Baharara J et al. (2016), Evaluation of the Anti-proliferative Effects of...

ECB-ART-44491

Avicenna J Med Biotechnol 2016 Jan 01;81:29-35.

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Evaluation of the Anti-proliferative Effects of Ophiocoma erinaceus Methanol Extract Against Human Cervical Cancer Cells.

Baharara J , Amini E , Namvar F .

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BACKGROUND: Marine organisms provide appreciable source of novel bioactive compounds with pharmacological potential. There is little information in correlation with anti-cancer activities of brittle star. In the present study, anti-neoplastic efficacy of Ophiocoma erinaceus methanol extract against human cervical cancer cells was investigated. METHODS: The HeLa cells were cultured and exposed to brittle star methanol extract for 24 and 48 hr. The anti-proliferative properties were examined by MTT assay and the type of cell death induced was evaluated through morphological changes, flow cytometry, Annexin kit and caspase assay. To assess the anti-metastatic activity, wound healing assay was conducted and photographs were taken from the scratched areas. Further, to understand molecular mechanism of cell apoptosis, the expression of Bax was evaluated. RESULTS: The morphological analysis and MTT assay exhibited that the brittle star methanol extract can exert dose dependent inhibitory effect on cells viability (IC50, 50 μg/ml). Flow cytometry and fluorescence microscopy demonstrated increment of sub-G1 peak, early and late apoptosis in HeLa treated cells. Wound healing migration assay showed that brittle star extract has anti-neoplastic efficacy by inhibiting cell migration. Caspase assay and RT-PCR analysis revealed that brittle star methanol extract induced caspase dependent apoptosis in HeLa cells through up-regulation of caspase-3 followed by up-regulation of Bax gene which is a hallmark of intrinsic pathway recruitment. CONCLUSION: These results represented further insights into the chemopreventive potential of brittle star as a valuable source of unknown therapeutic agents against human cervical cancer.

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Species referenced: Echinodermata
Genes referenced: bax LOC115919910 LOC580856

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References [+] :

Andersson, Biological activity of saponins and saponin-like compounds from starfish and brittle-stars. 1989, Pubmed, Echinobase


	Figure 2. Cytomorphological alterations of HeLa cells treated with brittle star alcoholic extract. A) Control (without treatment) indicated accumulation of HeLa cells uniformly. (B, C, D, E, F) HeLa cells treated with 12.5, 25, 50, 100, 200 μg/ml extract for 48 hr indicate apoptotic features such as cytoplasmic blebbing (orange arrows), round shape (black arrows) and apoptotic body formation (white arrows), respectively. Magnification=×400.
	Figure 3. Detection of apoptosis using flow cytometry. Histogram of untreated and treated HeLa cells with 50, 100 μg/ml brittle star extract indicated apoptosis induction in HeLa cells.
	Figure 4. Apoptotic morphological alterations in HeLa treated cells with Annexin V-PI staining. (A, B) HeLa cells untreated observed by inverted fluorescence microscope, respectively. C, D) The HeLa treated cells with 50 μg/ml brittle star methanol extract indicate externalization of phosphatidylserine to outer leaflet as one of the apoptosis main characteristic. Green color is indicating apoptotic cells (400×magnification).

	Figure 6. Inhibitory effect of brittle star alcoholic extract on cell migration of HeLa cells by wound healing assay. A) Control group, 48 hr after scratch formation B) Treatment group with 50 μg/ml of brittle star extract (IC50 value), which exhibited the brittle star extract effectively suppressed cell motility 48 hr after scratch formation.
	Figure 7. A) Qualitative assessment of the effect of brittle star methanol extract on expression of Bax mRNA (135 bp) in HeLa cells. B2M mRNA was used as an internal control gene (230 bp). B) The densitometry of gel quantitatively evaluated by image J indicates up-regulation of Bax pro-apoptotic gene in treated HeLa cells.