Yasuhide Kitagawa

Vascular Endothelial Growth Factor Contributes to the Prostate Cancer-Induced Osteoblast Differentiation Mediated by Bone Morphogenetic Protein

Human prostate cancer has a high predisposition to metastasize to bone, resulting in the formation of osteoblastic metastases. The mechanism through which prostate cancer cells promote osteoblastic lesions is undefined. Vascular endothelial growth factor (VEGF) has been implicated as a mediator of osteoblast activity. In the present study, we examined if prostate cancer cells promote osteoblastic activity through VEGF. We found that LNCaP and C4-2B prostate cancer cell lines and primary tumor and metastatic prostate cancer tissues from patients expressed VEGF. Bone morphogenetic proteins (BMPs), which are normally present in the bone environment, induced VEGF protein and mRNA expression in C4-2B cells. Furthermore, BMP-7 activated the VEGF promoter. Noggin, a BMP inhibitor, diminished VEGF protein expression and promoter activity in C4-2B cells. Conditioned media (CM) from C4-2B cells induced pro-osteoblastic activity (increased alkaline phosphatase, osteocalcin, and mineralization) in osteoblast cells. Both noggin alone and anti-VEGF antibody alone diminished C4-2B CM-induced pro-osteoblastic activity. Transfection of C4-2B cells with VEGF partially rescued the C4-2B CM-induced pro-osteoblastic activity from noggin inhibition. These observations indicate that BMPs promote osteosclerosis through VEGF in prostate cancer metastases. These results suggest a novel function for VEGF in skeletal metastases. Specifically, VEGF promotes osteoblastic lesion formation at prostate cancer bone metastatic sites.

Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of human telomerase and is a critical determinant of the enzymatic activity of telomerase. Expression of hTERT is known to be regulated mainly at the transcriptional level. In the present study, using transient expression assays, we identified a 400 bp silencer of the hTERT promoter between -776 and -378 upstream of the proximal core promoter. The inhibitory effects of this silencer were enhanced with cellular differentiation. A computer-assisted homology search identified multiple binding motifs for myeloid-specific zinc finger protein 2 (MZF-2) within this region. Mutation introduced in these sites resulted in significant activation of hTERT transcription. Gel shift assays demonstrated that MZF-2 proteins specifically bound to these sites. Overexpression of MZF-2 in cells led to down-regulation of hTERT transcription as well as telomerase activity. These findings suggest that the 400 bp region upstream of the hTERT core promoter that we identified functions as a negative regulatory region and that MZF-2 may be an effector of negative regulation of hTERT.

Vascular Endothelial Growth Factor Contributes to Prostate Cancer–Mediated Osteoblastic Activity

Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse pre-osteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium-induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B-induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer-induced osteoblastic activity in vivo.

Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system

Cytosine deaminase (CD) activates prodrug 5‐FC to 5‐FU and is used for suicide gene therapy (the CD/5‐FC system). E. coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that directly converts 5‐FU into 5‐fluorouridine monophosphate and improves the antitumoral effect of 5‐FU. This study demonstrates the effectiveness of transduction of the UPRT gene in addition to CD/5‐FC cancer suicide gene therapy.

Tranilast inhibits hormone refractory prostate cancer cell proliferation and suppresses transforming growth factor β1-associated osteoblastic changes

Tranilast is a therapeutic agent used in treatment of allergic diseases, although it has been reported to show anti‐tumor effects on some cancer cells. To elucidate the effects of tranilast on prostate cancer, we investigated the mechanisms of its anti‐tumor effect on prostate cancer.

Antitumor effect of reduction of 150-kDa oxygen-regulated protein expression on human prostate cancer cells

Background: The 150‐kDa oxygen‐regulated protein ORP150, a new member of the heat shock protein family that functions as a molecular chaperone in the endoplasmic reticulum, was found to increase in infiltrating cancer cells. Since enhancement of ORP150 expression and the presence of vascular endothelial growth factor (VEGF) in human prostate cancer glands were immunohistochemically demonstrated, we examined whether transduced antisense ORP150 cDNA can reduce angiogenicity and tumorigenicity through suppression of VEGF secretion.

EXPRESSION OF MESSENGER RNAS FOR MEMBRANE-TYPE 1, 2, AND 3 MATRIX METALLOPROTEINASES IN HUMAN RENAL CELL CARCINOMAS

PURPOSE Three different membrane-type matrix metalloproteinases (MT1, 2, and 3-MMP) that can activate proMMP-2 (progelatinase A) are thought to play an important role in invasion and metastasis by various human carcinomas. To further clarify this role, we examined mRNA expression of MT-MMPs in human renal cell carcinomas. MATERIALS AND METHODS mRNA was extracted from 25 clinical specimens of renal cell carcinoma and 23 specimens of normal renal parenchyma remote from the tumor. Reverse transcription polymerase chain reaction (RT-PCR) using specific primers was performed, and PCR products were hybridized to 32P-labeled internal probes and analyzed by a bioimage analyzer. RESULTS MT1, 2, and 3-MMP mRNA expression in carcinomas was significantly higher than in normal parenchyma. In terms of the pathologic stage, MT1-MMP mRNA expression in pT2 and pT3 tumors was significantly higher than those in pT1 tumors. Although the sample size was small, it was evident that MT3-MMP mRNA expression in clear cell subtype renal cell carcinomas was higher than in the group of tumors including the granular cell subtype. CONCLUSIONS These three MT-MMPs may play an important role in the pathogenesis of human renal cell carcinoma, and MT1-MMP in particular is important in invasion by carcinoma cells. It is interesting that the expression of MT3-MMP was higher in carcinomas, especially clear cell carcinoma, than in normal parenchyma, so that MT3-MMP may provide a clue an understanding of the molecular mechanism of carcinogenesis in human kidney.

Modified anatrophic partial nephrectomy with selective renal segmental artery clamping to preserve renal function: a preliminary report.

Objectives:  Partial nephrectomy is effective for preserving renal function, but temporary clamping of the renal artery for hemorrhagic control may impair renal function due to ischemia/reperfusion injury. Anatrophic partial nephrectomy (APNx) has been proposed to minimize renal ischemia/reperfusion injury by clamping only the feeding artery. We aimed to evaluate whether anatrophic partial nephrectomy (APNx) is useful in preserving renal function and to assess variations in renal artery anatomy to determine the feasibility of selective segmental artery clamping.

Free-to-total prostate-specific antigen (PSA) ratio contributes to an increased rate of prostate cancer detection in a Japanese population screened using a PSA level of 2.1–10.0 ng/ml as a criterion

BackgroundAlthough the free-to-total prostate-specific antigen (PSA) ratio (f/t ratio) is low in prostate cancer, its usefulness in mass screening remains unclear. We examined the clinical usefulness of the f/t ratio for screening in a Japanese population.MethodsSince 2000, we have performed PSA screening in Japanese men aged 55–69 years. From 2000 to 2002, patients with total PSA (tPSA) levels over 2.1 ng/ml were referred to the urological clinic for secondary screening, regardless of the f/t ratio. We analyzed both the tPSA level and the f/t ratio in prostate cancer patients, and since 2003, subjects with tPSA levels ranging from 2.1–10.0ng/ml and f/t ratios higher than 0.22 have not been referred for secondary screening. Here, we report the results of tPSA screening, comparing findings in the two periods (2000–2002 and 2003–2005).ResultsBetween 2000 and 2005, we performed the tPSA screening test in 27 730 men, and detected 214 cases of prostate cancer. Sixty patients (28.0%) showed tPSA levels between 2.1 and 4.0ng/ml. There were no differences in cancer detection rates between the two periods in the populations referred for an initial screening. However, the percentage of individuals referred for secondary screening decreased from 17.0% to 13.0% during the later period (P < 0.001). The cancer detection rate in all patients with biopsies rose from 13.5% in the earlier period to 22.7% in the later period for the group with tPSA between 2.1 and 10.0ng/ml (P < 0.001).ConclusionThe f/t ratio may be a useful additional screening parameter for patients showing tPSA levels between 2.1 and 10.0ng/ml on their initial screening examination.

Etiological role of human papillomavirus infection for inverted papilloma of the bladder

The status of human papillomavirus (HPV) infection in urothelial inverted papilloma was examined in the present study. Formalin‐fixed and paraffin‐embedded tissues from eight cases of inverted papilloma of the bladder were studied. The presence of HPV‐DNA was examined by modified GP5/6+PCR using archival tissue sections by microdissection. HPV genotype was determined with a Hybri‐Max HPV genotyping kit. Immunohistochemical analysis for p16‐INK4a, mcm7, HPV‐E4, and L1, and in situ hybridization for the HPV genome were performed. HPV was detected in seven of eight cases (87.5%) of inverted papilloma. Three cases were diagnosed as inverted papilloma with atypia, while the remaining five were typical cases. HPV‐18 was detected in two cases, including one inverted papilloma with atypia, and HPV‐16 was detected in four cases, including one inverted papilloma with atypia. Multiple HPV type infection was detected in one typical case and one atypical case. High‐risk HPV was present in all HPV‐positive cases. Cellular proteins, p16‐INK4a and mcm7, which are surrogate markers for HPV‐E7 expression, were detected in all HPV‐positive cases, and their levels were higher in inverted papilloma with atypia than in typical cases. In contrast, HPV‐E4 and L1, which are markers for HPV propagation, were observed in some parts of the typical inverted papilloma tissue. High‐risk HPV infection may be one of the causes of urothelial inverted papilloma, and inverted papilloma with atypia may have malignant potential. J. Med. Virol. 83:277–285, 2011.