Patterns of metastasis by the human prostate cancer cell line PC-3 in athymic nude mice

Cells from the PC-3 human prostate cancer cell line were evaluated in athymic nude mice in order to determine the influence of size of the primary tumor and site inoculation on the incidence and pattern of metastasis. At autopsy, all organs, including the skeleton, were evaluated for metastasis. Subcutaneous injections resulted in metastases to the draining axillary lymph node and lungs (56% and 13%, respectively), and were correlated with size of the primary tumor. Tail vein injection resulted in a high incidence of lung metastasis, while injection into the peritoneal space, spleen, and seminal vesicles resulted in intraabdominal tumor growth, liver metastasis, and large tumors within the seminal vesicles, respectively. Skeletal metastases were not observed in any of the animals studied. We conclude that injection of PC-3 cells into various sites results in different patterns of metastasis, but may not constitute an entirely suitable animal model of human prostate cancer due to the lack of metastasis to the skeleton.     

Transplantable human prostatic carcinoma (PC-82) in athymic nude mice. II. Tumor growth and androgen receptors

Androgen receptor (AR) levels were measured in PC-82 tumor tissue grown in hormonally manipulated nude mice. In the nuclei of tumor tissue from intact male mice a relatively low concentration (mean 25 fmol/mg protein) of androgen receptors (ARn) was found, while no receptors for estrogens or progestins were detected. The total number of androgen receptors in the PC-82 tumor tissue (measured in the nuclei 1 h after injection of a single high dose of testosterone (T)) was found to be 100 fmol/mg protein. The antiandrogen cyproterone acetate, administered in combination with the high dose of T, significantly lowered the amount of ARn in the tumor tissue. In the nuclei of tumor tissue from intact tumor-bearing male mice with T-containing Silastic implants, a 4-times higher amount of tightly associated AR was found. In addition, an increased growth rate of the tumor was observed following T implantation. This finding suggests that the increased growth rate of the PC-82 tumor is associated with a continuous occupancy of AR in the nuclei of the tumor tissue. Castration of tumor-bearing male mice, which arrests the growth of this tumor, did not affect the concentration of ARn in the tissue compared to that of tissue in the intact control situation. In addition, the total amount of AR measured after T injection was not affected by castration. Therefore, the availability of a sufficient and steady level of T in the plasma and consequently the duration of the presence of AR in the nucleus of the PC-82 tumor tissue, rather than the total concentration of AR, appear to be the limiting factors in the modulation of hormonal responses in this androgen target tissue.     

The multikinase inhibitor sorafenib induces caspase-dependent apoptosis in PC-3 prostate cancer cells

The present study investigated the effects of the multikinase inhibitor sorafenib on androgen-independent can- cer cells viability and intracellular signaling. Human androgen-independent PC-3 prostate cancer cells were treated with sorafenib. At concentration that suppresses extracellular signal-regulated kinase phosphorylation, sorafenib treatment reduced the mitochondrial transmembrane potential. Sorafenib also down-modulated the levels of mye- loid cell leukemia 1, survivin and cellular inhibitor of apoptosis protein 2. Sorafenib induced caspase-3 cleavage and the mitochondrial release of cytochrome c. However, no nuclear translocation of apoptosis inducing factor was detected after treatment and the pan-caspase inhibitor Z-VAD-FMK had an obvious protective effect against the drug. In conclusion, sorafenib induces apoptosis through a caspase-dependent mechanism with down-regulated antiapoptotic proteins in androgen-independent prostate cancer cells in vitro.     

槲皮素对前列腺癌PC-3细胞凋亡作用的研究

目的:研究槲皮素对人前列腺癌PC-3细胞的凋亡作用。方法:体外培养PC-3细胞,给予不同浓度(50、100、150、200、250μmol/L)的槲皮素处理,MTT法检测槲皮素对细胞抑制率;流式细胞仪检测细胞凋亡率;透射电镜观察细胞超微结构的变化。结果:槲皮素能抑制人前列腺癌PC-3细胞的体外生长,且呈时间与剂量依赖性,浓度由低到高,其24 h的抑制率(%)分别为3.01±1.32、4.84±1.73、20.35±1.30、16.78±1.89、27.25±4.01,48 h的抑制率(%)分别为10.18±1.16、6.22±0.04、24.29±4.19、22.4±4.26、41.42±5.43,当药物浓度>150μmol/L时P<0.05,具有统计学意义;流式细胞术显示PC-3细胞凋亡率随槲皮素浓度升高和作用时间延长而增加(P<0.05),其中浓度150μmol/L和200μmol/L组,24 h的凋亡率(%)分别19.10±0.28、26.55±0.78,48 h的凋亡率(%)分别为27.65±1.06、38.30±5.96;电镜观察到细胞的典型凋亡形态学变化。结论:在适当的条件下,槲皮素对人前列腺癌PC-3细胞增殖有明显的抑制作用,同时可诱导PC-3细胞凋亡,其具体作用机制有待进一步深入研究。     

Transplantable human prostatic carcinoma (PC-82) in athymic nude mice. III. Effects of estrogens on the growth of the tumor tissue

The transplantable human prostatic tumor model (PC-82) in nude mice was used to evaluate the indirect and possibly direct effects of estrogens on the growth of prostatic tumor tissue. High (pharmalogical) doses of plasma estradiol (E2) were achieved in tumor-bearing mice by using E2-containing Silastic implants of different lengths. In comparison with the situation in men, in mice much higher concentrations of circulating E2 (exceeding 3 nmol/liter) were necessary to attain (near)-castrate levels of plasma testosterone (T). Treatment of tumor-bearing mice with a high dose of E2 resulted in tumor growth arrest and a subsequent decline of the tumor volume, which equals the effects of castration. No evidence was found that either of the two doses of E2 applied had any additive inhibitory effect on tumor growth when compared to castration alone. It was inferred from these findings that in the PC-82 tumor model, estrogens, rather than having a direct effect on the tumor tissue, mainly act indirectly by their suppressive effect on T secretion in the host animal. A different and unexpected result was obtained in castrated tumor-bearing mice treated with a combination of E2 and T. With both doses of E2 this type of treatment led to a smaller increase of the tumor volume compared with mice receiving T only, the result of the high dose being statistically significant. This antagonistic effect of the two steroids on the PC-82 tissue was paradoxically associated with a sharp increase of nuclear androgen receptor levels and a higher concentration of dihydrotestosterone in the tumor tissue. Plasma and tissue concentrations of T appeared to be unaltered. The present study of the PC-82 prostate tumor shows that only by careful monitoring of plasma steroid levels in tumor-bearing mice can conclusions about the effectiveness of hormonal treatment regimens, such as estrogen therapy, be drawn.     

Transplantable human prostatic carcinoma (PC-82) in athymic nude mice: I. Hormone dependence and the concentration of androgens in plasma and tumor tissue

This paper is the first part of a series of three describing a number of observations made on the PC-82 human prostatic carcinoma, xenografted into nude mice. The previously described androgen-dependence, one of the main properties of this tumor, has been the subject of subsequent studies. The impact of hormonal manipulation on the growth of the tumor and on plasma and tissue concentrations of androgens is discussed in this first part of the series. The great variability of plasma testosterone (T) levels in intact male mice (range: 1-90 nmol/liter) has been levelled out by the use of T-containing Silastic implants, resulting in levels ranging from 18 to 35 nmol/liter. Moreover, this route of administration also facilitated hormonal manipulation of tumor bearing mice. Androgen withdrawal from male mice with growing PC-82 tumors caused 80% tumor regression at ten weeks after androgen deprivation; the decline of the tumor volume followed a biphasic course. Delayed androgen substitution in castrated male mice grafted with PC-82 30 days before resulted in growth of the tumor tissue. This indicates that cells do not die and keep the capability to respond to androgens. It was concluded that the growth of the PC-82 tumor is not compatible with plasma T levels lower than 1 nmol/liter. Variable concentrations of endogenous T and dihydrotestosterone (DHT) were detected in total homogenates of PC-82 tumor tissue. Androgen withdrawal from T-implanted, tumor-bearing female mice caused a rapid reduction (90% within one day) of the tissue-T and a slower decline (up to 90% within seven to ten days) of tissue-DHT concentrations.     

参附注射液对前列腺癌PC-3细胞凋亡诱导的影响

目的:研究参附注射液(SF)对人前列腺癌PC-3细胞凋亡的影响及可能机制。方法:实验设立对照组和SF 50、100、200μl/ml组,Annexin V/PI染色流式细胞术检测细胞凋亡,RT-PCR检测p53 mRNA表达。结果:与对照组比较,作用24、48、72 h后,SF 50、100、200μl/ml组PC-3细胞存活率显著减少(P均0.05)。SF各组24 h存活率分别为(93.76±2.63)%、(81.21±1.80)%、(18.01±3.84)%;48 h存活率分别为(94.67±1.11)%、(78.33±2.89)%、(10.34±1.44)%;72 h存活率分别为(91.30±0.47)%、(36.67±1.56)%、(1.33±0.32)%,呈浓度和时间依赖性。作用48 h后,p53 mRNA表达明显升高(P0.05)。结论:SF可以诱导PC-3细胞凋亡,其作用机制可能与p53表达增高相关。     

靶向沉默核干因子对前列腺癌PC-3细胞增殖能力的影响

目的:检测前列腺癌PC-3、LNCaP及DU145细胞中核干因子(Nucleostemin,NS)基因的表达,研究NS基因沉默后对PC-3细胞增殖能力的影响。方法:采用免疫细胞化学法及逆转录-聚合酶链反应(RT-PCR)分别检测NS蛋白及mRNA在3种前列腺癌细胞中的表达。用NS特异性小发夹RNA表达质粒转染PC-3细胞,分别用RT-PCR及Western印迹方法检测转染后细胞(简称NS-shRNA-PC-3)中NSmRNA及蛋白的变化。比较NS基因沉默前后PC-3细胞体外、裸鼠体内增殖能力及凋亡情况的变化。结果:3种细胞中均显示NS基因高表达。转染后NS-shRNA-PC-3细胞中NS表达显著降低,细胞增殖速度减慢,G0/G1期细胞百分率显著升高,早期凋亡细胞增多。体内致瘤实验显示,NS基因沉默后,PC-3细胞在裸鼠体内增殖能力显著降低。结论:NS在前列腺癌细胞系中呈高表达,RNA干扰沉默NS基因后PC-3细胞增殖能力显著降低,凋亡细胞增多。     

3-溴丙酮酸对前列腺癌PC-3细胞增殖、迁移及侵袭的影响

目的:观察3-溴丙酮酸(3-BrPA)对前列腺癌细胞PC-3增殖、迁移和侵袭能力的影响,并初步探讨其内在机制。方法:体外培养PC-3细胞,倒置显微镜下观察不同浓度3-BrPA对PC-3细胞形态学的影响;应用MTT法、细胞划痕和Transwell法检测不同浓度3-BrPA在不同时间段(24、48、72 h)对前列腺癌PC-3细胞增殖、迁移和侵袭的影响;应用Western印迹检测3-BrPA作用后各组PC-3细胞葡萄糖转运蛋白1(GLUT1)和基质金属蛋白酶14、9、2(MMP-14、MMP-9、MMP-2)蛋白表达的变化。结果:在一定浓度范围内,随着3-BrPA浓度的升高,细胞形态的改变愈明显。MTT结果表明,随着3-BrPA浓度的增加、作用时间延长PC-3细胞抑制率增大(P<0.01)。细胞划痕和Transwell细胞侵袭实验显示:与对照组相比,培养24 h后25、50、100μmol/L 3-BrPA浓度组细胞划痕迁移愈合率降低,细胞侵袭数目减少,且培养48 h与24 h相比,各组细胞迁移愈合率和侵袭细胞数目均降低,表明细胞体外迁移率和侵袭细胞数目与3-BrPA作用浓度、作用时间有关,具有剂量和时间依赖性(P<0.01)。Western印迹检测结果表明:与对照组相比,25、50、100μmol/L 3-BrPA浓度组中GLUT1、MMP-14、MMP-9、MMP-2蛋白表达量明显降低(P<0.01)。结论:糖酵解抑制剂3-BrPA降低PC-3增殖、迁移和侵袭能力,可能与下调GLUT1、MMP-14、MMP-9及MMP-2蛋白表达有关。     

维生素E琥珀酸酯诱导前列腺癌PC-3细胞凋亡的实验研究

目的：研究维生素E琥珀酸酯（VES）对非雄激素敏感的前列腺癌PC-3细胞株的凋亡诱导作用及分子机制。方法：以体外培养的PC-3细胞为研究对象,采用含不同浓度的VES培养液作用于细胞。采用噻唑兰（MTT）比色法检测细胞增殖活性;采用Wright—Giemsa染色、流式细胞术（FCM）检测细胞凋亡并测定Fas蛋白表达水平;采用酶联免疫法检测转化生长因子口（TGF-β）的表达水平的变化。结果：VES可显著抑制PC-3细胞的生长及增殖,光镜下可见到PC-3细胞呈典型凋亡的形态学改变。FCM细胞周期分析显示G2／M期细胞增加而s期细胞明显减少,Fas蛋白和TGF-β表达明显上调。结论：VES可诱导前列腺癌PC3细胞凋亡,其作用机制可能与其上调Fas、TGF-β的表达有关。     

基质交联分子1对前列腺癌PC-3细胞凋亡相关蛋白表达的影响

目的:观察抑制基质交联分子1(STIM1)对前列腺癌PC-3细胞凋亡相关蛋白表达的影响。方法:将携带STIM1基因的小干扰RNA(shRNA)慢病毒载体STIM1-pGCSIL-GFP转染人激素非依赖性前列腺癌PC-3细胞,3d后荧光倒置显微镜观察转染效率;1周后RT-PCR及Western印迹验证STIM1抑制表达有效性,并采用Western印迹检测PC-3细胞中凋亡相关蛋白Bcl-2、Bax,survivin、激活型Caspase-3的表达水平。结果:倒置显微镜观察发现PC-3细胞病毒转染效率80%。转染1周后,RT-PCR及Western印迹显示STIM1被有效抑制。抑制STIM1表达后,对照组Bcl-2/Bax比率为1.24,干扰组PC-3细胞Bcl-2/Bax比率为0.31,比率显著下降;干扰组PC-3细胞survivin表达明显降低,相对表达量为对照组的0.14倍;Caspase-3裂解激活相对表达量为对照组的1.52倍(P0.05)。结论:STIM1在前列腺癌PC-3细胞中可视为致癌基因,抑制其表达可通过下调Bcl-2/Bax比率,降低survivin表达,激活Caspase-3级联通路,诱导细胞凋亡。     

雷公藤内酯醇对前列腺癌细胞PC-3及血管内皮生长因子的影响

目的:探讨雷公藤内酯醇对雄激素非依赖性前列腺癌细胞株PC-3的抑制作用及对血管内皮生长因子(VEGF)表达的影响.方法:分别用0、6.25、12.5、25、50 nmol/L浓度的雷公藤内酯醇作用于PC-3细胞,24 h、48 h、72 h后,以MTT法检测细胞生长活性,24 h后流式细胞仪测定细胞周期及细胞凋亡的变化,透射电镜观察细胞超微结构变化;ELISA法测定培养上清液VEGF的水平.结果:雷公藤内酯醇能以剂量与时间依赖性的方式抑制PC-3细胞的生长,促进其凋亡;细胞周期主要阻滞于S期,部分细胞出现凋亡的形态学改变;VEGF表达较对照组明显降低.结论:雷公藤内酯醇能显著抑制PC-3细胞的体外生长,促进其凋亡,并降低VEGF的表达.     

靶向ADAM17基因siRNA对前列腺癌PC-3细胞增殖能力的影响

目的:探讨靶向抑制ADAM17基因对雄激素非信赖性前列腺癌PC-3细胞增殖的影响。方法:应用ADAM17 siRNA转染PC-3细胞后,通过RT-PCR、Western印迹方法分别检测ADAM17 mRNA和蛋白表达变化;MTT、BrdU掺入法检测下调ADAM17对PC-3细胞的增殖和DNA合成能力的影响;流式细胞术检测ADAM17 siR-NA对PC-3细胞细胞周期的影响;Western印迹检测下调ADAM17对PC-3细胞增殖相关基因表达的影响。结果:两对ADAM17 siRNAs均可有效地降低PC-3细胞ADAM17 mRNA和蛋白的表达;MTT结果显示与对照组(0.80±0.51)相比,两对ADAM17 siRNAs均可显著抑制细胞的生长(0.43±0.57、0.44±0.64,P均<0.05);Br-dU掺入实验显示与对照组(0.79±0.72)相比,ADAM17 siRNAs均能显著下调DNA的合成能力(0.48±0.43、0.54±0.59,P<0.05);流式细胞术结果显示,与对照组(41.38±1.53)%相比,ADAM17 siRNAs可显著增加G1期细胞数量[(61.83±2.41)%、(59.78±1.92)%,P均<0.05]、降低S期细胞数量[从(33.51±1.47)%减少到(23.64±2.56)%、(25.24±1.86)%,P均<0.05],同时伴随着cyclin D1蛋白的表达下降而p21蛋白的表达升高。结论:ADAM17 siRNA可以通过下调cyclin D1、上调p21的表达而抑制前列腺癌PC-3细胞增殖,ADAM17可能成为前列腺癌基因治疗的靶点。     

葡萄籽提取物对前列腺癌PC-3细胞的生长抑制作用

目的:观察葡萄籽提取物(GSE)对前列腺癌PC-3细胞的生长抑制作用。方法:经预试选用不同浓度(100、200、300μg/ml)的GSE分别作用PC-3细胞24、48、72h,以原代培养的1～3日龄SD大鼠肾细胞作为正常对照;采用四唑氮蓝(MTT)显色法检测GSE对PC-3细胞和SD大鼠肾细胞的生长抑制作用。结果:GSE以浓度和时间依赖性方式抑制PC-3细胞生长(P<0.01),对原代培养SD大鼠肾细胞生长仅有轻度抑制作用。结论:GSE可抑制前列腺癌PC-3细胞生长,有可能成为治疗前列腺癌的一种新药物。     

Akt特异性抑制剂鱼藤素对前列腺癌PC-3细胞生长的影响

目的:观察Akt抑制剂鱼藤素对前列腺癌PC-3细胞株的抑制作用并探讨其可能的作用机制。方法:用MTT法检测鱼藤素对PC-3细胞的增殖抑制率;流式细胞术(FCM)检测鱼藤素对细胞周期的影响;RT-PCR检测细胞中小鼠双微体2(MDM2)、糖元合成酶激酶3β(GSK3β)mRNA表达的变化;Western印迹法检测MDM2、GSK3β蛋白表达的变化。结果:MTT法显示,10、100、500、1 000 nmol/L的鱼藤素作用于前列腺癌PC-3细胞24、48、72 h后,对前列腺癌PC-3细胞增殖抑制率分别为(91.10±3.75)%、(86.39±1.16)%、(79.51±2.63)%;(82.46±3.65)%、(76.84±0.97)%、(69.69±2.30)%;(81.46±0.41)%、(75.56±1.12)%、(54.07±3.21)%;(66.77±2.82)%、(58.22±0.35)%、(39.34±2.40)%,均能显著抑制其增殖(P均<0.01);FCM检测显示各浓度的鱼藤素使前列腺癌PC-3细胞周期阻滞在G0/G1期比例增加[(53.4±2.3)%、(62.4±2.2)%、(63.6±1.1)%、(65.0±0.3)%、(66.5±1.9)%,P均<0.01],S期细胞比例减少[(26.9±1.7)%、(14.7±2.4)%、(11.1±5.2)%、(5.8±1.1)%、(7.0±0.6)%,P均<0.01];RT-PCR和Western印迹法结果显示鱼藤素上调了GSK3βmRNA和蛋白的表达水平,而下调了MDM2 mRNA和蛋白的表达水平。结论:Akt抑制剂鱼藤素能抑制前列腺癌PC-3细胞的增殖,其机制可能与影响Akt信号通路下游分子GSK3β、MDM2的表达相关。     

双氢青蒿素对前列腺癌细胞PC-3M生长的影响及其机制探讨

目的:观察双氢青蒿素对雄激素非依赖性前列腺癌细胞株PC-3M细胞凋亡和血管内皮生长因子(VEGF)表达的影响。方法:不同浓度(0、25、50、100μmol/L)的双氢青蒿素分别作用于PC-3M细胞48 h,MTT法检测细胞生长活性;流式细胞仪测定细胞凋亡率;分光光度法检测细胞凋亡过程中caspase-3、caspase-8活性变化;半定量RT-PCR法检测PC-3M细胞内VEGF mRNA的表达;Western印迹法检测细胞VEGF蛋白表达。结果:双氢青蒿素能显著抑制PC-3M细胞的增殖,与对照组(0μmol/L)的细胞凋亡率(2.92±0.45)%相比,各剂量组(25、50、100μmol/L)的细胞凋亡率[(8.85±0.74)%,(12.83±0.84)%,(18.65±1.24)%]显著增加,caspase-8[(0.47±0.05)U/μg vs(1.22±0.15)U/μg,(1.76±0.07)U/μg,(2.91±0.24)U/μg]、caspase-3[(0.44±0.07)U/μg vs(0.95±0.08)U/μg,(1.48±0.14)U/μg,(2.92±0.45)U/μg]活性显著增加,呈剂量依赖性(P<0.01)。PC-3M细胞内VEGF mRNA的表达和蛋白表达呈剂量依赖性降低。结论:双氢青蒿素能显著抑制体外PC-3M细胞的生长,并促进其凋亡,机制可能与增加凋亡蛋白酶和抑制VEGF表达有关。     

SNCG基因转染对PC-3细胞抗肿瘤药物作用效果的影响

目的:观察转染了质粒PCI-NEO-SNCG后的前列腺癌激素非依赖性细胞系PC-3细胞对抗肿瘤药物顺铂(DDP)、5-氟尿嘧啶(5-FU)、阿霉素(ADM)、长春新碱(VCR)、紫杉醇(TAX)的敏感性,探讨SNCG的表达对各种抗肿瘤药物作用效果的影响。方法:转染质粒PCI-NEO和PCI-NEO-SNCG至PC-3细胞,采用RT-PCR法检测PC-3细胞中SNCG的表达;MTT法检测各抗肿瘤药物对转染后PC-3细胞的抑制作用;流式细胞术分析转染细胞经TAX作用后的细胞周期及凋亡。结果:5种抗肿瘤药物对转染了空载体PCI-NEO质粒及PCI-NEO-SNCG质粒细胞的生长抑制作用均存在时间依赖性;转染PCI-NEO的PC-3细胞组与转染PCI-NEO-SNCG的PC-3细胞组中各种抗肿瘤药物的抑制效果比较显示,DDP、5-FU、ADM、VCR的抑制效果两组间没有显著性差异(P>0.05),而TAX对转染PCI-NEO-SNCG的细胞的抑制率较转染PCI-NEO的细胞显著降低(P<0.01);经TAX处理48 h后,在转染PCI-NEO质粒的细胞中,停留在G2-M期的细胞比例显著高于转染PCI-NEO-SNCG质粒的细胞(P<0.01),而停留在G0-G1期及S期的细胞比例,在转染PCI-NEO质粒的细胞中显著低于转染PCI-NEO-SNCG质粒的细胞(P<0.01);在转染PCI-NEO质粒的细胞中Caspase-3的表达显著高于转染PCI-NEO-SNCG质粒的细胞(P<0.01)。结论:TAX对转染了SNCG基因的PC-3细胞中的生长抑制作用明显降低,提示SNCG的表达可抑制TAX的作用效果,这一发现可为前列腺癌的个体化治疗提供理论依据和指导。     

ONO-AE3-208对裸鼠前列腺癌全身骨转移形成的抑制作用

目的:应用EP4受体拮抗剂ONO-AE3-208作用于裸鼠前列腺癌骨转移模型,观察其对前列腺癌骨转移形成的抑制作用。方法:将荧光素稳定转染前列腺癌PC3细胞构建PC3/LUC细胞,经小鼠左心室注射PC3/LUC细胞以构建全身转移模型。于构建术前1周开始按实验组及对照组分别给予ONO-AE3-208及双蒸水,应用活体生物发光成像系统观察术后两组小鼠模型转移形成时间、肿瘤荧光负荷及生存曲线的变化情况。结果:模型构建术后30d通过活体生物成像系统进行荧光负荷量的分析可见经双蒸水腹腔注射的对照组肿瘤负荷升高水平明显高于经ONO-AE3-208处理的实验组,且其升高水平与处理时间呈正比(P0.01);对照组小鼠转移形成率(93.3%)显著高于实验组(33.3%)(P0.01)。其中实验组的中位转移形成时间为29 d(95%CI=26.547~35.262),对照组仅有21 d(P0.01),经ONO-AE3-208作用可以显著延长小鼠前列腺癌转移形成时间。结论:EP4受体拮抗剂ONO-AE3-208对小鼠前列腺癌骨转移的形成有抑制作用。