Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4-9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P=0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in non-neoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (P<0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy.     

Identification of claudin-4 as a marker highly overexpressed in both primary and metastatic prostate cancer

In the quest for markers of expression and progression for prostate cancer (PCa), the majority of studies have focussed on molecular data exclusively from primary tumours. Although expression in metastases is inferred, a lack of correlation with secondary tumours potentially limits their applicability diagnostically and therapeutically. Molecular targets were identified by examining expression profiles of prostate cell lines using cDNA microarrays. Those genes identified were verified on PCa cell lines and tumour samples from both primary and secondary tumours using real-time RT-PCR, western blotting and immunohistochemistry. Claudin-4, coding for an integral membrane cell-junction protein, was the most significantly (P<0.00001) upregulated marker in both primary and metastatic tumour specimens compared with benign prostatic hyperplasia at both RNA and protein levels. In primary tumours, claudin-4 was more highly expressed in lower grade (Gleason 6) lesions than in higher grade (Gleason >or=7) cancers. Expression was prominent throughout metastases from a variety of secondary sites in fresh-frozen and formalin-fixed specimens from both androgen-intact and androgen-suppressed patients. As a result of its prominent expression in both primary and secondary PCas, together with its established role as a receptor for Clostridium perfringens enterotoxin, claudin-4 may be useful as a potential marker and therapeutic target for PCa metastases.     

Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate epithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour material when compared to non-malignant tissue (P < 0.05; Mann-Whitney U-test). Inhibin/activin betaA- and betaB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of betaB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While betaB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin alpha-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin betaB-subunit mRNA or by a decrease of ActRIB mRNA levels.     

Expression of bone morphogenetic proteins in human prostatic adenocarcinoma and benign prostatic hyperplasia.

There are important interactions between prostatic tumours and bone. This study was designed to examine whether prostatic tissue can express bone inductive factors, in particular, the Bone Morphogenetic Proteins (BMPs). The polymerase chain reaction (PCR) has been used to screen for the expression of BMPs one to six in the prostatic tissue of patients with benign prostatic hyperplasia (BPH), non-metastatic prostatic adenocarcinoma and metastatic prostatic adenocarcinoma. BMPs were expressed in both benign and malignant prostate tissue and in the prostate tumour cell lines, PC3 and DU145. BMPs were also expressed in ocular melanoma tissue, a tissue which rarely metastasizes to bone. BMP-6 expression was detected in the prostate tissue of over 50% of patients with clinically defined metastatic prostate adenocarcinoma, but was not detected in non-metastatic or benign prostate samples or in ocular melanoma tissue. These findings suggest that the BMPs may play a role in the osteoinductive activity of prostate metastases and that the pattern of expression of BMPs may be important in the pathogenesis of osteoblastic metastases associated with prostate adenocarcinoma.     

Androgen regulation of the human pseudoautosomal gene MIC2, a potential marker for prostate cancer

Using the differential display–polymerase chain reaction technique to identify androgen-responsive genes in the human prostatic tumor cell line LNCaP, we cloned an expression tag homologous to the human pseudoautosomal gene MIC2. The role of MIC2 in the prostate had not previously been studied. We used a series of cell lines derived from LNCaP that varied in their degree of differentiation and metastatic potential to assess the relationship between MIC2 expression and androgen responsiveness in prostate cancer. The expression of MIC2 mRNA and its product E2 was upregulated by androgen in a dose- and time-dependent manner in the parental LNCaP line and correlated with the expression of prostate-specific antigen. In the LNCaP sublines and an androgen-repressed invasive human prostate cancer cell line (ARCaP), MIC2 gene expression was not regulated by androgen and was associated with poorer differentiation, decreased androgen sensitivity, and higher metastatic potential. Immunohistochemical analyses indicated that E2 was expressed in tissues from patients with primary prostate cancer (16 of 20), in fetal prostatic tissues (low levels in all 10 fetal tissues assessed), and sporadically in benign prostatic hyperplasia tissues (one of four). The normal prostate tissues did not show positive E2 staining, with the exception of one central-zone section from one of the eight normal prostate samples assessed. These findings suggest that deregulation of expression of the human pseudoautosomal gene MIC2 occurred in the prostate. Mol. Carcinog. 23:13–19, 1998. © 1998 Wiley-Liss, Inc.     

The expression of a variant prostate-specific antigen in human prostate.

Although a splicing variant of prostate specific antigen (PSA-v) mRNA has been described previously, whether its protein (PSA-v or PSA-related protein 1, i.e., PSA-RP1) is actually expressed in human prostate cells remains elusive. We report that PSA-v protein is expressed in prostatic epithelia of both cancerous and benign tissues. Also, secreted PSA-v can be detected in the medium of a prostate cancer (PCa) cell line. Consistently, PSA-v mRNA is exclusively expressed in benign luminal epithelia and cancer cells of the prostate by in situ hybridization. Northern analysis of a cohort of 51 pairs of RNA samples from microdissected tissues showed that PSA-v mRNA levels remained constant in both benign and cancerous tissues, whereas PSA levels declined in cancerous areas. Our result suggests that it would be feasible to develop proper immunoassays for PSA-v to test whether PSA-v could have some clinical utility.     

Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas

Transferrin receptor (TFRC) is a membrane-bound protein expressed in larger amounts in proliferating, e.g., malignant, cells than in quiescent cells. The specific expression of TFRC can represent a diagnostic tool or a therapeutic target in solid tumours expressing this antigen. Whether TFRC is expressed in human pancreatic tumours is unknown. The aim of this study was the investigation of the expression of TFRC and transferrin in human pancreatic cancer and in neuroendocrine tumours of the pancreas. Fifty one specimens of human pancreatic cancer and 14 samples of pancreatic neuroendocrine tumours were obtained after surgery. The expression of TFRC, transferrin and cytokeratin was studied by standard immunohistochemistry. Flow cytometry was used for the investigation of TFRC expression in nine cell lines of ductal pancreatic cancer in vitro. In contrast to normal tissue, 93% of pancreatic tumour cells showed positive (82%) or heterogeneous (11%) expression of TFRC. It was strongly expressed by malignant epithelial cells; normal stromal and endothelial cells were not stained by anti-TFRC antibodies. Primary tumours and metastases showed a similar frequency of TFRC expression. Three neuroendocrine carcinomas showed positive expression of TFRC by malignant tumour cells. The expression of TFRC was negative in benign neuroendocrine tumours of the pancreas. The cell lines of pancreatic cancer were characterised by a low expression of TFRC in vitro. In contrast to normal pancreatic tissue and benign neuroendocrine tumours of the pancreas, pancreatic cancer and neuroendocrine carcinoma are therefore characterised frequently by high expression of TFRC. Hence, TFRC represents a marker of malignant transformation in the pancreas that could be applied as potential diagnostic and therapeutic target.     

Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines.

Both amplification and overexpression of c-erb B-2/neu have been associated with the progression and possible prognosis of a number of human cancers. In this study, we demonstrated that c-erb B-2/neu may also play an important role in human prostate cancer. Our conclusion is based on the following observations: (1) A monoclonal antibody raised against a peptide sequence from the C-terminal domain of the human c-erb B-2/neu gene product reacted positively with 68.7% (11 of 16) of the human prostatic cancer tissue extracts analyzed by western blot procedure. These results were supported by the immunohistochemical staining of the prostatic cancer specimens; 80% (12 of 15) showed positive staining, primarily around the plasma membranes of the prostatic cancer cells. c-erb B-2/neu oncoprotein was not detectable in normal prostate tissues (five examined by immunohistochemical staining and three by western blotting) or in human benign prostatic hyperplasia (two examined by immunohistochemical staining and six by western blotting) and was expressed less abundantly with lower intensity in "normal" human prostate tissues adjacent to cancerous prostate tissue (5 of 12 examined by immunohistochemical staining). We observed no evidence of c-erb B-2/neu gene amplification in 10 fresh human prostatic cancer specimens examined by Southern blotting and in the cultured human prostatic cancer cell lines PC-3, DU-145, and LNCaP. (2) The c-erb B-2/neu protein was detected in both androgen-receptor-positive (LNCaP) and -negative (PC-3 and DU-145) human prostate cancer cell lines. Positive immunostaining of c-erb B-2/neu protein was found to be associated predominantly with the plasma membranes of PC-3 cells, but was also found to be widespread in the cytoplasmic region of the LNCaP cells and in the perinuclear region of the DU-145 cells. (3) Like prostate-specific antigen (PSA) expression, c-erb B-2/neu mRNA expression was also positively regulated by androgen in androgen-receptor-positive LNCaP cells in vitro and LNCaP tumors in vivo. When LNCaP tumors were grown in castrated male hosts, levels of c-erb B-2/neu and PSA mRNA expression decreased initially, but rebounded at 3 wk to levels comparable to those expressed by tumors maintained in intact adult male hosts.     

Expression of dentin sialophosphoprotein in human prostate cancer and its correlation with tumor aggressiveness

Recent studies have demonstrated that two SIBLING family members, bone sialoprotein (BSP) and osteopontin (OPN), are overexpressed in human prostate cancer. The expression of these proteins is associated with the acquisition of a metastatic phenotype by cancer cells and a poor prognosis for the patient. Dentin sialophosphoprotein (DSPP) shares several structural and genetic features with OPN and BSP. The presence of DSPP has been recently established in salivary glands, indicating that its expression is not restricted to mineralized tissues. However, its potential expression in human tumors has not been addressed yet. In this study, we sought to evaluate the expression of DSPP in human prostate cancer. Immunohistochemistry was performed on 69 prostate cancer specimens using LFMb-21 anti-DSPP monoclonal antibody. All of the prostate cancer lesions examined expressed detectable levels of DSPP, as compared with no or low level of expression in adjacent normal glands (p < 0.0001). High grade prostatic intraepithelial neoplasia (HGPIN) glands generally displayed DSPP expression levels that were similar to those found in neighboring cancer glands. DSPP expression was significantly associated with the pathological stage (p = 0.0087) and the Gleason score (p = 0.0176) of the tumors. Western Blot was performed on 5 representative prostate tumor extracts and 3 prostatic tumor cell lines (PC3, LNCaP and DU145). All tumor extracts and cell lines analyzed have been found to express DSPP. In addition, in situ hybridization was used to assess the presence of DSPP mRNA. DSPP was detected at the RNA level in both HGPIN and tumoral glands. This study shows for the first time that DSPP is ectopically expressed in human prostate cancer. The expression of this SIBLING protein strongly correlates with conventional histopathological prognostic indicators of prostate cancer progression.     

蛇葡萄素通过Dermcidin蛋白调节肝癌细胞株HepG2侵袭活力及侵袭基因表达的实验

 目的 研究蛇葡萄素（AMP）对肝癌细胞株HepG2侵袭活力及侵袭基因表达的调节作用及分子机制。方法 培养肝癌细胞株HepG2后用不同剂量的AMP处理（20、40、60、80 μmol/L）、转染Dermcidin的siRNA,测定细胞的迁移能力、侵袭能力以及Dermcidin、侵袭基因mRNA表达量。结果 不同剂量AMP处理后细胞的迁移、侵袭数目及细胞中Dermcidin、基质金属蛋白酶（MMP）2、MMP9、MMP10 mRNA水平均低于对照组,且AMP剂量越大,细胞的迁移、侵袭数目及细胞中Dermcidin、MMP2、MMP9、MMP10 mRNA水平越低;80 μmol/L AMP联合dermcidin siRNA后,HepG2细胞的迁移、侵袭数目以及细胞内MMP2、MMP9、MMP10 mRNA表达水平均增多。结论 AMP通过下调Dermcidin蛋白来抑制肝癌细胞株HepG2的侵袭活力、降低侵袭基因的表达水平。     

Bone morphogenetic protein 6 in skeletal metastases from prostate cancer and other common human malignancies.

Prostatic adenocarcinoma commonly metastasizes to bone. Unlike most other bony secondaries, the majority of skeletal prostatic metastases are osteoblastic rather than osteolytic in nature. Several growth factors which are known to stimulate bone formation are expressed in benign and malignant prostate cells, but none has been specifically linked to osteosclerotic metastases. Bone morphogenetic proteins (BMPs) induce ectopic bone formation in vivo. We have reported previously that BMP-6 mRNA and protein are expressed in the majority of primary prostatic carcinomas with established skeletal metastases but rarely in clinically organ-confined tumours. This study examines the expression of BMP-6 mRNA in matched prostatic primary and secondary bony lesions and in isolated skeletal metastases from prostatic adenocarcinomas, as well as other common human malignancies, by in situ hybridization. BMP-6 mRNA was detected in 11 out of 13 bone metastases from prostate carcinoma and in three paired samples of primary prostate carcinoma and matching skeletal metastasis. Weak signals for BMP-6 were also present in 5 out of 17 skeletal deposits from non-prostatic malignancies. BMP-6 mRNA appears to be strongly expressed in prostatic adenocarcinomas, both in the primary tumour and in bone metastases. It is also expressed, though less frequently, in skeletal metastases from other human carcinomas. Our findings suggest that BMP-6 may hold potential as an attractive marker and possible mediator of skeletal metastases, particularly in prostate carcinoma.     

Reduced expression of the low affinity nerve growth factor receptor in benign and malignant human prostate tissue and loss of expression in four human metastatic prostate tumor cell lines.

In the human prostate, a low affinity (p75) nerve growth factor (NGF) receptor (NGF-R) localizes to the epithelia while a NGF-like protein localizes to the stroma. This NGF-like ligand, derived from prostate stromal cell cultures, has been shown to participate in paracrine mediated growth of a human tumor epithelial cell line (TSU-prl) in vitro. In order to investigate the role of the NGF-R in neoplastic growth we have examined the expression of the NGF-R in normal prostate tissues, benign prostatic hyperplasia tissues, adenocarcinoma tissues, and four metastatic tumor cell lines of the human prostate. In primary epithelial cell cultures of normal human prostate the p75 NGF-R was localized by immunocytochemistry to cytoplasmic vesicles. Furthermore, Western blot analysis of the NGF-R in subcellular fractions of normal prostate tissue identified an M(r) 75,000 immunoreactive protein in the microsomal fraction under nonreducing conditions of sodium dodecyl sulfatepolyacrylamide gel electrophoresis. However, microsomal preparations of five prostatic adenocarcinoma and five benign prostatic hyperplasia specimens showed varying immunoreactivity among samples, all of which expressed less of the p75 NGF-R than the normal tissue. Interestingly, microsomal preparations of the human prostatic epithelial cell lines, TSU-prl, DU-145, PC-3, and LNCaP did not show NGF-R expression by immunoblot analysis. Hence, expression of the p75 NGF-R in normal prostate tissue, partial loss of NGF-R expression in benign and malignant prostate tissue, and complete loss of NGF-R expression in the four metastatic tumor cell lines, suggests an inverse association of p75 NGF-R expression with the neoplastic progression of the human prostate.