Mechanism Of Gypenoside L And Gypenoside LI Extracted From Gynostemma Pentaphyllum In Inhibiting Proliferation And Inducing Apoptosis Of Breast Cancer Cells

Aim of the study:Gynostemma pentaphyllum(Thunb.)Makino is a traditional Chinese medicinal material and an ethnic medicine,widely used in ethnic minority areas in southwest of China.It is called "gocaetmbaw" in Zhuang medicine,is one of the commonly used drugs in Zhuang people,and is widely used to treat various diseases.Gypenoside L and gypenoside LI are the main components isolated from Gynostemma pentaphyllum,which are a pair of isomers.Studies have shown that gypenoside L and gypenoside LI have inhibitory effects on the growth of a variety of cancer cells.However,its pharmacological effects and mechanisms in breast cancer have not been reported yet.International Agency for Research on Cancer in 2020 showed that the incidence of breast cancer ranks first in the world,and women's lives and health are seriously threatened.Currently,the main treatment methods for breast cancer are surgery,endocrine therapy,chemotherapy,radiotherapy and targeted therapy.Due to the lack of effective targeted therapy,the treatment of highly invasive and metastatic triple-negative breast cancer(TNBC)is still challenging.E2F1,a transcription factor,belongs to the E2F family,is involved in cell cycle regulation and apoptosis.Clinical evidence shows that aberrant upregulation of E2F1 frequently occurs in various types of human cancers that are related to malignant progression and poor survival prognosis.Excision repair cross-complementation group 6 like(ERCC6L),is a gene of centromere-related SNF2 family ATPase and involved in chromatin remodeling,DNA recombination and DNA repair.Studies have reported that ERCC6L promotes the proliferation of a variety of cancers.Therefore,the purpose of this subject is to explore the inhibitory effects and mechanisms of gypenoside L and gypenoside LI on MDA-MB-231 and MCF-7 cells.Methods and results:The phenotypic experiments showed that gyenoside L and gypenoside LI inhibited the proliferation and migration of breast cancer cells,and induced cell apoptosis in endogenous pathway.Moreover,the effect of gypenoside LI was stronger than that of gypenoside L,especially for MDA-MB-231 cells.RNA-seq sequencing,KEGG and GSEA enrichment analysis found that both gypenoside L and gypenoside LI could play a role in regulating the cell cycle.Flow cytometry showed that gyenoside L could induce cell cycle arrest in G2/M phase,while gyenoside LI could arrest cell cycle in G0/G1 phase.GO analysis found that gypenoside L was related to cell division and chromosome segregation;while gypenoside LI mainly affects DNA replication.Through the analysis of metascape online tool,it was found that the significantly differentially expressed genes(DEGs)or cycle-related gene set down-regulated by gypenoside LI was mainly regulated by E2F1,while gypenoside L did not.We have confirmed that gypenoside LI arrested the cell cycle in G0/G1 phase by inhibiting E2F1,and also inhibited the expression of ERCC6L by down-regulating E2F1 through transfection assays and rescue assays.In addition,gypenoside LI and siE2F1 interact synergistically to induce apoptosis in breast cancer cells at low concentration.Although gyponoside L could not inhibit the expression of E2F1,it could down regulate ERCC6L,which may be related to mitosis.In addition,Western blot results showed that neither gypenoside L nor gypenoside LI could change the expression of p53.Therefore,the anti-tumor effect of this pair of isomers does not depend on the p53 pathway.Conclusion:This study demonstrated that gypenoside L and gypenoside LI could inhibit the proliferation,arrest cell cycle and induce apoptosis of breast cancer cells,and their antitumor effects were independent of p53 pathway.However,the mechanism is different:gypenoside L arrested the cell cycle in G2/M phase,while gypenoside LI arrested the cell cycle in G0/G1 phase.Gypenoside LI down-regulated the expression of ERCC6L through E2F1,but gypenoside L did not change the expression of E2F1.Combined with database analysis,it is speculated that down-regulation of ERCC6L may be related to the mitotic pathway.