小分子化合物水飞蓟素和酮康唑对肿瘤靶点作用机制的研究
摘要
研究背景：
恶性肿瘤已成为人类死亡的一个重要原因。近几十年，肿瘤的手术及放化疗取得了长足的进步，肿瘤患者生存时间明显延长，但对大部分恶性肿瘤的治疗尚未能达到令人满意的效果。当今抗肿瘤药物的研制方向主要有：(1)针对肿瘤发生发展的机制，寻找新的分子作用(酶、受体、基因)靶点等；(2)寻找小分子靶向性药物，包括从中草药等天然产物和已有的化合物库中寻找活性成分。研究环境生物因素等外因在细胞恶性转化过程中细胞分子改变事件，可为恶性肿瘤的药物治疗寻找新的分子靶点。近来Bert Vogelstein等人对11个乳腺癌和11个结直肠癌进行了DNA突变分析，发现其中存在着约15个主要的细胞信号传导通路的失调，并启示我们，如果通过阻断这些通路中的始动者“Driver”，而不是仅仅是阻断个别的从动者“Passenger”基因，则无论这些通路中的基因如何突变，都能起到真正抑制肿瘤生长的作用。由此我们可以认为，研制抑制主要通路中关键基因“Driver”的分子靶向药物或多靶点的药物，是靶向治疗今后的发展方向。Vogelstein等人的研究表明，PI3K／Akt信号通路是关键性的几个信号通路之一。在很多人类肿瘤中普遍存在Akt的高表达，包括肝癌、白血病等，提示Akt可能是一个肿瘤预防和治疗的重要靶点。
我国作为传统中药大国，一直以来在中成药和天然药物的开发和研究上具有很多的积累。中药和天然药物的疾病预防和治疗以及保健作用比较明确，研制新药的成功率比较高，潜力大，前景广。由于中药的独特优势，从中筛选高效、低毒的单体化合物用于肿瘤治疗和预防已成为当前肿瘤治疗研究的热点。在天然植物单体化合物库中进行筛选，筛选得到的化合物一般对人体细胞安全、低毒。
激素和生长因子与其受体组成的信号通路也是肿瘤生长的一个重要因素。雄激素和雄激素受体(AR)对于男性生殖系统及其它组织器官的发育、分化，以及男性肿瘤如前列腺癌的发生、发展密切相关，异常的AR活性被认为是前列腺癌进展的关键因素。由于雄激素受体在雄激素非依赖性前列腺癌(AIPC)中常扩增和过表达，抑制AR的表达可能是治疗前列腺癌的一个有效方案。酮康唑(KT)常用作抗真菌药物，已有多个临床试验将其用于治疗雄激素非依赖性前列腺癌，并能显著抑制PSA水平和改善病人生活质量。但其在体外对前列腺癌细胞的直接作用机理不明。
研究目的：
揭示正常上皮细胞向肿瘤细胞转化中的信号通路改变，并研究小分子化合物水飞蓟素和酮康唑对肿瘤细胞不同信号通路Akt和雄激素受体AR的作用机制，从而为临床应用提供指导。
研究结果：
本论文主要研究结果如下：
1微囊藻毒素诱导大肠Crypt细胞转化的分子机制
1．1转化细胞MTC中Akt，MAPK信号通路激活
微囊藻毒素为我国特有的环境生物因素，研究发现经微囊藻毒素作用的永生化正常大肠Crypt细胞出现细胞克隆，发生细胞转化。
用基因芯片技术对转化细胞进行分析，我们发现Akt通路的多个基因发生上调，包括MAPKAPK2，Akt，HER2，cyclin D1和cyclin D3上调大于两倍，PI3K上调大于1．5倍。Western blot结果显示，微囊藻毒素转化MTC细胞中Akt，cyclinD1，cyclin D3和HER2蛋白表达量比正常对照大肠Crypt(NCC)细胞明显增高。进一步研究发现，MTC细胞中PI3K，Akt和MAPKAPK2酶活性较NCC细胞明显提高。
除了Akt信号通路异常激活外，基因芯片结果显示，MTC细胞中MAPK信号通路也存在上调，多个MAPK通路相关基因上调大于两倍，包括R-Ras，B-Raf，JNK1，JNK2和p38等，而Erk1／2 MAPK都没有明显改变。Western blot结果显示MTC细胞中p38和JNK蛋白显著上调，p38和JNK激酶总活性也明显升高。
1．2 Akt，p38和JNK信号通路的持续性激活可能是细胞转化的原因以及有效的肿瘤治疗靶点
Akt，p38和JNK信号通路在MTC细胞中持续激活，具体表现为21代和30代MTC细胞中的酶活性与15代细胞没有明显差异，且均显著高于NCC细胞。用Akt酶的抑制剂17-AAG 50nM和p38酶抑制剂SB203580 10nM显著抑制了MTC细胞的增殖活性，抑制率分别达42％和33％(与DMSO对照组相比P＜0．01)，同样的，10nM的JNK酶抑制剂SP600125也显示出了很强的细胞生长抑制作用(P＜0．05)，而联用17-AAG和SB203580能更强烈的抑制细胞增殖。这些结果提示Akt，p38和JNK的酶活性可能是微囊藻毒素引起细胞转化所需，这些信号通路可能是微囊藻毒素诱导肠上皮癌变的治疗靶点。
2水飞蓟素是有效的Akt酶抑制剂
2．1多种中药单体对肿瘤细胞的Akt活性影响
选用9种中药化合物，体外筛选Akt酶抑制剂，结果发现，水飞蓟素，辣椒素，和厚朴酚以及儿茶素能明显下调肿瘤细胞Akt酶活性。进一步用Western blot研究发现，用10，50，100ug／ml水飞蓟素处理K562细胞，Akt磷酸化被显著抑制，而且呈浓度依赖性，而Akt总蛋白没有明显改变，提示水飞蓟素可能是通过影响Akt蛋白翻译后修饰而影响其磷酸化。
2．2水飞蓟素对K562细胞的生长抑制作用
K562细胞经过不同浓度水飞蓟素作用48小时后，较低浓度水飞蓟素(1ug／ml)对K562细胞的生长没有影响，随着浓度升高，K562细胞生长受到不同程度抑制，当浓度大于50ug／ml后，细胞生长受到强烈抑制，抑制率分别为26．5％(50ug／ml)和71．2％(100ug／ml)，呈现浓度依赖性。
2．3水飞蓟素诱导细胞凋亡
经100ug／ml水飞蓟素处理48h，Hoechst染色后在荧光显微镜下观察，发现K562细胞核出现染色质浓缩和“凋亡小体”；进一步采用流式细胞术检测发现，用不同浓度水飞蓟素(10，50，100ug／ml)处理48hr之后，AV染色阳性细胞显著增多，证明水飞蓟素有效诱导K562细胞发生凋亡。接着我们用Western Blot检测发现Caspase-3，Caspase-9和PARP的活化产物在水飞蓟素处理后条带明显增强，且呈浓度依赖性。提示水飞蓟素通过介导Caspase-9激活的线粒体途径诱导凋亡。
3酮康唑(KT)对雄激素受体AR信号通路的下调作用
3．1 KT对前列腺癌细胞生长抑制作用
前列腺癌细胞LNCaP，LNCaP-C81，LNCaP-Rec4，LNCaP-SF，Du145，PC3和22RV1经过不同浓度KT作用48小时后，生长受到不同程度抑制，均呈浓度依赖性。
3．2 KT下调了前列腺癌细胞的AR蛋白表达以及PSA表达，分泌和转录水平
经0，5，10，25ug／ml的KT作用24h和48h，结果发现，KT能不同程度抑制细胞PSA表达，且呈现剂量和时间依赖性。对培养基上清进行PSA测定，我们发现细胞PSA的分泌随着KT浓度的增加而逐渐减少，呈现浓度依赖性。用RealtimePCR测定KLK3(PSA的编码基因)的表达水平发现，KT能显著抑制前列腺癌细胞的KLK3基因表达，并且具有浓度梯度效应，与PSA蛋白表达变化基本一致。PSA是AR的靶基因，由于其mRNA水平受到KT的影响而下调，我们进一步观察AR是否被KT影响。Western blot结果显示，经KT处理后三种细胞的AR表达水平不同程度的下降，而随着KT浓度增高，AR表达不断减少，说明KT下调了AR信号通路。
3．3 KT对细胞周期的影响
用流式细胞术进行检测发现，用KT 10ug／ml处理22RVl细胞48hr之后，G0／G1期细胞增多，相应的S期和G2／M期细胞都减少，提示在KT的作用下22RV1细胞发生了G0／G1细胞周期阻滞。用Western blot进一步探讨细胞周期蛋白的变化，细胞周期蛋白依赖激酶Cdk4，Cyclin D1和D3蛋白受到KT的抑制而表达减少，Cip1／p21和Kip1／p27蛋白水平在KT处理后上调，呈浓度梯度依赖。
3．4 KT诱导细胞凋亡
DNA Ladder实验显示，25ug／ml KT处理后22RV1细胞可见明显DNA Ladder梯度，提示KT诱导22RV1细胞发生凋亡。Western Blot检测发现在KT处理后Caspase-9，Caspase-3的切割活化片段条带明显增强，PARP也受到活化。
研究结论：
1．微囊藻毒素导致大肠Crypt细胞转化过程中Akt等信号通路持续性激活，可能是细胞恶性转化的原因。
2．水飞蓟素能有效抑制Akt磷酸化而下调Akt信号通路，并能通过激活caspase-9，-3酶和PARP诱导K562细胞发生凋亡。
3．酮康唑通过下调雄激素受体AR蛋白，减少前列腺癌细胞的PSA表达，诱导G0／G1细胞周期阻滞和细胞凋亡，以及细胞生长抑制作用。
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 microcystin
1.1 Akt and MAPK signaling pathway were activated in transformed cells
Cell 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 activated
Akt,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 colorectalepithelial
2 Silymarin is a potent inhibitor ofAkt kinase
2.1 The inhibition ofAkt kinase of cancer cells by several natural compounds
We 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 cells
After 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 apoptosis
After 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 pathway
3.1 The inhibition of cell proliferation by KT
The 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 andtranscription
After 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 KT
Flow 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 apoptosis
Using 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.
Conclusion
1.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.
恶性肿瘤已成为人类死亡的一个重要原因。近几十年，肿瘤的手术及放化疗取得了长足的进步，肿瘤患者生存时间明显延长，但对大部分恶性肿瘤的治疗尚未能达到令人满意的效果。当今抗肿瘤药物的研制方向主要有：(1)针对肿瘤发生发展的机制，寻找新的分子作用(酶、受体、基因)靶点等；(2)寻找小分子靶向性药物，包括从中草药等天然产物和已有的化合物库中寻找活性成分。研究环境生物因素等外因在细胞恶性转化过程中细胞分子改变事件，可为恶性肿瘤的药物治疗寻找新的分子靶点。近来Bert Vogelstein等人对11个乳腺癌和11个结直肠癌进行了DNA突变分析，发现其中存在着约15个主要的细胞信号传导通路的失调，并启示我们，如果通过阻断这些通路中的始动者“Driver”，而不是仅仅是阻断个别的从动者“Passenger”基因，则无论这些通路中的基因如何突变，都能起到真正抑制肿瘤生长的作用。由此我们可以认为，研制抑制主要通路中关键基因“Driver”的分子靶向药物或多靶点的药物，是靶向治疗今后的发展方向。Vogelstein等人的研究表明，PI3K／Akt信号通路是关键性的几个信号通路之一。在很多人类肿瘤中普遍存在Akt的高表达，包括肝癌、白血病等，提示Akt可能是一个肿瘤预防和治疗的重要靶点。
我国作为传统中药大国，一直以来在中成药和天然药物的开发和研究上具有很多的积累。中药和天然药物的疾病预防和治疗以及保健作用比较明确，研制新药的成功率比较高，潜力大，前景广。由于中药的独特优势，从中筛选高效、低毒的单体化合物用于肿瘤治疗和预防已成为当前肿瘤治疗研究的热点。在天然植物单体化合物库中进行筛选，筛选得到的化合物一般对人体细胞安全、低毒。
激素和生长因子与其受体组成的信号通路也是肿瘤生长的一个重要因素。雄激素和雄激素受体(AR)对于男性生殖系统及其它组织器官的发育、分化，以及男性肿瘤如前列腺癌的发生、发展密切相关，异常的AR活性被认为是前列腺癌进展的关键因素。由于雄激素受体在雄激素非依赖性前列腺癌(AIPC)中常扩增和过表达，抑制AR的表达可能是治疗前列腺癌的一个有效方案。酮康唑(KT)常用作抗真菌药物，已有多个临床试验将其用于治疗雄激素非依赖性前列腺癌，并能显著抑制PSA水平和改善病人生活质量。但其在体外对前列腺癌细胞的直接作用机理不明。
研究目的：
揭示正常上皮细胞向肿瘤细胞转化中的信号通路改变，并研究小分子化合物水飞蓟素和酮康唑对肿瘤细胞不同信号通路Akt和雄激素受体AR的作用机制，从而为临床应用提供指导。
研究结果：
本论文主要研究结果如下：
1微囊藻毒素诱导大肠Crypt细胞转化的分子机制
1．1转化细胞MTC中Akt，MAPK信号通路激活
微囊藻毒素为我国特有的环境生物因素，研究发现经微囊藻毒素作用的永生化正常大肠Crypt细胞出现细胞克隆，发生细胞转化。
用基因芯片技术对转化细胞进行分析，我们发现Akt通路的多个基因发生上调，包括MAPKAPK2，Akt，HER2，cyclin D1和cyclin D3上调大于两倍，PI3K上调大于1．5倍。Western blot结果显示，微囊藻毒素转化MTC细胞中Akt，cyclinD1，cyclin D3和HER2蛋白表达量比正常对照大肠Crypt(NCC)细胞明显增高。进一步研究发现，MTC细胞中PI3K，Akt和MAPKAPK2酶活性较NCC细胞明显提高。
除了Akt信号通路异常激活外，基因芯片结果显示，MTC细胞中MAPK信号通路也存在上调，多个MAPK通路相关基因上调大于两倍，包括R-Ras，B-Raf，JNK1，JNK2和p38等，而Erk1／2 MAPK都没有明显改变。Western blot结果显示MTC细胞中p38和JNK蛋白显著上调，p38和JNK激酶总活性也明显升高。
1．2 Akt，p38和JNK信号通路的持续性激活可能是细胞转化的原因以及有效的肿瘤治疗靶点
Akt，p38和JNK信号通路在MTC细胞中持续激活，具体表现为21代和30代MTC细胞中的酶活性与15代细胞没有明显差异，且均显著高于NCC细胞。用Akt酶的抑制剂17-AAG 50nM和p38酶抑制剂SB203580 10nM显著抑制了MTC细胞的增殖活性，抑制率分别达42％和33％(与DMSO对照组相比P＜0．01)，同样的，10nM的JNK酶抑制剂SP600125也显示出了很强的细胞生长抑制作用(P＜0．05)，而联用17-AAG和SB203580能更强烈的抑制细胞增殖。这些结果提示Akt，p38和JNK的酶活性可能是微囊藻毒素引起细胞转化所需，这些信号通路可能是微囊藻毒素诱导肠上皮癌变的治疗靶点。
2水飞蓟素是有效的Akt酶抑制剂
2．1多种中药单体对肿瘤细胞的Akt活性影响
选用9种中药化合物，体外筛选Akt酶抑制剂，结果发现，水飞蓟素，辣椒素，和厚朴酚以及儿茶素能明显下调肿瘤细胞Akt酶活性。进一步用Western blot研究发现，用10，50，100ug／ml水飞蓟素处理K562细胞，Akt磷酸化被显著抑制，而且呈浓度依赖性，而Akt总蛋白没有明显改变，提示水飞蓟素可能是通过影响Akt蛋白翻译后修饰而影响其磷酸化。
2．2水飞蓟素对K562细胞的生长抑制作用
K562细胞经过不同浓度水飞蓟素作用48小时后，较低浓度水飞蓟素(1ug／ml)对K562细胞的生长没有影响，随着浓度升高，K562细胞生长受到不同程度抑制，当浓度大于50ug／ml后，细胞生长受到强烈抑制，抑制率分别为26．5％(50ug／ml)和71．2％(100ug／ml)，呈现浓度依赖性。
2．3水飞蓟素诱导细胞凋亡
经100ug／ml水飞蓟素处理48h，Hoechst染色后在荧光显微镜下观察，发现K562细胞核出现染色质浓缩和“凋亡小体”；进一步采用流式细胞术检测发现，用不同浓度水飞蓟素(10，50，100ug／ml)处理48hr之后，AV染色阳性细胞显著增多，证明水飞蓟素有效诱导K562细胞发生凋亡。接着我们用Western Blot检测发现Caspase-3，Caspase-9和PARP的活化产物在水飞蓟素处理后条带明显增强，且呈浓度依赖性。提示水飞蓟素通过介导Caspase-9激活的线粒体途径诱导凋亡。
3酮康唑(KT)对雄激素受体AR信号通路的下调作用
3．1 KT对前列腺癌细胞生长抑制作用
前列腺癌细胞LNCaP，LNCaP-C81，LNCaP-Rec4，LNCaP-SF，Du145，PC3和22RV1经过不同浓度KT作用48小时后，生长受到不同程度抑制，均呈浓度依赖性。
3．2 KT下调了前列腺癌细胞的AR蛋白表达以及PSA表达，分泌和转录水平
经0，5，10，25ug／ml的KT作用24h和48h，结果发现，KT能不同程度抑制细胞PSA表达，且呈现剂量和时间依赖性。对培养基上清进行PSA测定，我们发现细胞PSA的分泌随着KT浓度的增加而逐渐减少，呈现浓度依赖性。用RealtimePCR测定KLK3(PSA的编码基因)的表达水平发现，KT能显著抑制前列腺癌细胞的KLK3基因表达，并且具有浓度梯度效应，与PSA蛋白表达变化基本一致。PSA是AR的靶基因，由于其mRNA水平受到KT的影响而下调，我们进一步观察AR是否被KT影响。Western blot结果显示，经KT处理后三种细胞的AR表达水平不同程度的下降，而随着KT浓度增高，AR表达不断减少，说明KT下调了AR信号通路。
3．3 KT对细胞周期的影响
用流式细胞术进行检测发现，用KT 10ug／ml处理22RVl细胞48hr之后，G0／G1期细胞增多，相应的S期和G2／M期细胞都减少，提示在KT的作用下22RV1细胞发生了G0／G1细胞周期阻滞。用Western blot进一步探讨细胞周期蛋白的变化，细胞周期蛋白依赖激酶Cdk4，Cyclin D1和D3蛋白受到KT的抑制而表达减少，Cip1／p21和Kip1／p27蛋白水平在KT处理后上调，呈浓度梯度依赖。
3．4 KT诱导细胞凋亡
DNA Ladder实验显示，25ug／ml KT处理后22RV1细胞可见明显DNA Ladder梯度，提示KT诱导22RV1细胞发生凋亡。Western Blot检测发现在KT处理后Caspase-9，Caspase-3的切割活化片段条带明显增强，PARP也受到活化。
研究结论：
1．微囊藻毒素导致大肠Crypt细胞转化过程中Akt等信号通路持续性激活，可能是细胞恶性转化的原因。
2．水飞蓟素能有效抑制Akt磷酸化而下调Akt信号通路，并能通过激活caspase-9，-3酶和PARP诱导K562细胞发生凋亡。
3．酮康唑通过下调雄激素受体AR蛋白，减少前列腺癌细胞的PSA表达，诱导G0／G1细胞周期阻滞和细胞凋亡，以及细胞生长抑制作用。
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 microcystin
1.1 Akt and MAPK signaling pathway were activated in transformed cells
Cell 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 activated
Akt,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 colorectalepithelial
2 Silymarin is a potent inhibitor ofAkt kinase
2.1 The inhibition ofAkt kinase of cancer cells by several natural compounds
We 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 cells
After 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 apoptosis
After 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 pathway
3.1 The inhibition of cell proliferation by KT
The 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 andtranscription
After 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 KT
Flow 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 apoptosis
Using 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.
Conclusion
1.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.
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