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p22phox confers resistance to cisplatin, by blocking its entry into the nucleus.

Hung CC, Chien CY, Chiang WF, Lin CS, Hour TC, Chen HR, Wang LF, Ko JY, Chang CH, Chen JY - Oncotarget (2015)

Bottom Line: This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis.This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation.Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Cisplatin (CDDP) is a potent chemotherapeutic agent but resistance to the drug remains a major challenge in cancer treatment. To evaluate the efficacy of CDDP in oral squamous cell carcinoma (OSCC), we found that p22phox was highly expressed in CDDP-resistant OSCC specimens. Knockdown of p22phox sensitized OSCC cell lines to CDDP (P < 0.05). Stable overexpression of p22phox augmented CDDP resistance, as evidenced by the significantly higher IC50 values. This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis. Akt phosphorylation was increased in p22phox stable lines. However, blocking PI3K/Akt pathway only partially restored CDDP-induced apoptosis. In addition, the overexpressed p22phox in OSCC cells exhibited cytoplasmic localization with enhanced perinuclear expression, consistent with the localization pattern in OSCC specimens. Remarkably, CDDP entry into the nucleus was severely impaired in p22phox-overexpressing cells (P < 0.001), and cytoplasmically accumulated CDDP was co-localized with overexpressed p22phox. This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation. Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis. Such diminished apoptosis was further abolished by p22phox-activating PI3K/Akt pathway. Our work has suggested a novel biomarker and insight into the mechanism of CDDP resistance.

No MeSH data available.


Related in: MedlinePlus

Elevated PI3K/Akt activity contributed to inhibition of CDDP-induced apoptosis in p22phox stable lines(A) The endogenous expression level of phosphorylated Akt (p-Akt) in p22phox stable lines was revealed by Western blot analysis. The numbers below the blots were quantitative ratios of p-Akt (56 kDa) or total Akt (t-Akt, 56 kDa)/GAPDH band intensities in p22phox lines normalized to those of control line. (B) PI3K/Akt activity was blocked by wortmannin or SC66, and p-Akt, t-Akt and cleavage of caspase 3 and PARP were examined. Cells were pretreated with wortmannin (2 μM) overnight or SC66 (10 μM) for 1 h, followed by the combined treatments of CDDP (10 μM) and the inhibitors for another 24 h. Arrow heads and stars represent the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The numbers below the blots were quantitative ratios of p-Akt, t-Akt, cleaved caspase 3 or cleaved PARP/GAPDH band intensities normalized to those without the inhibitor treatments in control line. (C) The cells were transfected with Akt or scrambled siRNA oligos (100 nM) for 48 h, followed by treatment with CDDP for 24 h. The cell lysates were analyzed by Western blotting and the results were similarly quantified as in (B). Arrow heads and arrows indicate the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The experiments were repeated four times, and the representative data are shown.
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Figure 5: Elevated PI3K/Akt activity contributed to inhibition of CDDP-induced apoptosis in p22phox stable lines(A) The endogenous expression level of phosphorylated Akt (p-Akt) in p22phox stable lines was revealed by Western blot analysis. The numbers below the blots were quantitative ratios of p-Akt (56 kDa) or total Akt (t-Akt, 56 kDa)/GAPDH band intensities in p22phox lines normalized to those of control line. (B) PI3K/Akt activity was blocked by wortmannin or SC66, and p-Akt, t-Akt and cleavage of caspase 3 and PARP were examined. Cells were pretreated with wortmannin (2 μM) overnight or SC66 (10 μM) for 1 h, followed by the combined treatments of CDDP (10 μM) and the inhibitors for another 24 h. Arrow heads and stars represent the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The numbers below the blots were quantitative ratios of p-Akt, t-Akt, cleaved caspase 3 or cleaved PARP/GAPDH band intensities normalized to those without the inhibitor treatments in control line. (C) The cells were transfected with Akt or scrambled siRNA oligos (100 nM) for 48 h, followed by treatment with CDDP for 24 h. The cell lysates were analyzed by Western blotting and the results were similarly quantified as in (B). Arrow heads and arrows indicate the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The experiments were repeated four times, and the representative data are shown.

Mentions: We then asked how p22phox inhibited CDDP-induced apoptosis in OSCC cells. PI3K/Akt pathway was examined because it is known to transduce pro-survival and anti-apoptotic signals in cells. There were much higher levels of endogenous Akt phosphorylation (p-Akt; S473) in p22phox stable lines, implicating increased Akt activity (Figure 5A). To determine whether this high Akt activation contributed to CDDP resistance, we tested how p22phox stable lines would respond to CDDP in the presence of the PI3K/Akt inhibitor wortmannin, SC66 or 3-MA (Supplementary Figure 1). In both stable lines, CDDP-induced apoptosis was significantly rescued when Akt activity was inhibited, as evidenced by the marked decrease in p-Akt and the induction of cleaved caspase 3 and PARP (Figure 5B and Supplementary Figure 1). Noticeably, total Akt (t-Akt) levels in SC66-treated cells were also reduced presumably because of ubiquitination-mediated protein degradation when Akt was inactivated by the inhibitor [17]. Furthermore, consistent with the results in Figure 5B, the levels of cleaved caspase 3 and PARP were increased in response to CDDP treatment when Akt expression was knocked down by siRNA in p22phox stable lines (Figure 5C). Therefore, inhibition of CDDP-induced apoptosis in p22phox stable lines was, at least in part, mediated through PI3K/Akt signaling pathway.


p22phox confers resistance to cisplatin, by blocking its entry into the nucleus.

Hung CC, Chien CY, Chiang WF, Lin CS, Hour TC, Chen HR, Wang LF, Ko JY, Chang CH, Chen JY - Oncotarget (2015)

Elevated PI3K/Akt activity contributed to inhibition of CDDP-induced apoptosis in p22phox stable lines(A) The endogenous expression level of phosphorylated Akt (p-Akt) in p22phox stable lines was revealed by Western blot analysis. The numbers below the blots were quantitative ratios of p-Akt (56 kDa) or total Akt (t-Akt, 56 kDa)/GAPDH band intensities in p22phox lines normalized to those of control line. (B) PI3K/Akt activity was blocked by wortmannin or SC66, and p-Akt, t-Akt and cleavage of caspase 3 and PARP were examined. Cells were pretreated with wortmannin (2 μM) overnight or SC66 (10 μM) for 1 h, followed by the combined treatments of CDDP (10 μM) and the inhibitors for another 24 h. Arrow heads and stars represent the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The numbers below the blots were quantitative ratios of p-Akt, t-Akt, cleaved caspase 3 or cleaved PARP/GAPDH band intensities normalized to those without the inhibitor treatments in control line. (C) The cells were transfected with Akt or scrambled siRNA oligos (100 nM) for 48 h, followed by treatment with CDDP for 24 h. The cell lysates were analyzed by Western blotting and the results were similarly quantified as in (B). Arrow heads and arrows indicate the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The experiments were repeated four times, and the representative data are shown.
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Figure 5: Elevated PI3K/Akt activity contributed to inhibition of CDDP-induced apoptosis in p22phox stable lines(A) The endogenous expression level of phosphorylated Akt (p-Akt) in p22phox stable lines was revealed by Western blot analysis. The numbers below the blots were quantitative ratios of p-Akt (56 kDa) or total Akt (t-Akt, 56 kDa)/GAPDH band intensities in p22phox lines normalized to those of control line. (B) PI3K/Akt activity was blocked by wortmannin or SC66, and p-Akt, t-Akt and cleavage of caspase 3 and PARP were examined. Cells were pretreated with wortmannin (2 μM) overnight or SC66 (10 μM) for 1 h, followed by the combined treatments of CDDP (10 μM) and the inhibitors for another 24 h. Arrow heads and stars represent the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The numbers below the blots were quantitative ratios of p-Akt, t-Akt, cleaved caspase 3 or cleaved PARP/GAPDH band intensities normalized to those without the inhibitor treatments in control line. (C) The cells were transfected with Akt or scrambled siRNA oligos (100 nM) for 48 h, followed by treatment with CDDP for 24 h. The cell lysates were analyzed by Western blotting and the results were similarly quantified as in (B). Arrow heads and arrows indicate the pro-forms and the cleaved forms of caspase 3 and PARP, respectively. The experiments were repeated four times, and the representative data are shown.
Mentions: We then asked how p22phox inhibited CDDP-induced apoptosis in OSCC cells. PI3K/Akt pathway was examined because it is known to transduce pro-survival and anti-apoptotic signals in cells. There were much higher levels of endogenous Akt phosphorylation (p-Akt; S473) in p22phox stable lines, implicating increased Akt activity (Figure 5A). To determine whether this high Akt activation contributed to CDDP resistance, we tested how p22phox stable lines would respond to CDDP in the presence of the PI3K/Akt inhibitor wortmannin, SC66 or 3-MA (Supplementary Figure 1). In both stable lines, CDDP-induced apoptosis was significantly rescued when Akt activity was inhibited, as evidenced by the marked decrease in p-Akt and the induction of cleaved caspase 3 and PARP (Figure 5B and Supplementary Figure 1). Noticeably, total Akt (t-Akt) levels in SC66-treated cells were also reduced presumably because of ubiquitination-mediated protein degradation when Akt was inactivated by the inhibitor [17]. Furthermore, consistent with the results in Figure 5B, the levels of cleaved caspase 3 and PARP were increased in response to CDDP treatment when Akt expression was knocked down by siRNA in p22phox stable lines (Figure 5C). Therefore, inhibition of CDDP-induced apoptosis in p22phox stable lines was, at least in part, mediated through PI3K/Akt signaling pathway.

Bottom Line: This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis.This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation.Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Cisplatin (CDDP) is a potent chemotherapeutic agent but resistance to the drug remains a major challenge in cancer treatment. To evaluate the efficacy of CDDP in oral squamous cell carcinoma (OSCC), we found that p22phox was highly expressed in CDDP-resistant OSCC specimens. Knockdown of p22phox sensitized OSCC cell lines to CDDP (P < 0.05). Stable overexpression of p22phox augmented CDDP resistance, as evidenced by the significantly higher IC50 values. This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis. Akt phosphorylation was increased in p22phox stable lines. However, blocking PI3K/Akt pathway only partially restored CDDP-induced apoptosis. In addition, the overexpressed p22phox in OSCC cells exhibited cytoplasmic localization with enhanced perinuclear expression, consistent with the localization pattern in OSCC specimens. Remarkably, CDDP entry into the nucleus was severely impaired in p22phox-overexpressing cells (P < 0.001), and cytoplasmically accumulated CDDP was co-localized with overexpressed p22phox. This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation. Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis. Such diminished apoptosis was further abolished by p22phox-activating PI3K/Akt pathway. Our work has suggested a novel biomarker and insight into the mechanism of CDDP resistance.

No MeSH data available.


Related in: MedlinePlus