Proteomic analysis of dunning prostate cancer cell lines with variable metastatic potential using SELDI-TOF

BACKGROUND: Surface enhanced laser desorption and ionization-time-of-flight (SELDI-TOF) is an evolving proteomic technology for improving biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures generated from body fluids, cells, and/or tissues. SELDI--based profiling identifies unique, differentially expressed proteins relating to specific cancer-related disease states. We utilized SELDI-TOF following pre-processing with molecular separation and chemical fractionation of cell membrane extracts from three Dunning rat prostate cancer cell lines of varying metastatic potential to search novel proteins that are differentially expressed. METHODS: Dunning rat cell sublines of variable (%) metastatic potential; G (0%), AT-1 (20%), and Mat-Ly-Lu (100%) were cultured in two different laboratories. Cell lysis was performed in a homogenation buffer (320 mM sucrose/50 mM Tris/0.5 mM PSMF) using Dounce homogenation. After centrifugation, the membrane pellet was washed 2x and then solublized in 2% CHAPS/8 M urea. This sample was further processed using positive pressure molecular ultrafiltration at 30 kDa or precipitation with 50% ammonium sulfate. Next, each sample was applied to an IMAC3-Ni ProteinChip (Ciphergen Biosystems, Freemont, CA) and analyzed using Ciphergen's Protein Biology System with protein peak analysis software. RESULTS: SELDI-TOF analysis differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20,000 Da. The preparations from the three cells lines showed clear differences when the extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kDa (decrease), 12 kDa (significant decrease), 14 kDa (decrease), and 17 kDa (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate laboratory, and using different lots of SELDI ProteinChips. CONCLUSIONS: The application of SELDI-TOF to a series of Dunning rat prostate cancer cell lines illustrated apparent changes in protein profiles among the three cell lines with known differences in metastatic biologic activity. SELDI-TOF identified four reproducible changes in protein expression in the AT1 and MLL metastatic cell sublines. Three of the expression changes were manifested as decreases, but one protein (17 kDa) was over-expressed in the AT1 and MLL cell lines. Emphasis will be placed on the isolation, purification, and characterization of the 17 kDa over-expressed protein and its potential role in PCa metastasis.     

Role of endothelin axis in progression to aggressive phenotype of prostate adenocarcinoma

BACKGROUND: Mitogenic and anti-apoptotic actions of endothelin-1 (ET-1) are mediated through endothelin A (ET(A)) receptors. We investigated endothelin receptor expression in increasingly aggressive phenotype and in vivo effects of combination therapy using ET(A) antagonist with paclitaxel. METHODS: Dunning prostate cancer cells ranged in aggressiveness from non-tumorigenic G, to tumorigenic, non-metastatic AT-1, and to tumorigenic and metastatic MLL. Binding assays were performed alongside Q-PCR to assess receptor density. MLL xenografts were treated with vehicle, atrasentan, paclitaxel, and paclitaxel+atrasentan. RESULTS: Saturation binding assays demonstrated endothelin receptor density of MLL and AT-1 cells seven- and threefold higher than G cells, respectively. Q-PCR showed 9- and 4.5-fold greater ET(A) mRNA expression in MLL and AT-1 than G cells, respectively and no endothelin receptor B (ET(B)) expression. Combination therapy had significant effect on reduction of tumor volume than paclitaxel or atrasentan alone. CONCLUSIONS: ET(A) expression increases in aggressive prostate carcinoma. ET(A) blockade combined with paclitaxel may reduce tumor growth in advanced prostate carcinoma.     

There are multiple forms of glyceraldehyde-3-phosphate dehydrogenase in prostate cancer cells and normal prostate tissue

We analyzed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in normal and malignant human prostate tissues, normal rat prostate, and Dunning R-3327 rat prostate cancer cell lines. We detected multiple forms of GAPDH in Dunning cell lines by two-dimensional protein electrophoresis and Western analysis. Five forms of GAPDH that differed by isoelectric point were detected for each of the two metastatic Dunning cell lines, while four or fewer forms were detected for Dunning cell lines with low metastatic ability. We also detected multiple forms of GAPDH in normal and malignant human prostate specimens by two dimensional protein electrophoresis and immunohistochemical analysis. GAPDH was undetectable in normal human prostate secretory epithelium by immunohistochemistry, but was abundant in nuclei of normal basal cells and stromal cells. In human prostate cancer specimens, there was a rough correlation between cytoplasmic staining for GAPDH and tumor grade, but GAPDH staining was extremely heterogeneous. GAPDH was abundant in nuclei of some high-grade human prostate tumors. Both of the human prostate cancer bone metastases analyzed with immunohistochemistry had markedly elevated cytoplasmic GAPDH expression. We conclude that multiple forms of GAPDH may play diverse roles in normal prostate tissue and in prostate cancer. © 1996 Wiley-Liss, Inc.     

3144m／z蛋白峰在胃癌患者血清中的表达及其临床意义

目的探讨3144m／z蛋白峰在胃癌患者血清中的表达及其与胃癌临床病理特征和预后的关系。方法收集2006年2月至2008年10月间浙江省肿瘤医院收治的、具有完整随访资料的327例胃癌患者的临床资料。将所有患者术前血清标本利用表面加强激光解析电离飞行时间质谱（SELDI．TOF．MS）技术检测3144m／z蛋白峰的表达。结果3144m／z蛋白峰阳性率为33．9％（111／327）,明显高于CEA阳性率（21．1％,69／327）,差异有统计学意义（P〈0．01）;两者联合检测的阳性率为45．6％（149／327）。3144m／z蛋白峰表达与肿瘤临床分期（P〈0．01）、神经浸润（P〈0．01）、肿瘤大小（P〈0．01）、脉管瘤栓（P〈0．05）、淋巴结转移（P〈0．05）、CEA表达（P〈0．05）及浸润深度（P〈0．05）有关。3144m／z蛋白峰阳性与阴性表达患者术后3年生存率分别为44．7％和64．4％．差异有统计学意义（P〈0．01）;但经Cox比例风险模型进行多因素预后分析,3144m／z蛋白峰表达并未被证实为胃癌患者的独立预后因素（P=0．057）。结论3144m／z蛋白峰有可能成为胃癌患者的诊断及预后标记物。     

Effect of prostatic inhibin peptide (PIP) on prostate cancer cell growth in vitro and in vivo

Prostatic inhibin peptide (PIP), is a 94 amino acid protein which is secreted by the prostate gland in an androgen-independent manner. Previously, it has been demonstrated that PIP appears to inhibit follicle-stimulating-hormone (FSH) secretion by the pituitary and prostate glands. In vitro, the Dunning R3327 rat prostate cancer cell line MAT-LyLu (MLL) cells and the human prostate cancer cell line PC-3, are stimulated to grow in response to exogenous FSH and these effects are blocked by PIP. In vivo, PIP inhibits the growth of the highly metastatic MLL prostate cancer cell line. A comparison of hormone levels in control and PIP-treated rats demonstrates a significant inhibition of FSH in treated animals. It appears that, in vivo, PIP may inhibit prostate cancer growth by inhibiting FSH. PIP may represent a novel hormonal treatment for prostate cancer. © 1993 Wiley-Liss, Inc.     

Expression of ras proto-oncogenes in the Dunning R3327 rat prostatic adenocarcinoma system

Steady-state levels of c-Ha-ras mRNA were measured in eight sublines of the Dunning R3327 rat prostatic adenocarcinoma. As a control, normal dorsal prostate tissue was studied. Increased expression of c-Ha-ras is associated with tumor progression in one lineage of the Dunning R3327 system (H to AT1 to MAT-Lu and MAT-Ly-Lu). Here ras mRNA increases as the tumor advances from androgen dependence and a high degree of differentiation to an anaplastic aneuploid phenotype with high metastatic potential. However, in the other Dunning lineage (H to HI to HI-F to AT3), expression of c-Ha-ras is variable and does not correlate with tumor progression. Immunocytochemistry showed that levels of the c-Ha-ras p21 protein paralleled steady-state mRNA levels in variants. Transfection assays, using NIH/3T3 cells, suggested that the ras loci were not activated in the R3327 tumors. Levels of c-Ki-ras mRNA were also measured in the Dunning tumors; these did not correlate with tumor progression in either lineage. Expression of N-ras mRNA was not detected in the Dunning tumors.     

Vascular endothelial growth factor content in metastasizing and nonmetastasizing Dunning prostatic adenocarcinoma

BACKGROUND: Tumor angiogenesis is important in progressive tumor growth and metastasis. In the normal rat prostate and in androgen-sensitive prostate tumors androgen ablation causes an involution of the vasculature and a decrease in the vascular endothelial growth factor (VEGF) levels before regression of the prostate gland. To examine whether angiogenesis and metastasis are regulated by VEGF in androgen-insensitive and metastasizing prostate tumors, five Dunning rat prostate cancer sublines were tested; the androgen-sensitive, nonmetastasizing R3327 PAP, and the androgen-insensitive, low metastasizing AT-1, and the three androgen-insensitive, metastasizing AT-2, AT-3, and MatLyLu Dunning prostatic adenocarcinomas. METHODS: VEGF levels were quantified in the rat dorsolateral prostate and in the five Dunning sublines using competitive RT-PCR, Western blot, and Elisa. Vascular density was determined by factor VIII staining. RESULTS: VEGF mRNA was increased in all tumors compared with normal prostates. The two metastatic sublines AT-3 and MatLyLu and the nonmetastatic subline AT-1 showed the highest VEGF mRNA expression. VEGF protein levels in the prostate gland showed increased expression in the metastatic sublines, AT-2, AT-3, and MatLyLu, compared with the nonmetastatic AT-1 subline and the ventral prostate. VEGF proteins in serum were highest in the metastatic AT-3 subline. The vessel density was highest in the two highly metastatic sublines AT-3 and MatLyLu. CONCLUSIONS: Our results suggest that VEGF levels are associated with microvessel density and the previously established metastatic pattern of these rat prostate tumor systems.     

结直肠癌患者血清蛋白质谱检测的意义

目的 比较原发性结直肠癌与结直肠癌转移患者血清蛋白质谱,筛选与结直肠癌转移相关的蛋白标志物.方法 采用表面增强激光解析电离-飞行时间质谱技术测定219例结直肠癌患者和健康人血清标本.将标本随机分为训练组(无转移患者57例,转移患者63例,健康人42例)和盲法测试组(无转移患者26例,转移患者31例).分析比较训练组结直肠癌转移与无转移患者及健康人血清蛋白质谱差异,筛选与转移相关的蛋白标志物,并用单盲法验证筛选出差异标志物的灵敏度与特异性.结果 在平均分子质量为2000～30000范围内比较原发性结直肠癌患者与健康人血清蛋白质谱,发现31个蛋白质峰差异有统计学意义,按P值由小到大前5位的蛋白质峰分别为3240.7、9289.3、5334.4、4596.1和4792.4;比较结直肠癌转移患者与健康人血清,发现38个蛋白质峰差异有统计学意义,按P值由小到大前5位的蛋白质峰分别为3240.7、9289.3、9184.4、3191.5和9340.9;单独比较原发性结直肠癌与结直肠癌转移患者血清,筛选出两个蛋白质峰(9184.4和9340.9)差异有统计学意义,联合应用这两种蛋白可以将转移与无转移结直肠癌患者正确分组,正确分组率分别为93.6%和91.2%.经测试组盲法验证得出的灵敏度和特异性分别为90.3%和88.5%.结论 用表面增强激光解析电离-飞行时间质谱技术可以监测结直肠癌转移,血清蛋白质谱中的差异蛋白可能是转移相关蛋白.     

90K/Mac-2 binding protein is expressed in prostate cancer and induces promatrilysin expression

BACKGROUND: 90K/Mac-2 binding protein is a cell adhesive protein whose level of expression has been correlated with metastatic potential in many different tumor types. The purpose of this investigation was to examine 90K expression in prostate cancer and to determine a possible role for 90K in cancer progression. METHODS: 90K expression in prostate cell lines and tissue samples was evaluated by immunohistochemistry. Expression in cell lines was also evaluated by Western blot analysis and real-time RT-PCR. Induction of promatrilysin by 90K was evaluated by ELISA. RESULTS: Some of the human prostate cell lines studied expressed 90K. 90K was over-expressed in 38.8% of prostate cancer tumor samples, 7.14% of PIN lesions, and 18.6% of normal tissue. 90K was also shown to induce promatrilysin expression in the prostate cell line, LNCaP. CONCLUSIONS: These data demonstrate that 90K is over-expressed in a large fraction of malignant tumors. The fact that 90K can induce expression of promatrilysin indicates a possible role for 90K in cancer progression and metastasis. This suggests that 90K over-expression may be a useful marker for examining prostate cancer progression.     

Bone morphogenetic protein-6 expression in normal and malignant prostate

Summary Bone morphogenetic proteins (BMPs) have multiple biologic functions, including bone formation and embryonic induction. One of these proteins, BMP-6, was reportedly expressed at high levels in human prostate cancers that had also metastasized to bone. This study investigated both BMP-6 mRNA and protein expression in normal and malignant rat and human prostate tissues. BMP-6 was detected in both rat normal prostate and in Dunning rat-prostate adenocarcinoma sublines. The levels of BMP-6 mRNA and protein were similar for normal and malignant rat prostate, regardless of the metastatic potential. Moreover, castration had no apparent effect on BMP-6 production in rat normal ventral prostate, suggesting an androgen-independent gene regulation of this protein. BMP-6 mRNA and protein were also produced by normal and neoplastic human prostate cancer (radical prostatectomy specimens and human carcinoma cell lines DU145 and PC3). BMP-6 mRNA and protein expression, however, was higher in prostate cancer as compared with adjacent normal prostate, with higher-grade tumors (Gleason score of 6 or more) having greater BMP-6 immunostaining than the lower-grade tumors (Gleason score of 4 or less). Taken together, these results suggest that BMP-6 protein expression may serve as a potential marker for prostate cancer but not as a metastatic marker. Moreover, BMP-6 may contribute to prostate neoplastic behavior even in the absence of androgens.     

Dunning rat prostate tumors and cultured cell lines fail to express human prostate carcinoma-associated antigens.

The objective of this study was to determine if human prostate carcinoma-associated tumor markers were expressed by Dunning rat prostate carcinomas. Frozen and formalin-fixed paraffin-embedded tissues from 12 different sublines of Dunning tumors were evaluated for marker expression by immunoperoxidase staining by using a panel of 9 monoclonal antibodies, including antibodies against human PAP and PSA. None of the Dunning tumors were found to express any of the human prostate tumor markers. Both fixed and live immunofluorescent assays were performed on 5 cultured Dunning tumor cell lines, evaluated either as single cells or as monolayers. As with the Dunning tumor tissues, none of the cell cultures expressed any of the 9 human prostate tumor markers. The lack of antigen expression by the Dunning tumor tissues and cell lines suggests that these human prostate tumor markers are quite species specific. These results limit the use of the Dunning prostate tumors as models to explore the preclinical application of these human prostate carcinoma-associated monoclonal antibodies and their target antigens.     

Inhibition of prostate cancer growth by estramustine and colchicine

Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of estramustine and colchicine, two microtubule inhibitors, in hormone-refractory prostate cancer. In clinically achievable concentrations, the combination of estramustine and colchicine was cytotoxic to both the Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). Microtubule function was assessed in vitro to evaluate possible mechanisms of action. In motility and cell cycle analysis assays, estramustine and colchicine inhibited cellular motility but not cell cycle transit. In vivo, these two agents both inhibited the growth of implanted Dunning rat prostate adenocarcinoma MLL cells but did not appear to have additive effects. The use of oral colchicine in the treatment of hormone-refractory prostate cancer requires further investigation.     

Proteomic analysis of serum, outflow dialysate and adsorbed protein onto dialysis membranes (polysulfone and pmma) during hemodialysis treatment using SELDI-TOF-MS

AIMS: Alterations in the profiling of peptides and proteins in the serum, outflow dialysate and adsorbed protein on the dialysis membrane were investigated. METHODS: Alterations in the protein profiling of routine hemodialysis using polysulfone (TS-UL) and PMMA (moderate flux membrane of polymethylmethacrylate: BK-U) in 8 patients and that of adsorption onto polysulfone and PMMA membranes in 4 patients were evaluated by SELDI-TOF-MS and ProteinChip array. Mass-to-charge ratios (m/z) between 2,000 and 120,000 were analyzed. RESULTS: The protein with a relative intensity of m/z 11,730 measured by SELDI-TOF-MS was present in a small amount in the outflow dialysate and in a large amount in adsorption (identified as beta2-microglobulin) onto PMMA membrane. Unexpectedly, 68 molecular masses of peptides that were adsorbed more onto polysulfone than onto PMMA membrane were observed. There were more peptides less than m/z 11,730 adsorbed onto polysulfone membrane than onto PMMA membrane. Dominant peaks, m/z 6,629 and 6,431 adsorbed onto polysulfone membrane were identified as apolipoprotein CI and truncated apolipoprotein CI, respectively. 37 proteins with molecular weights larger than m/z 11,730 showed greater filtration through PMMA membrane than through polysulfone membrane. 149 molecular masses that were adsorbed onto PMMA or more onto PMMA membrane than onto polysulfone membrane were observed. CONCLUSION: This experiment suggests that membrane adsorption is an important mechanism for the removal of middle-molecular-weight proteins by hemodialysis using not only PMMA membrane but also polysulfone membrane. Adsorption of peptide or protein onto a dialysis membrane may depend not only on the membrane material, but also on the peptide or protein.     

Establishment and characterization of seven Dunning rat prostatic cancer cell lines and their use in developing methods for predicting metastatic abilities of prostatic cancers

In vitro cell lines were established from seven biologically distinct in vivo Dunning R3327 rat prostatic tumor sublines. Some of these in vitro cell lines (i.e., G, AT-1, AT-2) retain a low metastatic ability when inoculated back into syngeneic Copenhagen male rats, while others (i.e., AT-3, MAT-LyLu, MAT-Lu) retain a very high metastatic ability. A series of genetic (i.e., DNA content per cell, modal chromosomal number), as well as phenotypic parameters (i.e., morphology, 5 alpha-reductase, androgen receptor, estrogen receptor) were used to validate that the in vitro cell lines retained the major characteristics of the parental in vivo tumor sublines used for their respective establishment. A series of additional characteristics (i.e., morphology, growth rate, saturation density in surface culture, anchorage-dependent and -independent clonogenic potential) were compared between the high vs. the low metastatic in vitro cell lines to determine if a discriminatory parameter could be identified which reproducibly predicted the metastatic abilities of the particular prostatic cancer cell line. While the combination of the in vitro cell lines and their parental in vivo tumor subline will be a valuable tool for developing methods for predicting metastatic ability of prostate cancers, no single parameter yet measured is entirely successful in making this important distinction.     

High mobility group protein HMGI(Y) enhances tumor cell growth, invasion, and matrix metalloproteinase-2 expression in prostate cancer cells

BACKGROUND: The high mobility group protein HMGI(Y) has oncogenic properties and correlates with an aggressive phenotype in prostate cancer. The molecular mechanisms involved in transformation associated with HMGI(Y) overexpression remain unknown. METHODS: The HMG-I isoform was transfected and overexpressed in nonmetastatic Dunning prostate cancer cells (G cells) without detectable HMGI(Y). The assays of cell proliferation, tumor formation, in vitro invasion, and cDNA microarray were performed to assess the effect of HMGI(Y) overexpression in the transfected G cells. RESULTS: Overexpression of HMG-I in G cells significantly increases cell proliferation and tumor growth and also modestly enhances in vitro invasion compared to mock transfectant. cDNA microarray revealed that expression of the matrix metalloproteinase-2 (MMP-2) proform was increased eightfold in G cells overexpressing HMG-I. CONCLUSIONS: Overexpression of HMG-I in prostate cancer cells enhances cell growth, invasion, and expression of the proform of MMP-2, which may initiate early steps involved in the metastatic cascade.     

Vitamin D receptor-dependent antitumour effects of 1,25-dihydroxyvitamin D3 and two synthetic analogues in three in vivo models of prostate cancer

OBJECTIVE: To determine the in vitro and in vivo effects of 1,25-dihydroxyvitamin D3 (calcitriol) and two newer less hypercalcaemic analogues, EB1089 and CB1093 (as the use of calcitriol as a therapeutic agent in humans has been limited by hypercalcaemia) in three rodent models of prostate cancer. MATERIALS AND METHODS: The highly metastatic MAT LyLu Dunning prostate model, PAIII tumours in Lobund-Wistar rats and LNCaP xenografts in nude mice were used. Vitamin D receptor (VDR) expression and binding were assessed in all cell lines. The effects of calcitriol, EB1089 and CB1093 on tumour growth, cell cycle and angiogenesis in vitro, and growth and serum calcium levels in vivo, were assessed. RESULTS: The growth of prostate adenocarcinoma was inhibited by calcitriol, EB1089 and CB1093 in the Dunning prostate model. Although both analogues increased serum calcium levels, the levels were significantly less than in rats treated with calcitriol. Tumour growth was also inhibited in male athymic nu/nu mice with LNCaP tumour xenografts. PAIII cells failed to express functional VDR and were insensitive to calcitriol and its analogues, either in vitro or in vivo. The analogues of calcitriol did not inhibit angiogenesis in a rat aorta assay. CONCLUSION: This is the first report comparing the actions of calcitriol and its analogues in different in vivo models. The results suggest that the newer less hypercalcaemic analogues of calcitriol may offer a novel therapeutic option for treating prostate cancer. VDR-dependent growth inhibition and not the inhibition of angiogenesis is the main mechanism of action of these compounds in vivo.     

Tissue-type transglutaminase expression in the Dunning tumor

Summary Transglutaminases with different functions and tissue distribution patterns can be distinguished by specific antibodies and by inhibition of enzyme activity in the presence of guanosine triphosphate (GTP). The most common form is the so-called tissue-type transglutaminase that is apparently involved in membrane, stabilization processes, e.g. during apoptosis, and can be inhibited by incubation with GTP at low calcium concentrations. A secretory transglutaminase that cannot be inhibited by GTP is synthesized in an androgen-dependent manner in the dorsal prostate of the rat, the site suggested to represent the origin of the Dunning tumor used as an experimental model in prostate cancer research. Here we studied the expression of transglutaminases in different Dunning tumor lines — mainly in the highly differentiated H subline - and characterized the enzyme both biochemically and immunocytochemically. A very high enzyme activity was found only in the less well differentiated HI-F tumor line. Immunohistochemical reactions and Western blot analysis showed that there is no secretory transglutaminase present in any of the Dunning tumor lines studied. Transglutaminase activity of the Dunning tumor results from the so-called tissue-type enzyme that is nonorgan specific. The absence of a secretory form of transglutaminase does not suport the contention of a prostatic origin o the Dunning tumor.     

Inhibition of prostate cancer growth by vinblastine and tamoxifen

Hormone refractory prostate cancer continues to be an aggressive and fatal disease. Agents which inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of tamoxifen and vinblastine, two microtubule inhibitors, in hormone refractory prostate cancer. In clinically achievable concentrations, the combination of tamoxifen and vinblastine was cytotoxic to both Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). In cell cycle analysis assays, tamoxifen and vinblastine inhibited cell cycle transit. In vivo, these two agents inhibited the growth of implanted MLL cells. It appears that tamoxifen and vinblastine may be effective agents for the treatment of patients with hormone refractory prostate cancer.     

Vitamin d inhibition of prostate adenocarcinoma growth and metastasis in the dunning rat prostate model system

Objectives

Risk factors for prostate cancer (PCa)-related mortality include old age, black race, and residence in northern latitudes. The objectives of this study are to examine the in vitro and in vivo effects of 1, 25-dihydroxycholecalciferol (1,25-D₃) and less-hypercalcemic analogues on the Dunning rat prostate adenocarcinoma model.

Methods

To evaluate the effect of 1,25-D₃ on PCa in vitro, we used the highly metastatic Mat-lylu (MLL) and moderately metastatic R3327-AT-2 (AT-2) Dunning prostate cell lines, and examined effects on growth, clonogenicity, differentiation, and cell cycle. In vivo analysis included examination of the effects of these compounds on tumor growth and metastasis.

Results

Using both the 3-day MTT and 7-day clonogenic assay, 1,25-D₃ demonstrated a growth inhibitory effect with a concentration for 50% inhibition (IC₅₀) of approximately 20 μM for both MLL and AT-2. Cell cycle analysis of treated MLL cells (10 μM 1,25-D₃ for 48 hours) had 25% more cells in the G₀/G₁ phase than did control cells. To examine the in vivo effect of 1,25-D₃ and the less hypercalcemic vitamin D analogue, Ro25-6760 (or 6760), on MLL PCa growth and metastasis, tumors (5 × 10⁵ cells) were implanted subcutaneously into the flank of Copenhagen rats on the same day that treatment was initiated with 1,25-D₃ ( 1μg) or 6760 (1 or 5 μg); rats received treatment three times a week. After 3 weeks, 1,25-D₃ and 6760 (5 μg dosing) resulted in an inhibition of tumor volume and a reduction in the number and size of lung metastases.

Conclusions

These preclinical studies demonstrate the profound in vitro, or in vivo, or both antiproliferative and differentiating effects of 1,25-D₃ and 6760 on PCa and suggest that these drugs may have potential beneficial effects in the treatment of advanced PCa.     

柯萨奇腺病毒受体在人PCa细胞Du145和LNCaP中的差异表达及意义

目的:探讨柯萨奇腺病毒受体(CAR)在PCa转移潜能获得机制中的可能作用。方法:用TransweⅡ技术鉴定两种PCa细胞株(Du145和LNCaP)的不同转移潜能,进而应用Western印迹法鉴定这两种细胞株中的CAR表达差异情况,并初步分析其在PCa转移过程中所发挥的作用。结果:Du145的体外侵袭潜能显著高于LNCaP细胞(P<0.05);同时,CAR在低转移细胞株LNCaP中高表达,而在高转移细胞株Du145中表达缺失(P<0.01)。结论:CAR在转移潜能不同的PCa细胞株之间确实存在着有意义的差异表达,而这种表达差异可能在影响PCa转移过程中发挥重要作用。