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Chemosensitisation by manganese superoxide dismutase inhibition is caspase-9 dependent and involves extracellular signal-regulated kinase 1/2.

Yeung BH, Wong KY, Lin MC, Wong CK, Mashima T, Tsuruo T, Wong AS - Br. J. Cancer (2008)

Bottom Line: Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway.Akt activation was not affected.These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Hong Kong, Hong Kong, PR China.

ABSTRACT
Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

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Involvement of ROS production in the manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-mediated activation of caspase-9. SKOV-3 cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with N-acetyl cysteine (NAC, 5 mM) for 30 min followed by doxorubicin (DOX, 5 μM) or paclitaxel (PTX, 0.1 μM) addition for another 48 h. Cells were then analysed for expression of caspase-9 and caspase-8 proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control.
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fig10: Involvement of ROS production in the manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-mediated activation of caspase-9. SKOV-3 cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with N-acetyl cysteine (NAC, 5 mM) for 30 min followed by doxorubicin (DOX, 5 μM) or paclitaxel (PTX, 0.1 μM) addition for another 48 h. Cells were then analysed for expression of caspase-9 and caspase-8 proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control.

Mentions: To ascertain the role of caspase-9, we treated siRNA-transfected cells with a specific inhibitor of caspase-9 (Z-LEHD-FMK) and then examined their sensitivity to apoptosis. Under these conditions, the DOX- or PTX-induced apoptosis was clearly prevented by the caspase-9 inhibitor Z-LEHD-FMK (Figure 9B). Similarly, treatment the cells with the pan-caspase inhibitor (Z-VAD-FMK) totally inhibited DOX- or PTX-induced apoptosis. In contrast, the inhibitor of caspase-8 (Z-IETD-FMK) had no effect on apoptosis. To confirm these data, a dominant-negative mutant of caspase-9 was used, and it also effectively inhibited apoptotic response to DOX and PTX in MnSOD-depleted cells (Figure 9C). Furthermore, pre-treatment of GSH (data not shown) or its precursor NAC (Figure 10) prevented the activation of caspase-9 caused by MnSOD siRNA. Consistent with the results described above, there was no effect on caspase-8 (Figure 10). These data suggest that cellular oxidative status could affect MnSOD siRNA-induced apoptosis to DOX or PTX by regulating caspase, the central component of the apoptotic pathways, and in particular caspase-9 activation.


Chemosensitisation by manganese superoxide dismutase inhibition is caspase-9 dependent and involves extracellular signal-regulated kinase 1/2.

Yeung BH, Wong KY, Lin MC, Wong CK, Mashima T, Tsuruo T, Wong AS - Br. J. Cancer (2008)

Involvement of ROS production in the manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-mediated activation of caspase-9. SKOV-3 cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with N-acetyl cysteine (NAC, 5 mM) for 30 min followed by doxorubicin (DOX, 5 μM) or paclitaxel (PTX, 0.1 μM) addition for another 48 h. Cells were then analysed for expression of caspase-9 and caspase-8 proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2480972&req=5

fig10: Involvement of ROS production in the manganese superoxide disumutase (MnSOD) small-interfering RNA (siRNA)-mediated activation of caspase-9. SKOV-3 cells were transiently transfected with either the Ctrl or MnSOD siRNA for 48 h, and then pre-treated with N-acetyl cysteine (NAC, 5 mM) for 30 min followed by doxorubicin (DOX, 5 μM) or paclitaxel (PTX, 0.1 μM) addition for another 48 h. Cells were then analysed for expression of caspase-9 and caspase-8 proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control.
Mentions: To ascertain the role of caspase-9, we treated siRNA-transfected cells with a specific inhibitor of caspase-9 (Z-LEHD-FMK) and then examined their sensitivity to apoptosis. Under these conditions, the DOX- or PTX-induced apoptosis was clearly prevented by the caspase-9 inhibitor Z-LEHD-FMK (Figure 9B). Similarly, treatment the cells with the pan-caspase inhibitor (Z-VAD-FMK) totally inhibited DOX- or PTX-induced apoptosis. In contrast, the inhibitor of caspase-8 (Z-IETD-FMK) had no effect on apoptosis. To confirm these data, a dominant-negative mutant of caspase-9 was used, and it also effectively inhibited apoptotic response to DOX and PTX in MnSOD-depleted cells (Figure 9C). Furthermore, pre-treatment of GSH (data not shown) or its precursor NAC (Figure 10) prevented the activation of caspase-9 caused by MnSOD siRNA. Consistent with the results described above, there was no effect on caspase-8 (Figure 10). These data suggest that cellular oxidative status could affect MnSOD siRNA-induced apoptosis to DOX or PTX by regulating caspase, the central component of the apoptotic pathways, and in particular caspase-9 activation.

Bottom Line: Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway.Akt activation was not affected.These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, The University of Hong Kong, Hong Kong, PR China.

ABSTRACT
Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

Show MeSH
Related in: MedlinePlus