Combined effect of terazosin and finasteride on apoptosis, cell proliferation, and transforming growth factor-beta expression in benign prostatic hyperplasia

BACKGROUND: Medical treatment of benign prostatic hyperplasia (BPH) targets relief of symptoms by causing either relaxation of the prostatic smooth muscle with alpha1 adrenergic blockade, or shrinkage of the gland with 5alpha-reductase inhibitors. We recently demonstrated that alpha1-blockers, such as terazosin, induce apoptosis in prostatic cells. In this study, we examined the combined effect of finasteride and terazosin on the rate of apoptosis and cellular proliferation to investigate their potential synergy at the cellular level. METHODS: Prostate specimens were obtained from men who were treated with either finasteride (n = 24), terazosin (n = 42), or combination therapy (n = 10) for varying time periods (1 week to 36 months) for the relief of the symptoms of BPH. The proliferative and apoptotic indices of both stromal and epithelial prostatic cell populations were determined. Antibodies against TGF-beta1 and TbetaRII were used to examine the immunoreactivity of TGF-beta1 and TbetaRII, respectively, in all prostate tissue sections. RESULTS: The apoptotic index in both prostate cell populations was significantly higher following the combination treatment compared to terazosin or finasteride alone. There were no significant changes in the rate of cellular proliferation with any treatment. Furthermore, there was a significant increase in TGF-beta1 expression in the prostates of patients treated with terazosin or combination therapy, while there was no change in TbetaRII expression. CONCLUSIONS: These results support the concept that induction of prostate apoptosis is a potential molecular mechanism underlying the combination effect of alpha1 blockade with 5alpha-reductase inhibitors in the effective treatment of BPH. The upregulation of TGF-beta1 implies a role for this ligand as an effector of apoptosis induction in response to alpha1-blockade or finasteride therapy of BPH patients.     

MAPK and PI3K inhibition reduces proliferation of Barrett's adenocarcinoma in vitro

BACKGROUND: Esophageal adenocarcinoma often arises from Barrett's esophagus. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) play critical roles in cell survival. We hypothesized that inhibition of these pathways in Barrett's adenocarcinoma would decrease cell proliferation and alter apoptosis in vitro. MATERIALS AND METHODS: Two Barrett's-associated adenocarcinoma cell lines, SEG-1 (wild-type p53) and BIC-1 (mutant p53), were treated with MAPK (U0126) and PI3K (LY294002) inhibitors at 20 microm concentrations. After 24 and 72 h, cell viability was measured by MTT assay. Apoptosis and necrosis were evaluated by the Annexin V-FITC assay. Statistical analysis was performed by ANOVA. RESULTS: LY294002 and U0126 treatment produced significant reductions (range 15.7 to 62.0%, P < 0.05) in cellular proliferation at both 24 and 72 h in the SEG-1 cells. BIC-1 cell viability was reduced (39.3 to 56.4%, P < 0.05) at 72 h. Both early and late apoptotic activity were significantly increased (P < 0.05) in the SEG-1 cells using both inhibitors. Necrosis was significantly reduced (P < 0.05) using both inhibitors. No changes in either early or late apoptosis or necrosis were observed in the BIC-1 cells. CONCLUSIONS: Herein, we report significant antiproliferative effects against Barrett's adenocarcinoma by MAPK and PI3K inhibition in vitro. Pro-apoptotic mechanisms prevail in the wild-type p53 cells. Further investigation is warranted to advance the clinical treatment of this devastating disease.     

索拉菲尼对肝癌细胞HepG2自噬的抑制作用

目的：研究化疗药物索拉菲尼（Sorafenib）对肝癌细胞HepG2自噬的作用及自噬与细胞增殖、细胞凋r_的关系。方法：常规细胞培养,以10μmol／L的索拉菲尼作用不同时间,采用MDC染色,在荧光显微镜下观察自噬泡的情况：用Western印迹检测自噬相关蛋白LC3的动态变化;用3-MA抑制肝癌细胞中自噬的表达,并用CCK8法检测索拉菲尼及其联合3-MA对细胞生存率的影响,用AnnexinV／PI流式细胞仪检测抑制自噬后凋亡的变化。结果：HepG2经索拉菲尼作用后,自噬泡明显减少,LC3蛋白尤其是LC3-Ⅱ随着作用时间延长逐渐减弱;索拉菲尼联合3-MA可进一步抑制HepG2细胞中自噬的表达,抑制自噬后细胞死亡明显增加,特别是细胞凋亡明显增加。结论：索拉菲尼抗肝癌作用可能与抑制肝癌细胞自噬有关。     

Methylglyoxal-induced apoptosis in human prostate carcinoma: potential modality for prostate cancer treatment

OBJECTIVE: To examine the cellular effects of methylglyoxal (MG), a toxic physiological metabolite, on human prostatic cancer PC-3 cells. METHODS: The effects of MG on cell growth and viability were evaluated first, and then its effects on the cell cycle and the glycolytic process were analyzed by Western blots and specific assays. Possible MG-induced apoptosis was also assessed by DNA analysis using agarose gel electrophoresis. RESULTS: MG > or =3 mM caused severe growth inhibition, resulting in nearly 100% cell death by 24h. The time course study revealed that expression of cyclin D(1), cdk2, and cdk4 was significantly (>50%) downregulated in 3 h of MG (3 mM) exposure, followed by the dephosphorylation of retinoblastoma protein by 6 h. Both the glyceraldehyde-3-phosphate dehydrogenase activity and the cellular lactate level were also reduced by approximately 50 and 80%, respectively, following 6-hour MG exposure. Induction of apoptosis by MG was indicated by partial degradation of poly(ADP-ribose) polymerase and further confirmed by discrete DNA fragmentation detected on an agarose gel. CONCLUSION: MG is capable of inducing apoptosis in prostatic cancer PC-3 cells, due primarily to a blocking of the cell cycle progression (G(1) arrest) and glycolytic pathway. Therefore, MG could be a potent apoptosis inducer, which may have a potential for prostate cancer treatment.     

Sodium fluoride modulates caprine osteoblast proliferation and differentiation

The cellular and molecular pathways of fluoride toxicity in osteoblasts are not very well understood. Therefore, the objective of the present study was to evaluate the effects of sodium fluoride (NaF) on caprine osteoblasts cultured in vitro. Caprine osteoblasts at 2.0 x 10(-4) cells/ml were incubated in vitro with NaF at 0, 10(-8), 10(-7), 10(-6), 10(-5), 10(-4), 5.0 x 10(-4), and 10(-3) M, and then proliferation, differentiation, apoptosis, calcification, and alkaline phosphatase activity were examined. Also, the effect of NaF on osteoblastic cell viability and the molecular events leading to apoptosis were determined. Electron microscopy revealed cytoplasmic and nuclear alterations in the ultrastructure of osteoblasts exposed to various NaF concentrations. A cell-based quantitative evaluation of the MTT assay showed that NaF at concentrations of 10(-8) to 10(-5) M promoted cell proliferation, whereas at 10(-4) to 10(-3) M it suppressed cell proliferation and induced apoptosis. Alkaline phosphatase (ALP) activity and mineralization ability increased in cells treated at 10(-8) to 10(-5) M with sodium versus the controls, but decreased at 5.0 x 10(-4) to 10(-3) M dosage. The highest incidence of early apoptotic cells and late apoptotic cells was reached (3.33% and 2.92%, respectively) under NaF concentration of 10(-4) M. In conclusion, results of this study indicated that NaF modulates osteoblast proliferation and differentiation in a dose-dependent manner and modified osteoblast metabolism bidirectionally, suggesting NaF may play a significant role in osteoblast physiology.     

Aberrant response in vitro of hormone-responsive prostate cancer cells to antiandrogens

Antiandrogens are in use alone and in combination with other agents as hormonal therapy for prostate cancer. We conducted studies on the androgen-responsive human prostate cancer cell line LNCaP to determine the direct effects of three antiandrogens (hydroxyflutamide, RU23908, and cyproterone acetate) on hormone-responsive human prostate cancer cells in culture. Dihydrotestosterone (DHT) stimulated the growth of LNCaP cells in a dose-dependent fashion. These cells contained approximately 31,000 high-affinity (Kd = 9 x 10(-10) M) androgen binding sites per cell. In the absence of any androgenic stimulation, all three antiandrogens tested showed agonistic properties by increasing the cell number and uptake of [3H]-thymidine. Competitive uptake studies using [3H]-R1881, a nonmetabolized androgen, showed that the three antiandrogens inhibited specific R1881 uptake with IC50s of 0.9 x 10(-7) M for hydroxyflutamide, 2 x 10(-7) M for RU23908, and 1 x 10(-7) M for cyproterone acetate. It is not known whether these unexpected agonistic effects are due to an altered receptor, previously unmasked agonistic properties of the antiandrogens, or emergence of a hypersensitive clone of cells.     

Chimeric molecules facilitate the degradation of androgen receptors and repress the growth of LNCaP cells

Post-translational degradation of protein plays an important role in cell life. We employed chimeric molecules （dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]） to facilitate androgen receptor （AR） degradation via the ubiquitin-proteasome pathway （UPP） and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells. Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment. Cell counting and the 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyl tetrazolium bromide （MTT） cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells. AR was tagged for elimination via the UPP by DHT-PROTAC, and this could be blocked by proteasome inhibitors. Degradation of AR depended on DHT-PROTAC concentration, and either DHT or an ALAPYIP-（arg）8 peptide could compete with DHT-PROTAC. Inhibition of cell proliferation and decreased viability were observed in LNCaP cells, but not in PC-3 or 786-0 cells after DHT-PROTAC treatment. These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC, and that the growth of LNCaP cells is repressed after AR degradation.     

EGF receptor activity modulates apoptosis induced by inhibition of the proteasome of vascular smooth muscle cells

The observation that intracellular protein turnover rates participate directly in cell viability led to the development and clinical use of potent proteasome inhibitors. This study determined that the mechanism of apoptosis that is induced by inhibition of the proteasome of vascular smooth muscle cells (VSMC) was related to the intracellular accumulation of Bad, a BH3-only member of the Bcl-2 family of apoptosis regulators. Experiments confirmed that the apoptotic process was mitochondria- and caspase-dependent. Ubiquitination and accumulation of Bad in VSMC followed inhibition of the proteasome, and depletion of Bad using RNA interference prevented apoptosis that was induced by proteasome inhibition with PS-341. EGF receptor (EGFR) activation produced posttranslational modifications of Bad, providing the pro-survival signals that prevented apoptosis of smooth muscle cells during proteasome inhibition. Antagonists of the EGFR potentiated the apoptotic rate. In summary, the activities of the EGFR and the proteasome focused on Bad and the intrinsic apoptotic pathway and were involved integrally in determining viability of VSMC. These findings might prove useful in the management of diseases in which proliferation of vascular smooth muscle cells plays a central role.     

Ezrin shRNA联合HSP70对骨肉瘤细胞凋亡和增殖的影响

 目的 探讨Ezrin基因沉默联合热休克蛋白（heat shock protein, HSP）70诱导的免疫杀伤对骨肉瘤细胞增殖和凋亡的影响。方法 采用人成骨肉瘤MG63细胞系作为研究对象,将人HSP70和Ezrin-shRNA的DNA片段分别克隆至含CMV和pU6双启动子的表达载体pGFP-V-RS中构建含HSP70和Ezrin-shRNA的真核表达载体pGFP-V-RS-shRNA和pGFP-V-RS-shRNA-HSP70。重组质粒分别转染入细胞,筛选出稳定表达的细胞克隆。荧光显微镜观察细胞形态及转染效果;荧光定量RT-PCR及蛋白印迹western blot检测稳定转染细胞株Ezrin和HSP70基因及蛋白水平的变化;采用MTT、流式细胞术检测细胞增殖、凋亡能力变化,Western blot检测凋亡及周期相关蛋白表达量变化;MTT法检测HSP70刺激产生特异性细胞毒性T淋巴细胞（CTL）对靶肿瘤细胞的杀伤作用。结果 荧光图像及基因和蛋白表达分析证实,首次成功构建同时沉默Ezrin和过表达HSP70的特异性载体。较单纯沉默Ezrin,同时过表达HSP70会在一定程度上减弱沉默Ezrin蛋白促进细胞凋亡和抑制增殖的效果,但与对照细胞相比,M63细胞凋亡率仍明显上升,自11.01%±0.22%上升至24.28%±0.50%,增殖速度则自395.14%±2.24%减少至310.00%±2.83%。另外,Western blot检测发现Ezrin-shRNA可促进凋亡基因Bax的表达,相反可降低抗凋亡基因Bcl-2和细胞周期蛋白Cyclin D1的表达。而Ezrin-shRNA/HSP70组较Ezrin-shRNA组促Bax表达和降低Bcl-2、细胞周期蛋白Cyclin D1表达有所减弱,但较阴性对照组仍有明显效果。MG63细胞的CTL细胞毒杀伤效应显示各效靶浓度下CT+IL-2+HSP70组的杀伤活性高于CT+IL-2组,最高达56.33%±1.95%。结论 同时沉默Ezrin、过表达HSP70既可以促进骨肉瘤细胞凋亡抑制其增殖,并利用HSP70诱导的CTL,增强对靶肿瘤细胞的杀伤作用。     

Eicosapentaenoic acid and gamma-linolenic acid induce apoptosis in HL-60 cells

Enteral nutrition with eicosapentaenoic acid (EPA; 20:5 n-3) and gamma-linolenic acid (GLA; 18:3 n-6) decreased pulmonary inflammation by reducing neutrophil counts and chemotactic factors in bronchoalveolar lavage fluid during acute respiratory distress syndrome (ARDS). We hypothesize that the anti-inflammatory effects of EPA and GLA may be due, in part, to induction of neutrophil apoptosis. The purpose of this study was to determine whether EPA and GLA, alone or in combination, trigger apoptotic cell death in the human promyelocytic leukemia HL-60 cell line. HL-60 cells were incubated with 10, 20, 50, and 100 micromol/L EPA, GLA, or various combinations of EPA and GLA for 2, 4, 8, 12, and 24 hs. Oleic acid (18:1 n-9) was used as a fatty acid control. Flow cytometry using dual staining with propidium iodide and annexin V-FITC assessed apoptosis, necrosis, and viability. Apoptosis was verified by DNA fragmentation as assessed by agarose gel electrophoresis. EPA, GLA, and various combinations of EPA and GLA significantly induced apoptosis and reduced cell viability in HL-60 cells. Viability was significantly reduced to the same extent with the combination of 50 micromol/L EPA\20 micromol/L GLA compared with 100 micromol/L EPA. These data indicate that EPA and GLA, alone or in combination, reduce cell survival by induction of apoptosis. Thus, induction of apoptosis by select dietary n-3 (EPA) and n-6 (GLA) polyunsaturated fatty acids may be the mechanism of the resolution of pulmonary inflammation in ARDS.     

Effect of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages

Bisphosphonates (BP) are powerful inhibitors of bone resorption. Their mechanism of action, although still unclear, is now believed to be at the cellular level. In this study we investigated the effects of these compounds on proliferation, induced either by L-cell conditioned medium (CSF-1) or 4-phorbol 12-myristate 13-acetate (PMA) of bone marrow cells (BMC) and on CSF-1-induced proliferation and viability of bone marrow derived macrophages (BMDM phi). BMC proliferation, measured by [3H]-TdR incorporation or by clonal assay in soft agar, was significantly inhibited by 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) and 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) at 2.5 x 10(-7) M and by dichloromethylenebisphosphonate (Cl2MBP) at 2.5 x 10(-6) M. This inhibitory effect was also confirmed on the proliferation, measured by [3H]-TdR incorporation, of BMDM phi. In the absence of CSF-1, the viability of this latter cell population, estimated by DNA content per well and lactate dehydrogenase (LDH) released into the medium, was affected in the following order of concentrations: Cl2MBP, 1.0 x 10(-4) M; AHBuBP, 5.0 x 10(-5) M; and AHPrBP, 2.5 x 10(-5) M. Since osteoclasts and macrophages might share a common early progenitor cell, probably under the control of CSF-1, the effect exerted by BP on the proliferation of the macrophage precursors may also be extended to the osteoclast precursors.     

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.     

Additive effects of calcium antagonists on cyclosporin A-induced inhibition of T-cell proliferation

The purpose of this study was to investigate possible additive effects of calcium antagonists on the cyclosporin A (CsA)-induced inhibition of cellular immunity. Human T-cells were isolated using standard methods and stimulated with phytohaemagglutinin (PHA, n = 8), the monoclonal antibody OKT3 (n = 6), or mixed lymphocyte reaction (MLR, n = 5). Verapamil, nifedipine, nimodipine or diltiazem were added (5 x 10(-7) - 5 x 10(-5) M) to the cultures, either alone, or in combination with CsA (62.5, 125, and 250 ng/ml). 3H-thymidine uptake was measured to estimate the proliferative responses and dose response curves were constructed for the Ca antagonists and their combinations with CsA. A 50% inhibition of T-cell proliferation in the different stimulation assays was achieved with 3.2 x 10(-5) - 5.3 x 10(-5) M verapamil, 2.5 x 10(-5) -4.3 x 10(-5) M nifedipine, 3.7 x 10(-6) - 5 x 10(-6) M nimodipine, and greater than 5 x 10(-5) M diltiazem. In combination with CsA a dose-dependent additive inhibitory effect of the Ca antagonists on T-cell proliferation was observed. This effect was less pronounced in the OKT3 assay, intermediate after PHA stimulation and most pronounced in MLR. Even in low concentrations, which correspond to therapeutic serum concentrations, Ca antagonists have an additive inhibitory effect in MLR. We conclude that Ca antagonists exert a dose-dependent inhibitory effect on T-cell proliferation. A combination of CsA with verapamil, nifedipine, nimodipine, or diltiazem is more effective than each drug given alone. This additive effect of Ca antagonists and CsA may possibly contribute to a better graft survival in clinical transplantation.     

Caspase-3 drives apoptosis in pancreatic cancer cells after treatment with gemcitabine

Pancreatic cancer remains a highly chemoresistant malignancy. Gemcitabine, the most effective firstline agent available, acts by disrupting cellular replication. Caspases belong to a family of proteases that function as key components of the apoptotic death machinery. We investigated the mechanisms by which gemcitabine blocks proliferation and whether it can induce apoptosis in pancreatic cancer cells. Quiescent pancreatic cancer cells (BxPC-3) were stimulated to proliferate (10% fetal calf serum) with or without gemcitabine, PS-341 (26S proteasome inhibitor), or both. Proliferation was measured by MTT assay and apoptosis by propidium iodine staining. To determine activation of the apoptotic regulatory cell proteins, caspase-3 and cleavage of poly(ADP-ribose)polymerase (PARP) into its 85-kDa fragment were assessed by Western blotting. Gemcitabine at even low doses (10 μmol/L) significantly inhibited cellular proliferation, whereas PS-341 (10 nmol/L) had no effect. With combined treatment, PS-341 potentiated the antiproliferative effects of gemcitabine (P = 0.001). At 48 hours, the apoptotic fraction was greatly enhanced by the presence of PS-341 compared with gemcitabine alone. Caspase-3 accumulated as early as 30 minutes and was associated with cleavage of PARP to its apoptotic fragment. Gemcitabine, a nucleoside analogue, may in part exert its antiproliferative effects by directing pancreatic cancer cells to a default pathway of apoptosis. 26S proteasome inhibition potentiates this effect, suggesting its potential clinical value against chemoresistance in pancreatic cancer. Presented at the Fourth Biennial Congress of the American Hepato-Pancreato-Biliary Association (AHPBA), Miami Beach, Florida, February Presented at the Fourth Biennial Congress of the American Hepato-Pancreato-Biliary Association (AHPBA), Miami Beach, Florida, February Supported by grants from the American Hepato-Pancreato-Biliary Association, Ethicon Research Fellowship, and National Pancreas Foundation.     

藤黄酸对肾癌786-O细胞活力和对HUVEC细胞血管形成能力的抑制作用及其分子机制

目的观察藤黄酸(GA)对肾癌786-O细胞活力和对HUVEC细胞血管形成能力的影响及其分子机制。方法不同浓度的GA处理786-O细胞一定时间后,分别采用Alamar blue法、流式细胞术和免疫印迹,检测GA对786-O细胞活力、周期分布、凋亡率及凋亡标志物聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解片段的影响;GA对HUVEC细胞的作用除进行上述检测外,还利用划痕实验法和Transwell小室法检测细胞横向及纵向迁移能力变化,反映GA对HUVEC细胞血管形成能力的影响;免疫印迹检测P21Waf1/Cip1、Bax、缺氧诱导因子(HIF-1α)、血管内皮生长因子(VEGF)和泛素化蛋白;特异性荧光蛋白酶体底物肽降解法检测细胞内糜蛋白酶样蛋白酶体活性。结果 GA能够剂量依赖性抑制786-O和HUVEC细胞的活力、诱导细胞G2-M期阻滞和凋亡,同时增加P21Waf1/Cip1、Bax和泛素化蛋白水平;能够减弱HUVEC细胞横向和纵向迁移能力,伴随HIF-1α的稳定性增加和VEGF表达下调;能够抑制细胞内糜蛋白酶样蛋白酶体活性。结论 GA通过诱导细胞G2-M周期阻滞和凋亡而显著抑制786-O和HUVEC细胞活力,并能有效抑制HUVEC细胞血管形成能力,其机制可能与蛋白酶体活性抑制所导致的底物蛋白P21Waf1/Cip1、Bax和HIF-1α的稳定性改变,及HIF-1α调控的下游蛋白VEGF表达水平的变化有关。     

Stromal/epithelial interactions of murine prostatic cell lines in vivo: a model for benign prostatic hyperplasia and the effect of doxazosin on tissue size

BACKGROUND: One of the major constraints in elucidating the mechanisms involved in the etiology of benign prostatic hyperplasia (BPH) is the lack of suitable model systems that are readily manipulable in vitro and in vivo. To address this issue, we have used murine prostatic cell lines to establish a novel in vivo model for studying prostatic cell interactions. METHODS: Luminal, basal, and smooth muscle (SM) cell lines were inoculated alone or in combinations under the renal capsule of intact or castrated male mice, and the growth and composition of prostatic tissue in the absence or presence of doxazosin was determined. RESULTS: Both the luminal and basal cell lines reconstituted prostatic tissue if co-inoculated under the renal capsule with normal SM cells, whereas none of the lines formed significant tissue when inoculated alone. Luminal cells produced and secreted prostatic secretory products. The growth of prostatic tissue formed from co-inoculation of basal and SM cells was androgen responsive. In addition, a significant reduction in prostatic tissue was noted in animals treated with doxazosin. CONCLUSION: We have established an in vivo model that uses prostatic epithelial and SM cell lines for investigating cellular interactions between epithelial and SM cells that regulate prostatic growth and function. This model will be useful for delineating the mechanisms by which prostatic cells interact and in determining the efficacy of new approaches aimed at interfering with prostatic stromal/epithelial interactions that result in abnormal cellular proliferation.     

右美托咪定对咪达唑仑所致神经干细胞增殖、分化和凋亡改变的影响

目的 研究咪达唑仑对神经干细胞(neural stem cells,NSCs)增殖、分化及凋亡的毒性作用及右美托咪定(dexmedetomidine,Dex)能否缓解咪达唑仑的神经毒性作用. 方法 分离培养孕14～15 d大鼠胚胎大脑皮质NSCs,将NSCs接种于培养板中,培养24 h后,将其按照完全随机分组法分为3组:对照组(C组)、咪达唑仑组(M组)、Dex联合咪达唑仑组(M+D组).分别采用咪达唑仑、Dex联合咪达唑仑处理培养的第一、二代NSCs 24 h,采用噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT)]比色法检测细胞活力,溴脱氧尿苷(5-bromo-2-deoxyuridine,BrdU)掺入法检测细胞增殖,免疫细胞化学法观察NSCs分化情况,原位末端标记法(terminal dUTP nick-end labeling,TUNEL)检测细胞凋亡. 结果 与对照组比较,咪达唑仑干预NSCs 24 h可降低细胞活力(对照组0.214±0.006,咪达唑仑组0.187±0.002)、减少细胞增殖[(对照组(35.7±1.0)％,咪达唑仑组(27.6±1.0)％]、增加细胞凋亡[对照组(5.7±0.8)％,咪达唑仑组(7.8±1.1)％](P＜0.01),但对NSCs分化没有显著影响(P＞0.05);与咪达唑仑处理比较,Dex联合咪达唑仑干预NSCs 24 h,可增加细胞活力[M+D组0.233±0.007]和细胞增殖[M+D组(35.7±1.1)％]、减少细胞凋亡[M+D组(5.3±1.0)％](P＜0.01),但对NSCs向神经元和星形胶质细胞分化无明显影响(P＞0.05). 结论 Dex可缓解咪达唑仑抑制NSCs增殖、促进细胞凋亡的作用,但不影响其向神经元和星形胶质细胞方向分化.     

Androgen dependence of the Dunning R3327G cell line in monolayer culture

For optimal application of new treatment strategies for prostate cancer, the basic biologic effects of androgens on cell kinetics and DNA synthesis require detailed examination. An androgen-responsive prostate cancer cell line in monolayer culture provides a means to study the biochemical mechanisms mediating hormonal stimulation of cell proliferation. We chose to evaluate the proliferative response of the Dunning R3327G tumor cell line (Du-G cells) to 5 alpha-dihydrotestosterone (DHT) in monolayer culture. The DU-G cells grew more rapidly in the presence of increasing concentrations of DHT in the range of 10(-8)-10(-5) M than with vehicle control. At 10(-7) M DHT, 3H-thymidine incorporation increased from 400 +/- 34 counts/min/well to 751 +/- 77 (p less than .01). Effects of DHT were maximal when a plating density of 10,000 cells/well was employed. Androgen effects on cellular growth were reproducible but were limited in magnitude. Rapid metabolism of DHT in culture did not explain this phenomenon. Du-G cells were not completely dependent on androgen, since cells continued to grow in media containing less than 10(-11) M dihydrotestosterone and hydroxyflutamide.     

蛋白酶体抑制剂MG132对胆囊癌细胞杀伤作用的实验研究

目的 研究蛋白酶体抑制剂MG132抑制胆囊癌细胞增殖及诱导凋亡的机制。方法 采用CCK8和流式细胞术检测胆囊癌细胞增殖及凋亡情况;采用Western blotting和RT-PCR检测凋亡相关基因Caspase-8、Caspase-3和DR5的mRNA和蛋白质表达;建立荷瘤裸鼠模型,观察MG132干预下裸鼠瘤重及瘤体积的变化。结果 在本研究中发现MG132能有效抑制体外和体内胆囊癌细胞的增殖,其作用呈剂量依赖性（P＜0.01）。MG132能促使胆囊癌细胞发生G2/M期阻滞及诱导细胞亡。MG132诱导胆囊癌细胞凋亡主要通过激活外源性凋亡通路中DR5、Caspase-8和Caspase-3过表达。结论 MG132能对胆囊癌细胞起杀伤作用,其机制可能通过影响细胞周期阻滞及诱导细胞凋亡起作用。     

联合甲磺酸伊马替尼和ABT-737对胃肠间质瘤细胞凋亡的影响

目的 观察联合甲磺酸伊马替尼(IM)和ABT-737对胃肠间质瘤细胞凋亡的影响.方法 培养胃肠间质瘤细胞株GIST-882;噻唑蓝(MTT)比色法分析不同浓度ABT-737和不同处理时间对GIST-882细胞生长的影响;分析联合应用不同浓度IM和ABT-737对GIST882细胞生长的影响.Western blot法分析IM处理对bcl-2蛋白的影响;并分析联合应用IM和ABT-737对Caspase-3 蛋白表达的影响.结果 10 μmol/L的ABT-737作用细胞24、48、72 h后,细胞的生存率分别是70％、55％、38％;20 μmol/L的ABT-737作用细胞24、48、72 h后,细胞的生存率均小于10％.0.1μmol/L的IM联合0.1μmol/L的ABT-737可将GIST-882细胞的生存率抑制在20％以下,10 μmol/L的IM联合10 μmol/L的ABT-737可将GIST-882细胞的生存率抑制在10％以下.IM对GIST882细胞bcl-2蛋白的表达无明显影响;联合应用IM和ABT-737可以促进GIST-882细胞活化型Caspase-3 蛋白的表达.结论 联合应用ABT-737可以加强IM对GIST细胞的促凋亡作用.