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ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.

Zheng G, Peng C, Jia X, Gu Y, Zhang Z, Deng Y, Wang C, Li N, Yin J, Liu X, Lu M, Tang H, He Z - Mol. Cancer (2015)

Bottom Line: Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells.CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation.Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zhengguopei@126.com.

ABSTRACT

Background: Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear.

Methods: Bioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed.

Results: ZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients.

Conclusion: Our finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1-CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.

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ZEB1–CA9 prevents chemotherapy-induced caspase-3 activation. (A and B) PYM and cDDP induced caspase-3 activation in Tca8113 and SCC-25 cell lines, respectively, as measured by reporter assays and western blotting. Overexpression of ZEB1 prevented caspase-3 activation in response to chemotherapy, while knockdown of CA9 impaired the effects of ZEB1 overexpression. (C) Chemotherapy had no significant effect on caspase-3 activation in Tca8113/PYM cells. Knockdown of ZEB1 enhanced caspase-3 activation induced by chemotherapy, and overexpression of CA9 attenuated this effect. vs. no treatment, * p < 0.01.
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Fig4: ZEB1–CA9 prevents chemotherapy-induced caspase-3 activation. (A and B) PYM and cDDP induced caspase-3 activation in Tca8113 and SCC-25 cell lines, respectively, as measured by reporter assays and western blotting. Overexpression of ZEB1 prevented caspase-3 activation in response to chemotherapy, while knockdown of CA9 impaired the effects of ZEB1 overexpression. (C) Chemotherapy had no significant effect on caspase-3 activation in Tca8113/PYM cells. Knockdown of ZEB1 enhanced caspase-3 activation induced by chemotherapy, and overexpression of CA9 attenuated this effect. vs. no treatment, * p < 0.01.

Mentions: A decrease in pHi correlates with an increase in apoptosis and caspase-3 is a key component of the apoptotic pathway [18]. We therefore performed a caspase-3 activity assay and western blotting to detect the cleaved, active protein and therefore determine the functional state of caspase-3 in cultured cells. We found that PYM (80mg/L) or cDDP (5mg/L) treatment significantly promoted caspase-3 activation in Tca8113 and SCC-25 cells (Figure 4A and B) and this was accompanied by a decrease in pHi (Figure 3A and B). With respect to the change in pHi, we found that ectopic ZEB1 expression blocked PYM or cDDP-induced caspase-3 activation in Tca8113 and SCC-25 cells (Figure 4A and B), while the simultaneous knockdown of CA9 abolished this ZEB1-related effect (Figure 4A and B). Furthermore, in Tca8113/PYM cells (in which endogenous ZEB1 is upregulated) we found that knockdown of ZEB1 promoted caspase-3 activation in response to PYM or cDDP (Figure 4C). However, ectopic CA9 expression attenuated the effect of ZEB1 knockdown on caspase-3 activation (Figure 4C). These results suggest that CA9 is involved in the ZEB1-dependent inhibition of caspase-3 activation.Figure 4


ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.

Zheng G, Peng C, Jia X, Gu Y, Zhang Z, Deng Y, Wang C, Li N, Yin J, Liu X, Lu M, Tang H, He Z - Mol. Cancer (2015)

ZEB1–CA9 prevents chemotherapy-induced caspase-3 activation. (A and B) PYM and cDDP induced caspase-3 activation in Tca8113 and SCC-25 cell lines, respectively, as measured by reporter assays and western blotting. Overexpression of ZEB1 prevented caspase-3 activation in response to chemotherapy, while knockdown of CA9 impaired the effects of ZEB1 overexpression. (C) Chemotherapy had no significant effect on caspase-3 activation in Tca8113/PYM cells. Knockdown of ZEB1 enhanced caspase-3 activation induced by chemotherapy, and overexpression of CA9 attenuated this effect. vs. no treatment, * p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4404088&req=5

Fig4: ZEB1–CA9 prevents chemotherapy-induced caspase-3 activation. (A and B) PYM and cDDP induced caspase-3 activation in Tca8113 and SCC-25 cell lines, respectively, as measured by reporter assays and western blotting. Overexpression of ZEB1 prevented caspase-3 activation in response to chemotherapy, while knockdown of CA9 impaired the effects of ZEB1 overexpression. (C) Chemotherapy had no significant effect on caspase-3 activation in Tca8113/PYM cells. Knockdown of ZEB1 enhanced caspase-3 activation induced by chemotherapy, and overexpression of CA9 attenuated this effect. vs. no treatment, * p < 0.01.
Mentions: A decrease in pHi correlates with an increase in apoptosis and caspase-3 is a key component of the apoptotic pathway [18]. We therefore performed a caspase-3 activity assay and western blotting to detect the cleaved, active protein and therefore determine the functional state of caspase-3 in cultured cells. We found that PYM (80mg/L) or cDDP (5mg/L) treatment significantly promoted caspase-3 activation in Tca8113 and SCC-25 cells (Figure 4A and B) and this was accompanied by a decrease in pHi (Figure 3A and B). With respect to the change in pHi, we found that ectopic ZEB1 expression blocked PYM or cDDP-induced caspase-3 activation in Tca8113 and SCC-25 cells (Figure 4A and B), while the simultaneous knockdown of CA9 abolished this ZEB1-related effect (Figure 4A and B). Furthermore, in Tca8113/PYM cells (in which endogenous ZEB1 is upregulated) we found that knockdown of ZEB1 promoted caspase-3 activation in response to PYM or cDDP (Figure 4C). However, ectopic CA9 expression attenuated the effect of ZEB1 knockdown on caspase-3 activation (Figure 4C). These results suggest that CA9 is involved in the ZEB1-dependent inhibition of caspase-3 activation.Figure 4

Bottom Line: Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells.CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation.Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute of Guangzhou Medical University, Hengzhigang Road 78#, Guangzhou, 510095, Guangdong, China. zhengguopei@126.com.

ABSTRACT

Background: Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear.

Methods: Bioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed.

Results: ZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients.

Conclusion: Our finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1-CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.

Show MeSH
Related in: MedlinePlus