Synthesis,Anti-tumor Activity Evaluation And Mechanistic Studies Of Shikonin Ester Derivatives

Shikonin is an active naphthoquinone that isolated from Chinese herbal medicine Lithospermum erythrorhizon.It has mulitiple pharmacology activities,antiinflammatory,antibacterial,antiviral,antiHIV activities,etc.Most importantly,it shows significant antitumor activity in vitro and in vivo.Nevertheless,researchers found it had strong toxic side effects through the subsequent studies and thus greatly reduced its clinical application value.Consequently,how to solve this problem became the focus of our research.In this study,we expect to make an improvement on its toxic side effects.We designed three series of shikonin ester derivatives and obtained them by chemical methods.Subsequently,we screened out three good antitumor functional molecules from them by MTT assay with shikonin and positive control drugs for reference.Their antitumor mechanisms were determined by various experiments as well.The results indicated that the antitumor activities of shikonin cinnamic acyl esters derivatives which contain electron withdrawing groups are better than that with electron-donating groups.After comparison with shikonin,compounds PMMB020 and PMMB028 showed better antitumor activity and the former contains CF3,the latter contains two F atoms.They can all effectively inhibit the proliferation of human melanoma cell(A875)with IC50 values as 0.65 ±0.03 ?M and 0.55 ±0.03 ?M.But their cytotoxicity towards noncancer cell human renal epithelial cells(293T)has been significantly reduced.Moreover,compound PMMB020 can lead to cell apoptosis by activating caspase family members in the mitochondrial apoptosis pathway while has no effect on cell cycle arrest.In the second series,both aryl dihydrothiazol and shikonin showed good antitubulin effect.Thus,the aryl dihydrothiazol acyl shikonin ester derivatives(PMMB123)showed better antubulin activity and good antiproliferation activity against HeLa cells with an IC50 value as 3.14±0.21 ?M,and has lower toxicity towards noncancer cells,African green monkey kidney cell(VERO)and human liver cell(L02).Additionally,it can also suppress the adhensive ability of HeLa cells and promote tubulin polymerization,breaking the dynamic of polymerization and de-polymerzation through binding to the paclitaxel site of tubulin.Thus,most HeLa cells were arrested at G2/M phase and casued cell apoptosis.The inhibition effect of compound PMMB123 is different from our previous study(in the previous study,shikonin derivatives are good tubulin inhibitors through binding to the colchicine site,inhibiting tubulin polymerization),and the difference tell us that the introduction of aryl dihydrothiazol moiety changed not only the structure,but also the antitumor mechanisms of shikonin and need to be further studied.Based on the above studies,?-lipoic acid and its derivatives which influence pyruvic acid metabolism were introduced to shikonin to obtanined some lipoic acyl shikonin ester derivatives.PMMB266,the best one among them,showed better anti-proliferation activity towards HeLa cells(IC50=3.14±0.58 ?M)than shikonin(IC50=6.86 ±1.22 ?M).PMMB266 is a dual-target antitumor agent,not only inhibited microtubule polymerization,but also inhibited pyruvate dehydrogenase kinase(PDK-1)activity,increasing pyruvate dehydrogenase(PDH)activity,thereby forcing the cancer cells go through more aerobic metabolism and apoptosis process.Further,we also observed that the twin drug can significantly inhibit the growth of tumor in vivo and improve the survival rate of mice.These research works laid a solid foundation for development and utilization of shikonin as an effective antitumor drug lead compounds and anticancer drug.To sum up,our work will provide new ideas for studying the antitumor activity and effective target of shikonin.The work will also provide experimental basis for developing shikonin and its derivatives as a new type antitumor agent.