Study Of Artemisitene Selectively Kills Cancer Cells And Its Machenism

ObjectiveTo explore the anti-tumor effect and molecular mechanism of artemisitene,and provide scientific evidences for its development and utilization.Contents and methods1.The effect of artemisitene on selectively killing cancer cells(1)Detect the toxic effect of artemisitene on human cancer and normal cells using CCK-8 assay,colony formation assay and flow cytometry.(2)Tumor xenografts model were established by the subcutaneous transplantation of cancer cells into 4-week-old athymic nude mice.Then explore the anti-tumor effect of artemisitene in vivo through intraperitoneal injecting artemisitene.In addition,detect the effect of artemisitene on the expression of PARP using Western Blot.2.Artemisitene selectively induces DNA damage in cancer cells by inhibiting the expression of TOP2B and TOP3B.(1)Detect the effect of artemisitene on the expression of yH2AX using Western Blot and immunofluorescence staining assay.(2)Detect the effect of artemisitene on the mRNA and protein expression of TOP2B and TOP3B using qPCR assay and Western Blot assay.(3)Generate overexpression vectors of TOP2B and TOP3B by using molecular cloning technology,in order to establish TOP2B or TOP3B overexpression stable cancer cell lines following the steps of transfection,lentivirus packaging and infection.Detect the effect of artemisitene on the cell viability and yH2AX expression in TOP2B or TOP3B overexpression cancer cells by CCK-8 assay and Western Blot assay.3.Artemisitene selectively regulates c-Myc stability in cancer cells(1)Detect the effect of artemisitene on the mRNA and protein expression of c-Myc using qPCR assay and Western Blot assay.(2)Establish stable cell lines with low expression of c-Myc,then detect the effects of c-Myc knockdown on the cell viability and mRNA expression of TOP2B and TOP3B using CCK-8 assay and Western Blot assay.(3)Detect the effect of artemisitene on the half-life of c-Myc,the effect of c-Myc knockdown on ?H2AX expression,and the expression status of yH2AX in c-Myc knockdown and overexpression TOP2B or TOP3B cancer cells by Western Blot assay.4.Artemisitene regulates c-Myc by selectively upregulate the expression of NEDD4 in cancer cells(1)Detect the effect of artemisitene on the mRNA expression of ubiquitin ligases of c-Myc using qPCR assay.(2)Establish stable cell lines with low expression and overexpression of NEDD4,then detect the effects of NEDD4 knockdown and overexpression on c-Myc expression by Western Blot assay.Results and conclusions1.Artemisitene has broad anti-tumor activity,which can suppress proliferation of many cancer cell lines,but has no apparent adverse or little effects on normal cells.2.Artemisitene effectively suppresses tumor growth and induces apoptsis of cancer xenografts in vivo.3.Artemisitene induces DNA damage by reducing c-Myc stability and inhibiting the expression of TOP2B and TOP3B,while overexpression of TOP2B or TOP3B,to some extent,rescues the DNA damage and cytotoxicity induced by artemisitene.4.c-Myc induces the expression of TOP2B and TOP3B.c-Myc knockdown results in downregulation of TOP2B and TOP3B and induction of DNA damage,while overexpression of TOP2B or TOP3B parly reduces the DNA damage and cytotoxicity caused by c-Myc knockdown.5.The molecular mechanism of artemisitene selectively killing cancer cells is that artemisitene reduces c-Myc stability by upregulating the expression of NEDD4(ubiquitin ligase of c-Myc),which contributes to inhibition of TOP2B and TOP3B expression and reduction of DNA damage,and finally leads to cancer cell apoptosis.