Purpose Estrogen receptor (ER) and the Piwi-Like Protein 1 (PIWIL1) oncogene alter the expression and methylation of oncogenes and tumor suppressor genes. Potential cross-talk between ER and the methylated-DNA binding protein, Kaiso, and its downstream genes c-Myc and CDKN2A remains unexplored. Dual targeting of ER and PIWIL1 might be an excellent therapeutic approach. Methods Initially, we used in silico molecular docking to identify potential blockers for ER and PIWIL1 among seven natural previously reported anticancer compounds. The best candidate was tested in vitro for its influence on KAISO expression by RT-PCR, promoter methylation of c-Myc and CDKN2A by methylation-specific PCR, cell cycle and apoptosis by flowcytometry, and cytotoxicity by MTT assay in the ER(+) MCF-7, and ER(−) MDA-231-MB breast cancer cell lines. Results Betulinic acid (BA), docked ER-α and PIWIL1 perfectly at critical amino acids with the lowest binding energy. Moreover, ER and PIWIL1 docked Kaiso with root mean square deviation = 5.05 Å and 4.75 Å, respectively, at the critical amino acid Y537. Experimentally, BA caused a dose-dependent cytotoxicity in both MCF-7 and MDA-MB-231 cells (IC50= 21.3 ± 0.05 and 100 ± 0.34 µM), respectively. BA caused a significant dose-dependent G0/G1 cell cycle arrest, apoptosis induction, and upregulated Kaiso expression coincided with increased methylation of c-Myc and demethylation of CDKN2A promoter regions. Conclusion: We are proposing a novel anticancer mechanism of action for BA via potential inhibition of Estrogen-ER-α and PIWIL1 signaling, modulation of KAISO expression and selective alterations in methylation state of c-Myc and CDKN2A key cancer-associated genes.
Purpose Estrogen receptor (ER) and the Piwi-Like Protein 1 (PIWIL1) oncogene alter the expression and methylation of oncogenes and tumor suppressor genes. Potential cross-talk between ER and the methylated-DNA binding protein, Kaiso, and its downstream genes c-Myc and CDKN2A remains unexplored. Dual targeting of ER and PIWIL1 might be an excellent therapeutic approach.Methods Initially, we used in silico molecular docking to identify potential blockers for ER and PIWIL1 among seven natural previously reported anticancer compounds. The best candidate was tested in vitro for its in uence on KAISO expression by RT-PCR, promoter methylation of c-Myc and CDKN2A by methylation-speci c PCR, cell cycle and apoptosis by owcytometry, and cytotoxicity by MTT assay in the ER(+) MCF-7, and ER(−) MDA-231-MB breast cancer cell lines.Results Betulinic acid (BA), docked ER-α and PIWIL1 perfectly at critical amino acids with the lowest binding energy. Moreover, ER and PIWIL1 docked Kaiso with root mean square deviation = 5.05 Å and 4.75 Å, respectively, at the critical amino acid Y537. Experimentally, BA caused a dose-dependent cytotoxicity in both MCF-7 and MDA-MB-231 cells (IC 50 = 21.3 ± 0.05 and 100 ± 0.34 µM), respectively. BA caused a signi cant dose-dependent G0/G1 cell cycle arrest, apoptosis induction, and upregulated Kaiso expression coincided with increased methylation of c-Myc and demethylation of CDKN2A promoter regions. Conclusion:We are proposing a novel anticancer mechanism of action for BA via potential inhibition of Estrogen-ER-α and PIWIL1 signaling, modulation of KAISO expression and selective alterations in methylation state of c-Myc and CDKN2A key cancer-associated genes. MCF-7 cells (48). One explanation for the G0/G1 cell cycle arrest and the subsequent apoptosis may be due to BA's ability to induce early DNA damage via direct binding to and inhibition of DNA topoisomerase II in a dose-dependent manner (49).Evading apoptosis is a fundamental hallmark of cancer cells (50). Therefore, combating aggressive chemo-therapeutic-resistant cancers like ER (-) cells through targeting both apoptosis and an alternative cell death mechanism such as necroptosis could be adopted for the treatment of such cancers, indeed several studies are reporting on the concept of harnessing necroptosis for ghting aggressive cancers including triple-negative breast cancers with necroptosis-inducing agents (51-54).In agreement with the cell cycle arrest and morphology results, BA treatment has induced apoptosis in a dose-dependent in MCF-7 but not necrotic cells even at the low, 10% IC 50 dose. As to be expected, MDA-MB-231 cells were more resistant at low BA dose and responded relatively differently, only treatment with the high, IC 50 dose (100 µM) caused a signi cant increase in both apoptotic and necrotic cells. These data also explain the greater susceptibility of MCF-7 cells to lower doses of BA compared to MDA-MB-231 cells that were only affected using high doses which shifted the cell death mechanism from solely apoptosis to a c...
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