前列消癥汤治疗激素难治性前列腺癌及调控PI3K/Akt传导通路的分子机制
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摘要
1目的
前列腺癌(prostate cancer, PCa)是男性泌尿生殖系统最重要的癌症之一,虽然其发病率在中国明显低于欧美地区,但其发病率正在显著上升。在我国多数被确诊的前列腺癌患者已是肿瘤的中晚期,失去根治机会,只能采用内分泌治疗,但是经过中位时间14～30个月,几乎所有患者都将发展为激素非依赖性前列腺癌(AIPC)及激素难治性前列腺癌(HRPC),目前对于HRPC仍没有一个标准有效的治疗方案,西医的各种治疗效果欠佳,而应用中医药治疗HRPC取得了一定疗效。
前列消癥汤是我国中西医结合泌尿外科奠基人之一刘猷枋教授创制的治疗晚期前列腺癌的方剂,该方由炙黄芪、生薏米、黄精、莪术、土贝母、猪苓、白花蛇舌草七味中药组成,具有益肾健脾、清热解毒、活血利湿的功效。本方扶正与祛邪并用,通过健脾益气,益。肾阴填精髓来扶正祛邪,重视清热利湿与活血行气以祛邪抗癌。前列消癥汤临床应用已十余年。通过总结既往治疗晚期前列腺癌90例,发现前列消癥汤能明显缓解患者排尿困难、食欲减低、乏力等症状,能延长雄激素依赖性向非依赖性的发展时间,明显提高了患者的生活质量。本研究通过对前列消癥汤治疗35例激素难治性前列腺癌患者的临床观察和前列消癥汤含药血清作用人前列腺癌PC3细胞的体外实验,从PI3K/Akt信号通路探索前列消癥汤治疗激素难治性前列腺癌可能的分子机制,阐述其科学性,为总结和挖掘刘猷枋名老中医经验提供新思路。
2方法
2.1临床观察部分
共纳入临床病例72例,全部患者既往均采用药物去势治疗,年龄50～87岁；病程18～36个月,按患者意愿将其分为前列消癥汤联合内分泌治疗组(治疗组)及西医治疗组(对照组)两组。对照组采用药物去势治疗：即皮下注射醋酸戈舍瑞林缓释植入剂3.6mg或肌肉注射醋酸曲普瑞林3.75mg,每28-33天1次。治疗组采用与对照组相同的药物去势方案加用前列消癥汤。最终治疗组共剔除2例(加用化疗1例,放疗1例),对照组剔除4例(加用化疗1例,放疗1例,失访2例),最终有效病例66例,治疗组35例和对照组每组31例,治疗组和对照组平均年龄分别是77.2岁和74.19岁。在6个月的治疗过程中,分别在治疗前、治疗后第1月、3月、6月采集患者生活质量观察指标：FACT-P (Version4)量表评分、Karnofsky量表评分、中医症状量表评分及实验室指标：前列腺特异性抗原(PSA)、血清睾酮(T)、全血细胞分析白细胞数(WBC)、血红蛋白(HB)。数据采用SPSS19.0软件,计量资料分析用两样本均数比较的t检验,计数资料分析用卡方检验、秩和检验。
2.2体外实验部分
本实验选取人PC-3细胞作为研究对象,按人和大鼠体表面积折算的等效剂量比率人(70.0kg)：大鼠(200g)=1：0.018换算大鼠的等效剂量,分为低剂量组(大鼠与人等效剂量),中剂量组(十倍于低剂量组),高剂量组(二十倍于低剂量两组),及生理盐水组4组,将生理盐水及各组前列消癥汤汤剂浓缩后给大鼠灌胃,制备大鼠空白血清及低、中、高剂量组含药血清。分别运用胎牛血清,大鼠空白血清及低、中、高三个剂量的大鼠含药血清培养前列腺癌PC-3细胞24小时后。运用CCK8法测量细胞增值,细胞流式技术测量细胞周期,Transwell小室检测细胞侵袭能力,Western Blot印迹法检测PI3K/Akt通路上游分子PTEN的蛋白表达及PI3K、Akt、 mTOR、p-4E-BP1和p70S6K蛋白磷酸化及表达,RT-PCR法检测含药血清对PI3K/Akt通路下游分子mTOR. NF-κB基因及上游PTEN基因mRNA的表达。
3结果
3.1临床观察部分
卡式评分(Karnofsky Performance Status Scale, KPS)和FACT-P (Version4)量表评分是目前公认的用于评价肿瘤患者临床疗效的指标,本研究采用卡式评分、FACT-P (Version4)量表评分和中医症状评分对前列腺癌患者的生活质量进行评价。前列消癥汤联合西医治疗方案组(治疗组)患者卡式评分、FACT-P (Version4)量表评分及中医单项症状评分在治疗前比较均无统计学差异,而在疗后1月、3月、6月与同时间段西医治疗方案组(对照组)比较,均优于西医治疗组,均有统计学差异,疗后6月为显著性差异。
实验室指标方面,治疗组疗后1月、3月PSA保持平稳,而对照组呈现上升趋势,两组具有显著差异；治疗组疗后6月PSA值再次上升,但上升的趋势慢于同时段对照组,具有显著性差异；前列腺癌患者所采用的去势及抗雄治疗会抑制肾脏分泌促红细胞生成素,导致患者出现血红蛋白降低的现象,所以我们采用血红蛋白(HB)为观察指标之一,在治疗前两组患者血红蛋白数量无差异,治疗后1月、3月、6月,治疗组HB数保持平稳,在正常值下线小幅波动,明显高于同时间段对照组,而对照组HB数量下降,出现低于正常值的情况,具有显著性差异；治疗前,治疗后1月、3月、6月与治疗前比较,治疗组及对照组白细胞数量在统计学上无差异,均处于正常值范围；治疗组和对照组患者经6个月的治疗,血清睾酮均处于去势水平；对照组只有2例患者出现轻微的肝功能异常,未予治疗很快自行恢复。
3.2体外实验部分
细胞增殖试验(CCK-8法)发现,不同浓度的前列消癥汤大鼠含药血清对前列腺癌PC-3细胞作用24小时、48小时、72小时后,均显示出细胞增殖抑制作用,随着药物浓度的升高抑制作用增强,中、高剂量组与对照组比较有统计学差异；不同剂量前列消癥汤大鼠含药血清培养PC-3细胞24小时后,流式细胞仪测定各组PC3细胞周期,PC-3细胞呈现出明显的Go/G1期阻滞现象,随着药物浓度的升高,阻滞于Go/G1期的细胞比例逐渐上升,相应的S期细胞比例明显下降,中、高剂量组与对照组比较有差异；采用Transwell小室法检测胎牛血清组及各含药血清组培养PC-3细胞24小时后,PC-3细胞降解matrigel胶并穿过8um孔径的聚碳酸酯侵袭能力的改变,结果显示,前列消癥汤中、高剂量组侵袭细胞数量明显减少,抑制率大于30%,具有抗侵袭作用,可以使PC-3细胞侵袭力下降;Western blot法检测各组前列消癥汤大鼠含药血清培养PC-3细胞24小时后细胞中PI3K/Akt信号通路关键蛋白变化的结果为,前列消癥汤低、中、高剂量组细胞内p-Akt、p-mTOR、p-4E-BP1和p-p70S6K蛋白水平及p-p65(NF-κB)蛋白水平呈下降趋势,而p-PTEN蛋白水平呈现升高趋势。前列消癥汤中、高剂量组p-Akt、p-4E-BP1和p-p70S6K蛋白水平及NF-κB蛋白表达下降,而p-PTEN蛋白表达升高,与对照组比较具有统计学差异：RT-PCR法检测各组前列消癥汤大鼠含药血清培养PC-3细胞24小时后PC-3细胞中mTOR、PTEN、RelA (NF-κB)基因表达变化的结果显示,高剂量组mTOR基因及RelA (NF-κB)基因的表达被抑制,PTEN基因的表达得到增强,与对照组比较均有差异。
4结论
4.1临床观察部分
4.1.1前列消癥汤联合西医治疗方案能改善激素难治性前列腺癌患者的生活质量,提高卡式评分、改善晚期前列腺癌患者的身体状况、情感状况,使患者能更好的回归家庭及社会,改善和维持患者生存的各项基本功能。
4.1.2前列消癥汤联合西医治疗方案能改善前列腺癌患者出现的纳呆、乏力、失眠、便秘、排尿困难、尿血、水肿等症状。
4.1.3前列消癥汤联合西医治疗方案能延缓PSA增长速度。
4.1.4前列消癥汤联合西医治疗方案能提高患者外周血HB。
4.2体外实验部分
4.2.1前列消癥汤可以通过上调PTEN基因的表达及蛋白翻译,阻断Akt的过度活化,从而抑制PI3K/Akt信号通路,使Go/G1期细胞比例增加,细胞停滞于G1期,从而抑制细胞的增值；前列消癥汤可以抑制Akt、mTOR蛋白及其下游关键蛋白4E-BP1和p70S6K的活化,阻断Akt/mTOR通路,抑制PC-3细胞增殖。
4.2.2前列消癥汤可以抑制核转录因子NF-kB基因及蛋白的表达,阻断PI3K/Akt/NF-kB信号轴,抑制PC-3细胞溶解细胞周围基质,降低其浸润和转移的能力；前列消癥汤可以抑制Akt/mTOR/p70s6k通路中mTOR蛋白,p70s6k蛋白的活化,抑制肌动蛋白的细丝重构,降低细胞迁移能力。
通过以上实验可以推断,前列消癥汤治疗激素难治性前列腺癌效果肯定,其可能的分子机制是前列消癥汤通过抑制前列腺癌细胞PI3K/Akt通路各级关键蛋白的活化及表达,抑制前列腺癌细胞的增值,促进细胞凋亡,并降低其溶解细胞周围基质及运动的能力,抑制其转移。当然,本研究只是从PI3K/Akt通路的一个方面进行了初步的探索,其明确的机制尚需广泛深入的研究来揭示。
1Objective
Prostate cancer(PCa) is one of the most important cancer in men. In Asian countries such as China, although the incidence of PCa is lower than the European and American area, it increases significantly in China. In our country most PCa was found at the middle-late stage of the tumor and can not be cured at all. Although endocrine therapy to PCa is clearly effective at the beginning of the treatment, after a median time of14-30months, almost all PCa develop independence in prostate cancer for hormone (hormone independent PCa, HIPC). And the median surial in these patients is less than20months. For HIPC still there is not standard and effective treatment. And the application of traditional Chinese medicine treatment of HRPC has obtained the certain effect.
Qianliexiaozheng Doses is a TCM dose created by Pro.Liu Youfang, which can treat advanced prostate cancer effectively. This formula is composed of Seven Herbs:Radix Astragali, Rhizoma Polygonati, coix seed, rhizoma curcumae, Rhizoma Bolbostemmae, Polyporus and Hedyotis diffusa. The function of this formula is tonifying kidney and spleen, detoxification, promoting blood circulation and removing dampnesshis. This formula pays attention to regulate spleen and stomach function, strengthening the body resistance to eliminate cancer pathogenic factors through replenishing the spleen Qi and kidney essence, clearing heat, removing dampness and promoting blood and Qi circulation. Qianliexiaozheng Doses has used more than10years of in clinic. By summing up the experiments of90cases' treatment of advanced prostate cancer, Qianliexiaozheng Doses can be found that it can obviously alleviate the symptom such as dysuria, bad appetite, fatigue, prolong the development of androgen dependence to independence, improve the patient's quality of life. The objective of This research is to explore Qianliexiaozheng Doses treating prostate cancer possible molecular mechanism, expound its scientific nature, and summarize and provide new ideas to mining traditional Chinese medicine experience of Pro.Liu Youfang through clinically observing Qianliexiaozheng Doses to35cases with hormone refractory prostate cancer and exploring the PI3K/Akt signal transduction pathway by containing medicine serum human prostate carcinoma PC3cells in vitro experiment.
2Methods
2.1clinical observation
72cases clinical cases, all which were treated with drug castration in the past, aged50to87years old, course of18～36months, were divided into two groups:top Qianliexiaozheng Doses combined with endocrine therapy group (treatment group) and western medicine treatment group (control group). Control group was treated with Castration:subcutaneous injection of goserelin acetate sustained-release depot3.6mg or intramuscular triptorelin acetate3.75mg, once every28-33days. Treatment group was treated with Qianliexiaozheng Doses plus Castration. Final treatment group were removed2cases (1case with radiotherapy,1case with chemotherapy). Control group were removed4cases (1case with radiotherapy,1case with chemotherapy,2cases loss of follow up). The final effective66cases were composed of35cases in the treatment group and31cases control group each group. The average age of treatment group and control group is77.2years and74.19years respectively. During6months of treatment, respectively, before treatment and1,3,6months after treatment patients quality of life was observed though scales:FACT-p (Version4) scale score Karnofsky score, symptoms rating scale of TCM. And Laboratory indexes also were tested: prostate specific antigen (PSA), testosterone (T) in serum, blood cell analysis of white blood cell count (WBC), hemoglobin (HB). Measurement data was analysed with mean t test, counting data was analysed with chi-square test and rank and inspection by SPSS19.0software.
2.2In vitro experiment
This experiment selected human PC-3cells as the research object, used the method of serum pharmacology, and divided into low dose group (rat and human equivalent dose), middle dose group (ten times in the low dose group), high dose group (twenty times in the low dose group two), and saline group. Qianliexiaozheng Doses was fed to rats by gastric lavage method, drug-containing serum of rats prepared. Respectively using fetal bovine serum and high, medium and low doses of drug-containing serum of rats cultures prostate cancer PC-3cells after24hours. Using CCK8method measure cell proliferation and cell streaming technology measure the cell cycle. Cell invasion ability was detected by Transwell Chambers method. The protein expression of PI3K/Akt pathway, PTEN and PI3K, and Akt phosphorylation was detected by Western Blot method. The expression of PI3K/Akt pathway downstream molecular mTOR inhibitors, the nf-kappa B, PTEN gene mRNA were detected RT-PCR method.
3Results
3.1clinical observation
Karnofsky Performance Status Scale(KPS) and FACT-P (Version4) scale is currently accepted for clinical evaluation of patients with tumors of the index. In this study, prostate cancer patients quality of life was evaluated by KPS, FACT-P (Version4) scale score and TCM symptom score. Before treatment, type score, FACT-P (Version4) scale score and TCM symptom score of Patients in treatment group, had no significant differences compared with the control group, but after1months,3months,6months treatment compared with the control group, all scores in treatment group were better than control group, especially significant difference after treatment after6months.
Laboratory index:after treatment for1months and3months, PSA remained stable in the treatment group, while the control group showed a rising trend. The two groups have significant difference. In the treatment group, after treatment for6months, PSA value rose again, but the trend of rising was slow compared with the control group with significant difference. The ovariectomized and antiandrogens treatment will inhibit the secretion of erythropoietin, lead to patients with hemoglobin decrease, so the hemoglobin (HB) were observed in the two groups before treatment. There was no difference in hemoglobin quantity before treatment, but after1month,3months,6months treatment, HB quantity decreased in control group, and below the normal value. HB quantity in treatment group remained stable, fluctuated in the normal line, and was significantly higher than the control group. Before and after1months,3months,6months treatment. the quantity of white blood cells had no difference in statistics in two groups, and also were in the range of normal value. After6months of treatment, serum testosterone was in castrated levels in two groups, while in the control group only2patients had mild abnormal liver function, resumed soon without treatment.
3.2in vitro experiment
Though cell proliferation assay (CCK-8method), different concentrations of Qianliexiaozheng Doses contained serum on prostate cancer cell line PC-3for24hours,48hours,72hours after symptom Decoction in rats, the inhibition of cell proliferation was showed. And inhibition was enhanced with increasing concentration of the drug. There was a significant difference between the high, middle Qianliexiaozheng Doses group and control group. Cultured rat PC-3cells in medicated serum for24hours, with the increase of drug concentration, PC-3cells detected by flow cytometry showed a G0/G1arrested phenomenon obviously, and arrested PC-3cells in G0/G1phase were increased, compared with S phase cells ratio decreased significantly. There was difference compared with the control group. Messured by Transwell assay, cultured PC-3cells by fetal bovine serum group and the Qianliexiaozheng Doses group after24hours, the invasion ability of PC-3cells and degradation of polycarbonate Matrigel8um through the aperture of the change. The result showed, in Qianliexiaozheng Doses middle, high dose group, invasion inhibition rate decreased more than30%. So Qianliexiaozheng Doses middle, high dose had the anti invasion effects, and can make PC-3cells decreased invasiveness. Prostaglandin Western blot method was used to detect serum containing different doses of elimination in cultured rat PC-soup The components of PI3K/Akt signaling pathway in3cells24hours after the cells. The result showed that cells p-Akt, p-mTOR, p-4E-BP1and p-p70S6K protein level and p-p65(NF-κB) protein levels in Qianliexiaozheng Doses high,middle,low group decreased, while the level of p-PTEN protein increased. P-Akt, p-4E-BP1and p-p70S6K protein level and NF-κB protein expression Qianliexiaozheng Doses high, middle, low group decreased, while p-PTEN protein expression increased. And there was statistical significantly difference compared with the control group. Mesured by RT-PCR, cultured rat PC-3cells by containing herbs serum after24h, the results of PTEN, mTOR RelA (NF-κB) gene expression in PC-3cells showed that mTOR gene and RelA (NF-κB) gene expression in high doses group was inhibited, and the expression of the PTEN gene was enhanced. And there was statistical significantly difference compared with the control group.
4Conclusion:
4.1clinical observation
4.1.1Qianliexiaozheng Doses combined with western medicine can significantly improve life quality of HIPC patients, improve the card high ratings, and improve health in patients with advanced prostate cancer, emotional state, enable patients to return to family and society, maintain and improve survival of patients.
4.1.2Qianliexiaozheng Doses combined western medicine therapy can significantly improve the symptoms of prostate cancer patients, such as fatigue, insomnia, constipation, dysuria, bloody urine, swelling and so on.
4.1.3Qianliexiaozheng Doses combined western medicine therapy can delay the PSA growth.
4.1.4Qianliexiaozheng Doses combined with western medicine treatment can improve patients' peripheral blood hemoglobin.
4.2In vitro experiment
4.2.1Qianliexiaozheng Doses can increase the expression of PTEN gene and protein translation, block the excessive expression of Akt, thus suppress PI3K/Akt signal pathway, cell stagnation in G1period rise, G0/G1phase cell proportion increase, thus inhibiting cell value. Qianliexiaozheng Doses can inhibit the Akt, protein mTOR inhibitors and their downstream key protein4E-BP1and p70S6K activation, block Akt/mTOR pathway, PC-3cell proliferation inhibition.
4.2.2Qianliexiaozheng Doses can inhibit the nuclear transcription factor NF-kB gene and protein expression, block PI3K/Akt/NF-kB signal shaft, restrain the PC-3lyse cells surrounding matrix to reduce its ability to invasion and metastasis. Qianliexiaozheng Doses can inhibit the elimination p70s6k/AKT/mTOR pathway of protein mTOR inhibitors, p70s6k protein activation, inhibition of actin filaments refactoring, and decrease cell migration ability.
Through the above experiments can be concluded that Qianliexiaozheng Doses treatment of hormone refractory prostate cancer effect, its possible molecular mechanism is Qianliexiaozheng Doses by inhibiting prostate cancer cells PI3K/Akt pathway activation and expression of all levels of key proteins, inhibition of the appreciation of prostate cancer cells, promote cell apoptosis, and to reduce its ability to dissolve cells surrounding stroma and movement, and inhibit the metastasis. Of course, this research only from one aspect of the PI3K/Akt pathway has carried on the preliminary exploration, its specific mechanism still needs further research to reveal.
前列腺癌(prostate cancer, PCa)是男性泌尿生殖系统最重要的癌症之一,虽然其发病率在中国明显低于欧美地区,但其发病率正在显著上升。在我国多数被确诊的前列腺癌患者已是肿瘤的中晚期,失去根治机会,只能采用内分泌治疗,但是经过中位时间14～30个月,几乎所有患者都将发展为激素非依赖性前列腺癌(AIPC)及激素难治性前列腺癌(HRPC),目前对于HRPC仍没有一个标准有效的治疗方案,西医的各种治疗效果欠佳,而应用中医药治疗HRPC取得了一定疗效。
前列消癥汤是我国中西医结合泌尿外科奠基人之一刘猷枋教授创制的治疗晚期前列腺癌的方剂,该方由炙黄芪、生薏米、黄精、莪术、土贝母、猪苓、白花蛇舌草七味中药组成,具有益肾健脾、清热解毒、活血利湿的功效。本方扶正与祛邪并用,通过健脾益气,益。肾阴填精髓来扶正祛邪,重视清热利湿与活血行气以祛邪抗癌。前列消癥汤临床应用已十余年。通过总结既往治疗晚期前列腺癌90例,发现前列消癥汤能明显缓解患者排尿困难、食欲减低、乏力等症状,能延长雄激素依赖性向非依赖性的发展时间,明显提高了患者的生活质量。本研究通过对前列消癥汤治疗35例激素难治性前列腺癌患者的临床观察和前列消癥汤含药血清作用人前列腺癌PC3细胞的体外实验,从PI3K/Akt信号通路探索前列消癥汤治疗激素难治性前列腺癌可能的分子机制,阐述其科学性,为总结和挖掘刘猷枋名老中医经验提供新思路。
2方法
2.1临床观察部分
共纳入临床病例72例,全部患者既往均采用药物去势治疗,年龄50～87岁；病程18～36个月,按患者意愿将其分为前列消癥汤联合内分泌治疗组(治疗组)及西医治疗组(对照组)两组。对照组采用药物去势治疗：即皮下注射醋酸戈舍瑞林缓释植入剂3.6mg或肌肉注射醋酸曲普瑞林3.75mg,每28-33天1次。治疗组采用与对照组相同的药物去势方案加用前列消癥汤。最终治疗组共剔除2例(加用化疗1例,放疗1例),对照组剔除4例(加用化疗1例,放疗1例,失访2例),最终有效病例66例,治疗组35例和对照组每组31例,治疗组和对照组平均年龄分别是77.2岁和74.19岁。在6个月的治疗过程中,分别在治疗前、治疗后第1月、3月、6月采集患者生活质量观察指标：FACT-P (Version4)量表评分、Karnofsky量表评分、中医症状量表评分及实验室指标：前列腺特异性抗原(PSA)、血清睾酮(T)、全血细胞分析白细胞数(WBC)、血红蛋白(HB)。数据采用SPSS19.0软件,计量资料分析用两样本均数比较的t检验,计数资料分析用卡方检验、秩和检验。
2.2体外实验部分
本实验选取人PC-3细胞作为研究对象,按人和大鼠体表面积折算的等效剂量比率人(70.0kg)：大鼠(200g)=1：0.018换算大鼠的等效剂量,分为低剂量组(大鼠与人等效剂量),中剂量组(十倍于低剂量组),高剂量组(二十倍于低剂量两组),及生理盐水组4组,将生理盐水及各组前列消癥汤汤剂浓缩后给大鼠灌胃,制备大鼠空白血清及低、中、高剂量组含药血清。分别运用胎牛血清,大鼠空白血清及低、中、高三个剂量的大鼠含药血清培养前列腺癌PC-3细胞24小时后。运用CCK8法测量细胞增值,细胞流式技术测量细胞周期,Transwell小室检测细胞侵袭能力,Western Blot印迹法检测PI3K/Akt通路上游分子PTEN的蛋白表达及PI3K、Akt、 mTOR、p-4E-BP1和p70S6K蛋白磷酸化及表达,RT-PCR法检测含药血清对PI3K/Akt通路下游分子mTOR. NF-κB基因及上游PTEN基因mRNA的表达。
3结果
3.1临床观察部分
卡式评分(Karnofsky Performance Status Scale, KPS)和FACT-P (Version4)量表评分是目前公认的用于评价肿瘤患者临床疗效的指标,本研究采用卡式评分、FACT-P (Version4)量表评分和中医症状评分对前列腺癌患者的生活质量进行评价。前列消癥汤联合西医治疗方案组(治疗组)患者卡式评分、FACT-P (Version4)量表评分及中医单项症状评分在治疗前比较均无统计学差异,而在疗后1月、3月、6月与同时间段西医治疗方案组(对照组)比较,均优于西医治疗组,均有统计学差异,疗后6月为显著性差异。
实验室指标方面,治疗组疗后1月、3月PSA保持平稳,而对照组呈现上升趋势,两组具有显著差异；治疗组疗后6月PSA值再次上升,但上升的趋势慢于同时段对照组,具有显著性差异；前列腺癌患者所采用的去势及抗雄治疗会抑制肾脏分泌促红细胞生成素,导致患者出现血红蛋白降低的现象,所以我们采用血红蛋白(HB)为观察指标之一,在治疗前两组患者血红蛋白数量无差异,治疗后1月、3月、6月,治疗组HB数保持平稳,在正常值下线小幅波动,明显高于同时间段对照组,而对照组HB数量下降,出现低于正常值的情况,具有显著性差异；治疗前,治疗后1月、3月、6月与治疗前比较,治疗组及对照组白细胞数量在统计学上无差异,均处于正常值范围；治疗组和对照组患者经6个月的治疗,血清睾酮均处于去势水平；对照组只有2例患者出现轻微的肝功能异常,未予治疗很快自行恢复。
3.2体外实验部分
细胞增殖试验(CCK-8法)发现,不同浓度的前列消癥汤大鼠含药血清对前列腺癌PC-3细胞作用24小时、48小时、72小时后,均显示出细胞增殖抑制作用,随着药物浓度的升高抑制作用增强,中、高剂量组与对照组比较有统计学差异；不同剂量前列消癥汤大鼠含药血清培养PC-3细胞24小时后,流式细胞仪测定各组PC3细胞周期,PC-3细胞呈现出明显的Go/G1期阻滞现象,随着药物浓度的升高,阻滞于Go/G1期的细胞比例逐渐上升,相应的S期细胞比例明显下降,中、高剂量组与对照组比较有差异；采用Transwell小室法检测胎牛血清组及各含药血清组培养PC-3细胞24小时后,PC-3细胞降解matrigel胶并穿过8um孔径的聚碳酸酯侵袭能力的改变,结果显示,前列消癥汤中、高剂量组侵袭细胞数量明显减少,抑制率大于30%,具有抗侵袭作用,可以使PC-3细胞侵袭力下降;Western blot法检测各组前列消癥汤大鼠含药血清培养PC-3细胞24小时后细胞中PI3K/Akt信号通路关键蛋白变化的结果为,前列消癥汤低、中、高剂量组细胞内p-Akt、p-mTOR、p-4E-BP1和p-p70S6K蛋白水平及p-p65(NF-κB)蛋白水平呈下降趋势,而p-PTEN蛋白水平呈现升高趋势。前列消癥汤中、高剂量组p-Akt、p-4E-BP1和p-p70S6K蛋白水平及NF-κB蛋白表达下降,而p-PTEN蛋白表达升高,与对照组比较具有统计学差异：RT-PCR法检测各组前列消癥汤大鼠含药血清培养PC-3细胞24小时后PC-3细胞中mTOR、PTEN、RelA (NF-κB)基因表达变化的结果显示,高剂量组mTOR基因及RelA (NF-κB)基因的表达被抑制,PTEN基因的表达得到增强,与对照组比较均有差异。
4结论
4.1临床观察部分
4.1.1前列消癥汤联合西医治疗方案能改善激素难治性前列腺癌患者的生活质量,提高卡式评分、改善晚期前列腺癌患者的身体状况、情感状况,使患者能更好的回归家庭及社会,改善和维持患者生存的各项基本功能。
4.1.2前列消癥汤联合西医治疗方案能改善前列腺癌患者出现的纳呆、乏力、失眠、便秘、排尿困难、尿血、水肿等症状。
4.1.3前列消癥汤联合西医治疗方案能延缓PSA增长速度。
4.1.4前列消癥汤联合西医治疗方案能提高患者外周血HB。
4.2体外实验部分
4.2.1前列消癥汤可以通过上调PTEN基因的表达及蛋白翻译,阻断Akt的过度活化,从而抑制PI3K/Akt信号通路,使Go/G1期细胞比例增加,细胞停滞于G1期,从而抑制细胞的增值；前列消癥汤可以抑制Akt、mTOR蛋白及其下游关键蛋白4E-BP1和p70S6K的活化,阻断Akt/mTOR通路,抑制PC-3细胞增殖。
4.2.2前列消癥汤可以抑制核转录因子NF-kB基因及蛋白的表达,阻断PI3K/Akt/NF-kB信号轴,抑制PC-3细胞溶解细胞周围基质,降低其浸润和转移的能力；前列消癥汤可以抑制Akt/mTOR/p70s6k通路中mTOR蛋白,p70s6k蛋白的活化,抑制肌动蛋白的细丝重构,降低细胞迁移能力。
通过以上实验可以推断,前列消癥汤治疗激素难治性前列腺癌效果肯定,其可能的分子机制是前列消癥汤通过抑制前列腺癌细胞PI3K/Akt通路各级关键蛋白的活化及表达,抑制前列腺癌细胞的增值,促进细胞凋亡,并降低其溶解细胞周围基质及运动的能力,抑制其转移。当然,本研究只是从PI3K/Akt通路的一个方面进行了初步的探索,其明确的机制尚需广泛深入的研究来揭示。
1Objective
Prostate cancer(PCa) is one of the most important cancer in men. In Asian countries such as China, although the incidence of PCa is lower than the European and American area, it increases significantly in China. In our country most PCa was found at the middle-late stage of the tumor and can not be cured at all. Although endocrine therapy to PCa is clearly effective at the beginning of the treatment, after a median time of14-30months, almost all PCa develop independence in prostate cancer for hormone (hormone independent PCa, HIPC). And the median surial in these patients is less than20months. For HIPC still there is not standard and effective treatment. And the application of traditional Chinese medicine treatment of HRPC has obtained the certain effect.
Qianliexiaozheng Doses is a TCM dose created by Pro.Liu Youfang, which can treat advanced prostate cancer effectively. This formula is composed of Seven Herbs:Radix Astragali, Rhizoma Polygonati, coix seed, rhizoma curcumae, Rhizoma Bolbostemmae, Polyporus and Hedyotis diffusa. The function of this formula is tonifying kidney and spleen, detoxification, promoting blood circulation and removing dampnesshis. This formula pays attention to regulate spleen and stomach function, strengthening the body resistance to eliminate cancer pathogenic factors through replenishing the spleen Qi and kidney essence, clearing heat, removing dampness and promoting blood and Qi circulation. Qianliexiaozheng Doses has used more than10years of in clinic. By summing up the experiments of90cases' treatment of advanced prostate cancer, Qianliexiaozheng Doses can be found that it can obviously alleviate the symptom such as dysuria, bad appetite, fatigue, prolong the development of androgen dependence to independence, improve the patient's quality of life. The objective of This research is to explore Qianliexiaozheng Doses treating prostate cancer possible molecular mechanism, expound its scientific nature, and summarize and provide new ideas to mining traditional Chinese medicine experience of Pro.Liu Youfang through clinically observing Qianliexiaozheng Doses to35cases with hormone refractory prostate cancer and exploring the PI3K/Akt signal transduction pathway by containing medicine serum human prostate carcinoma PC3cells in vitro experiment.
2Methods
2.1clinical observation
72cases clinical cases, all which were treated with drug castration in the past, aged50to87years old, course of18～36months, were divided into two groups:top Qianliexiaozheng Doses combined with endocrine therapy group (treatment group) and western medicine treatment group (control group). Control group was treated with Castration:subcutaneous injection of goserelin acetate sustained-release depot3.6mg or intramuscular triptorelin acetate3.75mg, once every28-33days. Treatment group was treated with Qianliexiaozheng Doses plus Castration. Final treatment group were removed2cases (1case with radiotherapy,1case with chemotherapy). Control group were removed4cases (1case with radiotherapy,1case with chemotherapy,2cases loss of follow up). The final effective66cases were composed of35cases in the treatment group and31cases control group each group. The average age of treatment group and control group is77.2years and74.19years respectively. During6months of treatment, respectively, before treatment and1,3,6months after treatment patients quality of life was observed though scales:FACT-p (Version4) scale score Karnofsky score, symptoms rating scale of TCM. And Laboratory indexes also were tested: prostate specific antigen (PSA), testosterone (T) in serum, blood cell analysis of white blood cell count (WBC), hemoglobin (HB). Measurement data was analysed with mean t test, counting data was analysed with chi-square test and rank and inspection by SPSS19.0software.
2.2In vitro experiment
This experiment selected human PC-3cells as the research object, used the method of serum pharmacology, and divided into low dose group (rat and human equivalent dose), middle dose group (ten times in the low dose group), high dose group (twenty times in the low dose group two), and saline group. Qianliexiaozheng Doses was fed to rats by gastric lavage method, drug-containing serum of rats prepared. Respectively using fetal bovine serum and high, medium and low doses of drug-containing serum of rats cultures prostate cancer PC-3cells after24hours. Using CCK8method measure cell proliferation and cell streaming technology measure the cell cycle. Cell invasion ability was detected by Transwell Chambers method. The protein expression of PI3K/Akt pathway, PTEN and PI3K, and Akt phosphorylation was detected by Western Blot method. The expression of PI3K/Akt pathway downstream molecular mTOR inhibitors, the nf-kappa B, PTEN gene mRNA were detected RT-PCR method.
3Results
3.1clinical observation
Karnofsky Performance Status Scale(KPS) and FACT-P (Version4) scale is currently accepted for clinical evaluation of patients with tumors of the index. In this study, prostate cancer patients quality of life was evaluated by KPS, FACT-P (Version4) scale score and TCM symptom score. Before treatment, type score, FACT-P (Version4) scale score and TCM symptom score of Patients in treatment group, had no significant differences compared with the control group, but after1months,3months,6months treatment compared with the control group, all scores in treatment group were better than control group, especially significant difference after treatment after6months.
Laboratory index:after treatment for1months and3months, PSA remained stable in the treatment group, while the control group showed a rising trend. The two groups have significant difference. In the treatment group, after treatment for6months, PSA value rose again, but the trend of rising was slow compared with the control group with significant difference. The ovariectomized and antiandrogens treatment will inhibit the secretion of erythropoietin, lead to patients with hemoglobin decrease, so the hemoglobin (HB) were observed in the two groups before treatment. There was no difference in hemoglobin quantity before treatment, but after1month,3months,6months treatment, HB quantity decreased in control group, and below the normal value. HB quantity in treatment group remained stable, fluctuated in the normal line, and was significantly higher than the control group. Before and after1months,3months,6months treatment. the quantity of white blood cells had no difference in statistics in two groups, and also were in the range of normal value. After6months of treatment, serum testosterone was in castrated levels in two groups, while in the control group only2patients had mild abnormal liver function, resumed soon without treatment.
3.2in vitro experiment
Though cell proliferation assay (CCK-8method), different concentrations of Qianliexiaozheng Doses contained serum on prostate cancer cell line PC-3for24hours,48hours,72hours after symptom Decoction in rats, the inhibition of cell proliferation was showed. And inhibition was enhanced with increasing concentration of the drug. There was a significant difference between the high, middle Qianliexiaozheng Doses group and control group. Cultured rat PC-3cells in medicated serum for24hours, with the increase of drug concentration, PC-3cells detected by flow cytometry showed a G0/G1arrested phenomenon obviously, and arrested PC-3cells in G0/G1phase were increased, compared with S phase cells ratio decreased significantly. There was difference compared with the control group. Messured by Transwell assay, cultured PC-3cells by fetal bovine serum group and the Qianliexiaozheng Doses group after24hours, the invasion ability of PC-3cells and degradation of polycarbonate Matrigel8um through the aperture of the change. The result showed, in Qianliexiaozheng Doses middle, high dose group, invasion inhibition rate decreased more than30%. So Qianliexiaozheng Doses middle, high dose had the anti invasion effects, and can make PC-3cells decreased invasiveness. Prostaglandin Western blot method was used to detect serum containing different doses of elimination in cultured rat PC-soup The components of PI3K/Akt signaling pathway in3cells24hours after the cells. The result showed that cells p-Akt, p-mTOR, p-4E-BP1and p-p70S6K protein level and p-p65(NF-κB) protein levels in Qianliexiaozheng Doses high,middle,low group decreased, while the level of p-PTEN protein increased. P-Akt, p-4E-BP1and p-p70S6K protein level and NF-κB protein expression Qianliexiaozheng Doses high, middle, low group decreased, while p-PTEN protein expression increased. And there was statistical significantly difference compared with the control group. Mesured by RT-PCR, cultured rat PC-3cells by containing herbs serum after24h, the results of PTEN, mTOR RelA (NF-κB) gene expression in PC-3cells showed that mTOR gene and RelA (NF-κB) gene expression in high doses group was inhibited, and the expression of the PTEN gene was enhanced. And there was statistical significantly difference compared with the control group.
4Conclusion:
4.1clinical observation
4.1.1Qianliexiaozheng Doses combined with western medicine can significantly improve life quality of HIPC patients, improve the card high ratings, and improve health in patients with advanced prostate cancer, emotional state, enable patients to return to family and society, maintain and improve survival of patients.
4.1.2Qianliexiaozheng Doses combined western medicine therapy can significantly improve the symptoms of prostate cancer patients, such as fatigue, insomnia, constipation, dysuria, bloody urine, swelling and so on.
4.1.3Qianliexiaozheng Doses combined western medicine therapy can delay the PSA growth.
4.1.4Qianliexiaozheng Doses combined with western medicine treatment can improve patients' peripheral blood hemoglobin.
4.2In vitro experiment
4.2.1Qianliexiaozheng Doses can increase the expression of PTEN gene and protein translation, block the excessive expression of Akt, thus suppress PI3K/Akt signal pathway, cell stagnation in G1period rise, G0/G1phase cell proportion increase, thus inhibiting cell value. Qianliexiaozheng Doses can inhibit the Akt, protein mTOR inhibitors and their downstream key protein4E-BP1and p70S6K activation, block Akt/mTOR pathway, PC-3cell proliferation inhibition.
4.2.2Qianliexiaozheng Doses can inhibit the nuclear transcription factor NF-kB gene and protein expression, block PI3K/Akt/NF-kB signal shaft, restrain the PC-3lyse cells surrounding matrix to reduce its ability to invasion and metastasis. Qianliexiaozheng Doses can inhibit the elimination p70s6k/AKT/mTOR pathway of protein mTOR inhibitors, p70s6k protein activation, inhibition of actin filaments refactoring, and decrease cell migration ability.
Through the above experiments can be concluded that Qianliexiaozheng Doses treatment of hormone refractory prostate cancer effect, its possible molecular mechanism is Qianliexiaozheng Doses by inhibiting prostate cancer cells PI3K/Akt pathway activation and expression of all levels of key proteins, inhibition of the appreciation of prostate cancer cells, promote cell apoptosis, and to reduce its ability to dissolve cells surrounding stroma and movement, and inhibit the metastasis. Of course, this research only from one aspect of the PI3K/Akt pathway has carried on the preliminary exploration, its specific mechanism still needs further research to reveal.
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