1α,25-二羟维生素D_3对前列腺癌LNCaP细胞中E-cadherin调节作用的研究

为探讨1α，25－二羟维生素D_3与前列腺癌转移之间的关系，采用WesternBlot印迹及免疫荧光法分析了前列腺癌细胞林LNCaP细胞中E－cadherin的表达，用逆转录聚合酶链反应分析了E－cadherinmRNA的表达。结果当培养液中加入0．01、0．1和1．0nmol／L浓度的1α，25-二羟维生素D3时，LNCaP细胞中E－cadherin表达增强，当浓度增加到10和100nmol／L时，E－cadherin表达减少。E－cadherinmRNA变化与E－cadherin表达变化相一致。结果显示1α，25－二羟维生素D3在低浓度时能促进转移抑制物质E－cadherin的表达，高浓度时直接抑制癌细胞的增殖。认为1α，25－二羟维生素D3可能抑制前列腺癌细胞株LNCaP细胞的转移能力。     

雄激素对前列腺癌细胞的双向作用及其机制

目的探讨雄激素对前列腺癌细胞及其双向作用分子机制。方法在LNCaP细胞培养液中加入10~(-9)、10~(-10)、10~(-11)、10~(-12) mol/L的人工合成雄激素R1881,使用噻唑蓝(MTT)比色法和流式细胞术证明其双相作用。分别提取受R1881刺激和抑制的LNCaP细胞中的mRNA,反转录后行基因芯片分析,并使用逆转录-聚合酶链反应(RT-PCR)技术验证其结果。结果细胞被10~(-9) mol/L及以上浓度的R1881抑制,被10~(-10)mol/L及以下浓度的R1881刺激生长。与对照组比较,10~(-9)mol/L R1881作用组,320基因表达差异有意义,170基因表达下调,150基因表达上调。10~(-10) mol/LR1881作用组中,4608条基因表达差异有意义,2562基因表达下调,2046基因上调。结论R1881可在10~(-9)mol/L及以上浓度抑制LNCaP细胞生长,在10~(-10)mol/L及以下浓度刺激细胞生长。雄激素受体共调节物在R1881的双向作用中可能起一定作用。     

Ceramide-induced cell death in the prostate cancer cell line LNCaP has both necrotic and apoptotic features

BACKGROUND: Prostate cancer is the second leading cause of cancer death in men. The most common treatment of prostate cancer is androgen ablation therapy which leads to regression of the tumor due to increased cell death. However, at later stages, the tumor becomes resistant to androgen ablation. Ceramide is a lipid second messenger that mediates cell death in prostate cancer cells. Previous studies suggested that ceramide may cause either apoptosis or growth arrest in the androgen-responsive prostate cancer cell line LNCaP. However, the molecular details of ceramide-induced cell death in LNCaP cells remain to be elucidated. METHODS: To investigate the mechanisms of cell death in LNCaP cells, we used various methods, including cell viability assays, fluorescence image analysis, internucleosomal DNA fragmentation analysis, Western blotting, and protein kinase assays. RESULTS: Ceramide caused LNCaP cell death without exhibiting typical signs of apoptosis, such as internucleosomal DNA fragmentation and poly(ADP)-ribose-polymerase (PARP) proteolysis. In addition, the general caspase inhibitor z-VAD-fmk did not alter ceramide-induced cell death in LNCaP cells, whereas it efficiently inhibited thapsigargin-induced apoptosis under similar conditions. However, ceramide treatment of LNCaP cells resulted in nuclear fragmentation, which is characteristic of apoptosis. Ceramide induced a strong and prolonged activation of c-Jun N-terminal Kinase (JNK) that correlated very well with the time course of cell death. Whereas the PKC inhibitor bisindolylmaleimide prevented phorbol ester-induced apoptosis in LNCaP cells, it did not affect ceramide-induced cell death. These results suggest that LNCaP cell death induced by ceramide progresses through a novel pathway that is more necrotic than apoptotic.     

Murine androgen-independent neuroendocrine carcinoma promotes metastasis of human prostate cancer cell line LNCaP

BACKGROUND: Although neuroendocrine (NE) cells in prostate cancer have been speculated to accelerate the growth and progression of surrounding cancer cells, the evidence is as yet inconclusive. We investigated the effect of an NE allograft (NE-10) and its cell line, NE-CS, which were established from the prostate of the LPB-Tag 12T-10 transgenic mouse, on human prostate cancer cell line LNCaP. METHODS: The proliferation and pulmonary metastasis of LNCaP xenografts in athymic mice with and without NE-10 allografts were evaluated. Boyden chamber assay and microarray analysis were performed to investigate changes in invasion/migration and mRNA of LNCaP cells under the influence of the NE cells, respectively. RESULTS: NE-10 did not influence the proliferation of LNCaP. The pulmonary metastasis of LNCaP with NE-10 significantly increased compared to mice without it. The NE-CS cells accelerated the in vitro invasion/migration of adenocarcinoma cells. Increased expression of mRNA of gelsolin was observed in LNCaP cells incubated with the supernatant of NE-CS cells. CONCLUSIONS: The NE-10 allograft promotes pulmonary metastasis of subcutaneously inoculated LNCaP cells by facilitating cell invasion. Secretions from NE cells upregulate the expression of gelsolin, which is an actin-binding protein, resulting in acceleration of the migration of LNCaP cells.     

靶向TRPV6的siRNA对前列腺癌LNCaP细胞抑制作用的体外研究

目的:运用RNA干扰(RNAi)技术阻断前列腺癌LNCaP细胞中瞬时感受器电位离子通道蛋白V亚家族6(TRPV6)基因的表达,探讨TRPV6基因沉默对LNCaP细胞增殖、细胞周期及凋亡的影响。方法:针对TR-PV6基因,构建2条小干扰RNA(siRNA)序列;在脂质体介导下转染前列腺癌LNCaP细胞,用RT-PCR测定TRPV6mRNA的表达;MTT法和流式细胞技术测定siRNA对LNCaP细胞增殖、细胞周期及凋亡的影响。结果:两条siRNA序列均能有效地阻断LNCaP细胞中TRPV6基因在mRNA水平上的表达(P<0.01),且mRNA表达随着转染时间的延长而减少,转染72h后TRPV6mRNA的表达减少至对照组的27%和23%;siRNA转染能显著抑制LNCaP细胞增殖,转染48h细胞增殖抑制率最高,分别为34.53%和29.32%,与转染24h和72h比较有统计学意义(P<0.05);转染48h后,siRNA转染组G0～G1期细胞增多,S期细胞数量明显减少,与空白对照组及阴性对照组相比有统计学差异(P<0.01);siRNA转染组细胞的凋亡率分别为14.45%和12.73%,显著高于空白对照组及阴性对照组3.78%和5.22%(P<0.05)。结论:针对TRPV6的siRNA在体外能有效地抑制LNCaP细胞TRPV6mRNA的转录,同时能够抑制前列腺癌LNCaP细胞增殖,使细胞周期出现G0/G1期阻滞,细胞凋亡率显著增加。     

Lineage relationship between LNCaP and LNCaP-derived prostate cancer cell lines

BACKGROUND: LNCaP and its derivative cell lines, which include C4-2 (and the related C4-2B) and CL1, are used as models of prostate cancer. Unlike LNCaP, the other cell lines show features of progressed disease such as metastatic capability and hormone independence. Analyses were done to determine if C4-2 or CL1 cells were selected from pre-existent subpopulations in LNCaP. METHODS: Prostate cancer cells were characterized by cluster designation (CD) phenotyping. Specific cell populations were sorted by flow cytometry. DNA array analysis was used to probe differential gene expression. RESULTS: CD phenotyping showed that CL1 and C4-2 (and C4-2B) were very dissimilar, and C4-2 was more similar to LNCaP. One common difference between LNCaP and its derivatives was CD26, in which virtually all C4-2 or CL1 cells were CD26(+) but only approximately 10% of LNCaP cells were CD26(+). The CD26(+) subpopulation of LNCaP was isolated and cultured in vitro. After culture, a high percentage of the cells (descended from the sorted cells) were CD26(+), in contrast to those sorted by CD13 or CD44. The cultured CD13 and CD44 populations did not show a high percentage of CD13(+) and CD44(+) cells, respectively. CD13 and CD44 are markers, in addition to CD26, for CL1 but not for C4-2. CONCLUSIONS: C4-2 arose probably from CD26(+) LNCaP cells, while CL1 arose de novo.     

ETS rearrangements in prostate cancer

Prostate cancer is a clinically and molecularly heterogeneous disease. Understanding the biologic underpinning of prostate cancer is necessary to best determine how biology is associated with the risk of disease progression and how this understanding might provide insight into the development of novel therapeutic approaches. The focus of this review is on the recently identified common ETS and non-ETS gene rearrangements in prostate cancer. Although multiple molecular alterations have been detected in prostate cancer, a basic understanding of gene fusion prostate cancer should help explain the clinical and biologic diversity, providing a rationale for a molecular subclassification of the disease.     

Subtoxic concentration of doxorubicin enhances TRAIL-induced apoptosis in human prostate cancer cell line LNCaP

Most tumor cells are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis but sparing to normal cells, thus providing therapeutic potential for clinical use. Some tumor cells are resistant to TRAIL-induced cell death while the sensitivity could be recruited with the existence of some chemical agents. In this study, human prostatic cancer cell line LNCaP was found to be resistant to TRAIL-induced apoptosis while it could be restored to TRAIL sensitivity with combination treatment of low concentration of doxorubicin. TRAIL receptor-1 (DR4) and TRAIL receptor-2 (DR5) were upregulated under the treatment of doxorubicin and verified to be responsible for TRAIL-mediated signal transduction. Furthermore, caspase-8 and caspase-3 were activated and drove their autocleavage into programmed cell death. Interestingly, apoptosis-inhibitory protein c-FLIP, but not Bcl-2 and XIAP was downregulated after doxorubicin treatment. Taken together, these findings suggested that the pathway of cell apoptosis induced by TRAIL was intact but under negative control. Subtoxic concentration of doxorubicin effectively boosted TRAIL sensitivity via depletion of antiapoptotic protein. These findings support the new strategies for killing tumors with TRAIL and chemical agents.     

The mismatch repair gene hMSH2 is mutated in the prostate cancer cell line LNCaP

PURPOSE: Mismatch repair genes are responsible for the coordinated correction of misincorporated nucleotides formed during DNA replication. Inactivating and inherited mutations in the prototypic mismatch repair gene hMSH2 have been described in a cancer predisposition syndrome known as hereditary nonpolyposis colon cancer. Patients with hereditary nonpolyposis colon cancer are at increased risk for colon cancer and extracolonic cancers such as upper tract transitional cell carcinoma but not prostate cancer. We investigated expression of hMSH2 in prostate cancer cell lines using genetic and molecular analysis. MATERIALS AND METHODS: We used the 3 well described prostate cancer cell lines, DU145, LNCaP and PC3. Western blot analysis with monoclonal antibody to hMSH2 was used to assess expression. Southern blot and polymerase chain reaction of genomic DNA were used to identify genetic alterations in the hMSH2 gene. Single cell cloning, dinucleotide repeats and BAT-26 were used to assess the cell lines for microsatellite instability. RESULTS: The prostate cancer cell line LNCaP did not express hMSH2 and was found to have a homozygous deletion of hMSH2 exons 9 to 16, resulting in truncation of the protein. While microsatellite analysis did not reveal alterations at the BAT-26 locus, single cell cloning produced several LNCaP subclones with alteration at 1 dinucleotide repeat. CONCLUSIONS: The well described prostate cancer cell line LNCaP has a mutation in the hMSH2 gene, resulting in loss of expression and possible evidence of microsatellite instability. To our knowledge our finding is the first demonstration of a genetic alteration in hMSH2 in a prostate cancer cell line.     

Growth of an androgen-sensitive human prostate cancer cell line,LNCaP, in nude mice

This study was undertaken to establish an androgen-sensitive model of human prostatic carcinoma in nude mice. The androgen-sensitive prostatic carcinoma cell line, LNCaP, was suspended in reconstituted basement membrane (Matrigel) and injected subcutaneously into nude mice. The LNCaP cell line was chosen for the study, because the cell line is androgen-sensitive and secretes prostate specific antigen (PSA) into culture media. Following injection of 1 × 10⁶ LNCaP cells with 0.25 ml of Matrigel, 88. of mice exhibited palpable tumor burdens after 12 weeks of observation. In addition, significant levels of PSA were observed in the serum of LNCaP-bearing mice. Bilateral orchiectomy of mice resulted in tumor regression and stabilization of serum PSA levels, compared to testis-intact controls. A significant correlation of PSA to tumor volume and weight was observed. The castrate level of testosterone was confirmed by radioimmunoassay and was similar to testosterone levels in female nude mice. Matrigel allows for a conducive environment to propagate LNCaP cells in nude mice. Furthermore, the growth can be manipulated by castration, leading to involution of tumor and stabilization of serum PSA level. This in vivo model of hormone-sensitive human prostate cancer cell line will serve as a model for the study of prostate tumor biology and treatment. © 1993 Wiley-Liss, Inc.     

雌激素受体α上调前列腺癌细胞株LNCaP中L-Plastin启动子转录活性研究

目的鉴定雌激素受体在L-Plastin启动子上的结合位点，进一步阐明雌激素受体在激素依赖型前列腺癌中的作用机制。方法利用TFSEARCH软件分析L-Plastin启动子的序列，寻找可能调控L-Plastin表达的雌激素受体结合位点，利用PCR定点突变法构建删除该转录因子结合位点的重组子，检测切除该片段序列后荧光素酶活性，并用凝胶滞后实验和超滞后实验证实雌激素受体是否结合于该位点，研究雌激素受体在激素依赖型前列腺癌中调控L-Plastin表达的作用。结果TF SEARCH软件分析表明，在L-Plastin启动子上距转录起始点-85～-70bp处，含有雌激素受体结合位点ATTTCACTGTGACCT，删除该结合位点后L-Plastin启动子荧光素酶活性明显下降。凝胶滞后实验和超滞后实验表明雌激素受体α是结合于该位点的转录因子。结论雌激素受体α具有促进L-plastin在激素依赖型前列腺癌中表达的作用。     

LNCaP衍生的雄激素非依赖型前列腺癌细胞系C4-2对辐射的应答反应

放射治疗是治疗局灶性前列腺癌相对有效的方法,然而,30％的病人会产生辐射治疗抵抗。放射协同雄激素撤除为临床提供了一种改善疗效的治疗方法。雄激素撤除诱导的信号通路相应地也会介导前列腺癌细胞的放射敏感。C4—2细胞是由雄激素非依赖的LNCaP母体细胞在雄激素撤除条件下衍生而来的,C4-2细胞获得了雄激素非依赖生长的特性。我们分析了LNCaP和WC4－2对辐射的应答反应,克隆形成、细胞存活和细胞周期分析结果显示,C4－2细胞经照射后较LNCaP细胞更容易存活,对辐射处理表现出更强的抵抗能力。基因表达分析表明,一整套与细胞周期阻滞和DNA损伤相关的基因经过辐射处理后在LNCaP和C4．2细胞中差异表达,其结果与C4—2细胞的辐射抵抗性质一致,这些结果明显提示,前列腺癌细胞对辐射的抵抗与向雄激素非依赖发展的过程可能同步进行。LNCaP和C4-2细胞模型不仅可应用于研究前列腺癌向雄激素非依赖发展的过程,而且也用于分析前列腺癌细胞的辐射抵抗机制。     

Past and future impact of next‐generation sequencing in head and neck cancer

Progress in sequencing technology is intrinsically linked to progress in understanding cancer genomics. The purpose of this review was to discuss the development from Sanger sequencing to next‐generation sequencing (NGS) technology. We highlight the technical considerations for understanding reports using NGS. We discuss the findings of studies in head and neck cancer using NGS as well as The Cancer Genome Atlas. Finally we discuss future routes for research utilizing this methodology and the potential impact of this. © 2015 Wiley Periodicals, Inc. Head Neck 38 : E2395–E2402, 2016     

Emodin induces apoptosis in human prostate cancer cell LNCaP

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.