Antiproliferative effects of c-myc antisense oligonucleotide in prostate cancer cells: A novel therapy in prostate cancer

Objectives

To explore the possibility of using antisense oligonucleotide therapy for prostate cancer, we investigated the effect of c-myc-antisense-oligonucleotide (c-myc-As-ODN) in human prostate cancer cell lines such as LNCaP, PC3, and DU 145.

Methods

LNCaP, PC3, and DU145 cells were incubated in the presence of c-myc-As-ODN. Dose (0 to 10 μM) and time dependent (1 to 6 days) effects on proliferation and viability were examined by [³H]thymidine incorporation and MTT assay, respectively. Flow cytometry analysis was carried out to analyze cell cycle status by determining the DNA content in LNCaP cells. Control cultures received either c-myc-sense-ODN or scrambled (nonsense) nucleotides.

Results

Time- and dose-dependent decreases in DNA synthesis and cell viability were noted for all three prostate cancer cell lines after c-myc-As-ODN treatment. Further studies using LNCaP cells indicated that these changes were accompanied by an increase in the percentage of cells with less than 2N DNA content after c-myc-As-ODN treatment. The results suggest that c-myc-As-ODN induces cell death. Comparison of a c-myc-As-ODN-treated group with a group subjected to isoleucine deprivation revealed that thymidine incorporation was almost the same in c-myc-As-ODN-treated LNCaP cells and in LNCaP cells at early S phase.

Conclusions

These results suggest that c-myc-As-ODN inhibits prostate cancer cell growth and proliferation mainly by decreasing cell viability.     

SMO Expression in Colorectal Cancer: Associations with Clinical,Pathological, and Molecular Features

Background

Smoothened, frizzled family receptor (SMO) is an important component of the hedgehog signaling pathway, which has been implicated in various human carcinomas. However, clinical, molecular, and prognostic associations of SMO expression in colorectal cancer remain unclear.

Methods

Using a database of 735 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study, we examined the relationship of tumor SMO expression (assessed by immunohistochemistry) to prognosis, and to clinical, pathological, and tumor molecular features, including mutations of KRAS, BRAF, and PIK3CA, microsatellite instability, CpG island methylator phenotype (CIMP), LINE-1 methylation, and expression of phosphorylated AKT and CTNNB1.

Results

SMO expression was detected in 370 tumors (50 %). In multivariate logistic regression analysis, SMO expression was independently inversely associated with phosphorylated AKT expression [odds ratio (OR) 0.48; 95 % confidence interval (CI) 0.34–0.67] and CTNNB1 nuclear localization (OR 0.48; 95 % CI 0.35–0.67). SMO expression was not significantly associated with colorectal cancer-specific or overall survival. However, in CIMP-high tumors, but not CIMP-low/0 tumors, SMO expression was significantly associated with better colorectal cancer-specific survival (log-rank P = 0.012; multivariate hazard ratio, 0.36; 95 % CI 0.13–0.95; P _interaction = 0.035, for SMO and CIMP status).

Conclusions

Our data reveal novel potential associations between the hedgehog, the WNT/CTNNB1, and the PI3K (phosphatidylinositol-4,5-bisphosphonate 3-kinase)/AKT pathways, supporting pivotal roles of SMO and hedgehog signaling in pathway networking. SMO expression in colorectal cancer may interact with tumor CIMP status to affect patient prognosis, although confirmation by future studies is needed.     

Effect of keratinocyte growth factor and activin on cell growth in the human prostatic cancer cell line LNCaP

Keratinocyte growth factor (KGF) has paracrine properties in the human prostate which stimulate epithelial cell growth. Activins have profound effects on cell growth and function in the human prostate, and are expressed in LNCaP, DU 145 and PC3 cells. LNCaP cells were characterized by immuncytochemistry, an immunoassay and polymerase chain reaction. A 3[H]thymidine assay was used with 0.01–10 nM dihydrotestosterone, 10 µM flutamide, 1–100 ng/ml KGF and 3 nM activin. LNCaP cells expressed Ki67, PSA, cytokeratins (8, 18, 19, 14, 15) androgenreceptor but no KGF protein. LNCaP cells showed telomerase activity. Furthermore, ARmRNA (365 bp), but no KGF or KGFRmRNA were expressed. KGF ELISA detected no intracellular or secreted KGF. DHT (1, 10 and 100 nM) and KGF (10 and 100 ng/ml) significantly stimulated LNCaP cell proliferation. However, flutamide and 3 nM activin A significantly decreased cell proliferation in the presence and absence of KGF. The results of our experiments support the hypothesis that cell growth and proliferative characteristics of LNCaP cells are modulated by KGF and activin A.     

姜黄素对前列腺癌细胞核转录因子抑制蛋白表达的影响

目的观察姜黄素对前列腺癌细胞核转录因子抑制蛋白(IkBα)表达的影响,探讨姜黄素抑制前列腺癌细胞增殖的作用机制。方法分别用10、25、50、75和100μmol/L 浓度的姜黄素对雄激素依赖性及雄激素非依赖性前列腺癌细胞株 LNCaP 和 PC3进行干预,5、12和24 h 后采用噻唑蓝(MTT)比色法观察细胞增殖情况;采用流式细胞术测定24 h 后细胞周期变化;5 h 后 Western 印迹法检测细胞中 IkBα的表达。结果姜黄素显著抑制 LNCaP 及 PC3细胞的生长,呈剂量和时间依赖性;姜黄素将两种前列腺癌细胞阻滞于 G_2、M 期[LNCaP 与 PC3细胞,空白对照分别为(11.4±1.3)%与(17.3±1.7)%,100μmol/L 姜黄素作用后分别为(27.3±2.8)%与(33.4±4.0)%],从而诱导肿瘤细胞凋亡;姜黄素作用于 LNCaP 细胞后,细胞中 IkBα表达无变化(F=0.129,P>0.05);但作用于 PC3细胞后,细胞中 IkBα的表达明显增强,呈现出显著的剂量依赖性(F=31.618,P<0.05)。结论姜黄素通过活化 IkBα在 PC3细胞中的表达发挥抑制 PC3细胞增殖的作用。对于 LNCaP 细胞,姜黄素可能通过抗氧化、抑制细胞内代谢产物形成等方式抑制 LNCaP 细胞增殖。     

BCG directly induces cell cycle arrest in human transitional carcinoma cell lines as a consequence of integrin cross-linking

Background

Androgen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied.

Methods

We investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line.

Results

Androgen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number.

Conclusion

Increased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.     

The differential effects of prostate stromal cells derived from different zones on prostate cancer epithelial cells under the action of sex hormones

It is well known that prostate cancer (PCa) occurs predominantly in the peripheral zone (PZ), whereas benign prostatic hyperplasia (BPH) typically develops in the transition zone. To identify possible mechanisms underlying zonal differences, we compared the effects of prostate stromal cells derived from the peripheral zone (PZsc) and the transition zone (TZsc) on a PCa epithelial cell line (PC3) in the presence of sex hormones. First, we observed that androgen receptor (AR) mRNA was more highly expressed in PZsc than TZsc when the cells were treated with dihydrotestosterone (DHT) and β-oestradiol (E2) (P<0.05). By ELISA, we looked for differences in the secretion of peptide growth factors from PZsc and TZsc. We found that keratinocyte growth factor (KGF) secretion increased with increasing concentrations of DHT (P<0.01) and was higher in PZsc than TZsc. Under treatment with DHT plus E2, PZsc secreted more transforming growth factor-β1 (TGF-β1) than TZsc, but this pattern was reversed when the cells were treated with E2 only. With increasing concentrations of DHT, insulin-like growth factor-1 (IGF-1) secretion increased in PZsc but decreased in TZsc. To further characterize the effects of PZsc and TZsc on PC3 cells, we developed a coculture model and performed MTT assays, Western blot analysis and real-time RT-PCR. We found that PZsc promoted PC3 cell proliferation and progression better than TZsc, particularly when treated with 10 nmol l⁻¹ DHT plus 10 nmol l⁻¹ E2. In conclusion, our data suggest that PZsc may have a greater capacity to induce PCa development and progression than TZsc via growth factors regulated by sex hormones. These findings provide possible mechanisms underlying zonal differences in prostate diseases, which may aid the search for novel therapeutic targets for PCa.     

Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines

OBJECTIVE—Rodent insulinoma cell lines may serve as a model for designing continuously replicating human β-cell lines and provide clues as to the central cell cycle regulatory molecules in the β-cell.RESEARCH DESIGN AND METHODS—We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics.RESULTS—1) Despite their common T-antigen–derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat β-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate β-cell differentiation and function.CONCLUSIONS—These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human β-cells.Nutrients, growth factors, and physiological states such as obesity and pregnancy can induce β-cell replication. However, the molecular pathways that regulate DNA synthesis and mitosis in β-cells have only recently begun to be elucidated. Most insight into pancreatic β-cell cycle control is derived from studies in mice with targeted mutations in either the inhibitors or activators of cell cycle progression. For example, inactivating mutations in several cell cycle control molecules have revealed roles for p27, p18, p16, menin, pRb, p53, cyclin D1, cyclin D2, cdk4, E2F1, and E2F2 (1,2). These studies suggest that the G1/S checkpoint is especially important for the control of the cell cycle in pancreatic β-cells.Other attractive models in which to study β-cell cycle replication are rodent insulinoma cell lines, which are widely used as models for β-cell signaling, function, and replication (3). The four cell lines most widely used in β-cell research are MIN6, BTC3, INS1, and RINm5F cells. MIN6 and BTC3 cells are related lines that were generated by transgenic overexpression of the large T-antigen of the SV40 virus in mouse β-cells (3,4,5). INS1 and RINm5F are also related cell lines that were derived from radiation-induced tumors in rats (3,6,7). These cell lines vary in characteristics, but all are at least partially differentiated and proliferate more rapidly than normal β-cells. Surprisingly, despite their extensive use in the field of β-cell biology, there is almost no information regarding the mechanisms that lead to their loss of normal cell cycle control (3–7).The lack of a continuously growing human β-cell line suggests the possibility that lessons learned from rodent insulinoma lines may offer clues on how to generate corresponding human β-cell lines that may be useful for research and for cell replacement therapy of diabetes. We therefore examined the G1/S proteome of these four rodent insulinoma cell lines and compared them with primary islets in hope that these cell lines might reveal common mechanisms that lead to continuous proliferation of insulin-secreting cells and potential targets to induce proliferation in normal β-cells.As has been described previously in most human cancers (8–10), comprehensive analysis revealed multiple abnormalities in G1/S control in each of the four cell lines. Interestingly, despite their common origins (X-radiation in the rat cell lines or T-antigen overexpression in the mouse lines), insulinomas of common origin display very different G1/S profiles. In addition, several molecules that appear to be critical in maintaining arrest or allowing cell cycle progression in normal rodent islets, e.g., cyclin D2, are entirely dispensable in mouse and rat insulinoma lines. Finally, seven G1/S molecules that are capable of stimulating β-cell cycle progression were identified. These observations have important cautionary implications for the use of the four cell lines as models of “normal” β-cell replication. They also suggest strategies for developing continuously growing human β-cell lines.     

Effects of triptorelin, a gonadotropin-releasing hormone agonist, on the human prostatic cell lines PC3 and LNCaP

Some analogues of gonadotropin-releasing hormone (GnRH) influence the in vitro proliferation of cultured human cells by complex interactions that are only partially understood. This study explored the effect of Triptorelin, a GnRH agonist, on the LNCaP and PC3 prostatic cell lines, which are, respectively, responsive and unresponsive to androgen stimulation. The toxicity and cell cycle modifications induced by the drug were investigated by FACScan analysis; the effect on cell proliferation in different culture conditions was determined by counting in a Burker chamber; and the expression of binding sites for 125I-Triptorelin was revealed by displacement experiments. PC3 cell growth was completely unaffected by Triptorelin. The drug caused a double stimulatory-inhibitory action on the growth of actively proliferating LNCaP cells, depending upon the dose and environment. A significant inhibitory effect on proliferation, ranging from 25% to 65% compared with controls, was observed at a high dose (10(-4) M) according to the culture conditions; and a proliferative effect (42% compared with controls) was observed at a lower dose (10(-7) M) only in fetal bovine serum-supplemented medium. Displacement experiments revealed the expression of moderately high affinity and low affinity binding sites in LNCaP cells (Kd = 2.6 x 10(-8) and 7.7 x 10(-6) M) but only low affinity binding sites in PC3 cells (Kd = 2.7 x 10(-6) M), which suggests that the expression of binding sites with different affinity could be associated with a biological response to the drug. Proliferation studies in the presence of Cetrorelix, a GnRH antagonist, confirmed the different sensitivity of the 2 cell lines to GnRH analogues and showed that the proliferative effect of Triptorelin on LNCaP cells can be inhibited by the antagonist. Data confirm the cell specificity of Triptorelin's action and the peculiarity of its effects on prostatic cell proliferation in our experimental conditions.     

Apogossypolone, a novel inhibitor of anfiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro

Limited treatment options are available for aggressive prostate cancer. Gossypol has been reported to have a potent anticancer activity in many types of cancer. It can increase the sensitivity of cancer cells to alkylating agents, diminish multidrug resistance and decrease metastasis. Whether or not it can induce autophagy in cancer cells has not yet been determined. Here we investigated the antiproliferative activity of apogossypolone （ApoG2） and （-）-gossypol on the human prostate cancer cell line PC3 and LNCaP in vitro. Exposure of PC-3 and LNCaP cells to ApoG2 resulted in several specific features characteristic of autophagy, including the appearance of membranous vacuoles in the cytoplasm and formation of acidic vesicular organelles. Expression of autophagy-associated LC3-Ⅱ and beclin-1 increased in both cell lines after treatment. Inhibition of autophagy with 3-methyladenine promoted apoptosis of both cell types. Taken together, these data demonstrated that induction of autophagy could represent a defense mechanism against apoptosis in human prostate cancer cells.     

Tie2基因RNAi慢病毒载体的构建及其干预恶性黑色素瘤细胞的体外研究

目的 构建携带酪氨酸蛋白激酶受体2-小干扰RNA(Tie2-SiRNA)慢病毒载体,观察其对恶性黑色素瘤细胞的干扰作用.方法 将pSilencer 1.0-U6启动子-酪氨酸蛋白激酶受体2-小干扰RNA(pSilencer 1.0-U6-Tie2-siRNA)重组质粒经Xba Ⅰ酶切电泳鉴定后,与经Xba Ⅰ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒(pNL-EGFP)载体连接,产生加强绿色荧光蛋白的转移质粒-u6启动子-酪氨酸蛋白激酶受体2-Ⅰ(pNL-EGFP-U6-Tie2-Ⅰ)、pNL-EGFP-U6-Tie2-Ⅱ慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒,分别与水疱性口炎病毒G蛋白(pVSVG)包膜质粒和pHelper包装质粒组成慢病毒三质粒转染系统,再共转染293T细胞,产生pNL-EGFP-U6-Tie2-Ⅰ、pNL-EGFP-U6-Tie2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Tie2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Tie2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.8×103/ml.实时荧光定量RT-PCR结果显示:Tie2-SiRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Tie2基因的表达,其中较高者可达68.55%,并且2种慢病毒载体之间差异无统计学意义(t=0.362,P＞0.05).结论 成功构建了Tie2-SiRNA慢病毒载体,体外研究显示其能抑制Tie2 mRNA的表达,为下一步进行抑制肿瘤生成的动物实验研究奠定了基础.

Abstract：

Objective To construct lentivector carrying Tie2-Small interfering RNA(SiRNA),so as to study its influence on malignant melanoma cells.Methods Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with Xbal.ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-Ⅰ or pNL-EGFP-U6-Tie2-Ⅱ,and then the electrophoresis clones was sequenced.Plasmids of pNL-EGFP-U6-Tie2-1 and pNL-EGFP-U6-Tie2-Ⅱ were constructed and combined with pVSVG and pHelper,respcectively,to constitute lentiviral vector system of three plasmids.The Lentiviral vector svstem was transfected into 293T cell to produce pNL-EGFP-U6-Tie2-Ⅰ and pNL-EGFP-U6-Tie2-Ⅱ lentivirus.Then the supernatant was collected tO determine the titer.Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. Results The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing.And the titer of lentiviral vector was 8.8×103/ml,which was determined by 293T cell.The results of Reahime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells(P＜0.01).There was no significant difference in the expression level (P＞0.05)between the two lentiviral vectors of Tie2-RNAi.Conclusions Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly.The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.     

Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells

Background

ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.

Methods

The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.

Results

The activation of A₃AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 ^flox/?: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost.

Conclusion

The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

     

PIAS-NY稳定转染小鼠精母细胞株的构建与鉴定

目的:构建PIAS-NY基因慢病毒质粒,并将包装所得慢病毒感染小鼠精母细胞,获得稳定过表达PIAS-NY的细胞株。方法:应用PCR技术扩增目的基因,并将扩增产物插入慢病毒载体质粒pGC-FU上,对阳性克隆进行PCR筛选及测序鉴定。将重组质粒与两种辅助包装原件载体质粒共转染293T细胞,获得含慢病毒颗粒的细胞上清,用慢病毒感染小鼠精母细胞,并用Western印迹方法检测细胞中PIAS-NY蛋白的表达。结果:成功构建了pGC-FU-PIAS-NY慢病毒表达质粒,获得了稳定过表达PIAS-NY的小鼠精母细胞株。结论:PIAS-NY过表达慢病毒质粒及稳定转染小鼠精母细胞株的构建,为进一步体外研究PIAS-NY基因在精子发生中的功能奠定了基础。     

Expression of cellular adhesion molecules on human prostate tumor cell lines

By flow cytometric analysis we have examined the expression of cellular adhesion molecules (CAMs) on the surface of four different human prostate tumor cell lines: DU 145, from a brain metastasis; PC 3, from a bone metastasis; LNCaP.FGC, from a lymph node metastasis; and a primary tumor cell line, ND 1. The corresponding ligands for the expressed CAMs were, by and large, extracellular matrix proteins. We detected high-level expression of ICAM-1 on three of the four prostate cell lines, whereas LNCaP cells were negative. We observed unstable, heterogeneous expression of E-cadherin in the cell lines DU 145, PC 3, and ND 1. Flow cytometric cell sorting enabled us to divide PC 3 cells into negative and bright positive subpopulations but, after several cell divisions in culture, sorted cells returned to the original heterogeneous phenotype. The laminin-specific α₆β₄ integrin was not expressed by LNCaP, and was expressed at a low level and heterogeneously on DU 145 and PC 3 cell lines. In contrast, ND 1 cells, derived from a primary tumor, showed homogeneous and high-level expression of the α₆β₄ integrin. All of the prostate cell lines expressed the RGD-dependent binding of α₃β₁ and α₅β₁ integrins and did not reveal non-RGD-dependent α₄β₁ integrin expression. This finding provides a stimulus to investigate the inhibition capacity of RGD-containing peptides on the metastatic behavior of prostate tumor cells.     

Anti-apoptotic potencial role of p38 in prostate cancer

IntroductionTNF-α transduction pathway in prostate cancer seems to be diverted towards p38 activation. P38 may protect prostate tumoral cells from TNF-α apoptosis induced. The aim of this study was study the role of p38 in vivo (were evaluated some p38 downstream factors), as well as in vitro (in prostatic tumoral cell lines, LNCaP and PC3, pre-treated with TNF-α).Material and methodsTwo prostatic tumoral cell lines (LNCaP and PC3) were used in in vitro studies. Two different experiments were made: with TNF-α (several concentrations) and p38 specific inhibitor (SB203580). The apoptotic index were evaluated using DAPI staining and flow cytometry. P38 activation was measured by Western blot analysis. 15 normal samples (NP) and 27 prostate cancer samples (PC) were used in in vivo study, all of them were processed for immunohistochemistry and Western-blot.ResultsIn vitro, TNF-α induced apoptosis in LnCap when we increased its concentration but not in PC3. TNF-α stimulation led to increase a time-dependent p38 phosphorylation in two intermediate doses whereas in PC3 not changes were found. In LNCaP after its preincubation with SB203580 and TNF-α treatment showed a significative increasing of apoptosis.In vivo, all NP samples were found positives to p-Elk-1 and p-ATF-2 (nuclei of epithelial cells). In PC the expression of p-Elk-1 or p-ATF-2 increased and was located in the nucleus and cytoplasm of epithelial cells.ConclusionsOur data in vitro and in vivo suggest that p38 plays a very important role in prostatic tumour progression. These data suggest that the control activation of p38 might be a possible target to cancer prostate treatment.     

视黄醇类核内受体α基因慢病毒表达载体构建

目的 通过构建大鼠视黄醇类核内受体-α(RXR-α)基因慢病毒表达载体,获得可供转染的滴度.方法 大鼠RXR-α基因序列进行聚合酶链反应(PCR)扩增,与经AgeI酶切后的pCC-FU-3FLAG载体连接产生慢病毒载体表达质粒pGC-fu-3flag-Rxra,转化DH5α,PCR筛选阳性克隆,片段长为411bp.测序并转入293T细胞Western blot鉴定,90 KDr处有特征条带.将pGC-fu-3flag-Rxra、pHelper 1.0、pHelper 2.0三质粒共转染293T细胞,包装成慢病毒,测病毒滴度,1.00E-04μl组和Control组的Ct值存在差异(2.775).结果 DNA测序及Western blot鉴定证实构建的大鼠RXR-α基因慢病毒表达载体pGC-fu-3flag-Rxra正确,浓缩慢病毒悬液滴度为2×108TU/ml.结论 成功构建携带大鼠RXR-α基因的重组慢病毒表达载体.     

地高辛对前列腺癌LNCaP和PC3细胞增殖和凋亡的影响

目的 探讨地高辛对雄激素依赖和去势抵抗性前列腺癌细胞增殖和凋亡的影响及可能的机制.方法 设立地高辛的浓度和时间梯度,分别作用于正常前列腺细胞株、雄激素依赖性及去势抵抗性前列腺癌细胞株LNCaP和PC3,流式细胞仪检测凋亡指数,MTT法检测细胞增殖速率;应用Western Blot技术检测HIF-1 α和VEGF的蛋白水平.结果 地高辛对LN-CaP和PC3细胞均有良好的抑制增殖及促进凋亡的作用(P＜0.05);Western Blot法检测提示地高辛干预组HIF-1α和VEGF的蛋白水平较未干预组明显降低(P＜0.05).结论 地高辛可抑制雄激素依赖及去势抵抗性前列腺癌细胞增殖并促进其凋亡,作用机制可能是通过抑制HIF-1 α/VEGF这一信号通路来发挥作用.     

Inhibitory effects of digitalis on the proliferation of androgen dependent and independent prostate cancer cells

PURPOSE: Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. MATERIALS AND METHODS: Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2+ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. RESULTS: Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 microM. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 microM.) significantly increased intracellular Ca2+ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 microM. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. CONCLUSIONS: Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2+ and of apoptosis.