Study On The Efficacy,Mechanism And Toxicology Of Copper(Ⅱ) Complex Of Salicylate Phenanthroline In Inhibiting Hepatocellular Carcinoma

Objective: To provide scientific basis for the development of new chemotherapy drugs,we investigated the inhibitory effect and mechanisms of copper(II)complex of phenanthroline salicylate [Cu(sal)phen] against Hepatocellular Carcinoma(HCC)in vitro and in vivo.In addition,we evaluated Cu(sal)phen toxicity in the Balb/c mice.Methods: MTS assay and colony formation assay were used to detect the inhibitory effect of Cu(sal)phen on the viability and proliferation of HCC cells.Flow cytometry was used to analyze the effect of Cu(sal)phen on apoptosis,mitochondrial membrane potential and ROS production of HCC cells.Western Blot was used to detect the changes of apoptosis,DNA damage and JAK2-STAT5 signaling pathway related proteins in HCC cells treating with Cu(sal)phen.Xenografted animals were intraperitoneally injected with Cu(sal)phen,the body weight and tumor growth of BALB/c-nu mice were monitored.HE staining and immunohistochemistry were used to analyze the inhibitory effect and mechanism of Cu(sal)phen on the growth of hepatocellular carcinoma in vivo.BALB/c mice were intraperitoneally injected with different doses of Cu(sal)phen,and the in vivo toxicity of Cu(sal)phen was evaluated by detecting blood biochemical indicators and observing the morphological changes of organs in mice by hematoxylin-eosin staining.Results: In vitro,Cu(sal)phen inhibited the viability of HCC cells Hep G2 and HCC-LM9 in a time and concentration dependent manner.Colony formation assay further confirmed the inhibitory effect of Cu(sal)phen on the proliferation of HCC cells.Cu(sal)phen induced apoptosis,decreased mitochondrial membrane potential through induction of ROS accumulation in HCC cells in a dose-dependent manner.Pretreatment with NAC reduced the percentage of Cu(sal)phen induced apoptosis in HCC cells.Western Blot results showed that Cu(sal)phen up-regulated the expressions of pro-apoptotic proteins c-Caspase3 and apoptosis marker c-PARP,down-regulated the expressions of anti-apoptosis proteins Survivin and Bcl-2,and inhibited the activation of JAK2-STAT5 signaling pathway.At the same time,the expression of DNA damage-related protein gamma-H2 Ax was increased.Cu(sal)phen inhibited the growth of Hep G2 subcutaneous tumors in vivo,and down-regulated the expressions of Ki67,Bcl-2,Survivin proteins in tumor tissues.The results of toxicological experiment showed that the maximum dose of Cu(sal)phen just significantly reduced the body weight of BALB/c mice,and had no significant effects on blood biochemical and routine indexes.Meanwhile,lower doses of Cu(sal)phen treatment did not affect body weight and cause any significant damage to the organs(heart,liver,spleen,lung and kidney)of mice.Conclusion: Cu(sal)phen inhibits HCC cell viability and tumor growth in vitro and in vivo.The anti-HCC effect of Cu(sal)phen is related to the induction of excessive ROS in cells,which leads to mitochondrial damage and mitochondria-dependent apoptosis of HCC cells.Collectively,Cu(sal)phen is a relatively safe compound with inhibitory effect against liver cancer cells,and has the potential to be developed as a chemotherapy drug for liver cancer.