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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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ZHX2 represses MDR1 promoter activity via NF-YA-dependent interactions(A) HepG2 and HepG2.2.15 cells were co-transfected with pGL3-Mp along with pcDNA3.0 or pcZHX2 (left panel) or with pGL3-Mp and increasing amounts of pcZHX2 (right panel). (B) SMMC7721 cells were transfected with pGL3-Mp and siCON or ZHX2 siRNAs (left panel) or with pGL3-Mp and increasing amounts of ZHX2 siRNAs (right panel).(C) Diagram of the wild type and mutant type MDR1 promoters, showing the location of the Y box and the mutation used to generate pGL3-mMp. (D) HepG2 cells were co-transfected with pGL3-Mp or pGL3-mMp along with pcDNA3.0 or pcZHX2.(E) HepG2 cells were co-transfected with pGL3-Mp and pcDNA3.0 or pcZHX2(Left). In addition, cells were also transfected with siCON or NF-YA siRNA (siNF-YA, Right). Data are shown as the mean ± SD (n≥3); *p < 0.05, **p < 0.01,***p < 0.001.
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Figure 6: ZHX2 represses MDR1 promoter activity via NF-YA-dependent interactions(A) HepG2 and HepG2.2.15 cells were co-transfected with pGL3-Mp along with pcDNA3.0 or pcZHX2 (left panel) or with pGL3-Mp and increasing amounts of pcZHX2 (right panel). (B) SMMC7721 cells were transfected with pGL3-Mp and siCON or ZHX2 siRNAs (left panel) or with pGL3-Mp and increasing amounts of ZHX2 siRNAs (right panel).(C) Diagram of the wild type and mutant type MDR1 promoters, showing the location of the Y box and the mutation used to generate pGL3-mMp. (D) HepG2 cells were co-transfected with pGL3-Mp or pGL3-mMp along with pcDNA3.0 or pcZHX2.(E) HepG2 cells were co-transfected with pGL3-Mp and pcDNA3.0 or pcZHX2(Left). In addition, cells were also transfected with siCON or NF-YA siRNA (siNF-YA, Right). Data are shown as the mean ± SD (n≥3); *p < 0.05, **p < 0.01,***p < 0.001.

Mentions: Previous studies indicate that ZHX2 functions as a transcriptional repressor [9, 12, 13]. We therefore tested whether ZHX2 represses MDR1 promoter activity. Results of cotransfection and luciferase assays showed that ZHX2 overexpression significantly decreased MDR1 promoter activity in HepG2 and HepG2.2.15 cells (Figure 6A). Conversely, ZHX2 knock-down by siRNAs greatly enhanced the MDR1 promoter activity in SMMC7721 cells (Figure 6B). The decrease and increase in luciferase levels showed a dose-dependent response in cells that were cotransfected with ZHX2 expression vectors or ZHX2 siRNAs, respectively (Figure 6A and B).


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)

ZHX2 represses MDR1 promoter activity via NF-YA-dependent interactions(A) HepG2 and HepG2.2.15 cells were co-transfected with pGL3-Mp along with pcDNA3.0 or pcZHX2 (left panel) or with pGL3-Mp and increasing amounts of pcZHX2 (right panel). (B) SMMC7721 cells were transfected with pGL3-Mp and siCON or ZHX2 siRNAs (left panel) or with pGL3-Mp and increasing amounts of ZHX2 siRNAs (right panel).(C) Diagram of the wild type and mutant type MDR1 promoters, showing the location of the Y box and the mutation used to generate pGL3-mMp. (D) HepG2 cells were co-transfected with pGL3-Mp or pGL3-mMp along with pcDNA3.0 or pcZHX2.(E) HepG2 cells were co-transfected with pGL3-Mp and pcDNA3.0 or pcZHX2(Left). In addition, cells were also transfected with siCON or NF-YA siRNA (siNF-YA, Right). Data are shown as the mean ± SD (n≥3); *p < 0.05, **p < 0.01,***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: ZHX2 represses MDR1 promoter activity via NF-YA-dependent interactions(A) HepG2 and HepG2.2.15 cells were co-transfected with pGL3-Mp along with pcDNA3.0 or pcZHX2 (left panel) or with pGL3-Mp and increasing amounts of pcZHX2 (right panel). (B) SMMC7721 cells were transfected with pGL3-Mp and siCON or ZHX2 siRNAs (left panel) or with pGL3-Mp and increasing amounts of ZHX2 siRNAs (right panel).(C) Diagram of the wild type and mutant type MDR1 promoters, showing the location of the Y box and the mutation used to generate pGL3-mMp. (D) HepG2 cells were co-transfected with pGL3-Mp or pGL3-mMp along with pcDNA3.0 or pcZHX2.(E) HepG2 cells were co-transfected with pGL3-Mp and pcDNA3.0 or pcZHX2(Left). In addition, cells were also transfected with siCON or NF-YA siRNA (siNF-YA, Right). Data are shown as the mean ± SD (n≥3); *p < 0.05, **p < 0.01,***p < 0.001.
Mentions: Previous studies indicate that ZHX2 functions as a transcriptional repressor [9, 12, 13]. We therefore tested whether ZHX2 represses MDR1 promoter activity. Results of cotransfection and luciferase assays showed that ZHX2 overexpression significantly decreased MDR1 promoter activity in HepG2 and HepG2.2.15 cells (Figure 6A). Conversely, ZHX2 knock-down by siRNAs greatly enhanced the MDR1 promoter activity in SMMC7721 cells (Figure 6B). The decrease and increase in luciferase levels showed a dose-dependent response in cells that were cotransfected with ZHX2 expression vectors or ZHX2 siRNAs, respectively (Figure 6A and B).

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