The Research Of Anti-effect And Mechanisms Of 3-bromopyruvate Cholesterol Ester On Hepatoma

Ojective:As an inhibitor of energy metabolism in tumor cells,3-BP has a highly effective and broad-spectrum anti-tumor effect.However,the distribution of 3-BP in tumors is not specific and has serious toxic and side effects.The project plans to synthesize a p H responsive prodrug of 3-BP-CL through esterification reaction.By investigating its anti hepatoma effect and mechanism in vitro and in vivo,we will explore its inhibitory effect and mechanism on hepatoma,with a view to developing low toxicity and high efficiency 3-BP derived drugs.Methods:1 Synthesis and Characterization of 3-BP-CLCholesterol was connected with 3-bromopyruvate by esterification reaction to prepare compound 3-bromopyruvate-cholesterol ester and confirm its structure.The release of 3-bromopyruvate-cholesterol ester under acidic conditions was tested to determine whether it has p H responsiveness in the tumor microenvironment.2 Inhibition of 3-BP-CL on hepatoma cells and its mechanism2.1 Inhibition of 3-BP-CL on hepatoma cellsThe inhibitory effect of drugs on the proliferation and migration of SMMC-7721cells was detected by MTT assay,Calcein-AM/PI staining,colony cloning assay and scratch assay.2.2 Effect of 3-BP-CL on ferroptosis in hepatocellular cells.MTT assay was used to detect whether ferroptosis inhibitor Fer-1 could block the inhibitory effect of 3-bromopyruvate-cholesterol ester on hepatoma cells.To investigate the effect and mechanism of 3-bromopyruvate-cholesterol ester on ferroptosis in hepatocellular carcinoma cells.Lipid peroxide（LPO）fluorescence probe,flow cytometry and fluorescence microscopy were used to detect the production of LPO induced by SMMC-7721 cells.The effect of 3-bromopyruvate cholesterol ester on intracellular Fe²⁺level was detected by iron ion probe.The GSH depletion capacity of 3-bromopyruvate was determined by GSH detection kit.Western Blot was used to detect the effect of 3-bromopyruvate-cholesterol ester on the expression of HK-II and GPX4 in hepatoma cells.2.3 Induction effect of 3-bromopyruvate-cholesterol ester on apoptosis of hepatoma cells.Apoptosis detection kit,flow cytometry,JC-1 staining kit and Western Blot were used to determine the effect of 3-bromopyruvate-cholesterol ester on apoptosis of hepatocellular carcinoma cells.3 Inhibitory and toxic effects of 3-BP-CL on hepatocellular carcinoma transplantation model in nude mice.To establish a human hepatocellular carcinoma transplantation model in nude mice,observe the inhibitory effect and toxicity of 3-bromopyruvate-cholesterol ester on the growth of the model,and evaluate its inhibitory effect and safety on hepatocellular carcinoma in vivo.Results:1 FT-IR,DSC and ¹H-NMR spectra indicated the successful preparation of3-BP-CL.It responds to the acidic tumor microenvironment by releasing 3-bromopyruvate,a glycolytic inhibitor,and cholesterol,a lipid metabolite,through ester bond cleavage.2 3-BP-CL can inhibit the proliferation and metastasis of hepatocellular carcinoma cells,and the mechanism involves ferroptosis induction and apoptosis induction.2.1 3-BP-CL can inhibit the proliferation and metastasis of hepatoma cells.The results of MTT assay,Calcein-AM/PI staining assay,colony forming assay,scratch assay and DAPI staining assay all showed that 3-bromopyruvate-cholesterol ester had a good inhibitory effect on the proliferation and metastasis of hepatocellular carcinoma cells.2.2 3-bromopyruvate-cholesterol ester can induce ferroptosis in hepatoma cells.The LPO experiment showed that 3-bromopyruvate-cholesterol ester could increase the level of LPO in hepatoma cells and cause the accumulation of LPO in cells.Iron ion probe experiment showed that 3-bromopyruvate-cholesterol ester could increase the intracellular iron ions in hepatoma cells.The results of GSH consumption experiment showed that with the increase of 3-bromopyruvate-cholesterol ester concentration,the color of the solution gradually became lighter,and the consumption of GSH gradually increased.The results of Western Blot showed that the expression of HK-II and GPX4 in SMMC-7721 cells was significantly decreased with the increase of 3-BP-CL concentration.The expressions of HK-Ⅱand GPX4 were up-regulated after treatment with Fer-1.2.3 3-bromopyruvate-cholesterol ester could induce apoptosis of hepatoma cells.Flow cytometry and JC-1 staining showed that 3-bromopyruvate-cholesterol ester could induce more apoptosis.Western Blot results showed that with the increase of 3-bromopyruvate-cholesterol ester concentration,the expression of Bcl-2 was down-regulated,and the expression of Bax was up-regulated in SMMC-7721 cells.3 The 3-BP-CL inhibited the growth of hepatocellular carcinoma transplantation model in nude mice with little toxicity.In vivo experiments showed that the prepared 3-bromopyruvate-cholesterol ester had a better effect on slowing down tumor growth and could further induce ROS production,thereby further enhancing the anti-tumor effect through ferroptosis and apoptosis pathways.Conclusion:3-BP-CL can inhibit the proliferation and metastasis of hepatoma cells by inducing ferroptosis and apoptosis of hepatoma cells,and play an anti-liver cancer role.Due to the specific release of drugs in the acidic environment of tumor tissue,it has the characteristics of low toxicity and high safety.