PC-SPES Ⅱ对人前列腺癌激素非依赖型细胞株细胞周期和细胞凋亡的作用
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摘要
前列腺癌是西方国家男性患者中最常见的肿瘤,死亡率位居第二。在我国前列腺癌的发病率也在不断增加。随着患者不断增多,对这一疾病诊断和治疗方面存在的诸多问题,也显得日益突出,其中对于激素非依赖型前列腺癌的治疗,更为棘手。一种草药混合制剂PC-SPES(PC为prostate cancer的简写,SPES在拉丁语中有希望的意思)自1996年起在美国被用于治疗前列腺癌,它不仅能缓解前列腺癌患者的临床症状还能延缓前列腺癌的进展,更另人振奋的是其对雄激素非依赖型前列腺癌也有一定效果。但是2002年1月,美国加州卫生健康中心检测到该产品被微量的乙烯雌酚(DES)污染,故其疗效是源于草药还是源于微量DES污染颇受争议。另外PC-SPES对前列腺癌的治疗有类似雌激素药物的副反应,因此,有必要对这一草药制剂配方进行进一步的改进以及进一步明确其中草药制剂的作用。PC-SPES由七种中草药和一种墨西哥草药构成,而来源于墨西哥的草药沙巴棕(即锯叶棕)含有植物雌激素,因此,我们去除了源于国外的具有雌激素作用的沙巴棕成分,以PC-SPES中七种传统中草药为配方,精制提练而成PC-SPES Ⅱ,并经LC-MS检测,发现无DES污染。但这个纯中药制剂是否仍对激素非依赖型前列腺癌有效是一个值得研究的问题,在确证PC-SPES中草药成份抗肿瘤作用的同时,也希望PC-SPESⅡ能够去除PC-SPES雌激素样的副反应。为了解决这一问题,本研究从细胞周期和细胞凋亡入手,以前列腺癌激素非依赖型细胞株PC-3、DU145作为实验研究对象,体外研究PC-SPES Ⅱ对前列腺癌雄激素非依赖型细胞株的抗肿瘤作用。我们检测了前列腺癌分子生物学预后指标SLe~x在激素依赖型和非依赖型前列腺癌细胞株LNCaP和PC-3、DU145细胞表面的表达,并进行比较,进一步明确了各细胞株之间生物学行为的差别,从而可以更好地认知PC-SPES Ⅱ的抗肿瘤作用,更有力地证明PC-SPESⅡ抗癌作用的普遍性。另外,我们选取不同的细胞株,研究PC-SPES Ⅱ对不同前列腺癌细胞株的细胞周期和细胞凋亡的作用,找出它们分子生物学作用的共同机理。
全文共分三个部分:
第一部分 前列腺癌细胞株SLe~x抗原的表达及其酶学机制和意义
SLe~x抗原主要存在于细胞表面糖脂和糖蛋白的糖链上,是糖链末端的三糖结构性抗原。从正常细胞向肿瘤细胞转变时,细胞表面的糖链结构都会发生改变,
Prostate cancer is the leading cancer and the second most common cause of cancer-related death in men in the western countries. The incidence of the prostate cancer is continuously increasing in our country also. The problems of the diagnosis and therapy appeared sharper and sharper with the patients increasing. Among the problems, the therapy of the androgen-independent prostate cancer is more difficult than the others. Herbal mixture named PC-SPES has been used for therapy of prostate cancer since 1996 in the United States of America. Not only it can remit the clinic symptoms of patients, and also it can postpone the progression of the disease. Besides, the remarkable effects of the herbal mixture on the androgen-independent patients are more interested. But in 1. 2002, Hygiene and health center in California in the United States of America declared that the herbal mixture was contaminated with diethylstilbestrol (DES). So there appears an argument that which one, herbal mixture or DES gave rise to the effects on patients. In addition, PC-SPES has the side effect of estrogen-like in the course of therapy. So it is necessary to improve it. It was well known that PC-SPES includes seven Chinese herbs and a Mexican herb. Because the Mexican herb Saw Palmetto contains phytoestrogen, it possessing estrogen-like effect. Saw Palmetto was removed from PC-SPES and the seven traditional Chinese herbs prescription was remained and named PC-SPES Ⅱ. PC-SPES Ⅱ was refined from the reserved seven Chinese herbs and wasn't contaminated with DES detected by LC-MS. The problem that whether the Chinese herbal mixture PC-SPES Ⅱ still have antineoplastic effects deserves our researching. At the same time, avoiding the side effects of estrogen-like may be another advantage obtained from getting rid of Mexican herb. To resolve this problem, we verified the PC-SPES Ⅱ 's effects of arresting cell cycle and inducing the apoptosis on the androgen-independent prostate adenocarcinoma cell lines PC-3, DU145 in vitro. First of all, we detected the expression of SLe~x on the cell surface—a prognostic indicator for prostate cancer in the androgen-dependent and androgen-independent prostate cancer cell lines and further identified the difference of biological behaviour among cell lines. Accordingly, different antineoplasmic effects of PC-SPES Ⅱ on different biological behaviour cell lines was confirmed and then persuasively proved the catholicity of antineoplasmic
effects of PC-SPES II. Moreover, we can find out the common mechanism of molecular biology by comparing the way of arresting cell cycle and inducing apoptosis between the two cell lines.Part 1. Enzymology mechanism and significance of the expression of SLex antigen on prostate cancer cell surfaceSLex antigen mainly resides in sugar chains of glycolipid and glycoprotein on the cell surface. During normal cell convert into the tumor cell, the structure of glycan must change. So did that of the prostate cancer cells and the high expression of SLex is closely correlated with the adrogen-independence, metastasis and progression of the prostate adenocarcinoma. So it is an important prognostic indicator of prostate cancer. But how does it express in various prostate cancer cell lines is unknown by far. The results were as following:1. the cell surface of LNCap,PC-3 and DU145 cell all has the expression of SLex and the expressions of Slex among these three cell lines are different and gradually elevated in order of LNCaPGnT-I was the lowest in DU145 cells and the highest in LNCaP cells in order of LNCap>PC-3>DU145. However, the expressions of C2GnT-II among the three cell lines was contrary to C2GnT-I, the same as Slex, were in order of LNCap IValmost have not significant difference among the three cell lines.4. The whole expression of al,3FucT-III, al,3FucT-VI and al,3FucT-VII in LNCaP was the highest and remarkablely higher than in PC-3 and DU145. There is almost no difference of the whole expression between PC-3 and DU145.5. The growth velocity of each cell was in order of LNCaP structure of glycan on the cell surface is controlled by various glycosyltransferases. So does the expression of SLex. Our results show that C2GnT may be more important in the course of synthesis of Slex and C2GnT-II is not only the critical factor in the course of the synthesis of Slex in prostate carcinoma cell lines, but also is positively correlated with the degree of differentiation of tumor cells and may be foretelling the degree of malignancy of the prostate cancer.Part 2. Effect of Chinese herbal medicine PC-SPESII on cell cycle arrest in androgen-independent prostate carcinoma cell lines Although PC-SPES has effects on the androgen-dependent prostate carcinoma cell, hormone therapy is a more effective modality than the others. So there is no need to research the effects of PC-SPES II on the androgen-dependent prostate carcinoma cell line LNCaP. Because that arresting the tumor cell cycle can inhibit the growth of the tumor cells, in this part, the effects of PC-SPES II on cell cycle arrest in androgen-independent prostate carcinoma cell lines were mainly researched. The results were showed as following:1. PC-SPES II inhibited the growth of PC-3, DU145 cells detected by MTT.2. The cell cycles of PC-3 cells were mainly blocked at the Gl stage, while that of DU145 cells was blocked at both Gl and G2/M stages.3. The expression of pi 6, a cell cycle inhibitory protein, was elevated and the phosphorylated RB protein was decreased in the cells treated with PC-SPES II as compared with the non-treated control cells. However, the expressions of p21wafl, p27Kipl and cyclin Dl proteins were not altered.Herbal mixture PC-SPES II induced the growth inhibition of PC-3, DU145 by cell cycle arrest. Blocking cells at the Gl stage was the common pathway in both PC-3 and DU145 cell lines. So we considered that there may be a common mechanism of action of PC-SPES II arresting the cell cycle in androgen-independent prostate carcinoma cell lines, which is that the up-regulation of pi6 expression, in turn, inhibited the activity of cyclin dependent kinase (CDK), reduced the phosphorylation of RB, and finally blocked cells to get into S stage.Part 3. Effect of Chinese herbal medicine PC-SPES II on inducing the apoptosise of androgen-independent prostate carcinoma cell linesIn part two, we found that PC-SPES II not only inhibited the growth of PC-3, DU145 cells but also reduce the number of their cells. How did the number of tumor
cells be reduced? Caused By death or by apoptosis? In this part, we answered this question by researching the effects of PC-SPES II on inducing the apoptosis of androgen-independent prostate carcinoma cell lines PC-3, DU145. So that the effects of PC-SPES II on antineoplasia were further ascertained. The results were summarized as follows:1. PC-SPES II not only inhibited the growth of PC-3, DU145 cells but also induced death or apoptosis of them detected by MTT2. The apoptosis of PC-3, DU145 cells treated with PC-SPES II after 72 hours was showed by immunofluorescence staining of acricdine orange(AO).3. Apoptotic peak was showed by flow cytometry in PC-3 cells treated with PC-SPES II for 72 hours, but not in the non-treated control cells. Although apoptotic peak of DU145 cells treated with PC-SPES II for 72 hours was not showed by flow cytometry, the considerable cell fragments, which can also resulted from the cell apoptosis, were showed in the cells treated with PC-SPES II for 72 hours, but not in the control cells.4. The expressions of Bcl-2 and Bcl-xL, two antiapoptosis proteins, were decreased respectively in the PC-3, DU145 cells treated with PC-SPES II for 96 hours as compared with the non-treated control cells and Bax, a proapoptosis protein, was elevated only in PC-3 cells treated with PC-SPES II for 96 hours. The expression of Bax in DU145 cells was lost. Though there was no difference of the expression of PKB proteins between treated group and control group both in PC-3 and DU145 cells, the expressions of the activated PKB: the T308 and T473 phosphorylated PKB were elevated respectively in the PC-3, DU145 cells treated with PC-SPES II as compared with the non-treated control cells. Accordingly, the activated fragments of caspase-3 were also raised in both cell lines treated with PC-SPES II.On the basis of our findings, herbal mixture PC-SPES II induced the apoptosis of PC-3, DU145 cells, which was showed by detection of the apoptotic body, specific apoptotic peak and proteins related the apoptosis. It may result from the up-regulation of the expression of Bax only in PC-3 cells, down-regulation of expressions of Bcl-xL in both cell lines and down-regulation of expressions of Bcl-2 in both cell lines, which may be caused by down-regulation of the expression of activated PKB indirectly, in turn, indirectly induced the up-regulation of the activated fragmenst of caspase-3 by
全文共分三个部分:
第一部分 前列腺癌细胞株SLe~x抗原的表达及其酶学机制和意义
SLe~x抗原主要存在于细胞表面糖脂和糖蛋白的糖链上,是糖链末端的三糖结构性抗原。从正常细胞向肿瘤细胞转变时,细胞表面的糖链结构都会发生改变,
Prostate cancer is the leading cancer and the second most common cause of cancer-related death in men in the western countries. The incidence of the prostate cancer is continuously increasing in our country also. The problems of the diagnosis and therapy appeared sharper and sharper with the patients increasing. Among the problems, the therapy of the androgen-independent prostate cancer is more difficult than the others. Herbal mixture named PC-SPES has been used for therapy of prostate cancer since 1996 in the United States of America. Not only it can remit the clinic symptoms of patients, and also it can postpone the progression of the disease. Besides, the remarkable effects of the herbal mixture on the androgen-independent patients are more interested. But in 1. 2002, Hygiene and health center in California in the United States of America declared that the herbal mixture was contaminated with diethylstilbestrol (DES). So there appears an argument that which one, herbal mixture or DES gave rise to the effects on patients. In addition, PC-SPES has the side effect of estrogen-like in the course of therapy. So it is necessary to improve it. It was well known that PC-SPES includes seven Chinese herbs and a Mexican herb. Because the Mexican herb Saw Palmetto contains phytoestrogen, it possessing estrogen-like effect. Saw Palmetto was removed from PC-SPES and the seven traditional Chinese herbs prescription was remained and named PC-SPES Ⅱ. PC-SPES Ⅱ was refined from the reserved seven Chinese herbs and wasn't contaminated with DES detected by LC-MS. The problem that whether the Chinese herbal mixture PC-SPES Ⅱ still have antineoplastic effects deserves our researching. At the same time, avoiding the side effects of estrogen-like may be another advantage obtained from getting rid of Mexican herb. To resolve this problem, we verified the PC-SPES Ⅱ 's effects of arresting cell cycle and inducing the apoptosis on the androgen-independent prostate adenocarcinoma cell lines PC-3, DU145 in vitro. First of all, we detected the expression of SLe~x on the cell surface—a prognostic indicator for prostate cancer in the androgen-dependent and androgen-independent prostate cancer cell lines and further identified the difference of biological behaviour among cell lines. Accordingly, different antineoplasmic effects of PC-SPES Ⅱ on different biological behaviour cell lines was confirmed and then persuasively proved the catholicity of antineoplasmic
effects of PC-SPES II. Moreover, we can find out the common mechanism of molecular biology by comparing the way of arresting cell cycle and inducing apoptosis between the two cell lines.Part 1. Enzymology mechanism and significance of the expression of SLex antigen on prostate cancer cell surfaceSLex antigen mainly resides in sugar chains of glycolipid and glycoprotein on the cell surface. During normal cell convert into the tumor cell, the structure of glycan must change. So did that of the prostate cancer cells and the high expression of SLex is closely correlated with the adrogen-independence, metastasis and progression of the prostate adenocarcinoma. So it is an important prognostic indicator of prostate cancer. But how does it express in various prostate cancer cell lines is unknown by far. The results were as following:1. the cell surface of LNCap,PC-3 and DU145 cell all has the expression of SLex and the expressions of Slex among these three cell lines are different and gradually elevated in order of LNCaP
cells be reduced? Caused By death or by apoptosis? In this part, we answered this question by researching the effects of PC-SPES II on inducing the apoptosis of androgen-independent prostate carcinoma cell lines PC-3, DU145. So that the effects of PC-SPES II on antineoplasia were further ascertained. The results were summarized as follows:1. PC-SPES II not only inhibited the growth of PC-3, DU145 cells but also induced death or apoptosis of them detected by MTT2. The apoptosis of PC-3, DU145 cells treated with PC-SPES II after 72 hours was showed by immunofluorescence staining of acricdine orange(AO).3. Apoptotic peak was showed by flow cytometry in PC-3 cells treated with PC-SPES II for 72 hours, but not in the non-treated control cells. Although apoptotic peak of DU145 cells treated with PC-SPES II for 72 hours was not showed by flow cytometry, the considerable cell fragments, which can also resulted from the cell apoptosis, were showed in the cells treated with PC-SPES II for 72 hours, but not in the control cells.4. The expressions of Bcl-2 and Bcl-xL, two antiapoptosis proteins, were decreased respectively in the PC-3, DU145 cells treated with PC-SPES II for 96 hours as compared with the non-treated control cells and Bax, a proapoptosis protein, was elevated only in PC-3 cells treated with PC-SPES II for 96 hours. The expression of Bax in DU145 cells was lost. Though there was no difference of the expression of PKB proteins between treated group and control group both in PC-3 and DU145 cells, the expressions of the activated PKB: the T308 and T473 phosphorylated PKB were elevated respectively in the PC-3, DU145 cells treated with PC-SPES II as compared with the non-treated control cells. Accordingly, the activated fragments of caspase-3 were also raised in both cell lines treated with PC-SPES II.On the basis of our findings, herbal mixture PC-SPES II induced the apoptosis of PC-3, DU145 cells, which was showed by detection of the apoptotic body, specific apoptotic peak and proteins related the apoptosis. It may result from the up-regulation of the expression of Bax only in PC-3 cells, down-regulation of expressions of Bcl-xL in both cell lines and down-regulation of expressions of Bcl-2 in both cell lines, which may be caused by down-regulation of the expression of activated PKB indirectly, in turn, indirectly induced the up-regulation of the activated fragmenst of caspase-3 by
引文
[1]. Rter RE, Dekernion JB. Epimiology, etiology, and prevention of prostate cancer. In: Walsh PC, Retik AB, Vaughan ED, Wein AJ, eds. Campbell's urology, 8th ed. Philadelphia: Saunders, 2000. p: 303-324.
[2]. Marks LS, DiPaola RS, Nelson P, et al. PC-SPES: herbal formulation for prostate cancer. Urology. 2002; 60(3):369-375
[3]. DiPaola RS, Zhang H, Lambert GH. et al. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in prostate cancer. N Engl J Med. 1998 17; 339(12):785-791.
[4]. Porterfield H. UsToo PC-SPES surveys: review of studies and update of previous survey results. Mol Urol, 2000; 4:289-292.
[5]. De la Taille A, Buttyan R, Hayek O, et al. Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer. J Urol, 2000; 164:1229-1234.
[6]. Xu YM, Lu PC. Experimental studies on immunostimulatory effects of the Isatis indigotica polysaccharide. Chung Hsil Chieh Ho Tsa Chih, 19991;11: 357-359.
[7]. Li W, Asada Y, Yoshikawa T. Flavonoid constituents from Glycyrrhiza glabra root cultures. Phytochemistry, 2000;5: 447-456.
[8]. Tanaka O, Han EC, Yamaguchi H, et al. Saponins of plants of panax species collected in central Nepal, and their chemotaxonomical significance. Ⅲ. Chen pham Ball (Tokyo), 2000; 48:889-892.
[9]. Wang SY, Hsu ML, Hsu HC, et al. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer, 1997; 70:699-705.
[10]. Chan FL, Choi HL. Chen ZY, et al. Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin. Cancer. Lett. 2000; 160: 219-228.
[2]. Marks LS, DiPaola RS, Nelson P, et al. PC-SPES: herbal formulation for prostate cancer. Urology. 2002; 60(3):369-375
[3]. DiPaola RS, Zhang H, Lambert GH. et al. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in prostate cancer. N Engl J Med. 1998 17; 339(12):785-791.
[4]. Porterfield H. UsToo PC-SPES surveys: review of studies and update of previous survey results. Mol Urol, 2000; 4:289-292.
[5]. De la Taille A, Buttyan R, Hayek O, et al. Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer. J Urol, 2000; 164:1229-1234.
[6]. Xu YM, Lu PC. Experimental studies on immunostimulatory effects of the Isatis indigotica polysaccharide. Chung Hsil Chieh Ho Tsa Chih, 19991;11: 357-359.
[7]. Li W, Asada Y, Yoshikawa T. Flavonoid constituents from Glycyrrhiza glabra root cultures. Phytochemistry, 2000;5: 447-456.
[8]. Tanaka O, Han EC, Yamaguchi H, et al. Saponins of plants of panax species collected in central Nepal, and their chemotaxonomical significance. Ⅲ. Chen pham Ball (Tokyo), 2000; 48:889-892.
[9]. Wang SY, Hsu ML, Hsu HC, et al. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer, 1997; 70:699-705.
[10]. Chan FL, Choi HL. Chen ZY, et al. Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin. Cancer. Lett. 2000; 160: 219-228.