Limits...
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.

Show MeSH

Related in: MedlinePlus

ZHX2 suppresses MDR1 expression and increases ADM retention of HCC cells(A and B) ZHX2, MDR1 and β-actin mRNA levels(A) and protein levels (B) were determined by RT-PCR and western blot in HepG2 and HepG2.2.15 cells transfected with pcDNA3.0 or pcZHX2 and in SMMC7721 cells that were transfected with ZHX2 siRNAs or control siRNA (siCON). (C) ZHX2-EGFP expression and intracellular ADM were determined by fluorescence microscopy of HepG2 cells in pEGFP-ZHX2-transfected cells treated with ADM as described in Methods and Materials. Representative panels are shown. Red, autofluorescence of ADM ; Green, fluorescence of EGFP-ZHX2 ; Blue, DAPI staining. Bars, 10 μm. (D) ADM accumulation (left panels) and retention (right panels) in pEGFP-ZHX2 transfected HepG2 cells were analyzed by flow cytometry. (E) Left panel: ADM accumulation and retention based on data in Figure 2D. Right panel: ADM-releasing index was calculated based on accumulation and retention data from flow cytometry. Data are shown as the mean±SD (n=3); *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359216&req=5

Figure 2: ZHX2 suppresses MDR1 expression and increases ADM retention of HCC cells(A and B) ZHX2, MDR1 and β-actin mRNA levels(A) and protein levels (B) were determined by RT-PCR and western blot in HepG2 and HepG2.2.15 cells transfected with pcDNA3.0 or pcZHX2 and in SMMC7721 cells that were transfected with ZHX2 siRNAs or control siRNA (siCON). (C) ZHX2-EGFP expression and intracellular ADM were determined by fluorescence microscopy of HepG2 cells in pEGFP-ZHX2-transfected cells treated with ADM as described in Methods and Materials. Representative panels are shown. Red, autofluorescence of ADM ; Green, fluorescence of EGFP-ZHX2 ; Blue, DAPI staining. Bars, 10 μm. (D) ADM accumulation (left panels) and retention (right panels) in pEGFP-ZHX2 transfected HepG2 cells were analyzed by flow cytometry. (E) Left panel: ADM accumulation and retention based on data in Figure 2D. Right panel: ADM-releasing index was calculated based on accumulation and retention data from flow cytometry. Data are shown as the mean±SD (n=3); *p < 0.05.

Mentions: In order to further confirm the negative regulation of ZHX2 on MDR1 in HCC, we then did in vitro studies. ZHX2 and MDR1 mRNA levels were compared in several liver cancer cell lines. RT-PCR analysis showed an inverse correlation between MDR1 and ZHX2 expression: cells with higher MDR1 mRNA levels (HepG2 and HepG2.2.15 cells) had lower ZHX2 mRNA levels whereas those with lower MDR1 (SMMC7721 cells) had higher ZHX2 (Figure S1A). Interestingly, ZHX2 expression level correlated with CDDP sensitivity in HCC cells (Figure S1B), indicating that ZHX2 closely correlates with MDR1 expression and chemotherapy sensitivity of HCC cells. To explore further the relationship between these two genes, ZHX2 was overexpressed or knocked down by transient transfection. As shown in Figure 2A, ZHX2 overexpression led to decreased MDR1 mRNA levels in HepG2 and HepG2.2.15 cells, whereas ZHX2 knockdown with two different siRNAs (ZHX2-1674, ZHX2-2360) resulted in elevated MDR1 mRNA levels in SMMC7721 cells. This difference was also seen at the protein level as determined by western blot (Figure 2B and Figure S2). These data support the possibility that ZHX2 represses MDR1 expression in HCC cells.


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 suppresses MDR1 expression and increases ADM retention of HCC cells(A and B) ZHX2, MDR1 and β-actin mRNA levels(A) and protein levels (B) were determined by RT-PCR and western blot in HepG2 and HepG2.2.15 cells transfected with pcDNA3.0 or pcZHX2 and in SMMC7721 cells that were transfected with ZHX2 siRNAs or control siRNA (siCON). (C) ZHX2-EGFP expression and intracellular ADM were determined by fluorescence microscopy of HepG2 cells in pEGFP-ZHX2-transfected cells treated with ADM as described in Methods and Materials. Representative panels are shown. Red, autofluorescence of ADM ; Green, fluorescence of EGFP-ZHX2 ; Blue, DAPI staining. Bars, 10 μm. (D) ADM accumulation (left panels) and retention (right panels) in pEGFP-ZHX2 transfected HepG2 cells were analyzed by flow cytometry. (E) Left panel: ADM accumulation and retention based on data in Figure 2D. Right panel: ADM-releasing index was calculated based on accumulation and retention data from flow cytometry. Data are shown as the mean±SD (n=3); *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: ZHX2 suppresses MDR1 expression and increases ADM retention of HCC cells(A and B) ZHX2, MDR1 and β-actin mRNA levels(A) and protein levels (B) were determined by RT-PCR and western blot in HepG2 and HepG2.2.15 cells transfected with pcDNA3.0 or pcZHX2 and in SMMC7721 cells that were transfected with ZHX2 siRNAs or control siRNA (siCON). (C) ZHX2-EGFP expression and intracellular ADM were determined by fluorescence microscopy of HepG2 cells in pEGFP-ZHX2-transfected cells treated with ADM as described in Methods and Materials. Representative panels are shown. Red, autofluorescence of ADM ; Green, fluorescence of EGFP-ZHX2 ; Blue, DAPI staining. Bars, 10 μm. (D) ADM accumulation (left panels) and retention (right panels) in pEGFP-ZHX2 transfected HepG2 cells were analyzed by flow cytometry. (E) Left panel: ADM accumulation and retention based on data in Figure 2D. Right panel: ADM-releasing index was calculated based on accumulation and retention data from flow cytometry. Data are shown as the mean±SD (n=3); *p < 0.05.
Mentions: In order to further confirm the negative regulation of ZHX2 on MDR1 in HCC, we then did in vitro studies. ZHX2 and MDR1 mRNA levels were compared in several liver cancer cell lines. RT-PCR analysis showed an inverse correlation between MDR1 and ZHX2 expression: cells with higher MDR1 mRNA levels (HepG2 and HepG2.2.15 cells) had lower ZHX2 mRNA levels whereas those with lower MDR1 (SMMC7721 cells) had higher ZHX2 (Figure S1A). Interestingly, ZHX2 expression level correlated with CDDP sensitivity in HCC cells (Figure S1B), indicating that ZHX2 closely correlates with MDR1 expression and chemotherapy sensitivity of HCC cells. To explore further the relationship between these two genes, ZHX2 was overexpressed or knocked down by transient transfection. As shown in Figure 2A, ZHX2 overexpression led to decreased MDR1 mRNA levels in HepG2 and HepG2.2.15 cells, whereas ZHX2 knockdown with two different siRNAs (ZHX2-1674, ZHX2-2360) resulted in elevated MDR1 mRNA levels in SMMC7721 cells. This difference was also seen at the protein level as determined by western blot (Figure 2B and Figure S2). These data support the possibility that ZHX2 represses MDR1 expression in HCC cells.

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