casticin and Uterine-Cervical-Neoplasms

To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism.. Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L).. DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells.. CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.

Topics: Cell Line, Tumor; Female; HeLa Cells; Humans; Neoplastic Stem Cells; RNA, Small Interfering; Uterine Cervical Neoplasms

Casticin, one of the main components from Fructus Viticis, has been reported to inhibit the growth of various cancer cells, including the human cervical cancer cell line HeLa. The purpose of this study was to examine the apoptotic activity and molecular mechanism of casticin action on human cervical cancer cells. The apoptotic activity of casticin on human cervical cancer HeLa, CasKi, SiHa and peripheral blood mononuclear cells (PBMCs) was measured using a histone/DNA ELISA assay, flow cytometry with propidium iodide (PI) staining and DNA agarose gel electrophoresis. The mitochondrial membrane potential and reactive oxygen species (ROS) production were evaluated by flow cytometry analysis. Caspase activities were assayed using a caspase colorimetric activity assay kit. Protein expression levels of cytochrome c, Bax, Bcl-2, Bcl-xL and XIAP were analyzed by Western blotting. Casticin caused accumulation of the Sub-G1 cells and increased reactive oxygen species (ROS) production in HeLa, CasKi, SiHa cell lines, but not in PBMCs. Apoptosis of HeLa cells was induced by casticin via mitochondrial release of cytochrome c due to the reduction of mitochondrial trans-membrane potential, activation of caspase-3 and -9, and the production of reactive oxygen species. The pan caspase inhibitor zVAD-FMK, the caspase-9 inhibitor zLEHD-fmk and N-acetylcysteine suppressed casticin-induced apoptosis. Bax was upregulated, while expression levels of Bcl-xL and XIAP were downregulated. However, there was no change in the expression of Bcl-2 under the same treatment. Our results indicate that casticin-induced apoptosis of cervical cancer cells is mediated by ROS generation and mitochondrial signaling pathways.

Topics: Apoptosis; Caspases; Female; Flavonoids; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Signal Transduction; Uterine Cervical Neoplasms