Baharara J and Amini E (2015), The Potential of Brittle Star Extracted Polysac...

ECB-ART-44359

Avicenna J Med Biotechnol 2015 Jan 01;74:151-8.

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The Potential of Brittle Star Extracted Polysaccharide in Promoting Apoptosis via Intrinsic Signaling Pathway.

Baharara J , Amini E .

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BACKGROUND: Anti-cancer potential of marine natural products such as polysaccharides represented therapeutic potential in oncological researches. In this study, total polysaccharide from brittle star [Ophiocoma erinaceus (O. erinaceus)] was extracted and chemopreventive efficacy of Persian Gulf brittle star polysaccharide was investigated in HeLa human cervical cancer cells. METHODS: To extract polysaccharide, dried brittle stars were ground and extracted mechanically. Then, detection of polysaccharide was performed by phenol sulfuric acid, Ultra Violet (UV)-sulfuric acid method and FTIR. The anti proliferative activity of isolated polysaccharide was examined by MTT assay and evaluation of cell death was done through morphological cell changes; Propodium Iodide staining, fluorescence microscopy and caspase-3, -9 enzymatic measurements. To assess its underlying mechanism, expression of Bax, Bcl-2 was evaluated. RESULTS: The polysaccharide detection methods demonstrated isolation of crude polysaccharide from Persian Gulf brittle star. The results revealed that O. erinaceus polysaccharide suppressed the proliferation of HeLa cells in a dose and time dependent manner. Morphological observation of DAPI and Acridine Orange/Propodium Iodide staining was documented by typical characteristics of apoptotic cell death. Flow cytometry analyses exhibited the accumulation of treated cells in sub-G1 region. Additionally, polysaccharide extracted induced intrinsic apoptosis via up-regulation of caspase-3, caspase-9 and Bax along with down-regulation of Bcl-2 in HeLa cells. CONCLUSION: Taken together, the apoptosis inducing effect of brittle star polysaccharide via intrinsic pathway confirmed the anti tumor potential of marine polysaccharide. Therefore, these findings proposed new insight into anti cancer properties of brittle star polysaccharide as a promising agent in cervical cancer treatment.

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Species referenced: Echinodermata
Genes referenced: bax LOC115919910

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References [+] :

Albalasmeh, A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. 2013, Pubmed

	Figure 1. Schematic depiction of polysaccharide extraction method from O. erinaceus.
	Figure 2. A) Phenol-sulfuric acid reaction, production of furfural derivative characterized the carbohydrate content. (A=extracted polysaccharide, B= blank, C=D-GLC). B) FTIR spectra in the 4000-400 cm_1 region of D-GLC and O. erinaceus which confirmed the similar peak of carbohydrates in extracted polysaccharide.

	Figure 4. Morphological effect of O. erinaceus extracted polysaccharide on HeLa cells. Panel A) visualization with inverted microscopy. Panel B) AO/ PI double staining and Panel C) DAPI staining. Arrows in panel B from left to right point to live cells, apoptotic cells and necrotic cells and in panel C indicate chromatin condensation.
	Figure 5. Estimation of apoptotic effect of O. erinaceus crude polysaccharide on cervical cancer cells by flow cytometry. Flow cytometry histogram of untreated and treated HeLa cells showed that inhibitory concentrations of extracted polysaccharide (25, 50 μg/ml) increased sub-G1 peak demonstrating involvement of apoptotic cells in cytotoxicity of brittle star polysaccharide.

	Figure 7. HeLa cells were treated with O. erinaceus extracted polysaccharide and the mRNA expression of Bax and Bcl-2 were assessed. RT-PCR analysis indicated that isolated polysaccharide exerted pro apoptotic effect on HeLa cells.