Production and characterization of phycobiliproteins from cyanobacteria and their effects on HepG2 cancer cell lines

Phycobiliproteins have a wide range of promising applications in food, nutraceutical and biomedical industries because of their antibacterial, antifungal, anticancer and antioxidant properties. Due to the fact that synthetic drugs for treating cancer have some adverse effects, there is a need for alternative therapy by using natural compounds to inhibit the growth of cancer cells. Therefore, this study emphasized on the effect of cyanobacterial phycobiliproteins on the growth and proliferation of cancer cell lines. Marine and freshwater cyanobacteria were screened to compare the concentration of phycobiliproteins and purity of phycocyanin subjected to different drying methods. Results from oven dried showed that total phycobiliproteins production (208.1±3.14mg/g) and purity ratio of phycocyanin (1.2) was significantly (p<0.05) higher in marine periphytic Geitlerinema sp. compared to other cyanobacterial species. Therefore, Geitlerinema sp. was used for succeeding experiments. Phycobiliproteins production and purity ratio was significantly (p<0.05) higher in Geitlerinema sp. at 35ºC, 30 ppt and pH 8 compared to other temperature, salinity and pH levels. Under these conditions, protein concentration in phycobiliproteins crude extract from marine Geitlerinema sp. was 64.5 ± 0.2μg/ml with molecular weight approximately between 19 KDa to 26 KDa. In addition, crude phycobiliproteins extract of Geitlerinema sp. have the similar characteristics as commercial phycocyanin. In vitro screening of cytotoxic effects of crude phycobiliproteins extract (rich fraction of phycocyanin) found that IC50 (half maximal inhibitory concentration) value (3.8±1.26 μg/ml) lower in HepG2 cell line. In addition, concurrent study of the crude phycobiliproteins extract with paclitaxel showed IC50 value of crude phycobiliproteins extract similar to the paclitaxel and no negative effect on normal liver cell line (Chang liver cell line). Morphological changes of HepG2 treated with crude phycobiliproteins extract (rich fraction of phycocyanin) exhibited cell shrinkage, chromatin condensation, membrane blebbing, cytoplasomic extrusions and formation of apoptotic bodies. The Annexin V assay revealed apoptotic induction in HepG2 cells exposed to the crude phycobiliproteins extract in a timedependent manner, whereas DNA fragmentation of HepG2 cells were detected using 1.0% agarose gel electrophoresis. Cell cycle analysis showed that there was significant (p<0.05) G1 phase arrest at each time point from 12 to 72 h. Crude phycobiliproteins extract significantly (p<0.05) stimulated both caspase 9 and 3 activities. Results from the apoptotic gene expression showed significant (p<0.05) down-regulated expression level of anti-apoptotic Bcl2 gene, up-regulated expression level of Bax, Bak that increases mitochondrial membrane potential (MMP) and pore formation. Subsequently, cytochrome C and upregulated expression level of Apaf-1 were released. These effects were associated with the induction of caspase-9, -3 activities, and subsequent DNA fragmentation by blocking PARP followed by intrinsic pathway of apoptosis. Meanwhile, crude phycobiliproteins extract significantly (p<0.05) down-regulated or inhibited NF-kB2 and NF-kB1 respectively which may directly or indirectly regulate Bcl2 through the modulation of anti-apoptotic genes to induce intrinsic pathway of apoptosis. In addition, it was found that phycobiliproteins crude extract from the Geitlerinema sp. Significantly (p<0.05) inhibited TNF-α, FADD, TRADD, Interleukin-6, down regulation of IFN-gamma and JNK-1 gene but no activation of caspase 8 which strongly suggested that induction of apoptosis in HepG2 cells is without involvement of extrinsic pathway. Thus, crude phycobiliproteins extract (rich fraction of phycocyanin) from marine cyanobacteria Geitlerinema sp. has significant induction of apoptosis on HepG2 cells via multiple signal transduction pathways involving intrinsic pathway and may have potential therapeutic value as anticancer towards human hepatocellular carcinoma.

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