Silibinin sensitizes human prostate carcinoma DU145 cells to cisplatin- and carboplatin-induced growth inhibition and apoptotic death

In several recent studies, we have shown that silibinin inhibits the growth of human prostate cancer cells (PCA) both in vitro and in vivo. Here, we investigated the effect of silibinin in combination with cisplatin and carboplatin on human PCA DU145 cell growth and apoptosis. Cisplatin alone at 2 microg/ml dose produced 48% cell growth inhibition, whereas a combination with 50-100 microM silibinin resulted in 63-80% (p<0.05-0.001) growth inhibition. Similarly, compared to 68% growth inhibition at 20 microg/ml carboplatin, addition of 50-100 microM doses of silibinin caused 80-90% inhibition (p<0.005-0.001). In the studies assessing the effect of these combinations on cell cycle progression, a combination of cisplatin or carboplatin with silibinin resulted in a stronger G2-M arrest, compared to these agents alone showing a moderate G2-M and G1 arrests in case of cisplatin and silibinin, and a complete S phase arrest with carboplatin, respectively. A stronger G2-M arrest by these combinations was accompanied by a substantial decrease in the levels of cdc2, cyclin B1 and cdc25C. Silibinin/platinum compound combinations were also effective in inducing apoptosis where cisplatin and carboplatin when combined with silibinin enhanced apoptosis from 8 to 15% and from 20 to 40%, respectively. Apoptosis induction was further confirmed by PARP and caspases 3, 9 and 7 whose cleaved levels were also enhanced by combination treatment. In addition, there was a significant increase in cytochrome c release in the cytosol following treatment of DU145 cells with these combinations. Together, these results show a substantial increase in the efficacy of platinum compounds on human PCA cells, when combined with silibinin, which provide a rationale for further investigations with these combinations.     

葛根芩连汤拮抗D-半乳糖诱导大鼠糖耐量减退作用

目的 研究葛根芩连汤(gegenqinlian decoction,GGQLD)、二甲双胍(metformin,met)和水飞蓟宾(silibinin,sil)对D-半乳糖(D-galaetose,G-gal)诱导大鼠糖耐量减退(impaired glucose tolerance,IGT)的影响。方法 以ip D-gal 150 mg·kg-1,qd×56d诱导胰岛素抵抗(insulin resistance,IR),IGT大鼠模型,测其空腹血糖(fasting blood glucose,FBG)和口服葡萄糖耐量试验(o-ral glucose tolerance test,OGTT)后2小时血糖(2-hours blood glucose,2hBG)含量;并观察GGQL3个剂量、met和sil对病鼠FBG及OGTT后2hBG的效应。结果 正常对照组、GGQL3个剂量、met和sil与模型组比较,FBG均未见明显影响,差异未见显著性(P>0.05);而OGTY后2hBG,模型组明显高于正常对照组(P<0.01),说明D-gal至大鼠IGT模型成立;GGQL3个剂量、met和sil均能抑制OGTT后2hBG的升高,差异均有高度显著性(P<0.01),FBG与OGTT后2hBG间差值降低,差异均有高度显著性(P<0.01),说明各药均能改善D-gal致IR模型大鼠的IGT。结论 D-gal可诱导以IGT为表现的IR,其机制可能与D-gal诱导蛋白非酶糖基化-氧化应激-自由基损伤作用和直接干扰糖脂代谢有关;葛根芩连汤与met和sil一样,能改善D-gal诱导IR大鼠的糖耐量。     

Silibinin Impairs Constitutively Active TGFα-EGFR Autocrine Loop in Advanced Human Prostate Carcinoma Cells

PURPOSE: Epidermal growth factor (EGF) and transformation growth factor-alpha (TGFalpha) are potent mitogens that regulate proliferation of prostate cancer cells via autocrine and paracrine loops, and promote tumor metastasis. They exert their action through binding to the cell surface receptor, epidermal growth factor receptor (EGFR), and cause activation of Erk1/2 mediated mitogenic signaling in human prostate cancer (PCA) at both advanced and androgen-independent stages. Thus, we rationalized that inhibiting this mitogenic pathway could be useful in controlling advanced PCA growth. METHODS: LNCaP and DU145 human PCA cells were treated with silibinin (100-200 muM) for different time points, and the levels of TGFalpha, activated signaling molecules (EGFR, Erk1/2 and Jnk1/2) and Erk1/2 kinase activity were analyzed employing ELISA, immunoprecipitation and/or immunoblotting techniques. The mRNA levels of TGFalpha were analyzed by RT-PCR. RESULTS: Treatment of cells (LNCaP and DU145) with silibinin resulted in a decrease in TGFalpha protein at both secreted and cellular levels together with a decrease in its mRNA level. Silibinin also caused an inhibition of EGFR activation followed by that of Erk1/2 without any change in their protein levels. The kinase activity of Erk1/2 to Elk1 was also inhibited by silibinin at least in DU145 cells. In other study, silibinin caused strong inhibition of Jnk1/2 activation in LNCaP cells while in DU145 cells, a strong induction in Jnk1/2 activation was observed. These results suggest that silibinin impairs TGFalpha-EGFR-Erk1/2 signaling in both androgen-dependent (LNCaP) and -independent (DU145) advanced human prostate carcinoma cells. CONCLUSIONS: This study, for the first time, identifies the inhibitory effect of silibinin on constitutively active TGFalpha-EGFR autocrine loop in advanced human PCA cells, which plausible contributes to the strong efficacy of silibinin in PCA prevention and intervention, as reported in recent studies.     

水飞蓟宾导致慢性肝炎患者缺铁性贫血的研究

目的：研究水飞蓟宾对慢性肝炎患者缺铁性贫血的影响。方法：选取120例慢性肝炎患者分为两组（每组60例）,两组患者在性别、年龄、临床类型及肝功能等实验室检查指标上均无显著性差异。对照组服用多烯磷脂酰胆碱（易善复,赛诺菲安万特制药有限公司）,剂量为70~140 mg,口服,每日3次,12周为一个疗程;试验组服用水飞蓟宾（水林佳,天津天士力制药有限公司）,剂量为70~140 mg,口服,每日3次,12周为一个疗程。两组于治疗前、治疗2周、治疗结束后4周测定血红蛋白、血清铁蛋白、血清铁。结果：对照组60例中,服药前后,血红蛋白、血清铁蛋白、血清铁均无显著变化;试验组60例,给药后血红蛋白、血清铁蛋白、血清铁的差异有显著性意义（P<0.05）。结论：水飞蓟宾会导致慢性肝炎患者缺铁性贫血的发生。     

口服水飞蓟宾胶囊联合吡柔比星膀胱灌注对非肌层浸润性膀胱癌患者术后复发的影响

目的:观察口服水飞蓟宾胶囊联合吡柔比星膀胱灌注对非肌层浸润性膀胱癌患者术后复发的影响。方法:将非肌层浸润性膀胱癌患者124例患者随机分为对照组62例和观察组62例,对照组给予吡柔比星膀胱灌注,观察组给予口服水飞蓟宾胶囊联合吡柔比星膀胱灌注。单因素分析性别、年龄、血尿、吸烟、肿瘤数目、肿瘤大小、肿瘤病理分级、肿瘤分期、水飞蓟宾联合吡柔比星对膀胱肿瘤复发的影响。将单因素分析可能影响肿瘤复发的因素纳入多因素Cox回归分析独立影响肿瘤复发的因素。结果:单因素分析结果提示肿瘤数目、肿瘤大小、肿瘤病理分级、肿瘤分期、水飞蓟宾联合吡柔比星可能是影响膀胱肿瘤复发的因素（P＜0.05）;多因素Cox回归分析显示肿瘤多发、T1是非肌层浸润性膀胱癌的独立危险因素（P＜0.05）,水飞蓟宾联合吡柔比星是非肌层浸润性膀胱癌的独立保护因素（P＜0.01）。结论:口服水飞蓟宾胶囊联合吡柔比星膀胱灌注对非肌层浸润性膀胱癌TUR-BT术后复发的预防效果较好,可减少患者术后早期复发率,且不增加不良反应,较为安全,值得临床推广应用。     

水飞蓟宾对大鼠肝贮脂细胞HSC-T6增殖和胶原合成的影响

目的：观察水飞蓟宾对大鼠肝贮脂细胞ＨＳＣ－Ｔ６增殖和胶原合成的影响,探讨其保护肝脏及抗硬化机制。方法：采用结晶紫染色法测定细胞增殖,＾３Ｈ－脯氨酸掺入法测定胶原合成。结果：水飞蓟宾（６．２５～５０μｇ／ｍｌ）以浓度依赖方式抑制血清、巨噬细胞条件培养液以及血小板源生长因子或转化生长因子β１诱导的细胞增殖和胶原合成。结论：抑制贮脂细胞增殖和胶原合成可能是水飞蓟宾保护肝脏抗硬化机制之一。     

高压乳匀法制备中药固体脂质纳米粒

目的采用高压乳匀法将中药有效成分包载于固体脂质纳米粒(SLN),并研究制备的纳米粒的主要性质。方法选择水飞蓟宾(SIL)和汉防己甲素(TET)为模型药物,采用高压乳匀法将其分别包载于SLN。在电镜下观察其形态,以粒度分析仪和Zeta电位分析仪测定其粒径和Zeta电位,用葡聚糖凝胶柱层析法和HPLC测定其包封率和载药量,还观察了SLN的稳定性。结果高压乳匀法制备的SIL-SLN呈球状,形态规则,平均粒径为(157±8)nm,Zeta电位为(-35.36±2.68)mV,包封率为95.64%,载药量为4.63%;TET-SLN呈片状存在,不规则,粒径较小,平均粒径为(47±3)nm,Zeta电位为(-32.99±2.54)mV,包封率为97.82%,载药量为4.76%。SIL-SLN和TET-SLN有较高稳定性。结论高压乳匀法适于制备包载中药的SLN。     

水飞蓟宾对顺铂所致大鼠肾毒性的预防作用

观察了大鼠 ig水飞蓟宾 (SB)对顺铂 (CDDP)肾毒性的预防作用及可能机理 ,并初步探讨 SB对CDDP抗肿瘤效应的影响 .大鼠经 ig SB 2 0 0 mg·kg-1后 1 h,ip CDDP5mg· kg-13和 5d后与仅ip CDDP组比较 ,体重和谷胱甘肽过氧化物酶活性明显升高 ,而尿素氮含量 ,尿 N -乙酰 -β- D-氨基葡萄糖苷酶的排泄及丙二醛生成均明显降低 .ig SB30 0 mg· kg-1后 ip CDDP5mg·kg-1对荷 S180 小鼠的瘤重抑制率为 85.7% ,与仅 ip CDDP 5mg· kg-1组的瘤重抑制率 (81 .7% )无明显差别 ,ig SB30 0mg·kg-1组的瘤重抑制率为 30 % .表明 SB明显预防 CDDP所致大鼠肾毒性 ,但不影响 CDDP抗肿瘤作用 . SB预防肾毒性的部分机理可能与清除自由基 ,抑制脂质过氧化形成 ,并提高机体抗氧化能力有关 .SB对 CDDP抑制小鼠 S180 生长的效能没有明显影响     

水飞蓟宾联合阿法替尼抑制卵巢癌细胞增殖和侵袭的机制研究

目的 探讨水飞蓟宾联合阿法替尼对卵巢癌的治疗作用及其可能机制。方法 将不同浓度阿法替尼和水飞蓟宾单独或联合作用于卵巢癌SKOV3和CP70细胞48 h，采用CCK-8法检测水飞蓟宾和阿法替尼单独或联用对细胞增殖的影响，并计算半数抑制浓度(IC₅₀)；采用集落形成试验检测药物对细胞增殖的影响；采用Transwell试验检测药物对细胞侵袭的影响；采用免疫荧光和鬼笔环肽染色检测黏着斑激酶(focal adhesion kinase，FAK)和细胞骨架的影响；采用Western blotting检测药物对FAK及其磷酸化的影响；通过数据库分析FAK在卵巢癌中表达水平以及表达水平与预后之间的关联。结果 水飞蓟宾和阿法替尼均能抑制SKOV3和CP70细胞的生长，且抑制作用呈浓度依赖性，水飞蓟宾和阿法替尼对SKOV3的IC₅₀分别为81.2，1.9 μmol·L^-1，对CP70的IC₅₀分别为154.1，4.1 μmol·L^-1。两药联合应用能显著增加生长抑制率；两药均能抑制SKOV3细胞的侵袭能力，且两药联合作用能显著增加对SKOV3细胞侵袭的抑制作用；两药联用能抑制FAK表达水平及磷酸化。水飞蓟宾和阿法替尼均能够显著破坏细胞骨架的完整性，联用时对细胞骨架的破坏更强；数据库分析表明FAK在卵巢癌中的高表达，且FAK的高表达与卵巢癌预后呈负相关。结论 初步证明水飞蓟宾联合阿法替尼用药能显著抑制SKOV3和CP70的增殖，其作用机制可能是通过破坏细胞骨架和减少FAK的形成来抑制SKOV3细胞的侵袭能力。