status (parental p53 and knockout p53 HCT116 cells), indicating that MMC upregulates DR4 and DR5 expression via a p53-independent mechanism. In addition, our results also show that MMC induces apoptosis mediated through expression of death receptors in independent of Bax and Bim, indicating that the role of p53, Bax or Bim on induction of DR4 and DR5 depends on the nature of the stimulus.

DR4 and DR5 induction by MMC was reported previously by other groups. However, how MMC upregulates DR4 and DR5 has not been fully elucidated. It has been previously shown that JNK activation positively regulates DR5 and DR4 expression.^26,27 In our study, we found that MMC increases the levels of p-JNK and p-c-Jun, indicating that MMC also activates JNK signaling. Both gene silencing of JNK and JNK inhibitors SP600125 suppresses the expression of DR4 and DR5. It is clear that MMC induces JNK-dependent DR4 and DR5 expression.

Taken together, our results provide the first mechanistic evidence that MMC treatment results in JNK-mediated upregulation of DR4 and DR5, downregulation of anti-apoptotic proteins and upregulation of pro-apoptotic proteins, thus rendering cancer cells more sensitive to the cytotoxic activities of TRAIL. In addition, our studies also show that the combined treatment with MMC and TRAIL induces apoptosis in TRAIL-resistant colon cancer cells. Thus, these studies suggest that TRAIL can be given in combination with MMC, especially for those tumors that develop resistance to TRAIL.

Materials and Methods

Materials

Ten millimolar Mitomycin C (purchased from NCI and Sigma) was prepared in 100% dimethyl sulfoxide, stored as small aliquots at -80°C and then diluted as needed in cell culture medium. His-tagged recombinant human TRAIL (rhTRAIL) was produced and purified as described previously.³⁵ Streptomycin, Dulbecco’s modified Eagle’s medium, McCoy’s 5A and fetal bovine serum were obtained from Invitrogen. Antibodies against DR4, caspase-9, Bcl-XL, cIAP-1, Bid, Bax, p53 and caspase-3 were obtained from Santa Cruz Biotechnology. Antibodies against PARP, JNK, p-JNK, Bim, Bcl-2, c-IAP1, MEK, p-MEK, c-Jun, p-c-Jun, Mcl-1, Akt, p-AKT, caspase-3 and DR5 were purchased from cell signaling. Antibodies against Ran, XIAP and caspase-8 were purchased from BD Biosciences. Caspase inhibitors were purchased from R&D Systems. SP600125 were purchased from Sigma.

Cell lines

All cell lines were purchased from the American Type Culture Collection, unless otherwise stated, and maintained in their appropriate growth medium at 37°C and 5% CO₂. Colon adenocarcinoma HCT116, HCT116 variants with deletions in p53, bax and HT-29 human colon cancer cells were cultured in McCoy’s 5A medium, supplemented with 10% fetal calf serum and penicillin/streptomycin (Invitrogen). HFF cells were cultured in Dulbecco’s modified Eagle’s medium with 15% fetal calf serum.

Cell viability assay

To measure cell viability, we used the CellTiter-Glo® Luminescent Cell Viability Assay (Promega), which use a unique, stable form of luciferase to measure ATP as an indicator of viable cells, and the luminescent signal produced is proportional to the number of viable cells present in culture. Cells were pretreated with different concentrations of MMC for 12 h or 24 h, and then exposed to different concentrations of TRAIL for 12 h. An equal volume (100 µL) of CellTiter-Glo™ reagent was added and the solution was mixed gently for 2 min on an orbital shaker. Mixture was incubated at room temperature for 10 min to allow luminescent signal to stabilize, and then imaging was performed using the Xenogen IVIS system to quantify the cell viability.

Crystal violet assays

For crystal violet assays, cells were treated with MMC for 12 h or 24 h and then treated with combination of MMC and TRAIL for 12 h. Cells were washed with PBS, fixed with methanol, stained with crystal violet and imaged.

Propidium iodide (PI) staining for DNA fragmentation

Cell death was quantified by propidium iodide (PI) staining for DNA content and FACS. Cells were pretreated with MMC for 12 h or 24 h, and then exposed to TRAIL for 12 h. Floating and adherent cells were collected and fixed in 70% ethanol, followed by ribonuclease (RNase) A treatment and PI staining. A total of 20,000 events were analyzed by flow cytometry using an excitation wavelength set at 488 nm and emission set at 610 nm.

Analysis of DR4 and DR5 surface expression

Cells (3 × 10⁵) were treated with MMC and washed with PBS supplemented with 2% FBS after detachment with trypsinization. Cells were then stained with mouse monoclonal anti-human DR5 (Imgenex) or DR4 (Imgenex) for 20 min at room temperature before washing and resuspension in phycoerythrin (PE)-conjugated goat anti-mouse or rabbit antibody (Invitrogen) for 20 min at room temperature before wash and resuspension in PBS supplemented with 2% FBS for flow-cytometric analysis using an excitation wavelength of 488 nm. Mouse and rabbit IgG isotypes were used as isotype controls.

RNA analysis and reverse transcription

Polymerase Chain Reaction (RT-PCR)—DR5 and DR4 mRNA was detected using RT-PCR as follows. Total RNA was isolated from cells using Qiagen RNEasy kit as instructed by the manufacturer. One microgram of total RNA was converted to cDNA using Superscript reverse transcriptase (Invitrogen) and then amplified by fast-start Taq polymerase (Roche). The total RNAs were then amplified by PCR using the primers previously described³⁶: DR5 sense 5′-AAGACCCTTGTGCTCGTTGTC-3′, DR5 anti-sense 5′-GACACATTCGATGTCACTCCA-3′, DR4 sense 5′-CTGAGCAACGCAGACTCGCTGTCCAC-3′, DR4 antisense 5′-TCCAAGGACACGGCAGAGCCTGTGCCAT-3′, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense, 5′-GTCTTCACCACCATGGAG-3′ and GAPDH antisense 5′-CCACCCTGTTGCTGTAGC-3′. The reaction sequence consisted of 94°C for 2 min, and 94°C for 35 cycles of 30 sec each; 50°C for 30 sec and 72°C for 45 sec with an extension at 72°C for 10 min. PCR products were run on 1.5% agarose gel and then stained with ethidium bromide. Stained bands were visualized under UV light and photographed.

Transfection with siRNA

High-purity control (scrambled RNA), Bim, JNK1 and JNK2 small interfering RNA (siRNA) oligos were purchased from Santa Cruz Biotechnology. Transfection of these siRNA duplexes was conducted in 6-well plates using the Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s manual. After 24 h of transfection, cells were treated with MMC for 24 h. Whole-cell extracts were prepared for relevant protein analysis by western blotting.

Western blot analysis

Cells were lysed in a single-detergent lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 10 mM NaF, 1% Triton X-100] supplemented with protease (Sigma) and phosphatase (Calbiochem) inhibitor cocktails. Protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories) according to the manufacturer’s instructions. Equal amounts of protein were separated by 4 to 12% SDS–PAGE (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore) and then blotted with each antibody, and detected by an ECL reagent (GE Healthcare).

Xenograft studies

Nude mice were housed and maintained in accordance with the Institutional Animal Care and Use Committee and state and federal guidelines for the humane treatment and care of laboratory animals. Four- to 6-wk-old NCr nude mice were purchased from Taconic and injected subcutaneously with 1 × 10⁶ HCT116 (p53−/−) or 2 × 10⁶ HT-29 cells mixed with Matrigel (BD Biosciences). Tumors were developed in the absence of treatment until they are ~0.2 cm in diameter. The mice were then subjected to the MMC and TRAIL treatment regimen. Animals were treated with MMC (1 mg/kg) by intraperitoneal injection for 24 h, followed by one intravenous dose of purified rhTRAIL (100 ug). As a negative control, a subset of the mice were injected (i.p. and i.v.) with saline (vehicle) at the same frequency of treatment. Animals were treated for 3 consecutive weeks. The tumor size was monitored every week using caliper measurements of the tumor volume.

Statistical analysis

Data were analyzed using the Student's t-test for comparison between groups or ANOVA followed by Scheffe’s test for multiple comparisons. Significance was defined as p values less than 0.05.

Glossary

Abbreviations:

MMC

mitomycin C

TRAIL

tumor necrosis factor (TNF)-related apoptosis-inducing ligand

DR4

TRAIL receptor 1

DR5

TRAIL receptor 2

Supplemental Material

Supplemental material may be found here: www.landesbioscience.com/journals/cc/article/21643/