Studies On The Anti-Tumor Effect And The Mechanism Of Peperomia Ruizet Pav.

Natural products are the important source of anti-tumor drugs,at present more than forty percent of clinical medicines are from natural products and their derivatives,including paclitaxel vinblastine,podophyllotoxin and camptothecin.In some regions of Yunnan and Guizhou province in China the plant Peperomia Ruizet Pav(Peperomia)was described as anti-tumor folk medicine.But the mechanisms of anti-tumor effect in the plant Peperomia were rare reported.In this study,the anti-tumor effect of three plants of Peperomia was analyzed,and the plant Peperomia tetraphylla(P.tetraphylla)was used to explore the mechanism of apoptosis.In addition,the ethanol extract of P.tetraphylla was extracted and separated by screening active ingredient to determine the antitumor compound.1)The MTT assay was used to study the cytotoxic effect of the extracts from the three different Peperomia plants on human lymphoma U937 cells,the result showed that the three different Peperomia species dose-dependently inhibited cell proliferation.And the P.tetraphylla demonstrated the highest cytotoxic activity on U937 cells.We also tested the cytotoxic effect of ethyl acetate fraction(EAEPT)and the water fraction from P.tetraphylla,and demonstrated that EAEPT was the effective parts of anti-tumor effect.2)The mechanism of apoptosis induced by P.tetraphylla on U937 lymphoma cells.The ethanolic extracts and the ethyl acetate fraction of P.tetraphylla(EAEPT)could induce apoptosis measured with Hoechst 33342 stainning,and EAEPT induced apoptosis in a dose-dependent manner detedted by Annexin V-FITC/PI staining.EAEPT also caused U937 cell cycle arrest in a way of increasing S phase and decreasing G2/M and G1 phase.The treatment of EAEPT could down-regulate the expression of CCNB1,CCND1 and CDK1 in a dose dependent manner.Further research showed that EAEPT could activate Caspase-8,then the activated Caspase-8 sheared the structure of BH3 domain protein Bid,then activated the Caspase-3;On the other hand,EAEPT could increase the ROS level and induce loss of mitochondrial membrane potential,and could also increase the expression of Bax,induce the cleavage of the Caspase-9 and then activated Caspase-3.The apoptosis induced by EAEPT could be regulate by Caspase dependent and mitochondrial apoptosis pathway.3)Fractionation and identification of cytotoxic fractions from P.tetraphylla.To identify the active fraction which possesses cytotoxic activity on U937 cells in P.tetraphylla,the extract was partitioned with various solvent systems namely petroleum ether,ethyl acetate and n-butanol.And the ethyl acetate fraction chromatographically fractionated.Among these fractions,petroleum ether and ethyl acetate fraction showed cytotoxic activity on U937 cells.The ethyl acetate fraction was subjected to silica gel column chromatography,eluting with a gradient of chloroform and methanol,and seven fractions were obtained,and three sub-fractions(namely Sub1,Sub2 and Sub7)showed cytotoxic activity on U937 cells after 24 h treatment.Among these sub-fractions,Sub1 showed highest cytotoxic activity on U937 cells.The cytotoxic effect of the three compounds purified from P.tetraphylla on human lymphoma U937 cells,human melanoma cells A375,human skin squamous epidermis cells A431 and human bladder cancer cells T24 was observed by MTT assay.The results showed that the compound 1(Peperobtusin A)possessed the highest cytotoxic activity on several cell lines.And compounds 2 and 3 expressed a poor sensitivity of the tumor cell lines.To sum up,the results indicated that the Sub1 fraction was the active fraction in P.tetraphylla,and the compound 1(Peperobtusin A)could be the main active ingredients to anti-tumor.4)The mechanism of apoptosis induced by Peperobtusin A on U937 lymphoma cells.The apoptosis of U937 and A431 cells were detected with Annexin V-FITC/PI staining,and the U937 cells were more sensitive than A431 cells.Peperobtusin A also caused a S phase cell cycle arrest in U937 cells.Further research showed that the apoptosis induced by Peperobtusin A could increase the ROS level and induce loss of mitochondrial membrane potential,and increase the cleaved Caspase-8,Bid,Caspase-3,Caspase-9 and could also increase the expression of Bax,The Caspase Inhibitors Z-VAD-FMK could decrease the apoptosis mediated by Peperobtusin A in U937 cells.And combining with the Western blot results,the apoptosis induced by Peperobtusin A could be regulate by Caspase dependent and mitochondrial apoptosis pathway.MAPK signaling was related to apoptosis.The P38 and the active form P-P38 was detected by FCM and Western Blotting,and with adding the inhibitor SB203580 and Z-VAD-FMK confirmed that P38 MAPK was engaged in apoptosis.After the inhibition of Caspase,P38 MAPK and Caspase-9 may be the mainly regulator in the process of apoptosis.