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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

Reduced CDDP uptake into the nucleus, CDDP-DNA adduct formation and chk1-p53 activation in p22phox stable lines(A) CDDP distribution and localization was monitored by Alexa Fluor 488 fluorescence in p22phox stable lines and p22phox-overexpressing KB cells using confocal microscopy. Magnification: 1000X. The respective cytoplasmic and nuclear fluorescence intensity in each cell was measured by Olympus Fluoview Viewer (Ver. 3.0 software). The average cytoplasm-to-nucleus intensity ratios were determined from the randomly selected 24, 20, 21, 21 and 12 cells in control line, p22phox stable line #1, p22phox stable line #2, mock transfected KB and p22phox-DsRed transfected KB, respectively. The quantitative results and statistical analysis were shown in the right panels. (B) The p22phox stable lines and the control line were treated with 10 or 20 μM CDDP overnight, and genomic DNA was isolated and analyzed by dot blot assay using anti-CDDP adducts antibody. The arrows denote non-specific background signals. (C) The cells were treated with CDDP (20 μM) for 0, 2, 4 or 6 h and the lysates were analyzed by Western blot analysis using antibodies against t-chk1, p-chk1, t-p53 and p-p53. The numbers below the blots were quantitative ratios of p-chk1, t-chk1, p-p53 or t-p53/GAPDH band intensities normalized to those without CDDP treatment. Abbreviations: c, cytoplasm; n, nucleus; pos, positive control. The experiments were repeated four times, and the representative images or data are shown.
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Figure 7: Reduced CDDP uptake into the nucleus, CDDP-DNA adduct formation and chk1-p53 activation in p22phox stable lines(A) CDDP distribution and localization was monitored by Alexa Fluor 488 fluorescence in p22phox stable lines and p22phox-overexpressing KB cells using confocal microscopy. Magnification: 1000X. The respective cytoplasmic and nuclear fluorescence intensity in each cell was measured by Olympus Fluoview Viewer (Ver. 3.0 software). The average cytoplasm-to-nucleus intensity ratios were determined from the randomly selected 24, 20, 21, 21 and 12 cells in control line, p22phox stable line #1, p22phox stable line #2, mock transfected KB and p22phox-DsRed transfected KB, respectively. The quantitative results and statistical analysis were shown in the right panels. (B) The p22phox stable lines and the control line were treated with 10 or 20 μM CDDP overnight, and genomic DNA was isolated and analyzed by dot blot assay using anti-CDDP adducts antibody. The arrows denote non-specific background signals. (C) The cells were treated with CDDP (20 μM) for 0, 2, 4 or 6 h and the lysates were analyzed by Western blot analysis using antibodies against t-chk1, p-chk1, t-p53 and p-p53. The numbers below the blots were quantitative ratios of p-chk1, t-chk1, p-p53 or t-p53/GAPDH band intensities normalized to those without CDDP treatment. Abbreviations: c, cytoplasm; n, nucleus; pos, positive control. The experiments were repeated four times, and the representative images or data are shown.

Mentions: The unique localization pattern of p22phox when overexpressed propelled us to investigate whether this could contribute to p22phox-dependent CDDP resistance. Using green fluorescence (Alexa Fluor 488)-labeled CDDP to monitor CDDP uptake and distribution, we found that Alexa Fluor 488-CDDP signal was apparently more intense in the cytoplasm than the nucleus in p22phox stable lines. In contrast, the fluorescence signal was uniformly distributed throughout the entire cell in the control line. Furthermore, these observations were reproduced in KB carcinoma cell line transiently overexpressing p22phox (p22phox-DsRed). Quantitative analysis revealed that the average cytoplasm-to-nucleus ratio of Alexa Fluor 488 intensity was significantly higher in the p22phox-overexpressing cells; 22.31 and 16.58 vs 1.22, P < 0.001 in the stable lines and 8.47 vs 2.41, P < 0.001 in KB cells. More remarkably, the accumulated Alexa Fluor 488-CDDP in the cytoplasm coincided with the localization sites of the ectopically expressed p22phox, including those of the ring-like structure, in both cell lines (Figure 7A). To evaluate whether accumulation of CDDP in the cytoplasm would lead to decreased DNA damage in the nucleus, we examined CDDP-DNA adducts in p22phox stable lines. Indeed, dot blot analysis using anti-CDDP adduct antibody showed significant reduction of DNA adduct formation compared to the control line when the cells were treated with increasing doses of CDDP (Figure 7B). Furthermore, the activation of checkpoint kinase chk1 and p53, the two indicators of DNA damage response, was either attenuated or delayed during the 6-h CDDP treatment period in p22phox stable lines, as indicated by the decreased induction of p-chk1 (Ser345) and p-p53 (Ser15) (Figure 7C). These findings suggested that overexpression of p22phox might block the entry of CDDP into the nucleus, resulting in reduced formation of DNA adducts and activation of DNA repair signaling.


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)

Reduced CDDP uptake into the nucleus, CDDP-DNA adduct formation and chk1-p53 activation in p22phox stable lines(A) CDDP distribution and localization was monitored by Alexa Fluor 488 fluorescence in p22phox stable lines and p22phox-overexpressing KB cells using confocal microscopy. Magnification: 1000X. The respective cytoplasmic and nuclear fluorescence intensity in each cell was measured by Olympus Fluoview Viewer (Ver. 3.0 software). The average cytoplasm-to-nucleus intensity ratios were determined from the randomly selected 24, 20, 21, 21 and 12 cells in control line, p22phox stable line #1, p22phox stable line #2, mock transfected KB and p22phox-DsRed transfected KB, respectively. The quantitative results and statistical analysis were shown in the right panels. (B) The p22phox stable lines and the control line were treated with 10 or 20 μM CDDP overnight, and genomic DNA was isolated and analyzed by dot blot assay using anti-CDDP adducts antibody. The arrows denote non-specific background signals. (C) The cells were treated with CDDP (20 μM) for 0, 2, 4 or 6 h and the lysates were analyzed by Western blot analysis using antibodies against t-chk1, p-chk1, t-p53 and p-p53. The numbers below the blots were quantitative ratios of p-chk1, t-chk1, p-p53 or t-p53/GAPDH band intensities normalized to those without CDDP treatment. Abbreviations: c, cytoplasm; n, nucleus; pos, positive control. The experiments were repeated four times, and the representative images or data are shown.
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Figure 7: Reduced CDDP uptake into the nucleus, CDDP-DNA adduct formation and chk1-p53 activation in p22phox stable lines(A) CDDP distribution and localization was monitored by Alexa Fluor 488 fluorescence in p22phox stable lines and p22phox-overexpressing KB cells using confocal microscopy. Magnification: 1000X. The respective cytoplasmic and nuclear fluorescence intensity in each cell was measured by Olympus Fluoview Viewer (Ver. 3.0 software). The average cytoplasm-to-nucleus intensity ratios were determined from the randomly selected 24, 20, 21, 21 and 12 cells in control line, p22phox stable line #1, p22phox stable line #2, mock transfected KB and p22phox-DsRed transfected KB, respectively. The quantitative results and statistical analysis were shown in the right panels. (B) The p22phox stable lines and the control line were treated with 10 or 20 μM CDDP overnight, and genomic DNA was isolated and analyzed by dot blot assay using anti-CDDP adducts antibody. The arrows denote non-specific background signals. (C) The cells were treated with CDDP (20 μM) for 0, 2, 4 or 6 h and the lysates were analyzed by Western blot analysis using antibodies against t-chk1, p-chk1, t-p53 and p-p53. The numbers below the blots were quantitative ratios of p-chk1, t-chk1, p-p53 or t-p53/GAPDH band intensities normalized to those without CDDP treatment. Abbreviations: c, cytoplasm; n, nucleus; pos, positive control. The experiments were repeated four times, and the representative images or data are shown.
Mentions: The unique localization pattern of p22phox when overexpressed propelled us to investigate whether this could contribute to p22phox-dependent CDDP resistance. Using green fluorescence (Alexa Fluor 488)-labeled CDDP to monitor CDDP uptake and distribution, we found that Alexa Fluor 488-CDDP signal was apparently more intense in the cytoplasm than the nucleus in p22phox stable lines. In contrast, the fluorescence signal was uniformly distributed throughout the entire cell in the control line. Furthermore, these observations were reproduced in KB carcinoma cell line transiently overexpressing p22phox (p22phox-DsRed). Quantitative analysis revealed that the average cytoplasm-to-nucleus ratio of Alexa Fluor 488 intensity was significantly higher in the p22phox-overexpressing cells; 22.31 and 16.58 vs 1.22, P < 0.001 in the stable lines and 8.47 vs 2.41, P < 0.001 in KB cells. More remarkably, the accumulated Alexa Fluor 488-CDDP in the cytoplasm coincided with the localization sites of the ectopically expressed p22phox, including those of the ring-like structure, in both cell lines (Figure 7A). To evaluate whether accumulation of CDDP in the cytoplasm would lead to decreased DNA damage in the nucleus, we examined CDDP-DNA adducts in p22phox stable lines. Indeed, dot blot analysis using anti-CDDP adduct antibody showed significant reduction of DNA adduct formation compared to the control line when the cells were treated with increasing doses of CDDP (Figure 7B). Furthermore, the activation of checkpoint kinase chk1 and p53, the two indicators of DNA damage response, was either attenuated or delayed during the 6-h CDDP treatment period in p22phox stable lines, as indicated by the decreased induction of p-chk1 (Ser345) and p-p53 (Ser15) (Figure 7C). These findings suggested that overexpression of p22phox might block the entry of CDDP into the nucleus, resulting in reduced formation of DNA adducts and activation of DNA repair signaling.

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