Inhibition of proliferation of PC-3 human prostate cancer by antagonists of growth hormone-releasing hormone: lack of correlation with the levels of serum IGF-I and expression of tumoral IGF-II and vascular endothelial growth factor

BACKGROUND: Antagonists of growth hormone-releasing hormone (GHRH) such as JV-1-38 can inhibit androgen-independent prostate cancer directly by several mechanisms and/or indirectly by suppressing growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. To shed more light on the mechanisms involved, the effects of JV-1-38 on PC-3 human prostate cancer were compared with those of somatostatin analog RC-160 in vivo and in vitro. METHODS: Nude mice bearing PC-3 tumors received JV-1-38 (20 microg), RC-160 (50 microg) or a combination of JV-1-38 and RC-160. The concentration of IGF-I in serum and the expression of mRNA for IGF-II and vascular endothelial growth factor (VEGF) in tumor tissue were investigated. RESULTS: In vivo, the final volume of PC-3 tumors treated with JV-1-38 was significantly lowered by 49% (P < 0.01), whereas RC-160 exerted only 30% inhibition (NS), compared with controls. Combined use of both compounds augmented tumor inhibition to 63% (P < 0.001). Serum IGF-I levels were decreased only in mice treated with RC-160. JV-1-38 suppressed mRNA for IGF-II in PC-3 tumors by 42%, whereas RC-160 alone or in combination with JV-1-38 caused a 65% reduction. JV-1-38 and RC-160 used as single drugs decreased the expression of VEGF by 50%, and their combination caused a 63% reduction. In vitro, JV-1-38 inhibited the proliferation of PC-3 cells by 39%. This effect could be partially reversed by addition of IGF-I to the serum-free medium. RC-160 alone did not affect the PC-3 cell growth in vitro, but in combination with JV-1-38 it augmented the antiproliferative effect of the GH-RH antagonist to 72%. Exposure to JV-1-38 in vitro reduced the expression of mRNA for IGF-II in PC-3 cells by 55% but did not change VEGF mRNA levels, whereas RC-160 had no effect. CONCLUSIONS: The antiproliferative effect of JV-1-38 was not associated with the suppression of serum IGF-I and was only partially correlated with the expression of IGF-II and VEGF in PC-3 tumors, suggesting that other mechanisms play a role in the antitumor action of GHRH antagonists. Nevertheless, the stronger inhibition of tumor growth after combined treatment with JV-1-38 and RC-160 indicates that the interference with multiple local stimulatory factors leads to an enhanced inhibition of prostate cancer.     

蛙皮素对雄激素非依赖型前列腺癌PC-3细胞系的作用

目的观察蛙皮素(bombesin,BBS)对雄激素非依赖型前列腺癌PC-3细胞系的作用;检测PC-3细胞BBS受体及其 mRNA的表达。方法①MTT法检测BBS对PC-3细胞增殖的影响;②检测BBS处理后PC-3细胞贴壁率;③Millcell小室实验检测BBS对PC-3细胞播散能力的影响;④免疫荧光组织化学结合激光扫描共聚焦显微镜检测BBS处理的PC-3细胞角蛋白(CK)的表达[1];⑤用Fluo-3/AM荧光标记技术检测不同浓度BBS处理后PC-3细胞[Ca2 ]i浓度[2]。⑥免疫组化方法检测PC-3细胞中蛙皮素受体(BBS-R)蛋白表达;⑦利用逆转录聚合酶链反应(Rt-PCR)观察PC-3细胞BBS-R mRNA的表达。结果经BBS处理的PC-3细胞吸光度A值增高,细胞贴壁率增高,细胞播散能力提高,并呈现一定浓度依赖性;10-5mol/L浓度的BBS可促进PC-3细胞CK表达;BBS提高PC-3细胞[Ca2 ]i浓度;PC-3细胞中BBS-R蛋白表达呈阳性;Rt-PCR产物与预期的BBS-R的cDNA分子量完全相符。结论BBS可通过特异性受体介导致PC-3细胞系增殖、粘附、播散、伪足形成。一定浓度的BBS可明显提高PC-3细胞[Ca2 ]i浓度以及CK表达进而影响细胞骨架形态。     

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。     

Inhibition of growth of PC-82 human prostate cancer line xenografts in nude mice by bombesin antagonist RC-3095 or combination of agonist [D-Trp6]-luteinizing hormone-releasing hormone and somatostatin analog RC-160.

The effects of treatment with a bombesin receptor antagonist [D-Tpi6, Leu13 psi (CH2NH) Leu14]BN(6-14)(RC-3095) and the combination of an agonist of luteinizing hormone-releasing hormone [D-Trp6]-LH-RH and somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val- Cys-Trp-NH2 (RC-160) were studied in nude mice bearing xenografts of the hormone-dependent human prostate tumor PC-82. During the 5 weeks of treatment, tumor growth was decreased in all treated groups compared with controls. Bombesin antagonist RC-3095 and the combination of [D-Trp6]-LH-RH and RC-160 caused a greater inhibition of tumor growth than [D-Trp6]-LH-RH or RC-160 alone as based on measurement of tumor volume and percentage change in tumor volume. The largest decrease in tumor weight was also seen in the groups treated with the bombesin antagonist and with the combination of RC-160 and [D-Trp6]-LH-RH. Serum prostatic-specific antigen levels were greatly decreased, and insulin-like growth factor I (IGF-I) as well as growth hormone levels were reduced in all treated groups. Specific binding sites for [D-Trp6]-LH-RH, epidermal growth factor (EGF), IGF-I, and somatostatin (SS-14) were found in the tumor membranes. Receptors for EGF were significantly down-regulated by treatment with the bombesin antagonist or RC-160. Combination of LH-RH agonists with somatostatin analog RC-160 might be considered for improvement of hormonal therapy for prostate cancer. The finding that bombesin antagonist RC-3095 inhibits the growth of PC-82 prostate cancer suggests the merit of further studies to evaluate the possible usefulness of antagonists of bombesin in the management of prostatic carcinoma.     

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。     

Stimulatory effect of EGF and inhibitory effect of sialoadenectomy on growth of an EGF receptor-hyperproducing human gastric cancer xenograft in nude mice

We recently established epidermal growth factor (EGF) receptor-hyperproducing human gastric cancer xenografts in nude mice. The present study was designed to examine whether the growth of a xenograft having 1,098 ±276fmol/mg protein of EGF receptor would either be stimulated by the administration of EGF or inhibited by the removal of the submandibular glands (sialoadenectomy) which contain a large amount of EGF. A miniosmotic pump containing 2 g or 20 g of EGF was implanted on the back of the animals in the EGF stimulation experiments. The tumor growth was stimulated by the administration of EGF (P < 0.01), and the doubling time of the tumor was reduced relative to the controls (P < 0.01). Both the mitotic indices and the bromodeoxyuridine (BrdU)-labeling indices of the tumor were higher than those of the controls (P < 0.01). Tumor growth inhibited by the sialoadenectomy (P < 0.05) while the tumor doubling time was prolonged compared with the sham-operated mice (P < 0.05). These results suggest that the growth of a human gastric cancer xenograft may be modulated by EGF.     

Small interference RNA-mediated silencing of prostate stem cell antigen attenuates growth,reduces migration and invasion of human prostate cancer PC-3M cells

ObjectivesProstate stem cell antigen (PSCA), a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein, is highly expressed in both local and metastatic prostate cancer (CaP). Elevated PSCA expression has been shown to correlate with malignant phenotype and clinical progression. The purpose of the current study is to investigate the therapeutic potential of small interference RNA (siRNA) targeting PSCA on human CaP cells.Materials and methodsA set of two siRNAs directed different regions of human PSCA (siRNA-PSCA) were designed and transfected into a human CaP PC-3M cell line. The silencing effect was screened by RT-PCR and Western blotting. The biological effects of siRNA-PSCA on PC-3M cells were investigated by examining the cell proliferation through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle distribution through flow cytometry, and migration and invasion potencies through transwell invasion assay upon the PSCA silencing.ResultsPC-3M cells had positive PSCA expression on immunocytochemical assay. PSCA expression was depleted at 48 hours after transfection with siRNA-PSCA. Silencing of PSCA significantly suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of siRNA-PSCA was mediated by arresting cells in the G₀/G₁ phase rather than apoptosis. Furthermore, PSCA knockdown resulted in a marked decrease of cell migration and invasion capabilities in PC-3M cells.ConclusionsThe present study provides the first evidence that silencing PSCA using siRNA can inhibit the proliferation and invasiveness properties of human CaP cells, which may provide a promising therapeutic strategy for CaP and open a novel avenue toward the investigation of the role of PSCA overexpression in cancers.     

培养液中胆固醇缺乏对前列腺癌PC-3细胞生长和凋亡的影响

目的:探讨培养液中胆固醇水平对人前列腺癌PC-3细胞生长抑制及凋亡调控的作用。方法:前列腺癌PC-3细胞分别培养于普通和胆固醇缺乏培养液中,再分别加入不同剂量血小板源性生长因子PDGF或EGF作用后,倒置显微镜观察细胞形态,MTT法检测细胞的增殖抑制情况,流式细胞仪进行细胞凋亡率及细胞周期时相分析。结果:与普通培养液组比较,胆固醇缺乏培养液组细胞明显变圆、体积缩小、脱壁细胞增多。MTT法检测显示,胆固醇缺乏培养液组细胞增殖显著抑制,并呈剂量依赖性。流式细胞分析显示胆固醇缺乏培养液组细胞凋亡率同普通培养液组相比无显著差异。当加入PDGF或EGF刺激细胞增殖时,普通培养液组细胞数目显著增加,而在胆固醇缺乏培养液组脱壁细胞增加,细胞凋亡增多,同普通培养液组相比存在显著差异。流式细胞术分析细胞周期显示,同普通培养液组相比,胆固醇缺乏培养液组停滞在G0/G1期细胞增加,而S、G2/M期细胞减少。结论:胆固醇缺乏对PC-3细胞增殖有显著抑制作用,其作用机制并不是简单增加细胞凋亡,而可能是在不利增殖条件下,PC-3细胞的一种自我调节机制。     

多西紫杉醇对前列腺癌PC-3细胞的作用

目的 :观察多西紫杉醇对前列腺癌细胞系PC 3的体外作用 ,并探讨其作用机制。方法 :应用光镜形态学、MTT法、流式细胞仪和免疫细胞化学法观察了 1 0 -6mol/L、1 0 -7mol/L、1 0 -8mol/L浓度多西紫杉醇在体外对前列腺癌细胞系PC 3的作用和对细胞DNA含量及CyclinD1 表达的影响。结果 :1 0 -7mol/L以上浓度多西紫杉醇对前列腺癌细胞系PC 3有明显的的生长抑制作用 (抑制率≥ 4 7.5 % ,P <0 .0 5 ) ,诱导凋亡 (凋亡率≥1 6 .8% ,P <0 .0 5 ) ,下调CyclinD1 的表达 (表达率≤ 1 0 .8% ) ,与阳性对照组CyclinD1 表达率 2 5 .5 %相比有显著差异 (P <0 .0 5 )。结论 :多西紫杉醇对前列腺癌细胞系PC 3有明显的生长抑制和诱导凋亡作用 ,显示了多西紫杉醇有用于治疗激素非依赖性前列腺癌的可能性     

白花丹素体外诱导前列腺癌PC-3细胞凋亡的研究

目的 探讨白花丹素对前列腺癌PC-3细胞增殖、凋亡的作用及其和RelA(p65)表达的关系.方法 应用不同浓度梯度的白花丹素(1、5、1O、15、20 μmol/L)共同培养PC-3细胞24、48 h,噻唑蓝(MTT)比色法检测PC-3细胞增殖活力,双染流式细胞仪检测凋亡细胞,透射电镜观察超微病理变化,计算药物半数抑制浓度(IC50).逆转录-聚合酶链反应(RT-PCR)法扩增检测RelA(p65).结果 24 h组在10～20 μmol/L,48 h组在5～20 μmol/L时均出现生长抑制,IC50分别为12.88、3.71 μmol/L.双染流式细胞仪检测显示PC-3随作用浓度的上升凋亡率增加并呈现浓度依赖关系.透射电镜观察作用后PC-3呈现典型凋亡表现.RT-PCR结果提示其细胞凋亡率同Rel A(p65)表达呈负相关.结论 白花丹素体外实验可能通过抑制Rel A(p65)表达诱导PC-3的凋亡.     

±托泊苷联合紫杉醇对前列腺癌细胞株生长的协同抑制作用

目的 研究单独或联合使用±托泊苷(ETOP)和紫杉醇(TAXOL)对前列腺癌细胞株生长和细胞凋亡的影响.方法 前列腺癌细胞株经过不同浓度的ETOP 和TAXOL 单独或联合用药处理后,用MTT 法检测细胞抑制率,荧光显微镜观察细胞染色质形态的改变,DNA 梯度定性检测细胞凋亡的变化.结果 单独利用ETOP 或TAXOL 和联合用药,均能抑制前列腺癌细胞生长,并呈浓度和时间±赖关系,其中联合用药组抑制率明显高于单独用药组(P<0.05).ETOP 、TAXOL 及联合用药组细胞核出现典型的凋亡形态学的改变,细胞数目明显少于对照组,联合用药组细胞数目最少.ETOP、TAXOL 及联合用药组处理后的前列腺癌细胞的DNA,经琼脂糖凝胶电泳均出现典型"梯形"DNA 条带,联合用药组改变最明显.结论 ETOP 和TAXOL 均能够抑制前列腺癌细胞生长,联合用药能产生协同抑制作用.     

Regulation of prostate-specific antigen (PSA) gene expression and release in LNCaP prostate cancer by antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide

BACKGROUND: Prostate-specific antigen (PSA) is the best tumor marker for diagnosis and prognosis of prostatic carcinoma. The secretion of PSA from LNCaP human prostate cancer cells is influenced by acute stimuli such as vasoactive intestinal peptide (VIP), growth hormone-releasing hormone (GHRH), and chronic stimuli like androgens. METHODS: To study the regulation of basal and VIP/GHRH or androgen-stimulated secretion from LNCaP cells, we used a superfusion system, which allowed us to simultaneously measure PSA gene expression, PSA secretion, and cAMP release from the same cancer cells. LNCaP cancer cells were also implanted orthotopically into nude mice. RESULTS: VIP (30 pM-3 nM), GHRH (3 nM-300 nM), and dihydrotestosterone (100 nM) induced a significant increase in PSA gene expression, PSA secretion, and cAMP release. The dose and time-dependent effects of peptides were manifested only in the presence of androgens. At the end of continuous stimulation of cells with 1 nM VIP for 2 hr, large amounts of stored immunoreactive PSA still remained in the cells. Adenylate cyclase activator, forskolin (FSK), significantly increased PSA secretion and gene expression, and potassium, which causes nonspecific depolarization of membranes, augmented gene expression, and secretion of PSA, but did not influence cAMP release. This suggests that PSA secretion is regulated by cAMP-dependent as well as cAMP-independent pathways. In superfusion system, stimulatory effects of VIP and GHRH on PSA secretion were inhibited by VIP antagonist JV-1-53, and less by GHRH antagonist JV-1-38. In cell cultures, JV-1-38 had a stronger inhibitory effect on proliferation, indicating an involvement of the recently discovered tumoral GHRH receptors in this process. In nude mice, with orthotopically implanted LNCaP cancer cells, GHRH antagonist JV-1-38 alone or androgen ablation by castration had no effect on tumor growth and PSA levels. However, castration combined with treatment with GHRH antagonist, significantly decreased tumor growth and PSA secretion. CONCLUSIONS: Our findings suggest that the secretion of PSA is regulated rather than constitutive, contrary to previous reports. In addition, the effect of GHRH and VIP antagonists on PSA secretion from prostate cancer cells is not correlated with their antiproliferative action.     

The growth inhibitory effect of p21 adenovirus on androgen-dependent and -independent human prostate cancer cells

OBJECTIVE: To assess the potential of p21 as a gene therapy treatment for prostate cancer, by introducing p21 into both androgen-dependent (AD) and -independent (AI) human prostate cancer cell lines via a recombinant adenoviral vector, Ad5CMV-p21, carrying human p21 cDNA. MATERIALS AND METHODS: The LNCaP, DU145 and PC-3 human prostate cancer cell lines were cultured and infected with Ad5CMV-p21. Cell growth, cell-cycle progression and tumorigenicity were then assessed by thymidine incorporation into cellular DNA, and cell number, flow cytometry, and tumour growth after inoculating the cells into nude mice. RESULTS: Growth was inhibited in Ad5CMV-p21 viral-infected AD and AI prostate cancer cells. The effects were dose-dependent, regardless of the androgen status of the cell lines. Flow cytometric analysis showed that Ad5CMV-p21 arrested cell-cycle progression at G1/S with no appreciable effect on the levels of apoptotic cells. The tumorigenicity of cancer cells infected with Ad5CMV-p21 was greatly reduced in athymic mice. CONCLUSIONS: These results suggest that Ad5CMV-p21 may be a new therapeutic agent for human prostate cancer gene therapy.     

Antagonists of growth hormone releasing hormone (GHRH) and of bombesin/gastrin releasing peptide (BN/GRP) suppress the expression of VEGF, bFGF, and receptors of the EGF/HER family in PC-3 and DU-145 human androgen-independent prostate cancers

BACKGROUND: Antagonists of growth hormone releasing hormone (GHRH) as well as antagonists of bombesin/gastrin releasing peptide (BN/GRP) inhibit the growth of various malignancies (cancers) including prostate cancer. METHODS: We investigated the effects of GHRH antagonists MZ-J-7-118 and RC-J-29-18, BN/GRP antagonists RC-3940-II and RC-3940-Et and the combination of MZ-J-7-118 and RC-3940-II on the growth of PC-3 and DU-145 human androgen independent prostate cancers xenografted s.c. into nude mice. To elucidate the mechanisms of action of these analogs, growth factors like IGF-II (insulin-like growth factor-II), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor receptor/human epidermal growth factor receptor (EGF-R/HER) family were measured in tumors as well as IGF-I in serum. RESULTS: Antagonists of GHRH and BN/GRP alone or in combination significantly inhibited growth of PC-3 and DU-145 tumors, the greatest inhibition of tumor volume being achieved by combination of MZ-J-7-118 (5 microg/day) and RC-3940-II (10 microg/day). BN/GRP and GHRH antagonists and their combination also decreased the expression of VEGF significantly in PC-3 and non-significantly in DU-145, as measured by radioimmunoassay for VEGF protein and RT-PCR for mRNA levels of VEGF. GHRH and BN/GRP antagonists reduced bFGF concentrations and the maximal binding capacity of EGF receptors, and their mRNA levels in PC-3 and DU-145 tumors. mRNA levels for HER-2 and -3 were also diminished in PC-3 tumors by GHRH and BN/GRP antagonists. No changes in HER-4 were found after treatment. Serum IGF-I and tumoral IGF-II levels were not affected by the analogs. CONCLUSIONS: BN/GRP and GHRH antagonists inhibit growth of PC-3 and DU-145 prostate cancers by suppressing the expression of tumoral growth factors such as VEGF and bFGF as well as the receptors for EGF and related HER-2 and -3. Additive effects on tumor inhibition (TI) in vivo, but not on VEGF, bFGF, or members of the EGF/HER receptor family, can be achieved by the joint administration of both classes of analogs.     

粉防己碱对激素非依赖性人前列腺癌细胞株PC-3增殖和凋亡影响的研究

目的 探讨粉防己碱(TET)对激素非依赖性人前列腺癌细胞株PC-3细胞增殖抑制和凋亡作用及其机制。方法 应用CCK法观察不同浓度TET（1、2、4、8 μg/mL）对人前列腺癌细胞株PC-3生长的抑制作用;用Annexin-V与PI双染法流式细胞术分析TET对PC-3细胞凋亡的影响;RT-PCR检测TET对人前列腺癌细胞株PC-3 IL-8表达水平的影响。结果 TET对前列腺癌PC-3细胞株增殖有抑制作用,其抑制效应呈剂量依赖性。不同浓度TET作用48 h后,前列腺癌PC-3细胞株的凋亡率随着浓度的增高而升高,差异具有统计学意义（P<0.01）。不同浓度TET作用48 h后IL-8 mRNA表达水平随着浓度的增加而降低,差异具有统计学意义（P<0.01）。结论 TET能抑制人前列腺癌细胞株PC-3细胞增殖,并促进人前列腺癌细胞株PC-3的细胞凋亡,其作用机制可能与其下调IL-8 mRNA表达有关。