Preferential adhesion of prostate cancer cells to bone is mediated by binding to bone marrow endothelial cells as compared to extracellular matrix components in vitro.

We have demonstrated previously that the preferential adhesion of prostate cancer cells to human bone marrow endothelial (HBME) cells may contribute to their preferential metastasis to bone. Although a subject of debate, it has been postulated that the endothelial cells of the bone marrow are fenestrated. It is unknown therefore whether prostate cancer cells adhere preferentially to the extracellular matrix (ECM) or the endothelial cells. It has also been demonstrated in other organ systems that the types of cell adhesion molecules on the surface of endothelial cells lining the organ microvasculature are determined, in part, by the ECM of the organ. We investigated how prostate cancer cell adhesion to HBME cells is affected by growing HBME cells on selected organ-derived ECM proteins in vitro. Growth of HBME cells and immortalized human aortic endothelial cells on bone, kidney, and placenta ECM proteins significantly increased their ability to bind PC-3 cells. This increased adhesion was not dose dependent and was not demonstrated with human dermal microvascular endothelial cells. Scanning electron microscopic analysis demonstrated that prostate cancer cells adhered directly to the endothelial cells and not to the underlying substrata. These results suggest that unidentified cell adhesion molecules are expressed or up-regulated on the apical surfaces of human aortic endothelial cells and HBME cells grown on bone, kidney, and placenta ECMs. These results also strongly demonstrate that the adhesion of prostate cancer cells to bone may be initiated by direct binding to endothelial cells rather than direct binding to exposed ECM components.     

Inhibition of prostate specific antigen expression by genistein in prostate cancer cells

Recent studies have provided convincing evidence for the role of soy-isoflavones, particularly genistein, in the inhibition of prostate cancer cell growth. Prostate specific antigen (PSA) is a biological marker used to detect and monitor the treatment of prostate cancer patients. Previous studies have documented that isoflavones can inhibit the secretion of PSA in the androgen-dependent prostate cancer cell line, LNCaP, however, the effects of genistein on androgen-independent PSA expression has not been explored. In this study, we have utilized a prostate cancer cell line, VeCaP, which expresses PSA in an androgen-independent manner, to determine the effects of genistein on cell proliferation and PSA expression. Here we show that genistein inhibits cell growth similarly in both the LNCaP and VeCaP cell lines, but has differential effects on PSA expression. We demonstrate using concentrations of genistein that have been detected in the serum of humans consuming a soy-rich diet, that genistein decreases PSA mRNA, protein expression and secretion. Conversely, only high concentrations of genistein inhibited PSA expression in VeCaP cells. Additionally, we have demonstrated that genistein inhibits cell proliferation independent of PSA signaling pathways, providing further evidence to support the role of genistein as a chemopreventive/therapeutic agent for prostate cancer irrespective of androgen responsiveness.     

Co-expression and impact of prostate specific membrane antigen and prostate specific antigen in prostatic pathologies

Background

The present study was undertaken to relate the co-expression of prostate-associated antigens, PSMA and PSA, with the degree of vascularization in normal and pathologic (hyperplasia and cancer) prostate tissues to elucidate their possible role in tumor progression.

Methods

The study was carried out in 6 normal, 44 benign prostatic hyperplastic and 39 cancerous human prostates. Immunohistochemical analysis were performed using the monoclonal antibody CD34 to determine the angiogenic activity, and the monoclonal antibodies 3E6 and ER-PR8 to assess PSMA and PSA expression, respectively.

Results

In our study we found that in normal prostate tissue, PSMA and PSA were equally expressed (3.7 ± 0.18 and 3.07 ± 0.11). A significant difference in their expression was see in hyperplastic and neoplastic prostates tissues (16.14 ± 0.17 and 30.72 ± 0.85, respectively) for PSMA and (34.39 ± 0.53 and 17.85 ± 1.21, respectively) for PSA. Study of prostate tumor profiles showed that the profile (PSA+, PSMA-) expression levels decreased between normal prostate, benign prostatic tissue and primary prostate cancer. In the other hand, the profile (PSA-, PSMA+) expression levels increased from normal to prostate tumor tissues. PSMA overexpression was associated with high intratumoral angiogenesis activity. By contrast, high PSA expression was associated with low angiogenesis activity.

Conclusion

These data suggest that these markers are regulated differentially and the difference in their expression showed a correlation with malignant transformation. With regard to the duality PSMA-PSA, this implies the significance of their investigation together in normal and pathologic prostate tissues.     

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy.To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.     

Expression of prostate specific membrane antigen and three alternatively spliced variants of PSMA in prostate cancer patients

Prostate specific membrane antigen (PSMA) is a folate gamma glutamyl carboxypeptidase that is oriented on the plasma membrane of normal and prostate cancer cells. A cytosolic version of PSMA, PSM', results from alternative splicing of the PSMA gene. Two additional alternatively spliced variants of PSMA, PSM-C and PSM-D, have been described recently. The ratio of PSMA to PSM' mRNA was higher in a small number of prostate cancer specimens compared to normal prostate cancer and benign prostatic hypertrophy (Su et al. Cancer Res 1995;55:1441). The intent of our study was to measure the gene expression of PSMA and the 3 PSMA splice variants in a large number of patient's tissues. A real-time, quantitative PCR assay was developed to quantify PSMA, PSM', PSM-C and PSM-D. Discrimination among the variants was achieved by designing unique primers and TaqMan probes for each gene. Amplification and detection was specific for the desired splice variant and was sensitive to one gene copy per reaction. The assay was used to quantify the gene expression in specimens of normal, benign, primary and metastatic prostate cancer from 72 patients. The mean PSMA expression (relative to 18S rRNA) was 2- to 3-fold lower in normal prostate (n = 4) compared to primary (n = 55, p = 0.31) and metastatic (n = 20, p = 0.33) prostate cancer. There was no difference in the PSMA expression between benign and cancerous prostate tissue from the same patients (n = 35). The ratio of PSMA to PSM' was lowest in the normal prostate and increased with increasing Gleason score (p < 0.001). The increased ratio in these tissues was a reflection of both increasing PSMA levels and decreasing PSM' mRNA. The expression of PSM-C did not differ in any of the tissue categories studied. The expression of PSM-D was similar in normal and primary prostate cancer but was 2-fold higher in lymph node (p < 0.005) and bone metastases (p < 0.05) compared to the primary tumors. Our results of the first detailed quantitative analysis of PSMA mRNA expression in patient's tissues demonstrate that PSMA and the 3 PSMA splice variants are expressed in normal, benign, cancerous and metastatic prostate cancer. We note increased PSMA expression in some malignant tissues, however, these increases are modest in magnitude. We also report that the expression of a novel splice variant, PSM-D, is elevated in prostate cancer metastases.     

Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen

The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA) termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4⁺ and CD8⁺ T lymphocyte functions can be synergistically targeted against tumor cells.     

Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen

The development of immunotherapy for cancer, such as synthetic peptide-based vaccines, relies heavily on the identification of appropriate epitopes capable of eliciting antitumor T-cell responses. We have used a combination of computer-based algorithms to predict peptide sequences from prostate-specific membrane antigen (PSMA) capable of stimulating in vitro CTLs restricted by the HLA-A2 MHC molecule. Four of the five peptides that were predicted by these algorithms were capable of inducing antigen-specific CTLs that killed target cells that were pulsed exogenously with the corresponding peptides. However, only one of the four peptides, PSMA(27), induced CTLs that were effective at recognizing prostate tumor cells expressing the HLA-A2 and PSMA molecules. These results underline the importance of demonstrating antitumor reactivity of peptide-induced CTLs for the selection of epitopes destined to become immunotherapeutic for prostate cancer.     

GRP78-targeted nanotherapy against castrate-resistant prostate cancer cells expressing membrane GRP78

Glucose-regulated protein 78, GRP78, is a chaperone protein mainly located in the endoplasmic reticulum (ER) of normal cells. In stress conditions, GRP78 is overexpressed and in different cancer cell types, it is expressed at the cell surface, whereas it stays intracellular in non-cancerous cells. Therefore, it appears as a strategic target to recognize malignant cells. Prostate cancer is one of the most diagnosed cancers in men. The development of castrate resistant tumors and the resistance to chemotherapy frequently occur. The carboxy-terminal ER retention domain is defined by the KDEL amino acid sequence. We developed anti-KDEL functionalized polymeric nanoparticles (NPs) loaded with paclitaxel (Tx) to specifically target prostate cancer cells expressing GRP78. The sensitivity to Tx in different formulations was compared in three prostate cell lines: PNT1B, a normal cell line, PC3, a cancer cell line faintly expressing GRP78 at its surface, and DU145, a cancer cell line expressing GRP78 at its cell surface. Our results show that the targeted formulation significantly increases Tx sensitivity of cell line expressing GRP78 at its surface compared to other treatments suggesting the added value of GRP78 targeted therapy for castrate resistant tumor which expresses GRP78 at its cell surface.     

Antibody-dependent cytotoxic activity on human cancer cells expressing UK 114 tumor membrane antigen

The monoclonal antibody (P-3 mAb) and the rabbit immune sera (RIS) recognizing a 14 kDa perchloric acid soluble protein extracted from goat liver (UK 114), locate this antigen on the cell membrane of several human cancer cell lines. UK 114-positive cells (e.g. HT 29 and KATO VI cells) undergo antibody-dependent cytolysis bl vitro. In nu/nu mice bearing xenografted HT 29 cells, tumor growth was markedly impaired by peri-tumoral injection of anti-UK 114 antibodies. These experiments suggest that human tumors expressing UK 114 over the cell membrane may undergo antibody-mediated cytolysis and growth control.     

CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 +ve) and DU145 (CD44 -ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 +ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.     

Testosterone and prostate specific antigen stimulate generation of reactive oxygen species in prostate cancer cells.

Prostate specific antigen, the clinical marker for prostate cancer, is a neutral serine protease whose function is to lyse seminal proteins. Recent work by our laboratory has suggested that prostate specific antigen stimulates the generation of reactive oxygen species in prostate cancer cells. Using 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals, we found that prostate specific antigen markedly stimulated reactive oxygen species generation in LNCaP cells. The effect was concentration dependent and its specificity was supported by the fact that anti-prostate specific antigen antibodies abolished the response. Since testosterone stimulates the production of prostate specific antigen, we considered that the reactive oxygen species response to testosterone may be linked to prostate specific antigen. We found that the testosterone effect on reactive oxygen species was blocked by flutamide and by anti-prostate specific antigen antibody. Additionally, though PC3 and DU145 could not respond to testosterone, they readily increased reactive oxygen species in response to prostate specific antigen. Focusing on the mechanism of the prostate specific antigen effect, we tested two other serine proteases, trypsin and chymotrypsin, but found no effect on reactive oxygen species in LNCaP cells. Nevertheless, serine protease inhibitors, alpha(1)-antichymotrypsin, alpha(2)-macroglobulin and Bowman-Birk inhibitor, blocked reactive oxygen species generation stimulated by prostate specific antigen. This apparent paradox was investigated with the use of a specific anti-'prostate specific antigen' antibody which recognizes an epitope away from the catalytic site and which does not inhibit protease activity. Despite the lack of inhibition of proteolytic activity, this antibody blocked the effect of prostate specific antigen on reactive oxygen species generation. These findings suggest that although the integrity of the prostate specific antigen molecule is necessary for stimulating reactive oxygen species generation, its proteolytic activity is not. The underlying mechanism is currently under investigation.     

Growth in semisolid agar of prostate cancer cells obtained from bone marrow aspirates

Thirty-one bone marrow aspirations were performed on patients with prostatic carcinoma metastatic to bone. After separation over a Ficoll-Hypaque gradient viable nucleated cells were cultured in semisolid agar. Colony formation occurred in 14 of 27 (52%) nonbacterially contaminated cultures. Characterization of cells from the colonies showed them to be consistent with malignant prostate cells. After staining, these cells were periodic acid-Schiff positive, prostatic acid phosphatase positive, and prostatic specific antigen positive. Other studies demonstrated the cells to be karyotypically abnormal, ultrastructurally similar to epithelial cells, and capable of secondary colony formation. Three bone marrow aspirate specimens did not have metastatic prostatic carcinoma detected by standard methods but did demonstrate colony formation. However, colony formation was most frequently seen when a radionuclide scan was positive at the aspiration site and when tumor cells were microscopically detectable by Wright staining of a smeared aspirate. The potential utility of colony forming cultures in prostate cancer is discussed. In working with bone marrow aspirates, additional cell separation procedures may be required to calculate and maximize plating efficiencies.     

Abscisic acid regulates dormancy of prostate cancer disseminated tumor cells in the bone marrow

Prostate cancer (PCa) commonly metastasizes to the bone where the cells frequently undergo dormancy. The escape of disseminated tumor cells from cellular dormancy is a major cause of recurrence in marrow. Abscisic acid (ABA), a phytohormone, is known to regulate dormancy of plant seeds and to regulate other stress responses in plants. Recently, ABA was found to be synthesized by mammals cells and has been linked to human disease. Yet the role of ABA in regulating tumor dormancy or reactivation is unknown. We found that ABA is produced by human marrow cells, and exogenous ABA inhibits PCa cell proliferation while increasing the expression of p27, p21, and p16 and decreasing the expression of the proliferation marker, Ki67. Further, ABA significantly increased the percentage of PCa cells in the G₀ phase of the cell cycle as well as the duration the cells were arrested in G₀. We found that ABA regulates an increase of PPARγ receptor expression and suppressed phosphorylation of mTOR/p70S6K signaling and resulting in the induction of the cellular dormancy. We then confirmed that ABA regulates G₀ cell cycle arrest through PPARγ receptor signaling in vitro and under co-culture conditions with osteoblasts. Finally, we demonstrate that ABA regulates PCa dormancy in vivo following intratibial injection in an animal model. Together these data suggest that the ABA and PPARγ signaling pathways contribute to the establishment of PCa cellular dormancy in the bone marrow microenvironment. These findings may suggest critical pathways for targeting metastatic disease.     

应用前列腺特异抗原筛查诊断前列腺癌的临床意义

Objective To evaluate the clinical significance of prostate-specific antigen(PSA)screening in early detection of prostate cancer in Chinese men.Methods PSA screening was performed in 8562 asymptomatic men who had been enrolled for health checkup and all were ≥50 years old.Prostate biopsy was recommended for those with a serum PSA level≥4.0 ng/ml.The pathological and clinical features of the patients with prostate cancer detected by the PSA screening were compared with that of 82 clinically diagnosed prostate cancer patients during the same period.Results Of the 8562 asymptomatic men,719 had PSA levels ≥4.0 ng/ml and biopsy was performed in 295 of them.Fifty-eight prostate cancers were detected.The biopsy rate was 41.0% and positive detection rate was 19.7%.The overall age distribution in the screening group and the clinical groups was not significantly different(P = 0.176).However,41.4%(24/58)of the patients in screening group were ＞75 years old,and significantly more than that in the clinical group(25.6%,P = 0.0491).The proportion of the patients with PSA levels ≥20 ng/ml in the screening group was significantly less than that in the patients of the clinical group(44.8% vs.75.6%,P = 0.0002).Whether in the patients whose age was ＞ 75 years old(P ＜ 0.05)or ≤75 years old (P = 0.0002),the patients in the screening group had significantly lower Gleason scores ＜ 7(60.3% vs.34.1%,P =0.002),more T1 or T2 tumor(87.9% vs.26.8%,P ＜0.0001)and more chance to receive radical prostatectomy(50.0% vs.18.3%,P ＜ 0.0001)than the patients in the clinical group did.However,the distributions of PSA levels at diagnosis and biopsy Gleason scores were not significantly different between the above mentioned two groups(P ＞ 0.05).Conclusion Prostate-specific antigen (PSA)screening is useful for early detection of prostate cancer in Chinese men aged ≥ 50 years.The patients detected by PSA screening usually show a lower PSA level,Gleason scores and early clinical stage disease,and have more chance for radical prostatectomy than the clinically diagnosed patients.     

Evaluation of free-to-total prostate specific antigen ratio in the diagnosis of prostate cancer

It'sreportedthatfreetototalprostatespecificantigenration(f/tPSA)canprovidemorebenefitthanthesingleuseofprostatespecificantigen(PSA)inthediagnosisofprostatecancer(PCa).WemeasuredserumPSAandfPSAlevelsin62casesofbenignprostatichyperplasia(BPH)and40casesofPCausingradioimmunoassay,withpatients'agerange59y-89y.RESULTSPSA,fPSAandf/tPSAareshowninTable1.BoththesetwogroupsshowslinearcorrelationbetweenPSAandfPSA,correlationcoefficientofBPHis0.55(P<0.01),ofPCais0.44(P<0.01).Twoslopesha…     

应用前列腺特异抗原筛查诊断前列腺癌的临床意义

目的 探讨应用前列腺特异抗原(PSA)筛查诊断前列腺癌的临床意义.方法 对年龄≥50岁的8562例男性体检者进行PSA筛查,对血清PSA≥4.0 ng/ml者建议进行经直肠前列腺系统活检,活检病理确诊为前列腺癌的患者人选筛查组,记录其临床病理特点,并与同时期临床诊治的82例前列腺癌患者(临床组)进行比较.结果 在8562例进行血清PSA筛查的男性中,有719例血清PSA水平≥4.0 ng/ml,其中295例接受经直肠前列腺系统活检,共检出前列腺癌58例,活检率和活检阳性率分别为41.0%和19.7%.虽然两组患者的年龄分布差异无统计学意义(P=0.176),但筛查组中有41.4%(24/58)的患者年龄＞75岁,明显高于临床组(25.6%,P=0.0491).筛查组中血清PSA水平≥20.0 ng/ml的患者所占的比例为44.8%,明显低于临床组(75.6%,P＜0.0001).筛查组中活检Gleason评分＜7分的患者所占的比例为60.3%,明显高于临床组(34.1%,P=0.0020).筛查组中临床分期为T1和T2期(局限期)患者所占的比例为87.9%,明显高于临床组(26.8%,P＜0.0001).筛查组中接受根治性前列腺切除术的患者所占的比例为50.0%,明显高于临床组(18.3%,P＜0.0001).在年龄≤75岁的患者中,筛查组患者诊断时的血清PSA水平、活检Gleason评分和临床分期均显著低于临床组(均P＜0.05);在年龄＞75岁的患者中,筛查组患者的临床分期也明显低于临床组(P=0.0002),但两组诊断时血清PSA水平和活检Gleason评分的差异并无统计学意义(均P＞0.05).结论 应用血清PSA在我国50岁以上男性中进行前列腺筛查是有效的.筛查出的前列腺癌患者在血清PSA水平、活检Gleason评分、临床分期以及根治性切除的机会等方面均较临床组有明显优势.