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ZHX2 enhances the cytotoxicity of chemotherapeutic drugs in liver tumor cells by repressing MDR1 via interfering with NF-YA.

Ma H, Yue X, Gao L, Liang X, Yan W, Zhang Z, Shan H, Zhang H, Spear BT, Ma C - Oncotarget (2015)

Bottom Line: Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo.Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter.Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, P.R. China.

ABSTRACT
We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Here, we aim to test whether increased ZHX2 levels in HCC cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs. We showed evidence that increased ZHX2 levels correlated with reduced MDR1 expression and enhanced the cytotoxicity of CDDP and ADM in different HCC cell lines. Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo. Furthermore, immunohistochemical staining demonstrated the inverse correlation of ZHX2 and MDR1 expression in HCC tissues. Luciferase report assay showed that ZHX2 repressed the MDR1 promoter activity, while knockdown of NF-YA or mutating the NF-Y binding site eliminated this ZHX2-mediated repression of MDR1 transcription. Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter. Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

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Higher ZHX2 levels increase the sensitivity of HCC cells to the cytotoxic effects of CDDP and ADMHepG2 and HepG2.2.15 cells were transfected with pcDNA3.0 or pcZHX2 (A), whereas SMMC7721 cells were transfected with siCON or ZHX2-siRNAs (B). After 24 hours, cells were treated with CDDP (upper panels) or ADM (lower panels) and cultured for another 24 hours. The cytotoxicity was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=4); **p < 0.01, ***p < 0.001. (C and D) IC50 of CDDP (upper panels) or ADM (lower panels) in cell lines transfected as described above in A and B. The IC50 was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=3); *p < 0.05, **p < 0.01.
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Figure 3: Higher ZHX2 levels increase the sensitivity of HCC cells to the cytotoxic effects of CDDP and ADMHepG2 and HepG2.2.15 cells were transfected with pcDNA3.0 or pcZHX2 (A), whereas SMMC7721 cells were transfected with siCON or ZHX2-siRNAs (B). After 24 hours, cells were treated with CDDP (upper panels) or ADM (lower panels) and cultured for another 24 hours. The cytotoxicity was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=4); **p < 0.01, ***p < 0.001. (C and D) IC50 of CDDP (upper panels) or ADM (lower panels) in cell lines transfected as described above in A and B. The IC50 was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=3); *p < 0.05, **p < 0.01.

Mentions: The ability of ZHX2 to repress MDR1 led us to consider whether elevated ZHX2 levels would increase drug sensitivity in HCC cells. To test this, the cytotoxicity index of CDDP or ADM was determined in ZHX2-overexpressing cells or ZHX2-knockdown cells. In ZHX2-overexpressing cell lines (HepG2 and HepG2.2.15), the cytotoxicity index increased significantly after treatment with both CDDP and ADM (Figure 3A) compared to pcDNA3.0-transfected cells treated with these drugs. In accordance, knock-down of ZHX2 in SMMC7721 cells decreased the cytotoxicity index of both CDDP and ADM (Figure 3B). This is further supported by IC50 assay measured with increasing amounts of CDDP or ADM in different cell populations (Figure 3C and D). These data indicate that increased ZHX2 levels result in increased sensitivity of HCC cells to these chemotherapeutic drugs.


ZHX2 enhances the cytotoxicity of chemotherapeutic drugs in liver tumor cells by repressing MDR1 via interfering with NF-YA.

Ma H, Yue X, Gao L, Liang X, Yan W, Zhang Z, Shan H, Zhang H, Spear BT, Ma C - Oncotarget (2015)

Higher ZHX2 levels increase the sensitivity of HCC cells to the cytotoxic effects of CDDP and ADMHepG2 and HepG2.2.15 cells were transfected with pcDNA3.0 or pcZHX2 (A), whereas SMMC7721 cells were transfected with siCON or ZHX2-siRNAs (B). After 24 hours, cells were treated with CDDP (upper panels) or ADM (lower panels) and cultured for another 24 hours. The cytotoxicity was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=4); **p < 0.01, ***p < 0.001. (C and D) IC50 of CDDP (upper panels) or ADM (lower panels) in cell lines transfected as described above in A and B. The IC50 was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=3); *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359216&req=5

Figure 3: Higher ZHX2 levels increase the sensitivity of HCC cells to the cytotoxic effects of CDDP and ADMHepG2 and HepG2.2.15 cells were transfected with pcDNA3.0 or pcZHX2 (A), whereas SMMC7721 cells were transfected with siCON or ZHX2-siRNAs (B). After 24 hours, cells were treated with CDDP (upper panels) or ADM (lower panels) and cultured for another 24 hours. The cytotoxicity was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=4); **p < 0.01, ***p < 0.001. (C and D) IC50 of CDDP (upper panels) or ADM (lower panels) in cell lines transfected as described above in A and B. The IC50 was calculated as described in Materials and Methods. Data are shown as the mean±SD (n=3); *p < 0.05, **p < 0.01.
Mentions: The ability of ZHX2 to repress MDR1 led us to consider whether elevated ZHX2 levels would increase drug sensitivity in HCC cells. To test this, the cytotoxicity index of CDDP or ADM was determined in ZHX2-overexpressing cells or ZHX2-knockdown cells. In ZHX2-overexpressing cell lines (HepG2 and HepG2.2.15), the cytotoxicity index increased significantly after treatment with both CDDP and ADM (Figure 3A) compared to pcDNA3.0-transfected cells treated with these drugs. In accordance, knock-down of ZHX2 in SMMC7721 cells decreased the cytotoxicity index of both CDDP and ADM (Figure 3B). This is further supported by IC50 assay measured with increasing amounts of CDDP or ADM in different cell populations (Figure 3C and D). These data indicate that increased ZHX2 levels result in increased sensitivity of HCC cells to these chemotherapeutic drugs.

Bottom Line: Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo.Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter.Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of Ministry of Education and Department of Immunology, Shandong University School of Medicine, Jinan, Shandong, P.R. China.

ABSTRACT
We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Here, we aim to test whether increased ZHX2 levels in HCC cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs. We showed evidence that increased ZHX2 levels correlated with reduced MDR1 expression and enhanced the cytotoxicity of CDDP and ADM in different HCC cell lines. Consistently, elevated ZHX2 significantly reduced ADM efflux in HepG2 cells and greatly increased the CDDP-mediated suppression of liver tumor growth in vivo. Furthermore, immunohistochemical staining demonstrated the inverse correlation of ZHX2 and MDR1 expression in HCC tissues. Luciferase report assay showed that ZHX2 repressed the MDR1 promoter activity, while knockdown of NF-YA or mutating the NF-Y binding site eliminated this ZHX2-mediated repression of MDR1 transcription. Co-IP and ChIP assay further suggested that ZHX2 interacted with NF-YA and reduced NF-Y binding to the MDR1 promoter. Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in HCC cells both in vitro and in vivo.

Show MeSH
Related in: MedlinePlus