Targeted cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), AEZS-108 (AN-152), inhibits the growth of DU-145 human castration-resistant prostate cancer in vivo and in vitro through elevating p21 and ROS levels

Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone (LHRH) analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108, consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog, AEZS-108, on LHRHR positive castration-resistant prostate cancer cells.     

Down-regulation of Lsm1 is involved in human prostate cancer progression

Elucidation of genetic alterations is an approach to understanding the underlying molecular mechanisms of progression of human prostate cancers. We have searched for genes differentially expressed in advanced prostate cancers using cDNA-representational difference analysis, and thereby isolated the Lsm1 as one of down-regulated gene. An Lsm1 expression vector was transfected into PC3 cells, normally featuring down-regulated Lsm1, and four transfectants were established. No differences in morphology or cell proliferation were evident in comparison with parent PC3 or PC3/mock-transfectants. In contrast, significant suppression of invasive potential or metastatic ability of Lsm1 transfectants was observed in the Matrigel chemoinvasion assay and in nude mice, respectively. With human prostate cancers, almost all of informative prostatectomised cases without neoadjuvant therapy showed allelic retention in the Lsm1 region, whereas refractory cancers frequently showed allelic loss in this region. No critical gene mutations were found in open reading frame of Lsm1 in prostate cancers examined by PCR-SSCP analysis, including localised and refractory cancers. These results suggest that Lsm1 is deeply involved in prostate cancer progression through its down-regulation, independent of any gene mutation.     

Gene transfer of alpha1,3-fucosyltransferase increases tumor growth of the PC-3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC-3 with alpha1,3-fucosyltransferase III (FTIII) to obtain stable transfectants, PC-3-FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC-3-FTIII cells produced large prostate tumors, while mock-transfected PC-3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC-3-FTIII cells was inhibited by preincubation of the tumor cells with anti-sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC-3-FTIII cells and mock-transfected PC-3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC-3-FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock-transfected PC-3. Such adhesion was inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti-E-selectin, anti-P-selectin or anti-L-selectin antibodies did not inhibit the adhesion of PC-3-FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X.     

环王巴明诱导DU145细胞凋亡的机制研究

目的：研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制。方法：不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化。流式细胞术观察细胞凋亡率变化,RT—PCR检测5、10μmol／L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2mRNA表达水平差异。结果：当环王巴明浓度大于5μmol／L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变。环王巴明5μmol／L组Gli-1、Bcl-2 mRNA表达减弱,但同对照组差异无统计学意义（P〉0．05）,与空白及DMSO对照组相比10μmol／L环王巴明可以显著下调Gli—1、Bcl-2 mRNA表达（P〈0．01）。结论：环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2 mRNA表达,活化细胞凋亡的线粒体途径有关。     

Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin

Metastasis is a multi-step process wherein tumour cells detach from the primary mass, migrate through barrier matrices, gain access to conduits to disseminate, and subsequently survive and proliferate in an ectopic site. During the initial invasion stage, prostate carcinoma cells undergo epithelial-mesenchymal-like transition with gain of autocrine signalling and loss of E-cadherin, hallmarks that appear to enable invasion and dissemination. However, some metastases express E-cadherin, and we found close connections between prostate carcinoma cells and hepatocytes in a liver microtissue bioreactor. We hypothesise that phenotypic plasticity occurs late in prostate cancer progression at the site of ectopic seeding. Immunofluorescence staining for E-cadherin in co-cultures of hepatocytes and DU-145 prostate cancer cells revealed E-cadherin upregulation at peripheral sites of contact by day 2 of co-culture; E-cadherin expression also increased in PC-3 cells in co-culture. These carcinoma cells bound to hepatocytes in an E-cadherin-dependent manner. Although the signals by which the hepatocytes elicited E-cadherin expression remain undetermined, it appeared related to downregulation of epidermal growth factor receptor (EGFR) signalling. Inhibition of autocrine EGFR signalling increased E-cadherin expression and cell-cell heterotypic adhesion; further, expression of a downregulation-resistant EGFR variant prevented E-cadherin upregulation. These findings were supported by finding E-cadherin and catenins but not activated EGFR in human prostate metastases to the liver. We conclude that the term epithelial-mesenchymal transition only summarises the transient downregulation of E-cadherin for invasion with re-expression of E-cadherin being a physiological consequence of metastatic seeding.     

E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis

Circulating prostate cancer (PCa) cells preferentially roll and adhere on bone marrow vascular endothelial cells, where abundant E-selectin and stromal cell-derived factor 1 (SDF-1) are expressed, subsequently initiating a cascade of activation events that eventually lead to the development of metastases. To elucidate the roles of circulating PCa cells'' rolling and adhesion behaviors in cancer metastases, we applied a dynamic cylindrical flow-based microchannel device that is coated with E-selectin and SDF-1, mimicking capillary endothelium. Using this device we captured a small fraction of rolling PCa cells. These rolling cells display higher static adhesion ability, more aggressive cancer phenotypes and stem-like properties. Importantly, mice received rolling PCa cells, but not floating PCa cells, developed cancer metastases. Genes coding for E-selectin ligands and genes associated with cancer stem cells and metastasis were elevated in rolling PCa cells. Knock down of E-selectin ligand 1(ESL-1), significantly impaired PCa cells'' rolling capacity and reduced cancer aggressiveness. Moreover, ESL-1 activates RAS and MAP kinase signal cascade, consequently inducing the downstream targets. In summary, circulating PCa cells'' rolling capacity contributes to PCa metastasis, and that is in part controlled by ESL-1.     

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1-29)NH(2) re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1-29)NH(2) and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.     

Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells

Development of effective agents for treatment of hormone-refractory prostate cancer has become a national medical priority. We have reported recently that apigenin (4',5,7-trihydroxyflavone), found in many common fruits and vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin on androgen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53 and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-kappaB)/p65 and NF-kappaB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IkappaBalpha) in the cytosol. Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function, and (3) NF-kappaB inhibition.     

人前列腺癌细胞系DU 145中肿瘤干细胞样侧群细胞的分离

目的:分离人前列腺癌细胞系DU 145中的侧群(side population,SP)细胞,并初步分析其生物学特性。方法:采用荧光激活细胞分类(fluorescence activated cell sorting,FACS)技术,从DU 145细胞中分离出侧群细胞,并检测其比例;继而培养于无血清培养基中,观察其生长特性。采用反转录聚合酶链反应(RT-PCR)技术检测侧群细胞中ABCG2的表达水平。结果:DU 1 4 5细胞中存在含量极少的侧群细胞,比例约1.1%;培养于无血清培养基中成簇生长。和对应的母系DU 145细胞相比,DU 145侧群细胞的ABCG2表达增高。结论:人前列腺癌细胞系DU 145中存在具有肿瘤干细胞特性的侧群细胞。     

地塞米松对前列腺癌DU145细胞ERK1/2活性和cyclin D1表达的影响

目的: 〖HT5"SS〗 探讨糖皮质激素地塞米松抑制雄激素非依赖性前列腺癌细胞DU145的机制。〖HT5W〗方法: 〖HT5"SS〗通过细胞培养方法观察地塞米松及其受体阻断剂RU486对前列腺癌DU145细胞增殖的作用;采用流式细胞仪测定地塞米松对DU145细胞细胞周期的影响;利用Western blot技术检测DU145细胞受地塞米松作用后cyclin D1表达水平和ERK1/2活性的变化;用RTPCR技术检测DU145细胞中糖皮质激素受体mRNA的表达。〖HT5W〗结果: 〖HT5"SS〗 地塞米松明显抑制DU145细胞的增殖,并且有剂量依赖效应;地塞米松使细胞周期阻滞于G0/G1期。Western blot结果显示,地塞米松作用DU145细胞3 d后,细胞内ERK1/2的活性降低,cyclin D1表达量下降。RU486可以拮抗地塞米松对DU145细胞的作用效果。RTPCR结果显示DU145细胞有糖皮质激素受体mRNA的表达。〖HT5W〗结论: 〖HT5"SS〗地塞米松具有抑制前列腺癌DU145细胞增殖和阻滞细胞周期的作用,此作用与其降低ERK1/2活性、抑制cyclin D1合成有关,提示糖皮质激素对前列腺癌的治疗有重要意义。     

Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma

Background:

Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity.

Methods:

We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases.

Results:

In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3).

Conclusion:

CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.     

Sunitinib malate (SU-11248) alone or in combination with low-dose docetaxel inhibits the growth of DU-145 prostate cancer xenografts

The aim of the study was to evaluate the activity of the antiangiogenic agent SU-11248 (sunitinib malate, Sutent^®), alone or in combination with docetaxel. To this end, animals bearing DU-145 human hormone-refractory prostate cancer (HRPC) xenografts were treated with sunitinib (40 mg/kg daily, p.o.), docetaxel (10 or 30 mg/kg/week, i.v.), a combination of sunitinib (40 mg/kg daily) and docetaxel (10 mg/kg/week) or vehicle alone. At the end of the 3-week dosing schedule, single-agent treatment induced a tumor regression of 59%, 49% and 75% for sunitinib, docetaxel 10 mg/kg, and docetaxel 30 mg/kg, respectively. The combination of sunitinib with low-dose (10 mg/kg) docetaxel produced a tumor regression comparable to that obtained with high-dose (30 mg/kg) docetaxel, but tolerability was higher as indicated by mice weight. Both sunitinib and docetaxel inhibited tumor regrowth after initial treatment with the alternate drug. These results suggest that sunitinib alone or in combination with low-dose docetaxel may have a role in the treatment of HRPC.     

Treatment outcomes of small cell carcinoma of the prostate: a single-center study

BACKGROUND: The current study was conducted to determine the clinical characteristics and prognostic features associated with prostatic small cell carcinoma (SCC). METHODS: Between January 1985 and May 2005, 83 patients with SCC of the prostate were identified. Univariate and multivariate Cox proportional hazards modeling were used to assess the prognostic significance of the clinical parameters associated with disease-specific outcomes. RESULTS: Twenty-one patients had no evidence of distant metastasis at the time of the diagnosis of SCC, with the remaining patients demonstrating radiologic or biopsy-proven evidence of metastatic disease. Compared with patients with metastases, patients without metastases at the time of diagnosis were older (P = .001) and had a lower serum lactate dehydrogenase (LDH) level at the time of diagnosis (P = .002). On multivariate analysis, an elevated serum LDH level and low serum albumin at the time of SCC diagnosis was found to be predictive of inferior progression-free survival (P = .02 and P = .008, respectively) and inferior disease-specific survival (DSS) (P = .02 and P = .01, respectively). At the time of last follow-up, 72 patients (87%) had died of disease, with a median DSS duration of 13.1 months (range, 10.7-17.1 months). There was a statistically significant difference noted with regard to the median DSS of patients with nonmetastatic versus those with metastatic SCC (17.7 months [95% confidence interval (95% CI), 12.1-39.2 months] vs 12.5 months [95% CI, 8.1-16.1 months], respectively; P = .03). CONCLUSIONS: SCC of the prostate is a highly aggressive tumor, with serum LDH and albumin levels at the time of diagnosis believed to be predictive of disease-related outcomes. Although palliative, current systemic therapy does not result in cure and does not provide long-term survival for patients with metastases. For patients with nonmetastatic disease, a strategy utilizing systemic and local therapies should be evaluated further.     

前列腺干细胞抗原在前列腺癌诊断与治疗中的应用

人们对前列腺癌理想的诊断和治疗方式的探寻从未停止.近些年来,随着对前列腺干细胞抗原(prostate stem cell antigen,PSCA)研究地不断深入,特别是应用于前列腺癌诊治方面的新成果不断推出,使人们对PSCA充满期望.本文重点对PSCA的生物学特性及其在前列腺癌诊断方法和免疫治疗方面的新进展进行综述.     

EGF signalling in prostate cancer cell lines is inhibited by a high expression level of the endocytosis protein REPS2

In advanced prostate cancer, cellular changes occur leading to a transition from androgen-dependent to androgen-independent growth. During this transition, proliferation of androgen-dependent prostate cancer cells becomes more and more dependent on growth factors, like the epidermal growth factor (EGF). Endocytosis of growth factor receptors, one of the mechanisms that controls growth factor signalling, was observed to be markedly changed in advanced metastatic prostate cancer. Internalisation and signalling of EGF receptors was examined in different prostate cancer cell lines, in relation to the expression level of the endocytosis-related REPS2 gene. It was observed that a high level of REPS2 correlates with reduced EGF-internalisation. To investigate this more thoroughly, prostate cancer cells with inducible REPS2 expression were generated. Using these cells, it was found that REPS2-induction indeed results in reduction of EGF-internalisation. Furthermore, when EGF receptor signalling was evaluated, by examination of mRNA expression for several EGF-responsive genes (EGF receptor, EGR-1, Fos and Jun), it was observed that induced expression of REPS2 exerts an inhibiting effect on this signalling. From these experiments, it is concluded that increased REPS2 expression negatively affects EGF receptor internalisation and subsequent signalling. Therefore, decreased REPS2 expression during prostate cancer progression, observed in earlier work, may result in enhanced EGF receptor expression and signalling, which could add to the androgen-independent state of advanced prostate cancer.     

细胞黏附分子在肝癌复发转移中的作用研究进展

细胞黏附分子（CAMs）是位于细胞表面的糖蛋白,通过介导细胞与细胞、细胞与细胞外基质间的相互作用参与多种生理及病理过程。细胞黏附分子与包括肝癌在内的多种肿瘤的复发转移有着非常密切的关系。本文就细胞黏附分子在肝癌复发转移中作用的研究进展作一综述。