Study Of Molecular Mechanism And Sensitization Of β-elemene To 5FU In Triple-Negative Breast Cancer

OBJECTIVE: Triple-negative breast cancer refers to breast cancer with no expression of progesterone receptor,estrogen receptor,and human epidermal growth factor receptor 2.Its therapeutic effect and prognosis are relatively poor.Chemotherapy is one of its main therapies.5-fluorouracil is commonly used in the clinical treatment of triple-negative breast cancer,but the effect of 5-fluorouracil in actual clinical use is still unsatisfactory.β-elemene is an anti-tumor active monomer extracted from traditional Chinese medicine,and can exert anti-tumor effects by inhibiting PI3k/AKT,MAPK,NF-KB and other pathways.This study investigated whether β-elemene enhanced the inhibition of 5FU-mediated triple-negative breast cancer cell growth and proliferation,migration and invasion,apoptosis,globization ability and inhibition of PI3K/AKT pathway,MAPK pathway,NF-KB pathway.The aim of study was to investigate whether β-elemene may synergize with 5FU to enhance its chemotherapy effect.METHODS: 1.MTT assay was performed to detect the effects of different concentrations of 5-fluorouracil on two kinds of triple-negative breast cancer cells MDA-MB-231,cell viability after 48 hours of BT549,and 10 μ M 5-fluorouracil combined with 50μM β-elemene.Cell viability of cancer cells MDA-MB-231,BT549 after 48 hours.2.Clone formation assay The effect of β-elemene combined with 5-fluorouracil on the proliferation of triple-negative breast cancer cells.The effects of β-elemene combined with 5-fluorouracil on the growth morphology of triple negative breast cancer cells were observed.3.Scratch test,transwell Matrigel invasion test to detect the effect of β-elemene combined with 5-fluorouracil on the migration and invasion of triple-negative breast cancer cells.Western blot was used to detect the expression of major EMT proteins.4.Sphere formation assay,the effect of β-elemene combined with 5FU on the ability of triple-negative breast cancer cells to globulize was tested by sphere formation assay.The expression of main stemness proteins could be tested by Western blot.5.AO/EB staining assay were used to detect the apoptosis of triple-negative breast cancer cells induced by β-elemene combined with 5-fluorouracil.Wesrernblot detects the expression of apoptosis-related proteins.Laser confocal iμmunofluorescence assay was used to detect the in vitro release of cytochrome c mitochondria.6.Western blot analysis of the expression of major proteins in PI3K/AKT and RAF-MEK-ERK pathways in triple-negative breast cancer cells after 48 hours of combined β-elemene and 5-fluorouracil.MTT assay PI3 K inhibitor LY294002,MEK inhibitor U0126 pre-treatment and combined drug combination of three-negative breast cancer cells 48 hours after cell viability.7.β-elemene combined with 5-fluorouracil for three-fold breast cancer cells 48 hours later,we make use of western blot to test the expression of proteins which is related with NF-κB pathway in cytoplasm and nucleus,respectively.Laser confocal microscopy was used to detect the localization of p65 and p50 proteins in triple-negative breast cancer cells.Results: 1.β-elemene can promote the inhibition of 5-fluorouracil on the viability of triple-negative breast cancer cells.Combined with 20 μm β-elemene,the IC50 value of 5-fluorouracil was significantly reduced.2.β-elemene can promote the inhibition of 5-fluorouracil on the proliferation of triple-negative breast cancer cells.5 fluorouracil combined with β-elemene significantly changed the growth morphology of triple negative breast cancer cells.3.β-elemene can promote the inhibition of migration and invasion of triple-negative breast cancer cells by 5 fluorouracil,and the expression of EMT-related proteins is significantly reduced.4.β-elemene can promote the induction of apoptosis of triple-negative breast cancer by 5 fluorouracil,which activates the endogenous mitochondrial apoptosis pathway of triple-negative breast cancer cells.5.The combination of β-elemene and 5FU can enhance the ability of inhibiting the globulization of triple negative breast cancer cells,and can significantly reduce the expression of major stem-related proteins.6.β-elemene combined with 5 fluorouracil significantly inhibited PI3K/AKT,RAF-MEK-ERK and NF-κB signaling pathways in triple-negative breast cancer cells.Conclusion: These results suggest that β-elemene can enhance the inhibitory effect of 5FU on triple-negative breast cancer through PI3K/AKT,RAF-MEK-ERK and NF-kappa B signaling pathways in vivo.To provide a theoretical basis for the clinical application of β-elemene combined with 5FU in the treatment of triple negative breast cancer.