Effects Of Icariin On MELK Inhibiting Breast Cancer Cells And Signaling Pathway

Background:According to the International Organization for Cancer Research(IARC),the number of new cases of breast cancer in 2020 is 2.26 million,the largest in the world.With increasing levels of breast cancer diagnosis and treatment,the 5-year survival rate for breast cancer can reach approximately 90%.At present,western medicine in breast cancer mainly has surgery,radiation therapy,endocrinology,targeting,chemotherapy,immunotherapy and so on.With the in-depth understanding of genes and molecular mechanisms of breast cancer,regulation of apoptosis and proliferation of breast cancer cells by signaling pathways has become the hot spot of current research.MELK is a member of the Snfl/AMPK kinase family and is a conserved cyclin dependent kinase.Unlike other family members,MELK is not involved in the energy-metabolism balance,but rather in cell cycle regulation,cell proliferation,tumorigenesis,and apoptosis.Previous studies have found that MELK is expressed to varying degrees in various tumor cells and increased in breast cancer tissues,which is also related to poor prognosis of breast cancer,possibly because MELK participates in the Bcl-G signaling pathway and plays an anti-apoptotic role in breast cancerIn light of the tumor stem cell theory and the expression profile of MELK,MELK may be an important target for the treatment of breast cancer.Akt is a characteristic effector of phosphoinositide 3-kinase(PI3K)in the PI3K/Akt/mTOR pathway,and its dysregulation plays a key role in the pathogenesis of many human cancers.Many oncoproteins and tumor suppressors are cross-linked in the Akt pathway that causes cell proliferation,differentiation,inhibition of apoptosis,and remodeling of the actin cytoskeleton,which is abnormally activated in many human cancers and plays a crucial role in mammary tumor cell apoptosis,tumor growth and metastasis.Patchouliaceae patchouli,a commonly used herbal medicine,has immunologic,cardiovascular,osteoporotic,hematopoietic,endocrine,antioxidant,and antitumor properties.The major active ingredient of patchouli,Icariin,inhibits the growth of MDA-MB-231 and MCF-7 breast cancer cells by inducing G2/M arrest.Lutein patchouliside is a flavonoid compound extracted from Lutein patchouli and has gained increasing attention in recent years due to its antitumor activity.Studies have shown that patchouliside induces apoptosis of multiple tumor cells and attains antitumor effects by reducing invasion and migration of tumor cells.Objective:1.In vitro cell assays were performed to confirm the presence of MELK expression in tumor tissues and to screen patchouliside for its ability to inhibit and reduce MELK expression in breast cancer cells.2.To investigate the mechanism by which patchouliside inhibits breast cancer cells in vitro and in vivo and to identify regulatory pathways associated with MELK.Methods:The study was divided into three sections.The first section examined the tumour cell viability and expression of MELK by MTT,CRK-8 and Western blot assays,and examined whether patchoulidin could inhibit MCF-7 and MDA-MB-231 breast cancer cells and reduce MELK expression in breast cancer cells.The second part investigated the effect of patchoulidin on breast cancer cells by using CCK-8,flow cytometry to determine cell cycle distribution,Annexin V/PI staining to determine the rate of apoptosis,transwell cellmigration,real-time RT-PCR,Westem blot.In the third part,the effect of patchouliside on mouse breast cancor xenografts was studied in vivo.Results:First,1.Validation of MELK expression in all tumour cells;2.In several compounds patchoulidin is effective in suppressing,breast cancer cell growth and reducing MELK expression in breast cancer cells,comparable to the currently used MELK inhibitor OTSSP167 in clinical trials.Second,knockdown of MELK significantly reduced the expression of MELK in MCF-7 and MDA-MB-231 breast cancer cells,markedly reduced the expression of phosphorylated Akt,increased the expression of E-cadherin,decreased the expression of N-cadherin,and decreased the expression of vimentin,especially in MCF-7 breast cancer cells.2.patchouliside significantly suppressed the cell cycle of human breast cancer cells,resulting in decreased expression of Bcl-2,increased expression of Bax protein,and increased expression of Caspase-9 and cleaved-caspase-3,with a more pronounced effect on MCF-7 in human breast cancer cells.3.The transwell invasion assay demonstrates that patchouliside inhibits invasion of breast cancer cells with the lowest cell migration rate at a patchouliside concentration of 100μg/mL.Third,In vivo,patchoulidin and LY294002 significantly inhibited breast cancer growth,and patchoulidin was superior to LY294002.2.The Western blot assay demonstrated that patchoulidin reduced MELK expression,but not Akt phosphorylation,while suppressing metastasis,suggesting that patchoulidin could modulate MELK-mediated Akt signaling to inhibit breast cancer growth.Conclusions:This study found that icariin can inhibit proliferation,promote apoptosis,and inhibit migration/invasion of breast cancer.The apoptosis of breast cancer cells was induced by icariin through regulation of MELK.Icariin may inhibit breast cancer growth by regulating the MELK-mediated Akt signaling pathway.