Preliminary Study On The Mechanism Of Anti-triple Negative Breast Cancer Of Loonamycin From Deep Sea Microorganism

The International Agency for Research on Cancer of The World Health Organization(IARC)has released the latest global cancer burden data for 2020.Data show that the number of new cases of breast cancer is as high as 2.26 million,more than the lung cancers in 2.2 million.The breast cancer has replaced lung cancer and became the world’s largest cancer,besides,breast cancer is the malignant tumor with the highest mortality rate(15.5%).Triple negative breast cancer(TNBC)is a special type of breast cancer,accounting for about 15%-20%.The histopathological classification of triple-negative breast cancer is mostly above grade III,the histopathological classification of triple-negative breast cancer is mostly above grade III,with a high degree of deterioration,and more aggressive than other breast cancer subtypes.At the same time,triple-negative breast cancer contains a series of heterogeneous molecular structures,and its treatment progress has been lagging behind other molecular subtypes of tumors.Therefore,looking for new targets for triple-negative breast cancer and new chemotherapeutic drugs actively are still the target that we need to strive for.Loonamycin(LGY)is an analogue of Rebeccamycin with independent intellectual property rights isolated from Nocardia sp.by Ge Huimin’s team at Nanjing University.The results of previous studies have shown that LGY has the biological activity of inhibiting the proliferation of a variety of tumor cell lines.This study intends to detect the effects of LGY on the proliferation,cell cycle and apoptosis of two TNBC cells MDA-MB-231 and MDA-MB-468,and use transcriptome sequencing,Western Blot,flow cytometry and other experiments to analyze the molecular mechanism LGY,hope to further reveal the molecular mechanism of LGY inhibiting the proliferation of TNBC cell lines,and lay the foundation for the clinical application of LGY.In order to study the effect of LGY on the proliferation of TNBC cell lines,two TNBC cell lines MDA-MB-231 and MDA-MB-468 were chosen,and the Cell Counting Kit-8(CCK-8)experiment,flow cytometry Technology,DNA relaxation experiments and so on were used.The results of CCK-8 cell proliferation experiments show that LGY can strongly inhibit the proliferation of TNBC cell lines MDA-MB-231 and MDA-MB-468,and the inhibitory rate is time-dependent and dose-dependent.The experimental results of flow cytometry to detect apoptosis and cell cycle showed that: LGY has no significant effect on the apoptosis of two TNBC strains;LGY can cause cell cycle S phase arrest when acting on MDA-MB-231 cells;acting on MDA-MB-468 cells can cause cell cycle G2 arrest.The DNA relaxation experiments show that LGY has a significant inhibitory effect on topoisomerase I and is concentration dependent.In order to further study the molecular mechanism of LGY inhibiting the proliferation of TNBC cell lines,we use transcriptome sequencing technology to screen out the differentially expressed genes before and after treatment with the compound LGY,and conduct a comprehensive bioinformatics analysis.Through GO and KEGG enrichment analysis,LGY is used to annotate the potential biological functions and pathways that are sensitive to TNBC.The results found that the function of actin,cell adhesion molecules,and cytokine receptors are possible signal pathways for drug action.The enrichment of nerve signals and postsynaptic membrane suggests that LGY may be related to breast cancer neurophilic metastasis.PPI network analysis suggests that the two subunit proteins of G protein GNG7/11,chemokine ligand gene CXCL8 and adenylate cyclase ADCY2 may be regulatory sites.GSEA analysis suggests that NF-κB-mediated TNFα signaling pathway and P53 signaling pathway may be pathways for drug action,and the upstream proteins involved in regulation are PSMB5 and SETD7.The above research results indicate that LGY may affect the expression of GNG7,GNG11,CXCL8,ADCY2 and other genes by combining the gene expression regulatory proteins PSMB5 and SETD7,and ultimately up-regulate the NF-κB-mediated TNFαpathway and P53 pathway,leading to the G2/M cell cycle arrest in MDA-MB-468 cell line and exert anti-tumor effect.Through the research of this subject,we have got the following conclusions:(1)LGY inhibit the proliferation of TNBC cells by blocking the cell cycle;(2)LGY can cause cell cycle arrest by interacting with DNA-topoisomerase I and exert cytotoxicity;(3)Bioinformatics predicts that LGY may affect the expression of GNG7,GNG11,CXCL8,ADCY2 and other genes by combining gene expression regulatory proteins PSMB5 and SETD7,and ultimately up-regulate the NF-κB-mediated TNFα pathway and P53 pathway,leading to the cycle block TNBC cell line cells and exert cytotoxic effect.