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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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Increased ZHX2 levels in HepG2 cells enhance CDDP-induced apoptosis and activate the caspase pathwayHepG2 cells transfected with pcDNA3.0 or pcZHX2 were treated with CDDP for 24 hours. (A) Cells were incubated with PI and analyzed by flow cytometry. The percentage of cells with fragmented DNA (sub-G1 peak) are shown. (B) Staining of cells with Hoechst 33258 (upper images) and DAPI (lower images). Bars, 50 pixel. (C) Flow cytometry after staining with PI and Annexin V to determine the percentage of Annexin V-stained cells. (D) Western blot analysis to determine levels of ZHX2, Cytochrome C, full-length and cleaved caspase 3, cleaved caspase 9, full-length and cleaved PARP, and β-actin. Western blot data from three independent experiments are quantitated in the right panel, with levels if indicated protein shown relative to β-actin. *p < 0.05,**p < 0.01, ***p < 0.001.
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Figure 4: Increased ZHX2 levels in HepG2 cells enhance CDDP-induced apoptosis and activate the caspase pathwayHepG2 cells transfected with pcDNA3.0 or pcZHX2 were treated with CDDP for 24 hours. (A) Cells were incubated with PI and analyzed by flow cytometry. The percentage of cells with fragmented DNA (sub-G1 peak) are shown. (B) Staining of cells with Hoechst 33258 (upper images) and DAPI (lower images). Bars, 50 pixel. (C) Flow cytometry after staining with PI and Annexin V to determine the percentage of Annexin V-stained cells. (D) Western blot analysis to determine levels of ZHX2, Cytochrome C, full-length and cleaved caspase 3, cleaved caspase 9, full-length and cleaved PARP, and β-actin. Western blot data from three independent experiments are quantitated in the right panel, with levels if indicated protein shown relative to β-actin. *p < 0.05,**p < 0.01, ***p < 0.001.

Mentions: Since CDDP acts to increase apoptosis by DNA cross-linking, we next tested whether elevated ZHX2 would enhance CDDP-mediated DNA damage and apoptosis in HepG2 cells as judged by chromatin condensation and nuclear fragmentation [20]. ZHX2 overexpression by itself did not increase the number of apoptotic, sub-G1 cells compared to pcDNA3.0 transfected cells (Figure 4A). As expected, a distinct sub-G1 peak was detected in CDDP-treated HepG2 cells. Interestingly, ZHX2 overexpression significantly increased the sub-G1 population after CDDP treatment, indicating that elevated ZHX2 enhances CDDP-induced apoptosis. This increased apoptosis was further verified by Hoechst 33258 and DAPI staining in HepG2 cells, which showed increased chromatin condensation (Figure 4B) and flow cytometry after staining with Annexin V and propidium iodide (PI, Figure 4C).


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)

Increased ZHX2 levels in HepG2 cells enhance CDDP-induced apoptosis and activate the caspase pathwayHepG2 cells transfected with pcDNA3.0 or pcZHX2 were treated with CDDP for 24 hours. (A) Cells were incubated with PI and analyzed by flow cytometry. The percentage of cells with fragmented DNA (sub-G1 peak) are shown. (B) Staining of cells with Hoechst 33258 (upper images) and DAPI (lower images). Bars, 50 pixel. (C) Flow cytometry after staining with PI and Annexin V to determine the percentage of Annexin V-stained cells. (D) Western blot analysis to determine levels of ZHX2, Cytochrome C, full-length and cleaved caspase 3, cleaved caspase 9, full-length and cleaved PARP, and β-actin. Western blot data from three independent experiments are quantitated in the right panel, with levels if indicated protein shown relative to β-actin. *p < 0.05,**p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Increased ZHX2 levels in HepG2 cells enhance CDDP-induced apoptosis and activate the caspase pathwayHepG2 cells transfected with pcDNA3.0 or pcZHX2 were treated with CDDP for 24 hours. (A) Cells were incubated with PI and analyzed by flow cytometry. The percentage of cells with fragmented DNA (sub-G1 peak) are shown. (B) Staining of cells with Hoechst 33258 (upper images) and DAPI (lower images). Bars, 50 pixel. (C) Flow cytometry after staining with PI and Annexin V to determine the percentage of Annexin V-stained cells. (D) Western blot analysis to determine levels of ZHX2, Cytochrome C, full-length and cleaved caspase 3, cleaved caspase 9, full-length and cleaved PARP, and β-actin. Western blot data from three independent experiments are quantitated in the right panel, with levels if indicated protein shown relative to β-actin. *p < 0.05,**p < 0.01, ***p < 0.001.
Mentions: Since CDDP acts to increase apoptosis by DNA cross-linking, we next tested whether elevated ZHX2 would enhance CDDP-mediated DNA damage and apoptosis in HepG2 cells as judged by chromatin condensation and nuclear fragmentation [20]. ZHX2 overexpression by itself did not increase the number of apoptotic, sub-G1 cells compared to pcDNA3.0 transfected cells (Figure 4A). As expected, a distinct sub-G1 peak was detected in CDDP-treated HepG2 cells. Interestingly, ZHX2 overexpression significantly increased the sub-G1 population after CDDP treatment, indicating that elevated ZHX2 enhances CDDP-induced apoptosis. This increased apoptosis was further verified by Hoechst 33258 and DAPI staining in HepG2 cells, which showed increased chromatin condensation (Figure 4B) and flow cytometry after staining with Annexin V and propidium iodide (PI, Figure 4C).

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