Preliminary Study On The Mechanism Of Curcumin Analogue EF-24 Against Breast Cancer Cell Proliferation And SCRV Virus Replication

Curcumin analogs are a class of small chemical molecules based on curcumin,a natural polyphenol,that have received widespread attention for their anti-inflammatory,antitumor,and antiviral effects.5-bis{（2-fluorophenyl）methylene}-4-piperidone(molecular formula:C₁₉H₁₅F₂NO;molecular weight:311;abbreviation EF-24)is a curcumin analogue that has been identified and found to It has been identified to have diverse pharmacological functions.On this basis,we continued to investigate the anti-proliferative effect of EF-24 on breast cancer cells and the anti-replication of Siniperca chuatsi rhabdovirus（SCRV）.Breast cancer has become a major factor of tumor death in women worldwide and has now surpassed lung cancer as the world’s number one cancer,with diverse but ineffective therapeutic approaches,and the development of more compounds with tumor cell inhibitory effects and impact on tumorigenesis has become a hot concern.Therefore,in this study,EF-24（5μM）was firstly found to have anti-proliferative and anti-migratory activities against two breast cancer cell lines（MCF-7 and MDA-MB-231）by MTT,cell scratching and transwell assays.Further,autophagic vesicles were detected by acridine orange staining,and the double or multiple membrane structure of autophagic vesicles was observed by transmission electron microscopy.Meanwhile,up-regulated expression of autophagy key proteins LC3 and p62 was detected using Western blot.Immunofluorescence experiments also revealed that EF-24 could inhibit the degradation of autophagic vesicles in MCF-7 cells by blocking the fusion of autophagosomes with lysosomes.In contrast,in MDA-MB-231cells,EF-24 induced the initiation of autophagy rather than blocking the degradation of autophagic vesicles.To investigate the molecular mechanism of EF-24 against breast cancer cell proliferation in depth,Hoechst staining,flow cytometry and Western blot assays revealed that EF-24（5μM）induced apoptosis in MCF-7 and MDA-MB-231 cells.The autophagy inhibitor 3-MA further enhanced EF-24 induced apoptosis in MCF-7 cells,whereas in MDA-MB-231 cells,3-MA alleviated EF-24-induced apoptosis.Furthermore,EF-24treatment up-regulated the enzymatic activities of Caspase-3 and Caspase-9 in MDA-MB-231 cells and induced ROS accumulation and down-regulation of mitochondrial membrane potential.These results confirmed that EF-24 activated the mitochondrial apoptotic pathway in MDA-MB-231 cells.In addition,the antiviral effect of EF-24 was further investigated.In this paper,SCRV was used as the target,and firstly,the best inhibition effect of EF-24 co-incubation with SCRV on the progeny virus was found by CPE observation,virus titer assay,RT-q PCR and Western blot assay.In addition,flow cytometry experiments revealed that EF-24 could effectively reduce SCRV induced apoptotic signal in EPC cells.Further enzyme activity assay revealed that EF-24 treatment was able to down-regulate the caspase-3 and caspase-9enzyme activities provoked by SCRV infection,which tentatively confirmed the anti-SCRV effect of EF-24 by affecting the mitochondrial apoptotic pathway.In summary,the current study confirms that EF-24（5μM）has anti-proliferative and anti-SCRV viral replication effects in breast cancer cells.It provides a solid basis for the potential in vivo anticancer activity of this compound and the development of anti-viral drugs.