The Mechanism Of Cancer Targeting Effect Of Small Molecules Silymarin And Ketoconazole

Background:Cancer is one of the most important causes of human death.Great achievement ofcancer surgery and chemotherapy was obtained in those decades,leading to extendingof survival time of cancer patients.But the efficiency of those cancer therapies was notso good to satisfy patients.People want to develop anti-cancer drugs through:(1)findnew molecular target(enzymes,receptors and genes)involved in cancer development;(2)develop active molecules from traditional medicine or natural products.Extra factorsincluding environmental biology factors play important role in cell transformation.Revealing the mechanism might provide some new targets of cancer therapy.The BertVogelstein's group found that there was dysregulation of 15 main cell signalingpathways based on the analysis of DNA mutation of 11 breast and 11 colorectal cancersamples.It was suggested that it is important to block the initiator-"Driver"but not thefollower-"Passenger".We believe that developing some molecules targeting the keygenes of"Driver"of main signaling pathway is the future of target therapy.PI3K/Aktpathway is one of the most important pathways and is overexpressed in many humancancers including liver cancer and leukemia,suggesting that Akt is an important targetof cancer prevention and therapy.Chinese traditional medicine provides a good pool for cancer drug development.The advantages of new drugs made from traditional medicine and natural products arelow toxic and great activity.It becomes a research highlight of cancer drug development.Hormones and growth factors and their receptors are important to some tumordevelopment.Androgen and androgen receptor(AR)plays a important role indevelopment and differentiation of males and prostate cancer.Abnormal activity of ARsignaling is a key factor of prostate cancer advancement.Duplication andoverexpression is frequently observed in androgen-independent prostate cancer(AIPC),indicating that the suppression of AR expression might be an effective target oftreatment.Ketoconazole,which is an antifungal drug,was deployed in several clinicalstudies for AIPC treatment,with PSA decrease and improvement of life quality.Butthe mechanism of the direct effect of KT to prostate cancer cells remains unknown.Objects:To reveal the pathways involved in cell transformation of normal epithelial cells,study the mechanisms of silymarin and ketoconazole targeting Akt and androgenreceptor signaling pathway and provide some suggestions for clinical use.Contents:1 The mechanism of colon crypt cell transformation induced by microcystin1.1 Akt and MAPK signaling pathway were activated in transformed cellsCell clones were emerged after microcystin treatment of normal immortalized coloncrypt cells,indicating cell transformation occurred.Using genechip technology,wefound that many genes of Akt signaling pathway were upregulated,includingMAPKAPK2,Akt,HER2,cyclin D1 and cyclin D3 greater than 2 folds and PI3K morethan 1.5 folds.Western blot results showed that protein expression of Akt,cyclin D1,cyclin D3 and HER2 was increased in transformed cells(MTC)compared with normalcells(NCC).Further analysis demonstrated that kinase activities of PI3K,Akt and MAPKAPK2 in MTC cells were significantly enhanced.It was also shown that MAPK signaling pathway was activated,including R-Ras,B-Raf,JNK1,JNK2 and p38 upregulation more than 2 folds in MTC cells,except forErkl/2.Western blot results showed proteins of p38 and JNK were significantlyincreased.Further analysis showed that kinase activities of p38 and JNK were alsoenhanced.1.2 Akt,p38 and JNK MAPK signaling pathway were constitutively activatedAkt,p38 and JNK kinase activities were upregulated in 21 and 30 generations ofMTC cells and there was no significant decrease compared with 15 generations.Wewant to know if Akt,p38 and JNK kinase activities were the cause or consequence ofcell transformation.Therefore,we sought to determine whether the inhibition of Akt,p38 and JNK kinase activities could inhibit the proliferation of MTC cells.The mediumwas supplemented with 50nM(17-AAG),which is a potent inhibitor of Akt activation,10nM SB203580,as a selective inhibitor of p38 kinase and 10nM SP600125 toselectively inhibit JNK kinase.By this means,it was found that the proliferation ofMTC could be significantly decreased by each agent added separately compared withMTC treated with 10nM DMSO(control)(17-AAG and SB203580,all P＜0.01;SP600125,P＜0.05,Student's t-test),as detected by MTT assay,and to an even greaterextent if 17-AAG and SB203580 were added together.This suggested that Akt,p38 andJNK activities may be required for cell transformation and,therefore,these kinasesmight be useful therapeutic targets for blocking the carcinogenesis of colorectalepithelial2 Silymarin is a potent inhibitor ofAkt kinase2.1 The inhibition ofAkt kinase of cancer cells by several natural compoundsWe selected 9 compounds from traditional medicine or natural products for Aktkinase inhibition assay.The results showed that silymarin,capsaicin,honokiol andcatechin significantly inhibited Akt kinase acitivities.Western blot results showed that 10,50 and 100ug/ml silymarin inhibited the phosphorylation of Akt proteins in K562cells,withouth changing of total protein expression,indicating that silymarin mightaffect the post-translation of protein expression.2.2 Silymarin inhibited the cell proliferation of K562 cellsAfter treatment of silymarin for 48h,K562 cell proliferation was inhibited with adose-dependent manner,except for＜lug/ml dose.The inhibition rate was 26.5% and71.2% of 50 and 100ug/ml doses of silymarin,respectively.2.3 Silymarin induced cell apoptosisAfter 100ug/ml silymarin treatment for 48h,K562 nucleus showed chromosomecondenses by Hoechst staining.Flow cytometry showed AV positive cells weremarkedly increased after different doses of silymarin(10,50 and 100ug/ml)treatment,indicating cell apoptosis.Western blot results demonstrated that Caspase-3,Caspase-9and PARP were activated by silymarin.3 Ketoconazole(KT)down-regulated androgen receptor(AR)signaling pathway3.1 The inhibition of cell proliferation by KTThe cell proliferation of human prostate cancer cells LNCaP,LNCaP-C81,LNCaP-Rec4,LNCaP-SF,Du145,PC3 and 22RV1 was inhibited by KT for 48h with adose-dependent manner.3.2 KT downregulated AR protein expression and PSA expression,secretion andtranscriptionAfter treatment of 0,5,10 and 25ug/ml KT for 24h and 48h,PSA expression wasdecreased with a time-and dose-dependent manner.And the secretion of PSA wasinhibited by KT treatment based on the measurement of media PSA levels.RealtimePCR showed that transcription ofKLK3(PSA coding gene)was also inhibited.As PSAis a target gene of AR,we want to know if the expression of AR was affected or not.Western blot results showed that AR protein expression was decreased by KT treatmentin three different prostate cancer cells,suggesting the down-regulation of AR signaling by KT.3.3 Regulation of cell cycle by KTFlow cytometry results showed that G0/G1 phase cells increased by 10ug/ml KTtreatment for 48hr,and S and G2/M cells decreased,indicating a G0/G1 cell cycle arrestin 22RV1 cells.Western blot results demonstrated that several cell cycle proteins werechanged after KT treatment,including down-regulation of Cdk4,cyclin D1 and cyclinD3 and upregulation of Cip 1/p21 and Kip 1/p27 with a dose-dependent manner.3.4 KT induced cell apoptosisUsing DNA Ladder assay,we found that a clear DNA Ladder showed in 22RV1cells after 25ug/ml KT treatment for 48h,suggesting cell apoptosis in 22RV1 cells.Itwas shown that Caspase-9,Caspase-3 and PARP were activated after KT treatment byWestern blot determination.Conclusion1.Akt,p38 and JNK MAPK signaling pathway is constitutively activated in celltransformation of normal immortalized colon crypt cells by microcystin,and itmight be the cause of cell transformation.2.Silymarin is a potent inhibitor of Akt kinase,and induces apoptosis of K562 cellsthrough caspase-9,-3 and PARP activation.3.Ketoconazole inhibits AR signaling pathway including downregulation of ARprotein expression and PSA expression,secretion and transcription,accompanied byG0/G1 cell cycle arrest,apoptosis and inhibition of cell proliferation.