Experimental Study On Amorphophallus Rivieri Durieu Inhibiting Recurrence And Metastasis Of Triple Negative Breast Cancer Through PI3K/Akt Signaling Pathway

Objective To research the effects of Amorphophallus rivieri Durieu on proliferation and apoptosis of triple negative breast cancer cells,the regulations of Amorphophallus rivieri Durieu on PI3K/Akt signal pathway proteins and genes’ expression,so to investigate the effective molecular mechanism of Amorphophallus rivieri Durieu’s inhibition on breast cancer’s recurrence and metastasis.Methods ①The inhibitory effects of Amorphophallus rivieri Durieu medicated serum on proliferation of human breast cancer cell MDA-MB-231 in vitro were tested by MTT assay method,screening out the effective concentrations and effective time,determing the dose-effect and time-effect relationship.②The inhibitory effects of Amorphophallus rivieri Durieu medicated serum of different concentrations on the ability of cell migration and invasion were tested by scratch experiment and an invasion assay in vitro.③The MDA-MB-231 cellular morphological changes 48h after treated with Amorphophallus rivieri Durieu medicated serum of different concentrations were Inverted phase contrast microscope,transmission electron microscope was used to observe the changes of cellular structure and the phenomena of nuclear condensation,chromatin margination and apoptotic bodies formation.④The inhibitory effects of Amorphophallus rivieri Durieu medicated serum of different concentrations on MDA-MB-231 cellular apoptosis were deterined by flow cytometry 48h later.⑤The inhibitory effects of Amorphophallus rivieri Durieu medicated serum of different concentrations on MDA-MB-231 cellular mitotic cycle were deterined by flow cytometry 24h later.⑥The proteins expression of PI3K、Akt、Bad、Caspase9、NF-κB and mTOR of human breast cancer cell MDA-MB-231 treated with Amorphophallus rivieri Durieu medicated serum of different concentrations were tested by Western Blot.⑦The mRNA expression of PI3K、Akt、Bad、Caspase9、NF-κB and mTOR of human breast cancer cell MDA-MB-231 treated with Amorphophallus rivieri Durieu medicated serum of different concentrations were tested by RT-PCR.Results ①With the increased concentration of Amorphophallus rivieri Durieu medicated serum and prolonged treated time,the inhibitory effects of Amorphophallus rivieri Durieu medicated serum on the proliferation of human breast cancer cell MDA-MB-231 gradually enhanced,showing dose-effective and time-effective relationship.The inhibitory rate on cell proliferation of two Amorphophallus rivieri Durieu medicated serum with different concentrations were statistically different with controls at all of the 24h,48h,72h after treated(P<0.01 or P<0.05).②Scratch experiments indicated that compared with the normal control group,Amorphophallus rivieri Durieu medicated serum treated MDA-MB-231 cells showed decreased injury healing ability significantly,correlating with the concentration.The number of MDA-MB-231 cells through Millicell cell membrane decreased significantly(P<0.01)after treated with Amorphophallus rivieri Durieu medicated serum(P<0.01),correlating with the concentration.③Inverted phase contrast microscope observation showed that after treated with Amorphophallus rivieri Durieu medicated serum,MDA-MB-231 cells became gradually shrinked,rounded and floated,cell density decreased,widened the gap,cytoplasmic granules increased,decreased cell proliferation and cell body splited into several pieces.Transmission electron microscopy observation showed that 48h later,cell volume shrinked,cell surface fluff reduced,the nuclear membrane disappeared,nuclear atrophy and fragmentation appeared,apoptotic bodies formed in some cells,chromatin pyknosis partly appeared along with the nuclear membrane,forming a plurality of crumb edge set,cellular organelle extravagated and swelled,vacuole increased significantly.④48h after treated with Amorphophallus rivieri Durieu medicated serum of tow different concentrations,MDA-MB-231 cells showed varying degrees of apoptosis,and the apoptosis rate in the early and middle-late period showed apparently dose-effect relation,especially in the middle-late period.The total apoptosis rate of cells treated with Amorphophallus rivieri Durieu medicated serum of different concentrations were statistically significant compared with the controls(P<0.01).⑤24h after treated with Amorphophallus rivieri Durieu medicated serum of different concentrations,the percentage of Ml,M2 and M3 cycle cells were not significant increased compared with the control group(P>0.05).⑥Amorphophallus rivieri Durieu medicated serum of high concentrations can promote the proteins and mRNA expression while inhibiting the proteins and mRNA expression of PI3K、Akt、Bad、Caspase9、NF-κB and mTOR of human breast cancer cell MDA-MB-231,as proved by Western Blot and RT-PCR.Conclusion ①Amorphophallus rivieri Durieu can inhibit human breast cancer cell proliferation,and shows dose-effect and time-effect relationship.②Amorphophallus rivieri Durieu can inhibit human breast cancer cell migration and invasion in vitro.③Amorphophallus rivieri Durieu induced human breast cancer MDA-MB-231 cell apoptosis is not through the cell cycle effect.④Amorphophallus rivieri Durieu induced human breast cancer MDA-MB-231 cell apoptosis show does-effect and time-effect relation.⑤Amorphophallus rivieri Durieu can induce human breast cancer MDA-MB-231 cell apoptosis through PI3K/Akt signial path.Amorphophallus rivieri Durieu induce apoptosis by inhibitting the expression of antiapoptosis proteins:PI3K、Akt、mTOR and NF-κB while promoting the expression of proapoptosis proteins:Caspase9、Bad,showing a relation with concentration.