Cleistopholine isolated from Enicosanthellum pulchrum exhibits apoptogenic properties in human ovarian cancer cells

Background Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated. Hypothesis/purpose This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. Study design/methods Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay. Results The IC 50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptosis was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the treated cells compared with the untreated cells. In contrast, Bcl-2, Hsp70 and survivin decreased in expression upon cleistopholine treatment. Cell cycle was arrested at the G0/G1 phase and cell population percentage significantly increased to 43.5%, 45.4% and 54.3% in time-dependent manner in the cleistopholine-treated CAOV-3 cells compared with the untreated cells at 24, 48 and 72 h respectively. Conclusion The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian cancer disease.