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High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde.

Kim HB, Kim MJ, Kim DY, Lee JW, Bae JH, Kim DW, Kang CD, Kim SH - Mol. Cancer (2011)

Bottom Line: Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs.Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL.We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Pusan National University School of Medicine, Yangsan 626-870, South Korea.

ABSTRACT

Background: Tumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.

Results: PC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.

Conclusion: This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

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Combination effects of DMNB and TRAIL on DNA-PKcs/Akt molecules and caspase activity. (A) PC3 cells (left) and KM12 cells (right) were treated with the indicated doses of TRAIL in the presence or absence of 5 μM DMNB for 6 or 8 h, respectively, and assessed using Western blot analysis to monitor the protein levels of DNA-PKcs, pAkt and tAkt. The activation of caspases and Bax and the cleavage of PARP in cells treated with TRAIL and/or DMNB were determined by Western blot analysis. Actin was used as a loading control. (B) PC3 and KM12 cells were treated with 1 ng/ml TRAIL in the presence or absence of 5 μM DMNB for 2 h, and then incubated with an anti-DR5 antibody (1:100), and subsequently labeled with FITC-conjugated secondary antibodies (1:200) to determine the surface expression of DR5. Mouse IgG was used as an isotype control. The shaded and unshaded peaks correspond to control and specific staining, respectively.
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Figure 11: Combination effects of DMNB and TRAIL on DNA-PKcs/Akt molecules and caspase activity. (A) PC3 cells (left) and KM12 cells (right) were treated with the indicated doses of TRAIL in the presence or absence of 5 μM DMNB for 6 or 8 h, respectively, and assessed using Western blot analysis to monitor the protein levels of DNA-PKcs, pAkt and tAkt. The activation of caspases and Bax and the cleavage of PARP in cells treated with TRAIL and/or DMNB were determined by Western blot analysis. Actin was used as a loading control. (B) PC3 and KM12 cells were treated with 1 ng/ml TRAIL in the presence or absence of 5 μM DMNB for 2 h, and then incubated with an anti-DR5 antibody (1:100), and subsequently labeled with FITC-conjugated secondary antibodies (1:200) to determine the surface expression of DR5. Mouse IgG was used as an isotype control. The shaded and unshaded peaks correspond to control and specific staining, respectively.

Mentions: Since the suppression of DNA-PKcs level with siRNA induced the up-regulation of DR5 and the activation of caspases, we determined whether DMNB could potentiate TRAIL-induced cytotoxicity and apoptosis in TRAIL-resistant PC3 and KM12 cells and function as a TRAIL sensitizer. DMNB in combination with TRAIL sensitized PC3 cells (Figure 10A) and KM12 cells (Figure 10B) to TRAIL-induced cytotoxicity and apoptosis in a dose-dependent manner. We next examined whether the enhanced susceptibility of PC3 and KM12 cells to TRAIL following DMNB treatment was associated with caspase activation and the up-regulation of Bax through the inactivation of the DNA-PKcs/Akt signaling (Figure 11A). Co-treatment of PC3 or KM12 cells with TRAIL and DMNB resulted in a decrease in the levels of both DNA-PKcs and pAkt when compared to cells treated with TRAIL alone. The combination of DMNB and TRAIL was more effective for the activation of caspases, the inactivation of the DNA-PKcs/Akt signaling pathway, PARP cleavage, and the up-regulation of Bax than the treatment with TRAIL alone. In addition, combined treatment of DMNB and TRAIL increased surface expression of DR5 in both PC3 and KM12 cells, which did not respond to TRAIL alone (Figure 11B). These results suggest that the inactivation of the DNA-PKcs/Akt signaling pathway with siRNA or small molecules may be a useful strategy to increase the susceptibility of TRAIL-resistant solid tumor cells to TRAIL-induced cell death.


High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde.

Kim HB, Kim MJ, Kim DY, Lee JW, Bae JH, Kim DW, Kang CD, Kim SH - Mol. Cancer (2011)

Combination effects of DMNB and TRAIL on DNA-PKcs/Akt molecules and caspase activity. (A) PC3 cells (left) and KM12 cells (right) were treated with the indicated doses of TRAIL in the presence or absence of 5 μM DMNB for 6 or 8 h, respectively, and assessed using Western blot analysis to monitor the protein levels of DNA-PKcs, pAkt and tAkt. The activation of caspases and Bax and the cleavage of PARP in cells treated with TRAIL and/or DMNB were determined by Western blot analysis. Actin was used as a loading control. (B) PC3 and KM12 cells were treated with 1 ng/ml TRAIL in the presence or absence of 5 μM DMNB for 2 h, and then incubated with an anti-DR5 antibody (1:100), and subsequently labeled with FITC-conjugated secondary antibodies (1:200) to determine the surface expression of DR5. Mouse IgG was used as an isotype control. The shaded and unshaded peaks correspond to control and specific staining, respectively.
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Figure 11: Combination effects of DMNB and TRAIL on DNA-PKcs/Akt molecules and caspase activity. (A) PC3 cells (left) and KM12 cells (right) were treated with the indicated doses of TRAIL in the presence or absence of 5 μM DMNB for 6 or 8 h, respectively, and assessed using Western blot analysis to monitor the protein levels of DNA-PKcs, pAkt and tAkt. The activation of caspases and Bax and the cleavage of PARP in cells treated with TRAIL and/or DMNB were determined by Western blot analysis. Actin was used as a loading control. (B) PC3 and KM12 cells were treated with 1 ng/ml TRAIL in the presence or absence of 5 μM DMNB for 2 h, and then incubated with an anti-DR5 antibody (1:100), and subsequently labeled with FITC-conjugated secondary antibodies (1:200) to determine the surface expression of DR5. Mouse IgG was used as an isotype control. The shaded and unshaded peaks correspond to control and specific staining, respectively.
Mentions: Since the suppression of DNA-PKcs level with siRNA induced the up-regulation of DR5 and the activation of caspases, we determined whether DMNB could potentiate TRAIL-induced cytotoxicity and apoptosis in TRAIL-resistant PC3 and KM12 cells and function as a TRAIL sensitizer. DMNB in combination with TRAIL sensitized PC3 cells (Figure 10A) and KM12 cells (Figure 10B) to TRAIL-induced cytotoxicity and apoptosis in a dose-dependent manner. We next examined whether the enhanced susceptibility of PC3 and KM12 cells to TRAIL following DMNB treatment was associated with caspase activation and the up-regulation of Bax through the inactivation of the DNA-PKcs/Akt signaling (Figure 11A). Co-treatment of PC3 or KM12 cells with TRAIL and DMNB resulted in a decrease in the levels of both DNA-PKcs and pAkt when compared to cells treated with TRAIL alone. The combination of DMNB and TRAIL was more effective for the activation of caspases, the inactivation of the DNA-PKcs/Akt signaling pathway, PARP cleavage, and the up-regulation of Bax than the treatment with TRAIL alone. In addition, combined treatment of DMNB and TRAIL increased surface expression of DR5 in both PC3 and KM12 cells, which did not respond to TRAIL alone (Figure 11B). These results suggest that the inactivation of the DNA-PKcs/Akt signaling pathway with siRNA or small molecules may be a useful strategy to increase the susceptibility of TRAIL-resistant solid tumor cells to TRAIL-induced cell death.

Bottom Line: Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs.Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL.We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Pusan National University School of Medicine, Yangsan 626-870, South Korea.

ABSTRACT

Background: Tumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.

Results: PC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.

Conclusion: This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

Show MeSH
Related in: MedlinePlus