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

Overexpression of manganese superoxide disumutase (MnSOD) reduces ROS production in OVCAR-3. (A) Whole-cell lysates of MnSOD stably transfected clones (M5, M17, and M24), parental (WT), and vector (Neo) control cells were harvested and analysed for expression of MnSOD by western blotting. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, MnSOD-overexpressing clones; open histogram, Neo control. (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

fig2: Overexpression of manganese superoxide disumutase (MnSOD) reduces ROS production in OVCAR-3. (A) Whole-cell lysates of MnSOD stably transfected clones (M5, M17, and M24), parental (WT), and vector (Neo) control cells were harvested and analysed for expression of MnSOD by western blotting. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, MnSOD-overexpressing clones; open histogram, Neo control. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.

Mentions: We first transfected OVCAR-3 cells with empty vector (pcDNA3.1) or MnSOD-expressing vector. After transfection, cells were cultured in a medium containing 400 μg ml−1 of G418. Each colony that grew after G418 selection was picked and expanded. We obtained three independent clones, which expressed MnSOD. Notably, MnSOD was much expressed (3.7- to 3.9-fold) in all of three MnSOD-expressing OVCAR-3 clones (M5, M17, and M24) than in parental (WT) and vector (Neo) control clones (Figure 2A). It is important to note that the levels of MnSOD in these stable cell lines were less than those detected in MnSOD-overexpressing human ovarian carcinoma cell lines, including SKOV-3, indicating that expression was within normal levels (Figure 2A). Analysis (RT–PCR) showed that MnSOD mRNA was also significantly elevated in these stable transfectants (data not shown). Consistent with the western blots, all MnSOD-overexpressing clones showed decrease in ethidium fluorescence, showing that MnSOD is functionally active and able to reduce cellular ROS level (9.4–27.7%) in these cell lines. The representative result from one of them (M24) is shown in Figure 2B. To confirm the levels of MnSOD enzymatic activity, we performed MnSOD activity assays. As shown in Figure 2C, MnSOD-transfected clones had statistically significant increases in MnSOD activity compared with WT, and the activity of Neo was not different from that of WT. The values obtained from the MnSOD activity correlated well with the expression level and the O2•− content (P<0.05), suggesting that OVCAR-3 constitutively express active MnSOD.


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)

Overexpression of manganese superoxide disumutase (MnSOD) reduces ROS production in OVCAR-3. (A) Whole-cell lysates of MnSOD stably transfected clones (M5, M17, and M24), parental (WT), and vector (Neo) control cells were harvested and analysed for expression of MnSOD by western blotting. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, MnSOD-overexpressing clones; open histogram, Neo control. (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

fig2: Overexpression of manganese superoxide disumutase (MnSOD) reduces ROS production in OVCAR-3. (A) Whole-cell lysates of MnSOD stably transfected clones (M5, M17, and M24), parental (WT), and vector (Neo) control cells were harvested and analysed for expression of MnSOD by western blotting. β-Actin serves as a protein-loading control. (B) O2•− level was measured by flow cytometry analysis using dihydroethidium. Solid histogram, MnSOD-overexpressing clones; open histogram, Neo control. (C) MnSOD activity was measured. Values are mean±s.d. of three independent experiments. *P<0.05 vs WT and Neo control cells.
Mentions: We first transfected OVCAR-3 cells with empty vector (pcDNA3.1) or MnSOD-expressing vector. After transfection, cells were cultured in a medium containing 400 μg ml−1 of G418. Each colony that grew after G418 selection was picked and expanded. We obtained three independent clones, which expressed MnSOD. Notably, MnSOD was much expressed (3.7- to 3.9-fold) in all of three MnSOD-expressing OVCAR-3 clones (M5, M17, and M24) than in parental (WT) and vector (Neo) control clones (Figure 2A). It is important to note that the levels of MnSOD in these stable cell lines were less than those detected in MnSOD-overexpressing human ovarian carcinoma cell lines, including SKOV-3, indicating that expression was within normal levels (Figure 2A). Analysis (RT–PCR) showed that MnSOD mRNA was also significantly elevated in these stable transfectants (data not shown). Consistent with the western blots, all MnSOD-overexpressing clones showed decrease in ethidium fluorescence, showing that MnSOD is functionally active and able to reduce cellular ROS level (9.4–27.7%) in these cell lines. The representative result from one of them (M24) is shown in Figure 2B. To confirm the levels of MnSOD enzymatic activity, we performed MnSOD activity assays. As shown in Figure 2C, MnSOD-transfected clones had statistically significant increases in MnSOD activity compared with WT, and the activity of Neo was not different from that of WT. The values obtained from the MnSOD activity correlated well with the expression level and the O2•− content (P<0.05), suggesting that OVCAR-3 constitutively express active MnSOD.

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