Effect And Mechanism Of Dendrobium Nobile Lindl Alkaloids On Triple Negative Breast Cancer Cells

Objective: To explore the effects of Dendrobium nobile Lindl alkaloids on triple negative breast cancer cells and analyze the possible molecular mechanisms,and provide experimental evidence for the development of new anti-breast cancer drugs.Methods:1.Effects of Dendrobium nobile Lindl alkaloids on proliferation in human triple negative breast cancer cells: MDA-MB-231 and MDA-MB-453 cells were cultured,and the effects of different concentrations of DNLA on cell proliferation were detected by CCK-8.2.Effects of Dendrobium nobile Lindl alkaloids on apoptosis in human triple negative breast cancer cells: Flow cytometry was applied to discover the effect of DNLA on apoptosis of MDA-MB-231 and MDA-MB-453 cells.3.Effect of Dendrobium nobile Lindl alkaloids on migration and invasion in human triple negative breast cancer cells: After treatment with MDA-MB-231 and MDA-MB-453 cells for a certain time with a inhibition rate of less than 25%corresponding to DNLA,the wound healing assay analyzes the change of DNLA on cell migration ability,and Transwell assay further tests the effect of DNLA on cell migration and invasion.4.Molecular mechanism of Dendrobium nobile Lindl alkaloids on human triple negative breast cancer cells: Quantitative Real-time PCR to estimate the changes of PI3 K,AKT,caspase 3,caspase 9,E-cadherin and Vimentin m RNA expression by different concentrations of DNLA.The phosphorylation of PI3K/AKT pathway,activation of apoptotic proteins and expression of EMT-related proteins by DNLA were determined by Western blot using PI3 K inhibitors and agonists.Results:1.Different doses of DNLA were used to treat MDA-MB-231 and MDA-MB-453 cells respectively for different times(24,48,72h).DNLA inhibited the proliferation of cancer cells significantly in a dose-and-time dependent manner.2.With exposure to various concentrations of DNLA for 24 h,the apoptosis rate increased gradually with the increase of DNLA concentration,and there was significant difference between the high concentration group and the low concentration group.3.In the control group,MDA-MB-231 and MDA-MB-453 cells gradually recovered after different times(12,24,36h).After the drug treated with the cells,the wound healing ability was significantly weaker than that of the control group.That is to say,the migration ability of the cells was significantly weakened,and the cell migration ability decreased as the drug concentration increased.Regardless of the Transwell migration assay or the invasion assay,the number of MDA-MB-231 cells in the DNLA drug group across the microporous membrane was significantly reduced and showed a dose-dependent effect.However,it was found that MDA-MB-453 cells could not pass through the microporous membrane of the transwell plate during the experiment.The cells in the blank control group could not pass through the microporous membrane.After repeated experiments,no MDA-MB-453 cells were observed to pass through the microporous membrane.It might be related to the biological characteristics and growth pattern of MDA-MB-453 cells.4.DNLA increased the expression of caspase 3 and caspase 9 m RNA,and increased the expression of E-cadherin m RNA,which decreased the expression of vimentin m RNA.But DNLA did not significantly change the expression of PI3 K and AKT m RNA by q RT-PCR.Western blot analysis showed that DNLA group,LY294002 group and LY294002+DNLA group could decrease the expression of p-PI3 K and p-AKT protein,and increase the expression of apoptosis-related proteins active caspase 3 and active caspase 9.DNLA significantly up-regulated E-cadherin protein expression and inhibited vimentin protein expression.However,there was no significant difference between the IGF-1 group and the IGF-1+DNLA group,and no significant change in the combination group and the single drug group.Conclusion:1.A certain concentration of DNLA has a significant inhibitory effect on proliferation of human triple negative breast cancer cells in a dose-and time-dependent manner.2.DNLA may induce apoptosis of human triple negative breast cancer cells through mitochondrial pathway?3.DNLA may inhibit the EMT process of triple-negative breast cancer cells by up-regulating E-cadherin protein and down-regulating vimentin protein expression,and exert anti-metastatic effect.