The Activities And Mechanisms Of RLj-RD3 On Ovarian Carcinoma HeyA8 Cells

Ovarian carcinoma is a malignant tumor derived from ovary,which usually brings relative higher mortality rate in clinical studies due to its asymptomatic nature.At present,lots of deficiencies related to early diagnosis and treatment of ovarian carcinoma are still existent.Thus,further studies would focus on the pathogenesis of ovarian carcinoma and screening more effective anti-tumor drugs with fewer side effects.RGD?Arg-Gly-Asp?motif containing peptides usually have an arginine-glycine-aspartate sequence and could specially recognize integrins expressed on the cell surface.In recent years,a great deal of studies has concentrated on the anti-tumor application of RGD motif containing peptides.In the previous studies,recombinant RGD3 peptide?rLj-RGD3?from lamprey buccal glands was reported to inhibit platelet aggregation,as well as to induce the apoptosis of endothelial cells?ECV304?,liver and breast cancer cells.However,little work has been done on the effect of rLj-RGD3on the ovarian carcinoma cells?HeyA8?.In the present study,the inhibitory effect of rLj-RGD3 on the HeyA8 proliferation and its mechanisms were analyzed in detail.According to the MTT analysis,the proliferation of HeyA8 cells was significantly suppressed by rLj-RGD3 in a dose dependent manner with the IC₅₀ of 10?M.Furthermore,lactate dehydrogenase?LDH?assay indicated that rLj-RGD3could release LDH by destroying the cell membrane of HeyA8 cells.While the activity of rLj-RGD3 on LDH release is relative lower,indicated that the anti-proliferative effect of rLj-RGD3 on HeyA8 cells was partly due to its cytotoxic activity.In addition,both hoechst33258 and Annexin V/PI double staining assays showed that rLj-RGD3 could induce chromatin condensation in HeyA8 cells,and increase the percentage of early or late apoptotic cells,suggested that rLj-RGD3 is capable of inducing apoptosis in HeyA8 cells.Importantly,the present study proved that rLj-RGD3 could induce apoptosis of HeyA8 cells by changing the mitochondrial membrane potential,arrangement of cytoskeleton,expression level of apoptosis related proteins,concentration of calcium and phosphorylation level of proteins in MAPK signaling pathways by using flow cytometry and western blot.Furthermore,rLj-RGD3 could inhibit the adhesion of HeyA8 cells to extracellular matrix proteins including fibronectin,collagen IV and laminin by competitively binging to integrins,which indicated that rLj-RGD3 could recognize various types of integrin molecules.Finally,both transwell and western blot assays proved that rLj-RGD3 was able to inhibit the migration and invasion processes of HeyA8 cells by decreasing the phosphorylation level of focal adhesion kinase?FAK?.Therefore,the above results suggest that rLj-RGD3 is effective on the inhibition of HeyA8 cells proliferation,which provides significant theory strategies for ovarian carcinoma treatments in the future,and possesses potential economic benefits and application prospects.