Small molecule inhibitor YM155-mediated activation of death receptor 5 is crucial for chemotherapy-induced apoptosis in pancreatic carcinoma

Abstract

Despite much effort, pancreatic cancer survival rates are still dismally low. Novel therapeutics may hold the key to improving survival. YM155 is a small molecule inhibitor that has shown antitumor activity in a number of cancers by reducing the expression of survivin. The aim of our study is to understand the mechanisms by which YM155 functions in pancreatic cancer cells. We established the antitumor effect of YM155 with in vitro studies in cultured cells, and in vivo studies using a mouse xenograft model. Our data demonstrated that YM155 reduced the expression of survivin; however, downregulation of survivin itself is insufficient to induce apoptosis in pancreatic cancer cells. We showed for the first time that treatment with YM155 increased death receptor 5 (DR5) expression in pancreatic cancer cells. We found that YM155 induced apoptosis by broad-spectrum inhibition of IAP family member proteins (e.g., CIAP1/2 and FLIP) and induced proapoptotic Bak protein upregulation and activation; the antitumor effect of YM155 treatment with either the DR5 agonist lexatumumab or gemcitabine on pancreatic cancer cells was synergistic. Our data also revealed that YM155 inhibits tumor growth in vivo, without apparent toxicity to the noncancerous human pancreatic ductal epithelial cell line. Together, these findings suggest that YM155 could be a novel therapeutic agent for pancreatic cancer.

Figure 1. Survivin down-regulation is not sufficient to trigger apoptosis

(A), Chemical structure of YM155. (B), Panc-1 cells were treated with YM155 and cell lysates were prepared for Western blotting to detect survivin. β-actin were assessed as the control for equal loading of protein. (C), Panc-1 cells were transfected with either survivin-specific siRNA or scramble-siRNA as negative control. 48 h post-transfection, cell lysates were prepared for Western blotting to examine survivin. β-actin were assessed as the control for equal loading of protein. (D), Panc-1 cells were initially transfected with survivin-specific siRNA. 48 h post-transfection, cells were either treated with YM155 (10 nM) for an additional 24 h or not, control cells had neither YM155 treatment nor transfection with siRNA. Apoptosis was assessed by Hoechst 33258 staining (cells exemplifying apoptotic nuclei are demarcated by white arrows). (E), Panc-1 cells were treated as in Figure 1C, and the ratio of apoptotic cells was assessed by counting the number of cells with apoptotic nuclei. Each experiment was conducted in triplicate and repeated twice independently (*p<0.05). (F), Panc-1 cells were treated as in Figure 1C. Apoptosis was assessed by a DNA ladder assay. (G), Panc-1 cells were treated as in Figure 1C and cell lysates were prepared for Western blotting to detect survivin and cleaved Caspase 3. β-actin were assessed as the control for equal loading of protein.

Figure 2. YM155 induces death receptor 5 up-regulation

(A), Panc-1 cells were treated with YM155 for 48 h and cell lysates were prepared for Western blotting to detect DR4 and DR5. β-actin were assessed as the control for equal loading of protein. Band density was qualified by Image J software. (B), Panc-1 cells treated with YM155 and cell lysates were prepared for Western blotting to detect DR4 and DR5 expression. β-actin were used for equal total protein amounts loading control. Band density was qualified by Image J software. (C), Panc-1 cells treated with YM155 for 48 h were harvested and total mRNA was extracted. Reverse transcription PCR and quantitative real-time PCR were performed to examine DR5 mRNA levels with specific primers. Statistical analysis was performed to show the effects of YM155 on DR5 mRNA levels. Each experiment was conducted in triplicate and repeated twice independently (#p<0.05). (D), Panc-1 cells were treated and DR4 mRNA levels were examined as in Figure 2C (*p<0.05). (E), Panc-1 cells were transfected with survivin specific siRNA. 48 h post-transfection, cell lysates were prepared for Western blotting to detect survivin, DR4 and DR5 expression. β-actin were used for an equal protein loading control. (F), Cells were treated with DMSO (control), YM155 (10 nM), Lexa (1 μg/ml), or pre-treated with YM155 (10 nM) for 1 h followed by Lexa (1 μg/ml) for 12 h. Apoptosis was measured by Hoechst 33258 staining (representative apoptotic cells are demarcated by open arrows). (G), Cells were treated as in Figure 2F and apoptosis was tested by a DNA ladder assay. (H), Cells were treated as in Figure 2F and apoptosis was measured by Western blotting to detect Caspase 8, Bid, and Caspase 3 activation. (I), Cells were treated as in Figure 2F and apoptosis was measured. Statistical analysis was performed to determine the ratio of apoptotic cells by counting the number of apoptotic cells with condensed nuclei. Each experiment was conducted in triplicate and repeated twice independently (* p<0.05).

Figure 3. Altered expression of apoptotic proteins and oligomerization of Bak proteins

(A), Panc-1 cells were treated with YM155 for 48 h. Cell lysates were prepared for Western blotting to detect CIAP1/2, FLIP, survivin, Bcl-2, Mcl-1, Bak, and Bim expression. β-actin was used for an equal loading of protein. Band density was qualified by Image J software. Representative bands are shown and every experiment was repeated three times. (B), Cells were treated with YM155 (10 nM) or untreated for 48 h. Proteins were then cross-linked using DSS as described in ‘Materials and methods’. Bak protein was detected by Western blotting. A 26~28kDa band represents Bak monomers, and bands at 52~56 kDa detected represent Bak homodimers. (C), Panc-1 cells were treated with YM155 for 48 h. Cell lysates were prepared for non-reducing SDS-PAGE. Bak protein was detected by Western blotting. A 26~28 kDa band represents Bak monomers, and bands at 52~56 kDa detected represent Bak homodimers.

Figure 4. YM155 enhances gemcitabine-induced apoptosis in Panc-1 cells

(A), Cells grown in 96-well plate were treated with gemcitabine (4 mM) alone or in the presence or absence of YM155 (5 nM) for 24 h. Relative cell viability was measured via MTS assay (*P<0.05). (B), Cells were treated as in Figure 4A, and apoptosis was assessed by Hoechst 33258 staining. (C), Panc-1 cells grown in 6-well plate were either untreated (control) or treated with either: YM155 (5 nM), gemcitabine (4 mM), or a combination of YM155 (5 nM) with gemcitabine (4 mM) for 48 h. Cells were rinsed post treatment with fresh culture medium 3 times, cells were then cultured for another two weeks. Cell colony formation assays were performed with crystal violet staining.

Figure 5. YM155 has no apparent apoptotic cyto-toxicity to normal pancreas cells

(A), HPDE cells grown in 48-well plates were treated with YM155 (10 nM) for 48 h. Panc-1 cells treated under the same conditions were used as a positive control. Apoptosis was measured by Hoechst 33258 staining. (B), HPDE cells and Panc-1 cells (a positive control) grown in six-well plates were treated with YM155 for 48 h. Cell lysates were prepared for Western blotting to show Caspase 3 activation. β-actin were assessed as a control for equal protein loading.

Figure 6. YM155 induces tumor growth inhibition in vivo

(A), BALB/c severe combined immunodeficiency (SCID) mice were inoculated subcutaneously with 5×10⁶ Panc-1 cells. The mice were treated with 10 mg/kg body weight of YM155 via tail vein injection until day 14. At the end point of the experiment, mice were euthanized and tumors were excised. Pictures of excised tumors from xenograft mice show larger tumors from untreated (control) mice and smaller tumors from YM155-treated mice. (B), The mass of tumors from the control group and the YM155-treated group were compared. Statistical analysis was performed to show inhibitory effects of YM155 on the mass of implanted tumors (*P<0.05). (C), Xenograft pancreatic tumors from untreated groups and YM155-treated groups were collected and lysates were prepared. Western blotting was performed to detect DR5 expression. β-actin were assessed as an equal protein loading control. The five pairs of tissues tested were randomly selected sample pairs, from untreated or YM155-treated tumor mice.