Growth hormone-releasing hormone (GHRH) antagonists inhibit the proliferation of androgen-dependent and -independent prostate cancers

The antiproliferative effects of an antagonist of growth hormone-releasing hormone (GHRH) JV-1-38 were evaluated in nude mice bearing s.c. xenografts of LNCaP and MDA-PCa-2b human androgen-sensitive and DU-145 androgen-independent prostate cancers. In the androgen-sensitive models, JV-1-38 greatly potentiated the antitumor effect of androgen deprivation induced by surgical castration, but was ineffective when given alone. Thus, in castrated animals bearing MDA-PCa-2b cancers, the administration of JV-1-38 for 35 days virtually arrested tumor growth (94% inhibition vs. intact control, P < 0.01; and 75% vs. castrated control, P < 0.05). The growth of LNCaP tumors was also powerfully suppressed by JV-1-38 combined with castration (83% inhibition vs. intact control, P < 0.01; and 68% vs. castrated control, P < 0.05). However, in androgen-independent DU-145 cancers, JV-1-38 alone could inhibit tumor growth by 57% (P < 0.05) after 45 days. In animals bearing MDA-PCa-2b and LNCaP tumors, the reduction in serum prostate-specific antigen levels, after therapy with JV-1-38, paralleled the decrease in tumor volume. Inhibition of MDA-PCa-2b and DU-145 cancers was associated with the reduction in the expression of mRNA and protein levels of vascular endothelial growth factor. The mRNA expression for GHRH receptor splice variants was found in all these models of prostate cancer. Our results demonstrate that GHRH antagonists inhibit androgen-independent prostate cancers and, after combination with androgen deprivation, also androgen-sensitive tumors. Thus, the therapy with GHRH antagonist could be considered for the management of both androgen-dependent or -independent prostate cancers.     

Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion

Fractalkine displays features that distinguishes it from the other chemokines. In particular, besides its chemoattractant action it promotes, under physiologic flow, the rapid capture and the firm adhesion of a subset of leukocytes or intervenes in the neuron/microglia interaction. This study verified that indeed the human monocytic MonoMac6 cell line adheres to fibronectin-coated filters in response to soluble fractalkine (s-FKN). s-FKN stimulates, with distinct time courses, extracellular signal-related kinases (ERK1 and ERK2) and stress-activated protein kinases (SAPK1/JNK1 and SAPK2/p38). Both p60 Src and p72 Syk were activated under s-FKN stimulation with a rapid kinetic profile compatible with a downstream regulation on the mitogen-activated protein kinase (MAPK) congeners. The use of specific tyrosine kinase inhibitors revealed that the ERK pathway is strictly controlled by Syk, whereas c-Src up-regulated the downstream SAPK2/p38. In contrast, the SAPK1/JNK1 pathway was not regulated by any of these nonreceptor tyrosine kinases. The s-FKN-mediated increased adherence of MonoMac6 cells was partially inhibited by SB202190, a broad SAPKs inhibitor, PD98059, an MEK inhibitor, LY294002, a phosphatidyl inositol 3-kinase inhibitor, and a pertussis toxin-sensitive G protein. These data highlight that the integration of a complex array of signal transduction pathways is necessary to complete the full s-FNK-dependent adherence of human monocytic cells to fibronectin. (Blood. 2001;97:2031-2037)     

脑缺血后自噬的信号转导通路

脑缺血后自噬可被激活,且参与到缺血性脑损伤的发生和发展过程中,因此自噬相关信号转导通路的研究有可能为缺血性脑损伤提供新的治疗靶点。文章就脑缺血后自噬的信号转导通路进行了综述。     

睾酮和比卡鲁胺对雄激素依赖性前列腺癌细胞株LNcap-FCG生长的影响

目的 观察不同浓度的睾酮及雄激素拮抗剂比卡鲁胺对雄激素依赖性前列腺癌细胞株LNcap-FCG生长的影响。方法 体外培养前列腺癌细胞株LNcap-FCG，加入睾酮，使各组终浓度为10^-10、10^-9、10^-8、10^-7、10^-6、10^-5mol／L。另外，在以上浓度的各睾酮组中同时加入比卡鲁胺，使比卡鲁胺的终浓度为10、50μmol／L，应用5-溴脱氧尿核苷掺入法测定细胞的生长水平。结果 培养72h后，对照组LNcap-FCG细胞的吸光度（A）值为1.2411，睾酮在低浓度（10^-10、10^-9、10^-8、10^-7mol／L）时对LNcap-FCG细胞的生长起促进作用，A值分别为1.4247、1.5463、1.5110、1.4609；而在较高浓度（10^-6、10^-5mol／L）时，则对LNcap-FCG细胞的生长起抑制作用，A值分别为1.1239、0.9967；比卡鲁胺在两种浓度时对LNcap-FCG细胞的生长皆有抑制作用，而且浓度越高抑制作用越强，与对照组比较，差异均具有统计学意义。结论 在体外培养条件下，睾酮对LNcap-FCG细胞的生长依浓度关系呈双向作用，比卡鲁胺依浓度关系抑制LNcap-FCG细胞的生长。     

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.     

Signal transduction pathways mediating mucin secretion from intestinal goblet cells

Cholinergic stimulation of the HT29-18N2 goblet cell line increased mucin secretion as assessed: (1) with a mucin-specific immunoassay, (2) using whole-mount immunocytochemistry, or (3) by morphometric quantification of intracellular mucous granule stores. Cholinergic stimulation did not, however, result in the apical plasmalemmal membrane cavitation that is characteristic of recent compound exocytotic activity. The resposse was not dependent on protein kinase C activation since it was not inhibited by the kinase C antagonist H7 or potentiated by the diaacylglycerol kinase antagonist R59022. Calcium ionophore A23187 also accelerated mucin secretion by a noncompound exocytotic pathway. Activation of protein kinase C by phorbol 12-myristate 13-acetate, on the other hand, increased mucin secretion by a compound exocytotic pathway. The results provide insight into the signal transduction pathways underlying secretory responses of goblet cells observedin situ.This research was supported by funding from the National Institutes of Health (DK38587), the Cystic Fibrosis Foundation, and the Crohn's and Colitis Foundation of America. The image analysis system was purchased with the assistance of the University of Missouri Institutional Biomedical Research Support Grant RR07053 from the National Institutes of Health.     

Survivin regulates hematopoietic progenitor cell proliferation through p21WAF1/Cip1-dependent and -independent pathways

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 and Survivin enhance granulocyte macrophage colony-forming unit (CFU-GM) cell cycle and proliferation and have been implicated as antiapoptotic proteins. We investigated the relationships between p21 and Survivin in primary CFU-GM and c-kit+, lineage-negative (Lin-) cells and demonstrate p21-dependent and -independent pathways whereby Survivin regulates progenitor cell proliferation. Ectopic Survivin enhanced p21+/+ CFU-GM formation and expansion of c-kit+, Lin- cells, whereas p21 gene loss abrogated these effects, indicating a p21 requirement. A dominant-negative form of Survivin and p21 gene deletion accelerated the loss of CFU-GM upon growth factor deprivation, and wild-type Survivin overexpression inhibited apoptosis of p21+/+ CFU-GM and c-kit+, Lin- cells but not p21-/- cells, suggesting that both Survivin and p21 block apoptosis of progenitors and that Survivin-mediated antiapoptosis requires p21. In contrast to the p21-dependent antiapoptotic effects, Survivin increased the proportion of CFU-GM in S-phase in both p21+/+ and p21-/- cells. Furthermore, modulating Survivin expression increased polyploidy in c-kit+, Lin- cells, which was accentuated by p21 deficiency. These results suggest that the Survivin-p21 axis plays an important role in the proliferation of normal hematopoietic cells and that Survivin regulates apoptosis through a p21 WAF1/Cip1-dependent pathway but may control S-phase entry independent of p21.     

Signal transduction pathways participating in homeostasis and malignant transformation of the intestinal tissue

Intestinal homeostasis is a?complex and tightly regulated process governed by a?variety of signalling pathways that balance cell proliferation and differentiation. As revealed by extensive use of defined mouse models, perturbations within the signalling circuitry trigger initial expansion of premalignant cells. In this review, we attempt to summarise recent advances in the knowledge of the cellular signalling mechanisms that drive tumorigenesis in the human and mouse intestine. Keywords: colorectal cancer, epithelium, gut, intestine, mouse models, stem cells.     

TGF-β effects on airway smooth muscle cell proliferation, VEGF release and signal transduction pathways

Background and objective:   Airway smooth muscle (ASM) cell hyperplasia is a key feature of airway remodelling. Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) are key components in signal transduction associated with cell proliferation; MAPK consists of the extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH₂-terminal kinase (JNK). The effect of transforming growth factor (TGF)-β on the proliferation of ASM cells, the release of vascular endothelial growth factor (VEGF) by ASM cells and relevant signal transduction pathways were investigated.
Methods:   ASM cells were growth-arrested for 48 h then stimulated with platelet-derived growth factor (PDGF), TGF-β and dexamethasone. ASM cells were also treated with specific inhibitors of MAPK (PD98059), PI3K (wortmannin) and JNK (SP600125). Cell proliferation and VEGF concentrations were measured.
Results:   TGF-β neither augmented ASM cell proliferation nor showed a synergistic effect on PDGF-mediated ASM cell proliferation. Dexamethasone did not suppress ASM cell proliferation. VEGF release was augmented by TGF-β stimulation in a time-dependent manner, and was further enhanced by co-stimulation with PDGF and TGF-β. Dexamethasone suppressed VEGF release significantly. TGF-β enhanced PI3K phosphorylation, while PDGF augmented both ERK and PI3K phosphorylation. Wortmannin inhibited both TGF-β- and PDGF-stimulated VEGF release.
Conclusions:   TGF-β may facilitate airway remodelling by promoting VEGF release through the PI3K pathway, rather than via ASM cell proliferation.     

白细胞介素-8对前列腺癌细胞增殖及迁移的影响

目的研究白细胞介素(IL)-8及其受体在前列腺细胞系的表达及IL-8对前列腺癌细胞增殖及迁移的影响。方法免疫荧光和Western印迹技术分别检测IL-8及其受体在前列腺细胞系中的表达;利用CCK8、克隆形成、划痕实验检测IL-8对前列腺癌细胞生长和迁移的影响。结果免疫荧光和Western印迹结果显示PC-3、DU145和RWPE-1细胞中都有IL-8及其受体的表达,PC-3细胞中IL-8及其受体表达明显多于DU145和RWPE-1(P0.05);不同浓度的外源性IL-8能促进PC-3细胞增殖,随着浓度的增加细胞增殖效应也随之增加,浓度为80 ng/ml时增殖效果最明显,但浓度超过80 ng/ml后增殖效果有所下降,浓度为80 ng/ml时与其他组差异显著(P0.05);克隆形成和划痕实验结果显示80 ng/ml的外源性IL-8能明显促进PC-3细胞克隆形成和迁移能力(P0.01)。结论 IL-8可通过促进前列腺癌细胞生长、迁移而参与前列腺癌发生与演进过程。     

Signal transduction pathways involved in melatonin-induced neuroprotection after focal cerebral ischemia in mice

Because of its favorable action profile in humans, melatonin is a particularly interesting candidate as a neuroprotectant in acute ischemic stroke. Until now, the signaling mechanisms mediating melatonin's neuroprotective actions remained essentially uninvestigated. Herein, we examined the effects of melatonin, administered either orally for 9 wk as a stroke prophylactic (4 mg/kg/day) or intraperitoneally immediately after reperfusion onset (4 mg/kg), on the activation of signal transduction pathways in mice submitted to 90 min of intraluminal middle cerebral artery occlusion, followed by 24 hr of reperfusion. In these studies, melatonin significantly reduced ischemic infarct size by approximately 30-35%, as compared with animals receiving diluent (sham) treatment, independent of whether the indole was administered prior to or after ischemia. Under both conditions, animals receiving melatonin exhibited elevated phosphorylated Akt levels in their brains, as determined by Western blots. Additionally, phosphorylation levels of mitogen-activated protein kinase/extracellular-regulated kinase (ERK)-1/-2 and Jun kinase (JNK)-1/-2 were increased following prophylactic, but not acute, melatonin treatment. Our data suggest a role of phosphatidyl inositol-3 kinase/Akt signaling in acute melatonin-induced neuroprotection, while ERK-1/-2 and/or JNK-1/-2 rather appear to be involved in melatonin's long-term effects.     

Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology

Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 [CD31])-deficient mice have revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is expressed. In this review, we focus on the complex cytoplasmic domain of PECAM-1 and describe what is presently known about its structure, posttranslational modifications, and binding partners. In addition, we summarize findings that implicate PECAM-1 as an inhibitor of cellular activation via protein tyrosine kinase-dependent signaling pathways, an activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria. The challenge of future research will be to bridge our understanding of the functional and biochemical properties of PECAM-1 by establishing mechanistic links between signals transduced by the PECAM-1 cytoplasmic domain and discrete cellular responses.     

Noncanonical Wnt signaling mediates androgen-dependent tumor growth in a mouse model of prostate cancer

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.     

Androgen regulation of prostate morphoregulatory gene expression: Fgf10-dependent and -independent pathways

Androgens are essential and sufficient for prostate gland morphogenesis; however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone on the expression of several critical prostate morphoregulatory genes. Rat ventral prostates (VP) and lateral prostates (LP) were collected at birth, and contralateral lobes were cultured for 18 h in the presence or absence of 10 nM testosterone with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. Although expression of Fgf10, Nkx3.1, and Ptc was increased in both prostate lobes, other genes were regulated by testosterone in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus, in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Because previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.     

Intrinsic apoptotic and thioredoxin pathways in human prostate cancer cell response to histone deacetylase inhibitor

There is a great need to develop better mechanism-based therapies for prostate cancer. In this investigation, we studied four human prostate cancer cell lines, LNCaP, DU145, LAPC4, and PC3, which differ in response to the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (vorinostat), a new anticancer drug. Examining the role of intrinsic mitochondrial caspase-dependent apoptosis and caspase-independent, reactive oxygen species (ROS) facilitated cell death, has provided an understanding of mechanisms that may determine the varied response to the histone deacetylase inhibitor. We found striking differences among these cancer cells in constitutive expression and response to suberoylanilide hydroxamic acid in levels of antiapoptotic and proapoptotic proteins, mitochondria membrane integrity, activation of caspases, ROS accumulation, and expression of thioredoxin, the major scavenger of ROS. Identifying these differences can have predictive value in assessing therapeutic response and identifying targets to enhance therapeutic efficacy.