The Effects And Its Mechanism Of Peanut Skin Procyanidins On Prostate Cancer Cells

Prostate cancer is one of the most frequent cancers which affect men's health.In recent years,there is in an upward trend in China.At the same time,the efficacy is not obvious,but the treatment technology of prostate cancer has great by-effects on the human body and the treatment cost is also high,so the research related to prostate cancer treatment and prevention gradually attracted attention of researchers and the public.And plant Polyphenols have attracted increasing attention of researchers for the development of effective inhibitors against the development and formation of cancer caused by oxidative stress injury.Peanut skin is one of the main by-products of peanut,the yield is not only high,but the vast majority is also in a state of waste.The existing research shows that peanut skin is rich in procyanidins,which is mainly A-type procyanidin,and procyanidins are also widely used in the treatment and prevention of cancer because of their strong oxidant and antioxidant.However,the em PHasis on the relationship between oligomeric procyanidins'composition and structure and its inhibition of prostate cancer growth and prevention of prostate cancer caused by oxidative stress injury was rarely reported in the literature.Therefore,the androgen-independent prostate cancer cell line DU145 was used,and the different fractions PSPs(PSP-1?6)isolated from peanut skin procyanidins by gel were used as raw materials.First,the anti-tumor experiments were carried out in vitro.The effect of proliferation inhibition of prostate cancer DU145 cells by PSPs treatment was further studied by means of fluorescence microscopy,transmission electron microscopy,flow cytometry and western blot and fluorescence quantitative PCR.Then,we continued to use human prostate cancer DU145 cells to build H₂O₂-induced oxidative stress injury model in vitro,and investigatived the inhibitory effect of oxidative stress injury by PSPs.The superior fractions was selected and used to explore the mechanism of prostate protection by multi-index,multi-means and multi-way.Finally,clarifing the functional ingredient by mass spectrometry,which provided support for the development of raw materials and the treatment of prostate cancer subsequently.The main research contents and results are as follows:1.PSPs inhibited DU145 cells proliferation in vitroThere was a certain inhibitory effect on prostate cancer DU145 cells in a significant dose-dependent by PSP isolated from peanut skin and 6 fractions(PSP-1?6)isolated from PSP in a certain concentration range(12.5?g/m L;25?g/m L;50?g/m L;100?g/m L)in vitro,and the effect of PSP-2 was the most obvious among all fractions,which IC₅₀ was 48.565?g/m L.It can be further confirmed by optical microscopy and fluorescence microscopy and transmission electron microscopy that PSP-2 can induce apoptosis of prostate cancer DU145 cells.By flow cytometry,treatment with PSP-2caused significant apoptotic death in a dose-dependent manner at different concentrations,which reached 50.33%at the highest concentration.At the same time,PSP-2 induced S cell cycle arrest in DU145 cells.Further studies showed that the intracellular ROS level was elevated with the increase of drug concentration when PSP-2 triggered apoptosis in DU145 cells.As shown in the results of Western Blot and q RT-PCR,the p53 gene and protein expression was significantly increased in the PSP-2-treated group in a dose-dependent manner,while the expression of Bcl-2 gene and protein decreased,Bax gene and protein expression increased,So the PSP-2 treatment could obviously drop the ratio of Bcl-2/Bax,the Caspase-3 gene and protein expression activity was activated through a series of cascade reaction,so DU145 cells apoptosis,which maybe result in DU145 cells apoptosis.Thus,procyanidins separated from the peanut skin own the anti-prostate cancer effect in vitro.2.Inhibitory effect of PSPs on oxidative stress injury in prostate cancer DU145cellsThe cell viability was 50%approximately when 120?mol/L H₂O₂ stimulated DU145 cells for 2 h,and DU145 cells were injuryed significantly by H₂O₂-induced oxidative stress as intracellular ROS levels was increased,the levels of SOD,CAT and GSH were decreased.At the same time,H₂O₂-induced oxidative stress could induce cell cycle arrest in S phase and cell apoptosisin DU145 cells.The DU145 cell viability could be improved after pretreatment with PSP isolated from peanut skin and 6 fractions(PSP-1?6)isolated from PSP in a certain concentration range(0.2?g/m L;1?g/m L;5?g/m L)in vitro,and PSP was The most effective compound in protecting DU145 cells from H₂O₂-induced oxidative stress,followed by PSP-4 among all fractions.Further studies showed that PSP and PSP-4 could alleviate the cell cycle arrest and apoptosis caused by H₂O₂-induced oxidative stress,and enhance significantly the SOD level to decompose free radicals.Moreover,the level of CAT in the antioxidant enzyme system and the GSH level in the non-antioxidant enzyme system were both increased significantly in order to further decomposite of free radicals and decomposition products,and the intracellular ROS level was reduced ultimately,which protected DU145 cells against H₂O₂-induced oxidative stress.The results in Western Blot showed that PSP and PSP-4 pretreatment in a certain concentration range could inhibit the phosphorylation of ERK1/2 and JNK1/2 in DU145 cells.After a series of cell signals were transferred in DU145 cells,Bax protein level decreased,the level of Bcl-2 protein increased,which lead the increase of Bcl-2/Bax ratio.Finally,cleaved Caspase-9 and cleaved Caspase-3protease expression levels were dropped through the cascade reaction,which is contribute to reducing apoptosis and protecting ptostate from H₂O₂-induced oxidative stress and playing a protective role on the cells.3.Identification of the functional ingredients of anti-prostate cancer DU145cells in vitroPSP-2 and PSP-4 which possessed the better activity in different areas of research were identified by UPLC-Q-TOF-MS/MS.The results showed that PSP-2 isolated from peanut skin procyanidins by gel,with a strong pro-apoptotic effect on DU145 cells,was mainly composed of procyanidin B₃ and the other procyanidins dimer composed of a procyanidin monomer(catechins/epicatechin)linked to a luteolin/kaempferol,which the molecular weight is 574.1044.And PSP-4,with a strong oxidative stress injury inhibition on DU145 cells,was mainly composed of procyanidin A₁ and procyanidin A₂.