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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

Abolishment of CDDP-induced apoptosis in p22phox stable lines(A) Cells were treated with 10 μM CDDP for 24 h, and then subG1 population was evaluated by flow cytometry. The proportion of subG1 cells was presented as the percentage of the entire cell population. (B) Cells were treated with CDDP (10 μM) for 48 h and the expression of p22phox-DsRed fusion protein (50 kDa), caspase 3 (35 kDa), caspase 7 (35 kDa), caspase 9 (47 kDa) and PARP (116 kDa) was revealed by Western blot analysis. p22phox-DsRed fusion protein was detected using anti-DsRed antibody. Arrow heads and stars indicate the pro-forms and the cleaved forms of the caspase proteins and PARP, respectively. The numbers below the blots were quantitative ratios of cleaved caspase 3 (17 kDa), caspase 7 (20 kDa), caspase 9 (35/37 kDa) or PARP (89 kDa) /GAPDH band densities under CDDP treatment normalized to those without CDDP treatment. (C) DNA fragmentation of the CDDP-treated cells was detected by the TUNEL assay using fluorescence microscopy. Magnification: 200X. All experiments were repeated four times, and the representative data are shown.
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Figure 4: Abolishment of CDDP-induced apoptosis in p22phox stable lines(A) Cells were treated with 10 μM CDDP for 24 h, and then subG1 population was evaluated by flow cytometry. The proportion of subG1 cells was presented as the percentage of the entire cell population. (B) Cells were treated with CDDP (10 μM) for 48 h and the expression of p22phox-DsRed fusion protein (50 kDa), caspase 3 (35 kDa), caspase 7 (35 kDa), caspase 9 (47 kDa) and PARP (116 kDa) was revealed by Western blot analysis. p22phox-DsRed fusion protein was detected using anti-DsRed antibody. Arrow heads and stars indicate the pro-forms and the cleaved forms of the caspase proteins and PARP, respectively. The numbers below the blots were quantitative ratios of cleaved caspase 3 (17 kDa), caspase 7 (20 kDa), caspase 9 (35/37 kDa) or PARP (89 kDa) /GAPDH band densities under CDDP treatment normalized to those without CDDP treatment. (C) DNA fragmentation of the CDDP-treated cells was detected by the TUNEL assay using fluorescence microscopy. Magnification: 200X. All experiments were repeated four times, and the representative data are shown.

Mentions: Because CDDP is known to induce apoptosis in cancer cells [15, 16], we investigated whether such effect was attenuated in p22phox-overexpressing cells. Cell cycle analysis by DAPI staining showed that CDDP treatment in the control line caused a significant increase in subG1 cell population (4.66% to 32.23%), indicating the induction of apoptosis by CDDP. In sharp contrast, CDDP-induced subG1 accumulation was nearly abolished in p22phox stable lines (Figure 4A). We then examined whether this protective effect involved attenuation of apoptotic signaling. Whereas the cleaved forms of caspase 3, caspase 7 and caspase 9 were induced by CDDP in the control line, such induction was virtually abrogated in the two p22phox stable lines. The cleaved form of PARP, a well-known substrate for caspase 3 cleavage during apoptosis, was dramatically induced by CDDP in the control line. However, CDDP had no effect on the induction of PARP cleavage in the stable lines (Figure 4B). Furthermore, we showed that the majority of cells in CDDP-treated control line were TUNEL-positive, while no detectable TUNEL staining was observed in CDDP-treated p22phox stable lines (Figure 4C). Together, these results strongly suggested that overexpression of p22phox could protect OSCC cells from CDDP-induced apoptosis.


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)

Abolishment of CDDP-induced apoptosis in p22phox stable lines(A) Cells were treated with 10 μM CDDP for 24 h, and then subG1 population was evaluated by flow cytometry. The proportion of subG1 cells was presented as the percentage of the entire cell population. (B) Cells were treated with CDDP (10 μM) for 48 h and the expression of p22phox-DsRed fusion protein (50 kDa), caspase 3 (35 kDa), caspase 7 (35 kDa), caspase 9 (47 kDa) and PARP (116 kDa) was revealed by Western blot analysis. p22phox-DsRed fusion protein was detected using anti-DsRed antibody. Arrow heads and stars indicate the pro-forms and the cleaved forms of the caspase proteins and PARP, respectively. The numbers below the blots were quantitative ratios of cleaved caspase 3 (17 kDa), caspase 7 (20 kDa), caspase 9 (35/37 kDa) or PARP (89 kDa) /GAPDH band densities under CDDP treatment normalized to those without CDDP treatment. (C) DNA fragmentation of the CDDP-treated cells was detected by the TUNEL assay using fluorescence microscopy. Magnification: 200X. All experiments were repeated four times, and the representative data are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Abolishment of CDDP-induced apoptosis in p22phox stable lines(A) Cells were treated with 10 μM CDDP for 24 h, and then subG1 population was evaluated by flow cytometry. The proportion of subG1 cells was presented as the percentage of the entire cell population. (B) Cells were treated with CDDP (10 μM) for 48 h and the expression of p22phox-DsRed fusion protein (50 kDa), caspase 3 (35 kDa), caspase 7 (35 kDa), caspase 9 (47 kDa) and PARP (116 kDa) was revealed by Western blot analysis. p22phox-DsRed fusion protein was detected using anti-DsRed antibody. Arrow heads and stars indicate the pro-forms and the cleaved forms of the caspase proteins and PARP, respectively. The numbers below the blots were quantitative ratios of cleaved caspase 3 (17 kDa), caspase 7 (20 kDa), caspase 9 (35/37 kDa) or PARP (89 kDa) /GAPDH band densities under CDDP treatment normalized to those without CDDP treatment. (C) DNA fragmentation of the CDDP-treated cells was detected by the TUNEL assay using fluorescence microscopy. Magnification: 200X. All experiments were repeated four times, and the representative data are shown.
Mentions: Because CDDP is known to induce apoptosis in cancer cells [15, 16], we investigated whether such effect was attenuated in p22phox-overexpressing cells. Cell cycle analysis by DAPI staining showed that CDDP treatment in the control line caused a significant increase in subG1 cell population (4.66% to 32.23%), indicating the induction of apoptosis by CDDP. In sharp contrast, CDDP-induced subG1 accumulation was nearly abolished in p22phox stable lines (Figure 4A). We then examined whether this protective effect involved attenuation of apoptotic signaling. Whereas the cleaved forms of caspase 3, caspase 7 and caspase 9 were induced by CDDP in the control line, such induction was virtually abrogated in the two p22phox stable lines. The cleaved form of PARP, a well-known substrate for caspase 3 cleavage during apoptosis, was dramatically induced by CDDP in the control line. However, CDDP had no effect on the induction of PARP cleavage in the stable lines (Figure 4B). Furthermore, we showed that the majority of cells in CDDP-treated control line were TUNEL-positive, while no detectable TUNEL staining was observed in CDDP-treated p22phox stable lines (Figure 4C). Together, these results strongly suggested that overexpression of p22phox could protect OSCC cells from CDDP-induced apoptosis.

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