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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

Overexpression of p22phox led to its perinuclear localization in OSCC cells(A) The fluorescence signal of p22phox-DsRed in the stable lines or DsRed only in the control line was visualized by confocal microscopy. p22phox-DsRed fusion protein was mostly localized to the perinuclear region, yielding the ring-like pattern. In contrast, DsRed protein was localized throughout the whole cell in a diffuse fashion. (B) Overexpression of p22phox in the parental SAS cells by introducing a non-tagged p22phox expression construct (pcDNA3.0-p22phox) resulted in a similar perinuclear localization revealed by immunofluorescence microscopy. Magnification: 1000X. The experiments were repeated four times, and the representative images are shown.
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Figure 6: Overexpression of p22phox led to its perinuclear localization in OSCC cells(A) The fluorescence signal of p22phox-DsRed in the stable lines or DsRed only in the control line was visualized by confocal microscopy. p22phox-DsRed fusion protein was mostly localized to the perinuclear region, yielding the ring-like pattern. In contrast, DsRed protein was localized throughout the whole cell in a diffuse fashion. (B) Overexpression of p22phox in the parental SAS cells by introducing a non-tagged p22phox expression construct (pcDNA3.0-p22phox) resulted in a similar perinuclear localization revealed by immunofluorescence microscopy. Magnification: 1000X. The experiments were repeated four times, and the representative images are shown.

Mentions: The expression site of the ectopic p22phox protein in p22phox stable lines was examined. Since p22phox was tagged with the red fluorescence protein DsRed, we directly visualized the subcellular localization of p22phox by fluorescence microscopy in living cells. While there was diffuse DsRed fluorescence signal throughout the entire cell in the control line, the overexpressed p22phox-DsRed was predominantly localized in the cytoplasm, forming a ring-like pattern at the nuclear periphery in p22phox stable lines (Figure 6A). To confirm this observation, the parental SAS cells were transiently transfected with a non-tagged p22phox expression construct (pcDNA3.0-p22phox) and analyzed by immunofluorescence microscopy. Consistently, p22phox expression detected by anti-p22phox antibody displayed similar staining pattern around the nucleus (Figure 6B). More importantly, these findings are reminiscent of those in clinical samples in which p22phox was intensively localized to the perinuclear area when overexpressed in the CDDP-resistant specimens (Figure 1B–1D).


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)

Overexpression of p22phox led to its perinuclear localization in OSCC cells(A) The fluorescence signal of p22phox-DsRed in the stable lines or DsRed only in the control line was visualized by confocal microscopy. p22phox-DsRed fusion protein was mostly localized to the perinuclear region, yielding the ring-like pattern. In contrast, DsRed protein was localized throughout the whole cell in a diffuse fashion. (B) Overexpression of p22phox in the parental SAS cells by introducing a non-tagged p22phox expression construct (pcDNA3.0-p22phox) resulted in a similar perinuclear localization revealed by immunofluorescence microscopy. Magnification: 1000X. The experiments were repeated four times, and the representative images are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414176&req=5

Figure 6: Overexpression of p22phox led to its perinuclear localization in OSCC cells(A) The fluorescence signal of p22phox-DsRed in the stable lines or DsRed only in the control line was visualized by confocal microscopy. p22phox-DsRed fusion protein was mostly localized to the perinuclear region, yielding the ring-like pattern. In contrast, DsRed protein was localized throughout the whole cell in a diffuse fashion. (B) Overexpression of p22phox in the parental SAS cells by introducing a non-tagged p22phox expression construct (pcDNA3.0-p22phox) resulted in a similar perinuclear localization revealed by immunofluorescence microscopy. Magnification: 1000X. The experiments were repeated four times, and the representative images are shown.
Mentions: The expression site of the ectopic p22phox protein in p22phox stable lines was examined. Since p22phox was tagged with the red fluorescence protein DsRed, we directly visualized the subcellular localization of p22phox by fluorescence microscopy in living cells. While there was diffuse DsRed fluorescence signal throughout the entire cell in the control line, the overexpressed p22phox-DsRed was predominantly localized in the cytoplasm, forming a ring-like pattern at the nuclear periphery in p22phox stable lines (Figure 6A). To confirm this observation, the parental SAS cells were transiently transfected with a non-tagged p22phox expression construct (pcDNA3.0-p22phox) and analyzed by immunofluorescence microscopy. Consistently, p22phox expression detected by anti-p22phox antibody displayed similar staining pattern around the nucleus (Figure 6B). More importantly, these findings are reminiscent of those in clinical samples in which p22phox was intensively localized to the perinuclear area when overexpressed in the CDDP-resistant specimens (Figure 1B–1D).

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