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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 interacts with the MDR1 promoter via NF-Y(A) Co-IP of NF-YA and ZHX2 after transfection. pcZHX2 plasmid was transfected into HepG2 cells, followed by immunoprecipitations performed as the methods. (B and C) ChIP analysis of DNA from HepG2 cells transfected with pcEGFP-HA or pcZHX2-HA. (B) Conventional PCR amplification of DNA was performed with primers specific to the MDR1 promoter after immunoprecipitation with anti-HA, anti-NF-YA or IgG (control). (C) DNA enrichment was analyzed at the MDR1 promoter by real-time PCR and the results are presented as fold of enrichment over input. Data are shown as the mean ± SD (n=3); ***p < 0.001.
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Figure 7: ZHX2 interacts with the MDR1 promoter via NF-Y(A) Co-IP of NF-YA and ZHX2 after transfection. pcZHX2 plasmid was transfected into HepG2 cells, followed by immunoprecipitations performed as the methods. (B and C) ChIP analysis of DNA from HepG2 cells transfected with pcEGFP-HA or pcZHX2-HA. (B) Conventional PCR amplification of DNA was performed with primers specific to the MDR1 promoter after immunoprecipitation with anti-HA, anti-NF-YA or IgG (control). (C) DNA enrichment was analyzed at the MDR1 promoter by real-time PCR and the results are presented as fold of enrichment over input. Data are shown as the mean ± SD (n=3); ***p < 0.001.

Mentions: The transfection data described above led us to consider whether ZHX2 bound directly to the MDR1 promoter or inhibited NF-Y activity by an indirect mechanism. As previous report in HEK-293, Co-IP demonstrated the interaction of ZHX2 and NF-YA proteins in HepG2 cells (Figure 7A) [9]. To directly test whether ZHX2 bound to the MDR1 promoter, ChIP were performed using HepG2 cells transfected with pcZHX2-HA or pcEGFP-HA and oligonucleotides specific for the MDR1 promoter (Figure 6C). As shown in Figure 7B, a specific PCR amplification was detected in the anti-HA immunoprecipitation from HepG2 cells transfected with pcZHX2-HA but not in pcEGFP-HA, indicating that ZHX2 bound to the MDR1 promoter. Interestingly, a relative weak PCR amplification was detected in the anti-NF-YA immunoprecipitation from HepG2 cells transfected with pcZHX2-HA than that in pcEGFP-HA (Figure 7 B and C), suggested that the presence of transfected ZHX2 could influence the NF-YA binding to the MDR1 promoter. Taken together, these data suggest that ZHX2 interacts directly with NF-Y on the MDR1 promoter and that this interaction inhibits NF-Y-mediated activation of MDR1 transcription.


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 interacts with the MDR1 promoter via NF-Y(A) Co-IP of NF-YA and ZHX2 after transfection. pcZHX2 plasmid was transfected into HepG2 cells, followed by immunoprecipitations performed as the methods. (B and C) ChIP analysis of DNA from HepG2 cells transfected with pcEGFP-HA or pcZHX2-HA. (B) Conventional PCR amplification of DNA was performed with primers specific to the MDR1 promoter after immunoprecipitation with anti-HA, anti-NF-YA or IgG (control). (C) DNA enrichment was analyzed at the MDR1 promoter by real-time PCR and the results are presented as fold of enrichment over input. Data are shown as the mean ± SD (n=3); ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: ZHX2 interacts with the MDR1 promoter via NF-Y(A) Co-IP of NF-YA and ZHX2 after transfection. pcZHX2 plasmid was transfected into HepG2 cells, followed by immunoprecipitations performed as the methods. (B and C) ChIP analysis of DNA from HepG2 cells transfected with pcEGFP-HA or pcZHX2-HA. (B) Conventional PCR amplification of DNA was performed with primers specific to the MDR1 promoter after immunoprecipitation with anti-HA, anti-NF-YA or IgG (control). (C) DNA enrichment was analyzed at the MDR1 promoter by real-time PCR and the results are presented as fold of enrichment over input. Data are shown as the mean ± SD (n=3); ***p < 0.001.
Mentions: The transfection data described above led us to consider whether ZHX2 bound directly to the MDR1 promoter or inhibited NF-Y activity by an indirect mechanism. As previous report in HEK-293, Co-IP demonstrated the interaction of ZHX2 and NF-YA proteins in HepG2 cells (Figure 7A) [9]. To directly test whether ZHX2 bound to the MDR1 promoter, ChIP were performed using HepG2 cells transfected with pcZHX2-HA or pcEGFP-HA and oligonucleotides specific for the MDR1 promoter (Figure 6C). As shown in Figure 7B, a specific PCR amplification was detected in the anti-HA immunoprecipitation from HepG2 cells transfected with pcZHX2-HA but not in pcEGFP-HA, indicating that ZHX2 bound to the MDR1 promoter. Interestingly, a relative weak PCR amplification was detected in the anti-NF-YA immunoprecipitation from HepG2 cells transfected with pcZHX2-HA than that in pcEGFP-HA (Figure 7 B and C), suggested that the presence of transfected ZHX2 could influence the NF-YA binding to the MDR1 promoter. Taken together, these data suggest that ZHX2 interacts directly with NF-Y on the MDR1 promoter and that this interaction inhibits NF-Y-mediated activation of MDR1 transcription.

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