Synthesis Of Novel 1,4-naphthoquinone Derivatives Induce Reactive Oxygen Species-mediated Apoptosis In Human Hepatocellular Carcinoma Cells

ObjectiveHCC also known as hepatocellular carcinoma,it causes more than 1 million patients deaths per year.It is one of the most common malignant tumors in the world.It has strong metastasis and is extremely harmful to human health.Therefore,a new type of anticancer drug with low toxicity,low cost and high efficiency should be sought as soon as possible to improve the quality of life of patients with liver cancer.There are many organic compounds with antitumor activity in nature,and naphthoquinone compounds are among the excellent ones.However,naphthoquinones have strong toxic side effects generally,making them difficult to use in clinical treatment.Therefore,in order to improve the antitumor activity of 1,4-naphthoquinones and to reduce the side effects to some extent,we designed and synthesized two novel types of 1,4-naphthoquinone derivatives,2,3-dihydro-2,3-epoxy-2-propylsulfonyl-5,8-dimethoxy -1,4-naphthoquinone(EPDMNQ)and 2,3-dihydro-2,3-epoxy-2-nonylsulfonyl-5,8-dimethoxy -1,4-naphthoquinone(ENDMNQ)by Michael addition reaction and MCPBA oxidation reaction and a variety of liver cancer cells as research objects,from the cellular and molecular level,to explore the pharmacological effects of EPDMNQ and ENDMNQ on liver cancer cells,and its potential molecular mechanism and related Signal transduction pathway provides a strong theoretical basis for research and clinical treatment of liver cancer.MethodsIn this study,two novel types of 1,4-naphthoquinone derivatives,2,3-dihydro-2,3-epoxy-2-propylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone(EPDMNQ)and 2,3-dihydro-2,3-epoxy-2-nonylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone(ENDMNQ)were synthesized by Michael addition reaction and MCPBA oxidation reaction.To determine whether EPDMNQ and ENDMNQ had cytotoxic effects in liver cancer cells and cytotoxicity for normol cells,cell viabilities were determined by the MTT assay.The structure of synthetic compounds was identified by nuclear magnetic resonance(NMR)technique.The apoptotic effect of EPDMNQ and ENDMNQ on liver cancer cells was detected by Annexin V-FITC/PI double staining cell by fluorescence microscopy and flow cytometry.Western blotting was used to detect the regulation of EPDMNQ and ENDMNQ on the expression of apoptosis-related proteins in hepatoma cells,and the related signaling pathway proteins p38,JNK and ERK in hepatoma cells by EPDMNQ and ENDMNQ.The regulation of STAT3 phosphorylation;the regulation of EPDMNQ and ENDMNQ on the level of reactive oxygen species in hepatoma cells by flow cytometry and Western blotting,and the relationship between changes in reactive oxygen species and apoptosis.Results1?The result of nuclear magnetic resonance(NMR)technology showed that both hydrogen atoms at 3,5,6,7 and 8 positions,and alkane bonded to the fluorenyl group exhibited specific chemical shifts.The carbon spectrum shows that the chemical shifts of the carbon atoms of the two compounds are in line with expectations,and the mass spectrometry results indicate that the relative molecular masses of the two compounds are the same as expected;2?EPDMNQ and ENDMNQ inhibited liver cancer cells proliferation in a dose-dependent manner.The cytotoxic effect of EPDMNQ and ENDMNQ,and had significant difference compared with 5?FU.EPDMNQ and ENDMNQ exhibited lower cytotoxicity compared with 5?FU treatment in normal cell lines;3?EPDMNQ and ENDMNQ through increasing apoptotic proteins Bax,cle-caspase-3 and cle-PARP,decreasing the expression levels of anti-apoptotic protein Bcl-2,and induce apoptosis of human hepatoma Hep3 B cells;4?EPDMNQ and ENDMNQ can significantly increase the phosphorylation levels of p38 and JNK,decreased the expression of phosphorylation levels of ERK and STAT3 in Hep3 B cells;5?EPDMNQ and ENDMNQ can up-regulate the production of reactive oxygen species in Hep3 B cells and induce cell apoptosis.Conclusion2,3-dihydro-2,3-epoxy-2-propylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone(EPDMNQ)and 2,3-dihydro-2,3-epoxy-2-nonylsulfonyl-5,8-dimethoxy-1,4-naphthoquinone(ENDMNQ)has a good killing effect on 3 kinds of liver cancer cells,The specific mechanism of action may be that EPDMNQ and ENDMNQ play a regulatory role in MAPKs and STAT3 signaling pathways by increasing the level of intracellular reactive oxygen species,causing a cascade of caspase,leading to mitochondria-dependent apoptosis in liver cancer cells.