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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 combined with CDDP act cooperatively to inhibit xenograft tumor growth in nude miceNude mice containing subcutaneous HepG2.2.15 tumors were randomized to receive intra-tumor injections of pcDNA3.0 or pcZHX2, and half of each group were also injected with CDDP. (A) Tumor volume was calculated every other day for the entire 16-day study. Data are shown as the mean±SD ( Two-way ANOVA, ***p < 0.001). (B) Tumor weights in four different cohorts were measured at the end of the experiment. Data are shown as the mean±SD(n≥4); *p <0.05, **p<0.01. (C) Immunohistochemical analysis of ZHX2 expression in tumor samples. Bars, 20 pixel. (D) TUNEL staining as a measure of apoptosis in tumor samples indicate greatest staining in pcZHX2 plus CDDP injected tumors. Bars, 50 μm.
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Figure 5: ZHX2 combined with CDDP act cooperatively to inhibit xenograft tumor growth in nude miceNude mice containing subcutaneous HepG2.2.15 tumors were randomized to receive intra-tumor injections of pcDNA3.0 or pcZHX2, and half of each group were also injected with CDDP. (A) Tumor volume was calculated every other day for the entire 16-day study. Data are shown as the mean±SD ( Two-way ANOVA, ***p < 0.001). (B) Tumor weights in four different cohorts were measured at the end of the experiment. Data are shown as the mean±SD(n≥4); *p <0.05, **p<0.01. (C) Immunohistochemical analysis of ZHX2 expression in tumor samples. Bars, 20 pixel. (D) TUNEL staining as a measure of apoptosis in tumor samples indicate greatest staining in pcZHX2 plus CDDP injected tumors. Bars, 50 μm.

Mentions: In order to detect whether ZHX2 and CDDP could cooperate to inhibit tumor growth in vivo, HepG2.2.15 xenograft tumors were treated with CDDP and/or plasmid DNA (pcDNA3.0 or pcZHX2). As seen in previous studies [16], either CDDP treatment or ZHX2 overexpression alone inhibited xenograft growth significantly (Figure 5A). However, the combination of pcZHX2 and CDDP cooperatively inhibited tumor growth to a greater extent than treatment with pcZHX2 or CDDP alone (Figure 5A). Consistent with the diminished growth curves, tumor weights at time of sacrifice were also significantly reduced in mice with combined treatment compared to mice treated with pcZHX2 or CDDP alone (Figure 5B). Immunohistochemistry staining verified the ZHX2 overexpression in tumors injected with pcZHX2 (Figure 5C). TUNEL staining confirmed that ZHX2 promoted CDDP-induced apoptosis of tumor cells (Figure 5D). These results indicated that ZHX2 overexpression enhances the ability of CDDP to inhibit HepG2.2.15 growth in vivo.


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 combined with CDDP act cooperatively to inhibit xenograft tumor growth in nude miceNude mice containing subcutaneous HepG2.2.15 tumors were randomized to receive intra-tumor injections of pcDNA3.0 or pcZHX2, and half of each group were also injected with CDDP. (A) Tumor volume was calculated every other day for the entire 16-day study. Data are shown as the mean±SD ( Two-way ANOVA, ***p < 0.001). (B) Tumor weights in four different cohorts were measured at the end of the experiment. Data are shown as the mean±SD(n≥4); *p <0.05, **p<0.01. (C) Immunohistochemical analysis of ZHX2 expression in tumor samples. Bars, 20 pixel. (D) TUNEL staining as a measure of apoptosis in tumor samples indicate greatest staining in pcZHX2 plus CDDP injected tumors. Bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: ZHX2 combined with CDDP act cooperatively to inhibit xenograft tumor growth in nude miceNude mice containing subcutaneous HepG2.2.15 tumors were randomized to receive intra-tumor injections of pcDNA3.0 or pcZHX2, and half of each group were also injected with CDDP. (A) Tumor volume was calculated every other day for the entire 16-day study. Data are shown as the mean±SD ( Two-way ANOVA, ***p < 0.001). (B) Tumor weights in four different cohorts were measured at the end of the experiment. Data are shown as the mean±SD(n≥4); *p <0.05, **p<0.01. (C) Immunohistochemical analysis of ZHX2 expression in tumor samples. Bars, 20 pixel. (D) TUNEL staining as a measure of apoptosis in tumor samples indicate greatest staining in pcZHX2 plus CDDP injected tumors. Bars, 50 μm.
Mentions: In order to detect whether ZHX2 and CDDP could cooperate to inhibit tumor growth in vivo, HepG2.2.15 xenograft tumors were treated with CDDP and/or plasmid DNA (pcDNA3.0 or pcZHX2). As seen in previous studies [16], either CDDP treatment or ZHX2 overexpression alone inhibited xenograft growth significantly (Figure 5A). However, the combination of pcZHX2 and CDDP cooperatively inhibited tumor growth to a greater extent than treatment with pcZHX2 or CDDP alone (Figure 5A). Consistent with the diminished growth curves, tumor weights at time of sacrifice were also significantly reduced in mice with combined treatment compared to mice treated with pcZHX2 or CDDP alone (Figure 5B). Immunohistochemistry staining verified the ZHX2 overexpression in tumors injected with pcZHX2 (Figure 5C). TUNEL staining confirmed that ZHX2 promoted CDDP-induced apoptosis of tumor cells (Figure 5D). These results indicated that ZHX2 overexpression enhances the ability of CDDP to inhibit HepG2.2.15 growth in vivo.

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