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。     

Effects of quercetin on the heat-induced cytotoxicity of prostate cancer cells

BACKGROUND: It has been demonstrated that prostate cancer cells are relatively sensitive to heat stress. We have reported that heat treatment at 43 degrees C increases the expression of heat shock protein 70 (hsp70) in prostate cancer cells, leading to apoptosis. Hsp70 is a protein that protects cells against heat damage. Cells with lower levels of hsp70 have been shown to have a higher sensitivity to heat stress. Therefore, downregulation of hsp70 is expected to enhance heat-induced inhibitory effects on cell growth. Quercetin has been reported to be an agent that inhibits hsp70 expression. The present study was undertaken to investigate the effects of quercetin and/or heat on the growth of prostate cancer cells in vitro. METHODS: Three human prostate cancer cell lines were used: Lncap; PC-3; and JCA-1. The cells were treated with quercetin and/or heat. Alterations in the cell cycle and hsp70 expression were examined by means of flow cytometry (FCM). The apoptotic cells were detected by FCM using fluorescein isothiocyanate (FITC) labeled annexin V. RESULTS: Treatment with quercetin alone resulted in an apparent decrease of hsp70-positive cells and an increase of subG1 cells in JCA-1 and LNcap cells. Quercetin inhibited an increase of hsp70 expression after heat treatment and increased the number of subG1 cells with lower levels of hsp70 in JCA-1 and LNcap cells. Quercetin was found to enhance heat-induced inhibitory effects on cell growth and heat-induced apoptosis in both JCA-1 and LNcap cells. CONCLUSION: These results suggest that quercetin may enhance heat-induced cytotoxicity in prostate cancer cell lines through the inhibition of hsp70 production.     

HIF‐1α‐induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF‐1α)‐dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl₂, which induces HIF‐1α, to simulate hypoxia, or transfected with siRNAs targeting HIF‐1α (si‐HIF‐1α) and HSP70 (si‐HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF‐1α under hypoxia or simulated hypoxia, but not in the presence of si‐HIF‐1α. Hypoxia‐induced overexpression of ECM genes was significantly suppressed by si‐HIF‐1α or si‐HSP70. Cell viability positively correlated with hypoxia, but transfection with si‐HIF‐1α or si‐HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside‐treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si‐HIF‐1α or si‐HSP70 almost completely blocked these effects. These findings indicated that HIF‐1α‐induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF‐1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:975–980, 2014.     

Abrogation of heat-shock protein （HSP）70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC3m cells were treated with 0-16μmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10μmol/L antisense HSP70 oligomer for 48 hr or 8μtmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10μtmol/L antisense HSP70 oligomer treatment for 48 hr or 8μtmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abro-gates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression,in turn inducing apoptosis and inhibiting cell growth in PC-3m cells. (Asian JAndro12004 Dec;6:319-324)     

Hyperbaric oxygen treatment prevents nitric oxide‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia‐induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin‐1β (IL‐1β)‐induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT‐PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL‐1β‐treated chondrocytes. To clarify that the HSP70 was necessary for anti‐iNOS and anti‐apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO‐induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 376–384, 2013     

Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins

Androgens are critical to the growth and differentiation of prostate epithelial cells. Removal of androgen normally results in apoptosis, but androgen-independent tumours have developed mechanisms that allow cells to survive the loss of androgen. The caspases are central mediators of cell death. An important area for research involves manipulating caspases by novel mechanisms to induce apoptosis. However, such mechanisms as diethylmaleate priming are limited by an inability to selectively target tumour cells. Inhibitors of apoptosis proteins (IAPs) are recently identified anti-apoptotic caspase regulators. Each IAP homologue has a different mechanism of action. Because more than one member of the IAP family may be overexpressed in prostate cancer, successful treatment strategies will be defined by the ability to block all of the IAP expressed. Anti-sense oligonucleotide strategies have been shown to decrease IAP expression and increase prostate cancer cell susceptibility to apoptotic induction, although not by mitochondrial-mediated pathways. Fully understanding the basic apoptotic pathway and its regulation in prostate cancer will lead to more targets for manipulation, which can be translated into novel therapies. This article focuses on the role of the caspases and IAP in developing a rational approach to using apoptosis as a therapeutic target.     

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.     

Human prostate cancer cell death by novel anticancer compounds, apoptosis-inducing nucleosides from CD57+ HLA-DR(bright) natural suppressor cell line

BACKGROUND: We validated the induction of apoptosis in human prostate cancer PC3 cells by apoptosis-inducing nucleosides (AINs) released from the CD57(+)HLA-DR(bright)-natural suppressor (57.DR-NS) cell line. We analyzed the molecular signaling pathway during AINs-induced apoptosis in PC3 cells. METHODS: Direct and indirect co-cultures between 57.DR-NS and PC3 cells were performed. AINs were isolated by high-performance liquid chromatography (HPLC) from 57.DR-NS cell cultures. Apoptosis in PC3 cells was analyzed by DNA fragmentation, sub-G(1) DNA content with flow cytometry, and terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) method. The DNA strand breaks and activation of caspase-3 in PC3 cells were measured by DNA unwinding and flow cytometry assay. RESULTS: The 57.DR-NS cell line generated apoptosis in PC3 cells via AINs. AINs isolated from 57.DR-NS cell cultures induced apoptosis in PC3 cells. Furthermore, we found DNA strand breaks followed by activation of caspase-3 during AINs-induced apoptosis in PC3 cells. CONCLUSIONS: The data obtained here indicated that AINs could induce apoptosis in PC3 cells through DNA strand breaks and activation of caspase-3.     

谷氨酰胺诱导大鼠热休克蛋白70mRNA的表达

热休克蛋白(heat shock protein,HSP)是感染或非感染(如创伤、休克)等致病因素作用于机体后,诱发机体细胞合成的一组高度保守的蛋白质,谷氨酰胺(Glu-tam ine,Gln)作为体液中最丰富的氨基酸之一,对机体多个脏器有保护作用。一些研究表明,Gln在体外可促进果蝇Kc细胞HSP的表达[1];Gln诱导的HSP表达对肠道上皮细胞有保护[2]。但Gln是否能在动物的各个脏器引起HSP的表达及时间和量效关系如何,尚未见明确报道。本研究对此进行了实验观察,拟为临床应用提供理论依据。1材料和方法1.1主要试剂力太(批号J20020081,Fresennius Kabi公司,德国),…     

HSP27 and HSP70 interact with CD10 in C4-2 prostate cancer cells

BACKGROUND: CD10 is an approximately 100 kDa transmembrane metallo-endopeptidase. CD10 is strongly expressed by normal prostate epithelium. While only 30% of primary prostate tumors express CD10, it is strongly expressed by most lymph node metastases. The function of CD10 and the interaction between CD10 and other cellular proteins in prostate cancer (CaP) is not well defined. Cellular context may ultimately determine its biologic function in CaP. In this study, we compared CD10 mRNA and protein expression between benign and malignant prostate cells and employed proteomic analysis to identify proteins that interact with CD10 in C4-2 prostate cancer cells. METHODS: CD10 mRNA and protein expression was compared using RT-PCR and Western blotting. CD10-protein complexes were isolated by immunoprecipitation using anti-CD10 monoclonal antibodies. Eluted fractions were combined, trypsinized, and the resulting peptides analyzed by microLC-ESI-MS/MS. The parent proteins were identified by searching MS/MS spectra against a human protein database using SEQUEST. RESULTS: There were no differences in CD10 mRNA length or CD10 protein molecular weight between normal tissue and CaP. We identified 75 proteins unique to or heavily enriched in the CD10 immunoprecipitates by proteomic analysis. The 27 kDa heat shock protein (HSP27) and HSP70 were identified in three separate precipitations. Protein identification by proteomics was confirmed by Western blotting. Protein complexes immunopurified from C4-2 protein extracts using anti-HSP27 and anti-HSP70 antibodies were found to contain CD10. CONCLUSIONS: The function of CD10 in prostate cancer is largely unknown. In the C4-2 CaP cell line, CD10 was found to interact with both HSP27 and HSP70.     

Cryoablative response of prostate cancer cells is influenced by androgen receptor expression

OBJECTIVE

To investigate in prostate cancer cells the consequences of androgen‐insensitivity (AI) development on the cellular and molecular responses to freezing, as a challenge in prostate cancer treatment occurs when the androgen‐sensitive (AS) phenotype switches to an AI phenotype, the latter of which is often refractory to many therapies.

MATERIALS AND METHODS

PC‐3 (AI) and LNCaP (AS) were each genetically altered to express the opposite phenotype and subjected to an in vitro freezing model. Viability, caspase inhibitor and Western blot studies were used to determine the basis of the differential responses of AI and AS cells.

RESULTS

LNCaP high‐passage cells, formed by repeated passage of LNCaP (AS) cells, were AI and showed a phenotypic shift to freeze resistance matching the freeze response of PC‐3 cells (AI). While stably transfected androgen receptor (AR)‐transfected cells (PC‐3 AR) had a freezing sensitivity similar to that of the LNCaP (AS) cell line. Importantly, AI cell lines survived and recovered from freezing exposure to temperatures as low as ?40 °C whereas AS cell lines did not. Caspase inhibition studies and related fluorescent probes showed an elevated level of apoptotic involvement in both AS cell lines after freezing compared with their AI counterparts. Western blot analysis showed that AR expression was modified after exposure to freezing.

CONCLUSION

This study suggests that AS cancers may be far more sensitive to a freezing insult and this might be linked to elevated apoptosis and caspase activity. As such, cryoablation may prove most effective in cancer cells that have not yet progressed to a more resistant AI phenotype, but both generic variants can be fully ablated at sufficiently low temperatures.     

Galectin-1 and galectin-3 expression in human prostate tissue and prostate cancer

Galectin-1 and galectin-3, two β-galactoside-binding proteins, have been suggested to play a role in the development and progression of cancer. We have studied the expression of these molecules in normal human prostate tissue and prostate adenocarcinoma. Immunohistochemistry was used to examine formalin-fixed, paraffin-embedded sections of seven normal human prostates, eight cases of prostatic intraepithelial neoplasia (PIN), 20 primary adenocarcinomas of the prostate, and 12 prostate cancer metastases. Galectin-1 was expressed in most cases of all four histologic types. In contrast, galectin-3 expression was significantly decreased in primary carcinoma and metastatic disease compared with normal and premalignant tissue. Galectin-3 expression in primary tumors tended to be less than that of surrounding normal glands. We conclude that loss of galectin-3 expression may be associated with the progression of prostate cancer. Received: 24 May 1998 / Accepted: 8 April 1999