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Avicenna J Med Biotechnol
2019 Jan 01;113:208-214.
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The p53 Modulated Cytotoxicity of Ophiocoma scolopendrina Polysaccharide Against Resistance Ovarian Cancer Cells.
Amini E
,
Baharara J
,
Afzali M
,
Nikdel N
.
Abstract
Background: Marine environment is a valuable source of bioactive compounds with variable medicinal properties. Previously, it was shown that Ophiocoma erinaceus extracted polysaccharide has prominent cytotoxic effect on HeLa human cervical cancer cells. In the present study, the anti-cancer properties of polysaccharide extracted from Ophiocoma scolopendrina (O. scolopendrina) were examined in comparison with paclitaxel as a conventional drug against resistant ovarian cancer; also, its related mechanism against A2780cp ovarian cancer cells was investigated.
Methods: The A2780cp cancer cells and NIH3T3 normal cells were cultured and treated with different concentrations of polysaccharide extracted from O. scolopendrina for 24 hr and 48 hr. Then, cell toxicity was studied by MTT assay, morphology of cells was observed under inverted microscopy and the type of induced cancer cell death was assessed by annexin V-FITC, propodium iodide and acridine orange staining. Finally, the apoptosis pathway was determined by measurement of caspase-3 and caspase-9 activity and assessment of p53 and Bcl-2. The statistical analysis was performed by SPSS software, one way ANOVA and p<0.05 was considered significant.
Results: Our observations from MTT assay and morphological assessment exhibited that O. scolopendrina isolated polysaccharide inhibited proliferation of ovarian cancer cells with IC50 of 35 μg/ml, while paclitaxel suppressed tumor cell growth with IC50=10 μg/ml. In contrast, MTT observations revealed low cytotoxicity of these chemotherapeutic agents against NIH3T3 normal cells. Also, the analysis correlated with induced cell death elucidated that concurrent treatment of polysaccharide plus paclitaxel had a further anti-cancer effect against A2780cp cells mainly through restoration of p53 and mitochondrial apoptosis cell death induction.
Conclusion: Taken together, our research supports the finding that application of polysaccharide extracted from O. scolopendrina can be considered a promising marine chemotherapeutic approach for advancing efficacy of paclitaxel in treatment of resistant ovarian cancer. Additional in vivo experiments are required to elucidate the role of brittle star polysaccharides in animal and clinical trials.
Figure 2. Morphological alterations of A2780cp ovarian cancer cells and NIH3T3 fibroblast cells under exposure to inhibitory concentrations of O. scolopendrina polysaccharide and taxol using MTT assay. Reduction of the amount of formazan crystals in cancer cells compared with the untreated cells demonstrated anti-proliferative effect under inverted microscope (Magnification ×200). The emergence of high formazan crystals in NIH3T3 treated cells revealed the effect of extracted polysaccharide and taxol on ovarian cancer cells. A, E) Control, B, F) 10 μg/ml taxol, C, G) 30 μg/ml polysaccharide, D, H) 10 μg/ml taxol +12.5 μg/ml polysaccharide.
Figure 3. DAPI staining, changes in cell nucleus indicating nuclear fragmentation as clear features of apoptosis in treated cells with 50% inhibitory concentrations of polysaccharide and taxol, under fluorescence microscopy (Magnification ×400). A) Control, B) 10 μg/ml taxol, C) 30 μg/ml polysaccharide, D) 10 μg/ml taxol +12.5 μg/ml polysaccharide.
Figure 4. Acridine orange/propodium iodide staining indicated the apoptosis induction under treatment with polysaccharide and taxol. The untreated cells are green and indicator of live cells, yellow and orange color indicates apoptosis (Magnification ×400). A) Control, B) 10 μg/ml taxol, C) 30 μg/ml polysaccharide, D) 10 μg/ml taxol +12.5 polysaccharide μg/ml.
Figure 5. A) Histograms obtained by the annexin V-FITC / PI kit indicating apoptosis induction in A2780cpcells treated with O. scolopendrina polysaccharide and paclitaxel. B) PI assay demonstrated sub-G1 peak that is one of most important indicators of apoptosis induced by polysaccharide and paclitaxel. Co-treatment: 10 μg/ml taxol +12.5 μg/ml polysaccharide.
Figure 6. The measurement of caspase-3 and -9 activity in A2780cp cells treated with O. scolopendrina polysaccharide and paclitaxel. To determine whether apoptosis induction is caspase-dependent, caspas-3 and caspase-9 assay was used. Histogram represented that the caspase-3 and -9 activity increased under incubation with brittle star polysaccharide and paclitaxel as compared to untreated cells. As shown, the increment of caspase-3 and caspase-9 activity revealed that the O. scolopendrina polysaccharide can improve the cytotoxic effect of taxol via intrinsic apoptosis pathway.
Figure 7. A2780cp cells were treated with O. erinaceus extracted polysaccharide, taxol and synergistic treatment. The mRNA expressions of Bax and Bcl-2 were assessed by RT-PCR that demonstrated cytotoxic effect of polysaccharide, taxol and co treatment via intrinsic pathway (con= control or untreated cells, poly=30 μg/ml brittle star polysaccharide, tax=10 μg/ml taxol or paclitaxel, poly+tax=co treatment of 10 μg/ml taxol and 12.5 μg/ml polysaccharide).
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