Flowjo x compensation5/26/2023 For example, p53 modulates HR and NHEJ repair signaling. Both p53 and PI3KCA can regulate DNA damage response or repair. Notably, p53 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA) are commonly mutated in oral cancer. peruviana-derived withanolide (PHA) remains unclear. HSP90 can regulate DNA repair proteins and apoptosis. A molecular docking study for several withanolide analogs identified heat shock protein 90 (HSP90) as a potential target. We found that PHA inhibited the DNA repair process by blocking mRNA expressions of DNA repair genes, but the interaction between PHA and DNA repair enzymes is unclear. PHA Induces Oxidative Stress-Dependent DNA Damage and Inhibits Oxidative Stress-Dependent DNA Repair in Oral Cancer Cells Accordingly, PHA causes selective apoptosis in an oxidative stress-dependent manner in oral cancer cells. Finally, both PHA-induced oxidative stress and apoptosis were suppressed by NAC. Moreover, these extrinsic, intrinsic, and executor proteins for apoptosis signaling in oral cancer cells were further confirmed by Cas 3/7 assays using the specific Cas 8, Cas 9, and Cas 3 inhibitors ( Figure 4C). In addition, the pancaspase inhibitor (ZVAD) supported that PHA-induced apoptosis contributed to PHA-induced antiproliferation in oral cancer cells ( Figure 4B). In contrast, PHA did not activate Cas 3/7 activity in non-malignant oral cells, suggesting that PHA induces selective apoptosis in oral cancer cells rather than in non-malignant oral cells. Similarly, PHA induced apoptosis ( Figure 3) and triggered apoptosis signaling for c-PARP in western blotting and Cas 3/7 activity assays ( Figure 4). Therefore, PHA generates selective antiproliferation, oxidative stress, and apoptosis associated with DNA damage induction and DNA repair suppression in oral cancer cells. Moreover, the PHA-induced changes were alleviated by the oxidative stress inhibitor N-acetylcysteine. In contrast, the mRNA expressions for DNA repair signaling, including homologous recombination (HR) and non-homologous end joining (NHEJ)-associated genes, were inhibited by PHA in oral cancer cells. In flow cytometry and immunofluorescence assays, PHA induced γH2AX expressions and increased the γH2AX foci number of DNA damages in oral cancer cells. PHA also demonstrated selective apoptosis in oral cancer cells rather than non-malignant cells in annexin V/7-aminoactinmycin D and caspase 3/7 activity assays. Moreover, PHA induced other oxidative stresses in oral cancer cells, such as mitochondrial superoxide generation and mitochondrial membrane potential depletion. This selective antiproliferation of PHA was associated with the selective generation of reactive oxygen species (ROS) in oral cancer cells rather than in non-malignant oral cells, as detected by flow cytometry. During an ATP assay, PHA provided high cytotoxicity to two oral cancer cell lines (CAL 27 and Ca9-22) but no cytotoxicity to two non-malignant oral cells (HGF-1 and SG). This study examined the selective antiproliferation ability of PHA and explored detailed mechanisms of apoptosis, DNA damage, and repair. Either drug-induced apoptosis and DNA damage or DNA repair suppression may effectively inhibit cancer cell proliferation. The selective antiproliferation to oral cancer cells of Physalis peruviana-derived physapruin A (PHA) is rarely reported.
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