Limits...
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.

Show MeSH

Related in: MedlinePlus

Suppressing manganese superoxide disumutase (MnSOD) expression by small-interfering RNA (siRNA) increases ROS production in SKOV-3. (A) Cells transfected with non-specific control (Ctrl siRNA) or MnSOD siRNA were analysed for expression of MnSOD, Cu/Zn-SOD proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, untransfected and Ctrl siRNA-transfected cells; open histogram, MnSOD siRNA-transfected cells. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2480972&req=5

fig4: Suppressing manganese superoxide disumutase (MnSOD) expression by small-interfering RNA (siRNA) increases ROS production in SKOV-3. (A) Cells transfected with non-specific control (Ctrl siRNA) or MnSOD siRNA were analysed for expression of MnSOD, Cu/Zn-SOD proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, untransfected and Ctrl siRNA-transfected cells; open histogram, MnSOD siRNA-transfected cells. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.

Mentions: Subsequently, we used RNA interference to reduce MnSOD expression in SKOV-3 ovarian cancer cell line, which expressed high levels of endogenous MnSOD. Western blot analysis revealed that transfection with MnSOD siRNA specifically suppressed the protein expression of MnSOD and did not affect the expression of Cu/Zn-SOD or β-actin. The non-specific (Ctrl) siRNA exhibited no effect on the expression of all these molecules tested (Figure 4A). Moreover, flow cytometric analysis showed that there was an 11% increase in O2•− content in cells transfected with MnSOD siRNA, whereas Ctrl siRNA did not cause any significant change in cellular O2•− level (Figure 4B). Consistently, MnSOD activity was significantly lower in cells transfected with MnSOD siRNA from the Ctrl siRNA (Figure 4C). Figure 5A shows an ∼70% decrease in cell viability compared with the control. In accordance with this, 5 μM DOX or 100 nM PTX induced 2.5 and 9.6% apoptosis respectively, whereas a combination of DOX or PTX and MnSOD siRNA induced 15.6 and 32.7% apoptosis respectively (P<0.001 vs untransfected control and Ctrl siRNA; Figure 5B). Untreated and MnSOD siRNA-transfected cells had a very minimal number of apoptotic cells (Figure 5B). We also examined the reactivity to annexin V-FITC in conjunction with PI to detect exposure of dislocated phosphatidylserine to the external face of the plasma membrane, a process regarded as a marker of apoptosis. In view of the cells that are positive for both annexin V and PI may also be cells that are undergoing necrosis, only the percentage of early apoptotic cells (annexin V positive and PI negative) was quantified from three individual experiments and shown in Figure 5C. MnSOD siRNA increased the fraction of apoptotic cells to 12.6 and 23.1% respectively. This magnitude of change was comparable with the fold induction detected by TUNEL and DAPI assays. The slight variation using different techniques can be accounted for by their different sensitivity and detection of specific markers in the apoptotic pathway. Together, these results indicate that DOX or PTX, when used in combination with MnSOD siRNA, induce more efficient (3.5- to 6-fold) anti-cancer effects than each drug alone.


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)

Suppressing manganese superoxide disumutase (MnSOD) expression by small-interfering RNA (siRNA) increases ROS production in SKOV-3. (A) Cells transfected with non-specific control (Ctrl siRNA) or MnSOD siRNA were analysed for expression of MnSOD, Cu/Zn-SOD proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, untransfected and Ctrl siRNA-transfected cells; open histogram, MnSOD siRNA-transfected cells. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Suppressing manganese superoxide disumutase (MnSOD) expression by small-interfering RNA (siRNA) increases ROS production in SKOV-3. (A) Cells transfected with non-specific control (Ctrl siRNA) or MnSOD siRNA were analysed for expression of MnSOD, Cu/Zn-SOD proteins by western blotting using the respective antibodies. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, untransfected and Ctrl siRNA-transfected cells; open histogram, MnSOD siRNA-transfected cells. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.
Mentions: Subsequently, we used RNA interference to reduce MnSOD expression in SKOV-3 ovarian cancer cell line, which expressed high levels of endogenous MnSOD. Western blot analysis revealed that transfection with MnSOD siRNA specifically suppressed the protein expression of MnSOD and did not affect the expression of Cu/Zn-SOD or β-actin. The non-specific (Ctrl) siRNA exhibited no effect on the expression of all these molecules tested (Figure 4A). Moreover, flow cytometric analysis showed that there was an 11% increase in O2•− content in cells transfected with MnSOD siRNA, whereas Ctrl siRNA did not cause any significant change in cellular O2•− level (Figure 4B). Consistently, MnSOD activity was significantly lower in cells transfected with MnSOD siRNA from the Ctrl siRNA (Figure 4C). Figure 5A shows an ∼70% decrease in cell viability compared with the control. In accordance with this, 5 μM DOX or 100 nM PTX induced 2.5 and 9.6% apoptosis respectively, whereas a combination of DOX or PTX and MnSOD siRNA induced 15.6 and 32.7% apoptosis respectively (P<0.001 vs untransfected control and Ctrl siRNA; Figure 5B). Untreated and MnSOD siRNA-transfected cells had a very minimal number of apoptotic cells (Figure 5B). We also examined the reactivity to annexin V-FITC in conjunction with PI to detect exposure of dislocated phosphatidylserine to the external face of the plasma membrane, a process regarded as a marker of apoptosis. In view of the cells that are positive for both annexin V and PI may also be cells that are undergoing necrosis, only the percentage of early apoptotic cells (annexin V positive and PI negative) was quantified from three individual experiments and shown in Figure 5C. MnSOD siRNA increased the fraction of apoptotic cells to 12.6 and 23.1% respectively. This magnitude of change was comparable with the fold induction detected by TUNEL and DAPI assays. The slight variation using different techniques can be accounted for by their different sensitivity and detection of specific markers in the apoptotic pathway. Together, these results indicate that DOX or PTX, when used in combination with MnSOD siRNA, induce more efficient (3.5- to 6-fold) anti-cancer effects than each drug alone.

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