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

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. This study aims to isolate cleistoph...

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Published in:Phytomedicine (Stuttgart) 2016-04, Vol.23 (4), p.406-416
Main Authors: Nordin, Noraziah, Majid, Nazia Abdul, Mohan, Syam, Dehghan, Firouzeh, Karimian, Hamed, Rahman, Mashitoh Abdul, Ali, Hapipah Mohd, Hashim, Najihah Mohd
Format: Article
Language:English
Subjects:
Angiogenesis Inhibitors - pharmacology
Angiogenesis Inhibitors - therapeutic use
Annexin A5 - metabolism
Annonaceae - chemistry
Anthraquinones - isolation & purification
Anthraquinones - pharmacology
Anthraquinones - therapeutic use
Antineoplastic Agents, Phytogenic - pharmacology
Antineoplastic Agents, Phytogenic - therapeutic use
Apoptosis
Apoptosis - drug effects
Aza Compounds - isolation & purification
Aza Compounds - pharmacology
Aza Compounds - therapeutic use
Cancer cells
CAOV-3
Caspase 9 - metabolism
Caspases - metabolism
Cell Cycle - drug effects
Cell Cycle Checkpoints - drug effects
Cell Line, Tumor
Cell Proliferation
Cleistopholine
Computer industry
Cytochromes c - metabolism
DNA Fragmentation
Enicosanthellum pulchrum
Exhibitions
Female
Humans
Membrane Potential, Mitochondrial - drug effects
Mitochondria - drug effects
Mitochondria - metabolism
Natural product
Ovarian cancer
Ovarian Neoplasms - drug therapy
Ovarian Neoplasms - metabolism
Phytotherapy
Plant Extracts - pharmacology
Plant Extracts - therapeutic use
Plant Roots
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Summary: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. 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 72h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. 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. The IC50 of cleistopholine (61.4µM) was comparable with that of the positive control cisplatin (62.8µM) at 24h of treatment. Apoptosis was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48h, 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 72h respectively. The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian ca
ISSN:0944-7113
1618-095X
DOI:10.1016/j.phymed.2016.02.016