Molecular Mechanism Of Two L-type O-quinone Tanshinone Analogs Against Prostate Cancer

Objective:To study the anti-prostate cancer activity of two L-type o-quinone tanshinone analogues in vitro and in vivo,and to elucidate the molecular mechanism of their regulation on the growth,invasion and migration of prostatic carcinoma cells.Methods:MTT assay was used to detect the inhibition of two tanshinone analogues on the proliferation of prostate cancer cells.Flow cytometry was used to detect the effects of compounds on apoptosis,cycle,mitochondrial membrane potential,ROS and Ca²⁺levels of prostate cancer cells.Transwell assay was used to detect the effects of compounds on the invasion and metastasis of prostate cancer cells.Western Bot was used to detect the expression regulation of tanshinone analogues on proliferation,apoptosis and metastasis-related proteins of prostate cancer cells.In vivo tumorigenes-is assay was used to detect the inhibitory effect of the compound on the growth of prostate cancer cells in mice and the toxicity of the compound on heart,liver,spleen,lung and kidney.Results:1.Tanshinone analogs TD21 and TD22 had inhibitory effects on the proliferation of PC3 and LNCa P cells,and the inhibitory effects increased with the increase of concentration.MTT results showed that the IC₅₀values of tanshinone and TD22decreased significantly over time in PC3 cells（p<0.01）.The IC₅₀values of THE two cells did not change significantly in PC3 cells,except for TD21,which was1.047±0.200μM at 48 h（p<0.01）.According to cell morphology observation,PC3and LNCa P cells treated with the two compounds for 24 h showed obvious shrinkage,surrounding vesicles,and some cells became round and floated to death with the increase of concentration.2.Hoechst 33258 staining and flow cytometry showed that TD21 and TD22could induce apoptosis of prostate cancer cells.The flow cytometry results showed that the mitochondrial membrane potential of PC3 and LNCa P cells decreased,the intracellular Ca²⁺level of PC3 and LNCa P cells decreased,and the intracellular ROS level of both cells increased.The apoptosis rate of TD21 was higher than TD22 at the highest concentration.By WB assay,tanshinone analogues TD21 and TD22 could regulate PC3 and LNCa P-related apoptotic proteins Cytc,BCL-2,caspase-3,caspase-9,cleaved-caspase-9,GRP78,PERK,P-EIF2A,CHOP,and regulate the phosphorylation level of P38.3.Flow cytometry was used to detect that the two compounds could block the cell cycle in G2/M phase and regulate the expression of cyclin P21,CDC2,Cyblin B1 and PLK1 in G2/M phase.4.TD21 and TD22 could inhibit the invasion and migration of PC3 and LNCa P cells,and TD22 had higher invasion and migration numbers of PC3 cells than TD21,and TD21 had higher invasion and migration numbers of LNCa P cells than TD22.It can also regulate the expression of MMP1,MMP9 and VEGF related transfer proteins.5.AO staining showed that TD21 and TD22 could induce autophagy in PC3 and LNCa P cells,and TD21 and TD22 could regulate autophagy-related proteins such as AKT,m-TOR,LC3I and LC3II.6.TD21 and TD22 compounds inhibited the growth of prostate cancer cells in mice,and tumor volume decreased significantly with the increase of treatment days of TD21 and TD22.Conclusions:1.Two tanshinone analogues inhibit the proliferation of prostate cancer cells by inducing apoptosis and blocking cell cycle.Studies on the molecular mechanism of tanshinone analogues show that the two compounds mainly regulate mitochondrial pathway and endoplasmic reticulum stress pathway.2.The two tanshinone analogues inhibit the invasion and migration of prostate cancer cells by regulating the VEGE/MMP9/MMP1 pathway.Autophagy of prostate cancer cells can be induced by regulating AKT,m-TOR,LC3I and LC3II.3.Two tanshinone analogues can inhibit tumor formation of prostate cancer cells in vivo with low toxicity.