BAG3 down-modulation sensitizes HPV18(+) HeLa cells to PEITC-induced apoptosis and restores p53.
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ABSTRACT:
SUBMITTER: Cotugno R
PROVIDER: S-EPMC7116956 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: NDRG2, a member of N-Myc downstream regulated gene family, plays some roles in cellular stress, cell differentiation and tumor suppression. We have found that NDRG2 expression in cervical cancer Hela cells increases significantly upon stimulation with cisplatin, the most popular chemotherapeutic agent currently used for the treatment of advanced cervical cancer. This interesting phenomenon drove us to evaluate the role of NDRG2 in chemosensitivity of Hela cells. METHODS: In the present study, RNA interference was employed to down-regulate NDRG2 expression in Hela cells. RT-PCR and Western blot were used to detect expression of NDRG2, Bcl-2 and Bax in cancer cells. Real-time PCR was applied to detect miR-15b and miR-16 expression levels. Drug sensitivity was determined with MTT assay. Cell cloning efficiency was evaluated by Colony-forming assay. Apoptotic cells were detected with annexin V staining and flow cytometry. RESULTS: In vitro drug sensitivity assay revealed that suppression of NDRG2 could sensitize Hela cells to cisplatin. Down-regulation of NDRG2 didn't influence the colony-forming ability but promoted cisplatin-induced apoptosis of Hela cells. Inhibition of NDRG2 in Hela cells was accompanied by decreased Bcl-2 protein level. However, Bcl-2 mRNA level was not changed in Hela cells with down-regulation of NDRG2. Further study indicated that miR-15b and miR-16, two microRNAs targetting Bcl-2, were significantly up-regulated in NDRG2-suppressed Hela cells. CONCLUSIONS: These data suggested that down-regulation of NDRG2 could enhance sensitivity of Hela cells to cisplatin through inhibiting Bcl-2 protein expression, which might be mediated by up-regulating miR-15b and miR-16.
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Project description:The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.
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Project description:The p53 tumor suppressor induces cell growth arrest and apoptosis in response to DNA damage. Because these functions are achieved largely by the transcriptional properties of p53, nuclear localization of the protein is essential. Indeed, the tumors with aberrant cytoplasmic localization of wild-type p53 often exhibit an impaired response to DNA damage. In this study, we report that Thr-55 phosphorylation induces the association of p53 with the nuclear export factor CRM1, leading to p53 nuclear export. We further show that MDM2 also promotes the CRM1-p53 association and Thr-55 phosphorylation is required for this process. Interestingly, inhibition of Thr-55 phosphorylation by a dietary flavonoid, apigenin, specifically blocks the CRM1-p53 association, restores p53 nuclear localization, and sensitizes tumor cells with cytoplasm localized wild-type p53 to DNA damage. These data provide insights into the regulation of p53 nuclear localization by post-translational modification and suggest an avenue for targeted therapy for cancers caused by aberrant cytoplasm localization of wild-type p53.
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Project description:TP53 mutations are found in 50% of all cancers and mutated TP53 status is considered poor for treatment. However, some TP53 mutations exhibit only partial loss-of-function (LOF), meaning they retain residual transcriptional and non-transcriptional activities that are potentially beneficial for therapy. Earlier we have characterized a knock-in mouse model for the partial LOF mutant Trp53E177R (p53RR). Reduced DNA binding cooperativity of this mutant led to the loss of p53-dependent apoptosis, while p53 functions in cell cycle control, senescence, metabolism, and antioxidant defense remained intact. Concomitantly, tumor suppression was evident but strongly compromised compared to wild-type mice. Here we used the Trp53E177R mouse as a model to investigate whether residual functions of mutant p53 can be engaged to induce cell death, which is considered the most desirable outcome of tumor therapy. We made use of Mdm2 knock-out in developing embryos as a sensitive tool for detecting remaining p53 activities. Genetic ablation of Mdm2 led to embryonic lethality in Trp53E177R/E177R homozygotes at days 9.5-11.5. This effect was not rescued by concomitant p21-knockout, indicating its independence of p21-mediated cell cycle arrest. Instead, immunohistochemical analysis showed widespread apoptosis in tissues of defective embryos accompanied by persistent accumulation of p53RR protein. This led to partial restoration of the mutant's proficiency in transcriptional induction of the pro-apoptotic genes Bbc3 (Puma) and Bax. These data indicate that increased quantity can compensate for qualitative defects of p53 mutants and suggest that Mdm2-targeting (potentially in combination with other drugs) might be effective against cells bearing p53 partial LOF mutants.
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Project description:p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.
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