RA-C1,An Novel Analogue Of Rosmarinic Acid,suppresses Cell Proliferation Of Human Nasopharyngeal Carcinoma Cells Line CNE-1 Through Mitochondrion-dependent Signaling Pathway

Background : Due to low toxicity and attractive anticancer activity,Rosmarinic acid has become a promising candidate compound for cancer therapy.However,the application of RA is limited by poor water solubility and low bioavailability.What's more,because of the poor prognosis of NPC,new therapies are urgently required.Hence we performed structural modification of RA by replacement of ester with amide to increase the solubility and esterification of carboxylic acid to improve membrane permeability.Objective:This study was mainly to investigate the effects of RA-C1 on human nasopharyngeal carcinoma cell line CNE-1 in vitro,which investigate the effects of RA-C1 on cell proliferation and apoptosis of CNE-1 cells.We conclude that RA-C1 inhibits CNE-1 cells proliferaion by inducing apoptosis and this antitumor effect of RA-C1 is possibly mediated by the mitochondrion-dependent signaling pathway.Methods: Although rosmarinic acid is very well known for its anticancer activity,it shows some drawbacks.Here,we discovered a novel rosmarinic acid analogue,(R,E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxy-phenyl)acrylamido)propanoate(RA-C1),which was cytotoxic against the human nasopharyngeal carcinoma(NPC)cells tested.The MTT cell viability assay showed that treatment with RA-C1 selectively inhibited NPC CNE-1,CNE-2 and NPC HONE-1 cancer cells proliferation compared to that of normal human vascular endothelial cells(HUVEC)and human embryonic kidney 293 cells(HEK 293).The present study was designed to explore the molecular mechanism underlying the anticancer effect of RA-C1 in NPC CNE-1 cells.The extent of cell apoptosis was determined by Hoechst 33342 staining and Annexin-V FITC/PI double-staining assay.Then,the changes of mitochondrial transmembrane potential were examined by a JC-1 kit.The expressions of relative apoptotic protein,such as Cytochrome C,Bax,Bcl-2,cleaved caspase-3 and cleaved caspase-9 were detected by western blotting analysis.Result: The results showed that,both the Hoechst 33342 staining and Annexin-V FITC/PI double-staining assay confirmed the pro-apoptotic effect of RA-C1 on CNE-1 cells with the concentration rising up.In addition,RA-C1 treatment induced the attenuation of mitochondrial membrane potential,accompanied by the release of Cyt-c and the activation of caspase-9 and caspase-3.Consistent with this finding,an antioxidant,N-acetyl-L-cysteine(NAC),hindered RA-C1-induced apoptosis and attenuated the sensitivity of NPC CNE-1 cells to RA-C1.Furthermore,RA-C1 decreased the protein expression of Bcl-2,whereas increased the protein expression of Bax after RA-C1 treatment.Conclusion: These results indicate that RA-C1 can effectively inhibit the proliferation and induces the apoptosis of CNE-1 cells by interact with the mitochondrion-dependent signaling pathway.