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A 36-year-old male with right scrotal induration visited a local physician and ultrasonography showed a mass in the right testicle. He was referred to our hospital, where an additional ultrasonography examination revealed a 1×1-cm mass with clear borders, a heterogeneous interior, slight hyperintensity, and abundant blood flow in the upper part of the right testis. Contrast-enhanced computed tomography results indicated a massive lesion with an uneven contrast effect in the right testis and no evidence of metastasis, while magnetic resonance imaging showed the tumor with bleeding and internal heterogeneity. All tumor markers were negative. Under a diagnosis of primary germ cell tumor of the testis without metastasis, a high orchiectomy was performed. The pathological diagnosis was sertoli cell tumor. Histopathologically, the tumor was benign and no additional treatment was performed. Three years after the operation, the patient was well and without complications.

A 66-year-old woman who had been receiving medication for hypertension and hyperlipidemia was referred to our hospital for evaluation of a left adrenal tumor (12×8 mm) that was incidentally detected on computed tomography. Her 24-hour urinary catecholamine level was elevated, and metaiodobenzylguanidine (MIBG) scintigraphy revealed increased uptake in the area around the left adrenal gland, necessitating laparoscopic adrenalectomy for preoperative diagnosis of left adrenal pheochromocytoma. Intraoperatively, we detected a para-aortic tumor behind the adrenal gland, and this lesion was excised together with the adrenal gland. However, manipulation of the para-aortic tumor led to elevation in the blood pressure to 170 mmHg. Histopathological examination of the resected specimens revealed an adrenocortical adenoma and a para-aortic ganglioneuroma, consisting of ganglion cells, nerve fibers, and Schwann cells. The patient's blood pressure normalized immediately postoperatively, and MIBG scintigraphy revealed a negative result. Endocrine active ganglioneuromas are rare, and to our knowledge, currently only 8 cases (including ours) have been reported in the Japanese and English literature.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies, and an effective therapy is lacking for metastatic RCC. Anisomycin is known to inhibit protein synthesis and induce ribotoxic stress. The aim of this study was to explore whether anisomycin enhances the cytotoxic effects of mapatumumab, a human agonistic monoclonal antibody specific for death receptor 4 (DR4), in human RCC cells. We examined the cytotoxicity of anisomycin alone and in combination with mapatumumab in human RCC cell lines and primary RCC cell cultures. RCC cells treated with anisomycin showed cytotoxicity in a dose-dependent manner. Anisomyin in combination with mapatumumab showed a synergistic effect not only in two human RCC cell lines but also in five primary RCC cell cultures. The synergy between anisomycin and mapatumumab for cytotoxicity was also observed for apoptosis. Interestingly, anisomycin significantly increased DR4 expression at both the mRNA and the protein level. Furthermore, the combination-induced cytotoxicity was significantly suppressed by a human recombinant DR4:Fc chimeric protein. The combination of anisomycin and mapatumumab also enhanced the activity of caspases 8 and 3, the downstream molecules of death receptors. These findings indicate that anisomycin sensitizes RCC cells to DR4-mediated apoptosis through the induction of DR4, suggesting that combinational treatment with anisomycin and mapatumumab might represent a novel therapeutic strategy for the treatment of RCC.

Lysophosphatidic acid (LPA) may enhance diverse biologic activities in prostate cancer. This study was conducted to analyze expression levels of LPA-producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK), in prostate cancer with relevance to clinicopathological parameters. Real-time RT-PCR and western blotting were performed for ATX and AGK in non-neoplastic prostate cells (PrECs and PrSCs) and prostate cancer cell-lines (DU-145, PC-3, LNCaP, and AILNCaP). Immunohistochemical analyses were conducted in tissue specimens of 132 localized prostate cancer patients who underwent radical prostatectomy between 2001 and 2007 (median observation period, 22 months). Both enzymes were negatively expressed in PrECs and PrSCs at mRNA and protein levels. ATX expression was higher than AGK in AILNCaP, DU-145, and PC-3 cell-lines, while AGK was mainly expressed in LNCaP cells. Immunohistochemically, ATX and AGK expressions were negative in non-neoplastic epithelia, while both were weakly expressed in the majority of high-grade intra-epithelial neoplasia (HG-PIN). In cancer foci, ATX and AGK expressions were strong in 49% and 62%, weak in 40% and 32%, and negative in 11% and 6%, respectively. Expressions of both enzymes were significantly correlated with primary Gleason grade of cancer foci (P

Objectives Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies, and an effective therapy is lacking for metastatic RCC. Vitamin E (VE) has been intensively studied as a chemopreventive agent for various cancer types. Preclinical investigations have suggested that VE succinate (VES) is the most effective analog of VE in cancer cells; however, no study of VES in RCC has been done. We investigated the anticancer activity of VES against RCC. Methods Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell morphologic changes and cell viability were evaluated using phase-contrast microscopy and the trypan blue dye-exclusion test, respectively. Caspase activity was measured with a quantitative colorimetric assay. Results VES exerted dose- and time-dependent cytotoxicities against ACHN, a human RCC cell line, but VE and VE acetate did not. The cytotoxic effect was also observed in 2 other RCC cell lines, Caki-1 and Caki-2, and in primary RCC cells derived from 8 patients. Hoechst 33258 staining and DNA ladder analysis demonstrated that VES induced apoptosis in RCC cells. However, VES did not affect activation of caspase-3, -6, -8, or -9. Furthermore, inhibitors specific to caspase-8, -9, -6, and -3 did not block VES cytotoxicity and neither did the general caspase inhibitor VAD. Conclusions VES might induce apoptosis and cytotoxicity against RCC cells in a caspase-independent manner and has potential in vivo applications in the treatment of drug-and/or immunotherapy-resistant RCC.

OBJECTIVE To elucidate gene expression profiles of lysophosphatidic acid (LPA)-related molecules in cancer, pre-cancerous lesion, and benign hyperplasia of the prostate. MATERIALS AND METHODS Prostate tissue samples were surgically obtained from 10 patients with localized prostate cancer and seven patients with invasive bladder cancer. Cancer cells and the corresponding stromal cells from normal prostate, high grade intraepithelial neoplasia (HGPIN), benign hyperplastic glands were isolated by laser capture microdissection. mRNA levels of three LPA receptors, LPA1, LPA2, LPA3, two LPA-synthesizing enzymes, autotaxin (ATX), acylglycerol kinase (AGK), and a LPA-degradation enzyme, prostatic acid phosphatase (PAP), were quantitatively assessed. The expression levels of the same genes were also determined in three human prostate cancer cell lines LNCaP, PC-3, and DU-145. RESULTS LPA1 mRNA level was significantly decreased in HGPIN and cancer epithelia when compared to the benign glands. LPA3 mRNA level was elevated in cancer epithelia compared to benign glands. LPA3, AGK, and PAP were predominantly expressed in LNCaP cells while LPA1 and ATX gene expressions were found in PC-3 and Du-145 cells. In BPH, AGK was abundantly expressed in the stroma while PAP was predominant in epithelial cells. CONCLUSIONS By acting via LPA3, LPA may play an important role in the development of prostate cancer. Switching of LPA receptor expression from LPA3 to LPA1, may be involved in prostate cancer progression and/or androgen independence. LPA may also play a key role in the development of benign prostatic hyperplasia. Prostate 69:283–292, 2009. © 2008 Wiley-Liss, Inc.

There is accumulating evidence suggesting that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2 is a promising molecular target for cancer therapy. Therefore, we investigated the effect of chemotherapeutic agents on TRAIL-R2-mediated apoptosis and cytotoxicity in various human solid cancer cells. Treatment of the ACHN human renal cell carcinoma (RCC) cell line with agonistic TRAIL-R2 antibody (lexatumumab) in combination with 5-fluorouracil, vinblastine, paclitaxel, or docetaxel did not overcome resistance to these agents. However, treatment with lexatumumab in combination with doxorubicin had a synergistic cytotoxicity. Synergy was also achieved in two other human RCC cell lines, Caki-1 and Caki-2, and in eight primary RCC cell cultures. Sequential treatment with doxorubicin followed by lexatumumab induced significantly more cytotoxicity than reverse treatment or simultaneous treatment. Low concentrations of doxorubicin (0.1 and 1 microg/mL) significantly increased TRAIL-R2 expression at both the mRNA and protein levels. Furthermore, the combination of doxorubicin and lexatumumab significantly enhanced caspase 8 activity, Bid cleavage, Bcl-xL decrease, release of cytochrome c, and caspase 9 and caspase 3 activity, and induced synergistic apoptosis. The activation of caspases and apoptosis induced with lexatumumab and doxorubicin was blocked by the human recombinant DR5:Fc chimeric protein. In addition, synergistic cytotoxicity was also observed in human prostate, bladder, and lung cancer cells, but was inhibited by the DR5:Fc chimeric protein. These findings suggest that doxorubicin sensitizes solid cancer cells to TRAIL-R2-mediated apoptosis by inducing TRAIL-R2 expression, and that the combination treatment with lexatumumab and doxorubicin might be a promising targeted therapy for cancers, including RCC, prostate, bladder, and lung cancers.

OBJECTIVES Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. In this study, we investigated the susceptibility of human prostate cancer and bladder cancer cells to HGS-ETR2, a human monoclonal agonistic antibody specific for TRAIL-R2. METHODS The cell surface expression of TRAIL-R1 and TRAIL-R2 on prostate cancer and bladder cancer cells was determined using flow cytometry. Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and caspase activities were measured by a quantitative colorimetric assay. RESULTS HGS-ETR2 effectively induced apoptotic cell death in DU145, PC3, and LNCaP human prostate cancer cells and J82 and T24 human bladder cancer cells. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell surface expression of the two TRAIL receptors, in that TRAIL-R2, but not TRAIL-R1, was frequently expressed in the prostate cancer and bladder cancer cells. HGS-ETR2 significantly activated the caspase cascade, including caspase-3, -6, -8, and -9, which were the downstream molecules of the death receptors in prostate cancer cells. Caspase-3, -6, and -9 were also significantly activated with HGS-ETR2-induced apoptosis in the bladder cancer cells. CONCLUSIONS These findings suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent against prostate cancer and bladder cancer.

Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ERα mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ERα in MCF-7 cells, selenium increased ERβ mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ERα and ERβ in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.

Background : Lung resistance-related protein ( LRP ), like multidrug resistance gene 1 ( MDR1 ) and multidrug resistance-associated proteins ( MRP ), has been associated with intrinsic therapeutic resis- tance in various malignancies. To date, there has been no study on the expression of LRP in urothelial carcinomas of the renal pelvis and ureter. We investigated the protein and mRNA expression levels of LRP , MDR1 and MRP1 in this malignancy and the clinical significance of their expression was evaluated. Methods : Forty urothelial carcinomas of the renal pelvis and ureter and 31 normal upper urothelial samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction to determine the protein and mRNA levels of the multidrug resistance-related genes, respectively. Results : The positive staining rates and mRNA levels of LRP were the highest among these multi- drug resistance-related genes in both normal urothelium and carcinoma examinations. In contrast to the up-regulated expression of MDR1 , the expression of LRP tended to be down-regulated in carcinomas. Moreover, the expression of LRP inversely correlated with tumor grades, but this correlation was not found for the other two genes. However, there was no correlation among the expression of the three genes observed. Conclusion : Lung resistance-related protein was strongly expressed in urothelial carcinomas of the renal pelvis and ureter, particularly in well-differentiated carcinomas.

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the tumor necrosis factor family. The present study investigated whether anthracyclines enhance TRAIL-induced apoptosis and cytotoxicity in renal cell carcinoma (RCC) cells. Methods: Cytotoxicity was measured using the microtiter assay. Apoptosis was monitored using DNA ladder analysis. Caspase activity was determined using a quantitative colorimetric assay. Results: Treatment of ACHN and Caki-1 human RCC lines with TRAIL, in combination with subtoxic concentrations of epirubicin (EPI) or pirarubicin (THP), enhanced induction of apoptosis and cytotoxicity. Sequential treatment with EPI followed by TRAIL induced significantly more cytotoxicity than the inverse treatment. The combined cytotoxicity of TRAIL and EPI was significantly inhibited by the TRAIL-neutralizing fusion protein DR5:Fc, although EPI did not affect the mRNA expression of DR4, DR5, DcR1 or DcR2. The combination treatment with TRAIL and EPI activated caspase-6 and -3, which were downstream molecules of the death receptor. Furthermore, the combined cytotoxicity of TRAIL and EPI was almost completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-DMQD-CHO. Conclusion: These findings indicate that anthracyclines sensitize RCC cells to TRAIL-induced apoptosis and cytotoxicity through activation of caspases, suggesting that TRAIL, in combination with anthracyclines, has a therapeutic potential in the treatment of RCC.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies in humans. We show that adriamycin (ADR) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L have a synergistic cytotoxic effect against RCC cells. This synergistic cytotoxicity was obtained in ACHN, A704, Caki-1 and Caki-2 human RCC cell lines and freshly derived RCC cells from 6 patients. This synergistic effect, however, was not achieved in 5 samples of freshly isolated normal kidney cells. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of TRAIL/Apo2L and ADR was realized by inducing apoptosis. Sequential treatment with ADR followed by TRAIL/Apo2L induced significantly more cytotoxicity than the reverse treatment. ADR increased the expression of DR4 and DR5 in RCC cells, but not in the normal kidney cells. Furthermore, the synergistic cytotoxicity was significantly inhibited by DR4:Fc and DR5:Fc fusion proteins, which inhibit TRAIL/Apo2L-mediated apoptosis. In addition, caspase activity assays and treatment of caspase inhibitors demonstrated that the combination treatment with ADR and TRAIL/Apo2L activated caspase cascade, including caspase-9, -8, -6 and -3, which were the downstream molecules of death receptors. These findings indicate that ADR sensitizes RCC cells to TRAIL/Apo2L-mediated apoptosis through induction of DR4 and DR5, suggesting that the combination therapy of TRAIL/Apo2L and ADR might be effective for RCC therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells by engaging the death receptors 4 (DR4) and 5 (DR5). We investigated the effect of chemotherapeutic drugs on DR4-mediated apoptosis in human bladder cancer cells, using a human monoclonal agonistic antibody specific for DR4, mapatumumab. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Treatment of human bladder cancer T24 cells with mapatumumab in combination with mitomycin C, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with mapatumumab in combination with epirubicin (EPI) had a synergistic cytotoxic effect. Synergy was also obtained in KU7 and RT112 human bladder cancer cells. A synergistic effect was also observed with mapatumumab in combination with pirarubicin. The synergy obtained in cytotoxicity with mapatumumab and EPI was also achieved in apoptosis. EPI markedly increased DR4 expression in the bladder cancer cells at both the mRNA and protein levels. Furthermore, the combination-induced cytotoxicity was significantly suppressed by the DR4:Fc chimeric protein. The combination of EPI and mapatumumab significantly activated the caspase cascade, including caspase-8, -9 and -3, which are the downstream molecules of death receptors. These findings indicate that EPI sensitizes bladder cancer cells to DR4-mediated apoptosis through induction of DR4 and activation of caspases, suggesting that the combination therapy of EPI and mapatumumab may be effective for bladder cancer therapy.

We previously reported that detection of telomerase activity in urinary exfoliated cells obtained from urothelial cancer patients by telomeric repeat amplification protocol (TRAP) assay is a more sensitive method of diagnosis than conventional urine cytologic examination, particularly in patients with grade 1 tumors. To establish this method as a noninvasive screening test for the diagnosis of urothelial cancers, we performed the semi-quantitative analysis of telomerase activity using Telomerase PCR ELISA™. Spontaneous voided urine samples were obtained from 65 urothelial and 58 non-urothelial cancer patients. When the mean + 2 standard deviation of telomerase activity in urine sediments of non-urothelial cancer patients was arbitrarily determined as a cut-off, the sensitivity of TRAP enzyme-linked immunosorbent assay (ELISA) and the conventional cytology were 57% and 35%, respectively. Detection rate was significantly higher in semi-quantitative TRAP assay than in conventional cytologic examination in grade 1 cancer patients (52% vs. 5%, p = 0.00195). False positives were detected in 5% of non-urothelial cancer patients without pyuria and in 11% of non-urothelial cancer patients with pyuria (p = 0.395). Telomerase activity was enhanced in some cases after phenol extraction or extracting epithelial cells by using Dynabeads of macroscopic hematuria and pyuria, indicating that hematuria and pyuria might contribute to false negatives. In conclusion, the TRAP-ELISA method is superior to the standard TRAP assay in quantitativeness and simplicity of the experimental procedure. Detection of telomerase activity in urine sediments is particularly useful for the diagnosis of low-grade tumors. However, telomerase activity in patients with high grade tumors often might be underestimated due to the excessive amount of exfoliated epithelia with necrotic tissues, hematuria, and pyuria.

Lexatumumab, a human agonistic monoclonal antibody against tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor-2 (TRAIL-R2), is a promising molecular-targeted therapeutic agent. Our past study indicated that low concentrations of doxorubicin sensitized renal cell carcinoma (RCC) cells to lexatumumab-mediated apoptosis. The present study was designed to examine the cellular and molecular effects of lexatumumab and anthracyclines in RCC cells. The treatment of human RCC cells with lexatumumab in combination with anthracyclines, epirubicin, and pirarubicin had a synergistic cytotoxicity. A marked synergistic apoptosis was induced by lexatumumab in combination with epirubicin or pirarubicin. Epirubicin and pirarubicin significantly increased the TRAIL-R2 expression at both the mRNA and the protein levels. The combination-induced cytotoxicity was significantly suppressed by the human recombinant DR5:Fc chimeric protein. To further explore the molecular mechanisms in this synergistic cytotoxicity with lexatumumab and anthracyclines, the changes in 84 apoptosis-related genes were evaluated by a quantitative polymerase chain reaction (PCR) array. Among these genes, 18 (CD40LG, FASLG, LTA, TNSF7, FAS, BAG3, BAK1, BAX, BID, BIK, BCL10, caspase-1, caspase-5, caspase-6, caspase-10, TNF receptor-associated factor 1, PYCARD, and CIDEA) were significantly upregulated and eight (TNF receptor-associated factor 4, TNFRSF11B, TNF, BCL2, BCL2L1, BNIP3L, caspase-9, and DAPK1) were downregulated at mRNA levels in RCC cells cotreated with lexatumumab and epirubicin. Furthermore, the upregulation of mRNA levels of PYCARD and CIDEA was confirmed using real-time reverse transcriptase-PCR analysis. The present study demonstrates that anthracylines sensitize RCC cells to lexatumumab-mediated apoptosis by inducing TRAIL-R2 expression, and the utility of PCR array to elucidate the mechanism of synergistic apoptosis.

Purpose: This study was designed to evaluate the apoptotic effect of mapatumumab or lexatumumab, human agonistic antibodies that target the tumor necrosis factor–related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2), in combination with chemotherapeutic agents, against human solid cancer cells. Experimental Design: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Results: Treatment of ACHN human renal cell carcinoma cells with cisplatin combined with mapatumumab did not overcome resistance to these agents. However, treatment with cisplatin in combination with lexatumumab had a synergistic cytotoxicity. Synergy was also achieved in six primary renal cell carcinoma cell cultures. Lexatumumab and cisplatin also synergistically enhanced apoptosis. Pretreatment with cisplatin followed by lexatumumab resulted in high cytotoxicity compared with the reverse sequence. Cisplatin significantly increased TRAIL-R2 expression at both the mRNA and the protein levels. Furthermore, the combination of lexatumumab and cisplatin significantly enhanced caspase-8 activity, Bid cleavage, up-regulation of Bax, cytochrome c release, and caspase-9, caspase-6, and caspase-3 activities. Importantly, the activation of caspase-8 was significantly abrogated by the specific inhibitors of caspase-9, caspase-6, and caspase-3. Furthermore, combination-induced cytotoxicity was significantly suppressed by the DR5:Fc chimeric protein and the specific inhibitors of caspase-8, caspase-9, caspase-6, and caspase-3. A similar effect was observed in prostate cancer, bladder cancer, lung cancer, and cervical cancer cells. Conclusions: Cisplatin sensitizes solid cancer cells to lexatumumab-induced apoptosis by potentiation of the extrinsic and intrinsic apoptotic pathways that lead to amplification of caspase activation, particularly caspase-8, suggesting the combination treatment of solid cancers with cisplatin and lexatumumab might overcome their resistance.

OBJECTIVE To investigate the expression of thymidylate synthase (TS), a key enzyme in DNA synthesis that is over-expressed in several cancer cells, in bladder cancer and its association with patient prognosis and the response to adjuvant therapy. PATIENTS AND METHODS In all, 67 bladder tissue specimens were obtained from patients who had undergone transurethral resection (TUR). TS expression in bladder cancer and normal bladder tissue was analysed by immunohistochemistry. RESULTS Of the 67 bladder tissue specimens, 47 (70%) and 10 (15%) had positive expression for TS in cancer and normal tissues, respectively. TS expression was greater in patients with Grade 3 (16/17, 94%) than in Grade 1 and 2 (31/50, 64%; P = 0.002). It was also greater in Stage T1 (14/14) than in Stage Ta (33/53, 62%; P = 0.001). Furthermore, patients with negative TS expression had a longer postoperative recurrence-free survival (RFS) than those with positive expression during the 5 year follow-up (P = 0.028). In the patients with positive TS-expressing tumours, adjuvant therapy significantly improved RFS (P

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P

Interstitial cystitis remains a poorly understood urological condition characterized by chronic pelvic pain and increased urinary frequency in the absence of any known etiology. Urothelial dysfunction and other abnormalities are presumed to be involved in the disease. Uroplakins that are expressed by urothelial cells are thought to have an important role as major barrier proteins on the apical surface of the urothelium.Gene expression of uroplakin Ia, Ib, II, III and III-delta4 was quantitatively measured in bladder biopsy samples from 29 patients with interstitial cystitis and 16 control subjects using real-time reverse transcriptase-polymerase chain reaction.The mRNA levels of the uroplakin Ia, Ib and II genes were relatively low and uroplakin III was relatively high in interstitial cystitis bladders compared to normal controls, although not significantly. Uroplakin III-delta4, a splicing variant of uroplakin III, was significantly up-regulated in interstitial cystitis samples (p0.001). When patients with interstitial cystitis were divided into those with and without ulcerative changes, the uroplakin III and III-delta4 genes were significantly up-regulated only in patients with nonulcerative interstitial cystitis. Even more interesting was the finding that up-regulation of uroplakin III-delta4 was much more prominent than that of uroplakin III, that is 26.5 vs 5.6-fold compared to the median values of normal subjects.Although the clinical implications of the over expression of uroplakin III and III-delta4 in nonulcerative interstitial cystitis bladders remains to be clarified, from the diagnostic viewpoint uroplakin III-delta4 is a potential marker for identifying nonulcerative interstitial cystitis.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells by engaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediated apoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific for death receptor 4.Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis.Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined with doxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line, Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1 combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity with HGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1 induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p0.05). Doxorubicin remarkably increased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin and HGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream molecules of death receptors.These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediated apoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy of doxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a recently identified member of the TNF ligand family that selectively induces apoptosis in tumor cells in vitro and in vivo but not in most normal cells. Chemotherapeutic drugs induce apoptosis and the upregulation of death receptors or activation of intracellular signaling pathways of TRAIL. Numerous chemotherapeutic drugs have been shown to sensitize tumor cells to TRAIL-mediated apoptosis. Studies from our laboratory have also shown that TRAIL-resistant renal cell carcinoma, prostate gland cancer, and bladder cancer cells are sensitized by subtoxic concentrations of chemotherapeutic drugs including doxorubicin, epirubicin, pirarubicin, and cisplatin. TRAIL, particularly in combination with chemotherapeutic agents, is thus potentially promising in the treatment of cancer. This review addresses the putative role of TRAIL in cancer treatment and discusses the molecular basis of the synergistic effect of TRAIL and chemotherapeutic drugs.

BACKGROUND Macrophage inhibitory cytokine-1 (MIC-1) is a member of transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily. Despite its potential role in prostatic regulation, little is known about its biological activity. METHODS Expression profiling using 42K Affymetrix HuGeneFL® array was conducted to compare symptomatic benign prostatatic hyperplasia (BPH), histological BPH without symptoms, and normal prostate samples from donors. MIC-1 gene expression was analyzed by RT-PCR in pure culture of prostate epithelial and stromal cells, and prostate cancer cells, LNCaP, PC-3, DU-145. Influence of androgens on MIC-1 expression in LNCaP cells was analyzed by Northern blot. Enhancement of promoter activity of MIC-1 by androgens was examined using reporter assays. RESULTS In contrast to normal prostates, MIC-1 gene was down-regulated in BPH samples with symptoms and histological BPH obtained from cystoprostatectomy specimens (P

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a recently identified member of the TNF ligand family that selectively induces apoptosis in tumor cells in vitro and in vivo but not in most normal cells. Chemotherapeutic drugs induce apoptosis and the upregulation of death receptors or activation of intracellular signaling pathways of TRAIL. Numerous chemotherapeutic drugs have been shown to sensitize tumor cells to TRAIL-mediated apoptosis. Studies from our laboratory have also shown that TRAIL-resistant renal cell carcinoma, prostate gland cancer, and bladder cancer cells are sensitized by subtoxic concentrations of chemotherapeutic drugs including doxorubicin, epirubicin, pirarubicin, and cisplatin. TRAIL, particularly in combination with chemotherapeutic agents, is thus potentially promising in the treatment of cancer. This review addresses the putative role of TRAIL in cancer treatment and discusses the molecular basis of the synergistic effect of TRAIL and chemotherapeutic drugs.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells. The anthracycline doxorubicin (DOX) can sensitize several types of cancer cells to TRAIL-mediated apoptosis. Here we report that DOX enhances TRAIL-induced apoptosis and cytotoxicity against prostate cancer cells. Cytotoxicity was determined by a MTT assay. Synergistic effect was assessed by isobolographic analysis. Caspase activity was determined by a quantitative colorimetric assay. The combination treatment with DOX and TRAIL resulted in a synergistic cytotoxic effect on LNCaP, LNCaP-Bcl-2, PC-3, and PC93 human prostate cancer cell lines, but not on normal human prostatic stromal cells. Synergistic cytotoxicity was also obtained even when the exposure time was shortened from 24 to 8 or 2 h. A similar effect was achieved with TRAIL in combination with epirubicin, pirarubicin, or amrubicin. The synergy obtained in cytotoxicity with TRAIL and DOX was also achieved in apoptosis. DOX treatment significantly activated caspase-8, -6, and -3 in LNCaP cells. Furthermore, the synergistic cytotoxicity of TRAIL and DOX was completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-IETD-CHO, Ac-DQTD-CHO, or Ac-DMQD-CHO. These findings indicate that DOX enhances TRAIL-induced apoptosis and cytotoxicity in prostate cancer by activation of caspase cascades, and suggest that TRAIL in combination with DOX have a therapeutic potential in the treatment of prostate cancer.

The caspase family of proteases is speculated to have a crucial role in apoptosis. The effect of treatment with adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), vinblastine (VLB), IFN-alpha, or IFN-gamma on the activation of caspase-3, -6, -8, and -9 in renal cell carcinoma (RCC) cells was investigated, to clarify the mechanisms of chemo- and immunotherapeutic agent-mediated apoptosis. Caspase activity was determined by a quantitative colorimetric assay. Apoptosis was monitored by acridine-orange staining assay. Treatment of ACHN cells with CDDP, VLB, IFN-alpha, or IFN-gamma did not activate caspase-3, but its activity was increased 7.2-fold (p = 0.0001) with ADR and 2.8-fold (p = 0.0385) with 5-FU in comparison with control. Furthermore, when the ADR treatment time was shortened from 24 to 8 or 2 h, the same caspase-3 activation occurred. Activation of caspase-3 was also observed in six freshly isolated human RCC cells after the treatment with ADR. Of the six freshly derived RCC cells treated with 5-FU, caspase-3 activity was increased 3.1-fold (p = 0.0051) and 2.4-fold (p = 0.0346) in two of them, respectively. Epirubicin and pirarubicin, compounds closely related to ADR, also respectively enhanced 4.2-fold (p = 0.0052) and 2.8-fold (p = 0.0147) caspase-3 activity in ACHN cells. The activation of caspase-3 observed with a colorimetric assay was confirmed with immunocytochemical analysis using the anti-active caspase-3 mAb, which specifically recognizes the active form of caspase-3. Furthermore, both active caspase-3 and apoptosis triggered by either ADR or 5-FU were inhibited significantly by the general caspase inhibitor Z-VAD-FMK, or a specific caspase-3 inhibitor DMQD-CHO. These findings provide a mechanistic explanation for anthracyclines and 5-FU induced-apoptosis.

Objectives. To investigate the effect of anti-Fas monoclonal antibody (mAb) on the intracellular concentration of doxorubicin in renal cell carcinoma (RCC) cells. Little is known about the influence of anti-Fas mAb on the intracellular concentration of chemotherapeutic agents. Methods. The concentration of intracellular doxorubicin was determined by high-performance liquid chromatography. The mRNA and protein levels of multidrug resistance-associated protein gene were evaluated by reverse transcriptase-polymerase chain reaction and immunocytochemistry, respectively. Results. An increased concentration of doxorubicin inside the cells was found: 2.4-fold in ACHN cells (a human RCC cell line) after treatment with doxorubicin combined with anti-Fas mAb compared with doxorubicin alone. Of the five cases of freshly derived RCC cells treated with doxorubicin and anti-Fas mAb, the intracellular concentration of doxorubicin was increased 2.3 and 2.7-fold in two of them, respectively. Furthermore, both the mRNA and the protein levels of the multidrug resistance-associated protein gene were downregulated after treatment of ACHN cells with anti-Fas mAb. Treatment of ACHN cells with a combination of anti-Fas mAb and doxorubicin resulted in a potentiation of the doxorubicin-mediated cytotoxicity. Conclusions. The increased intracellular concentration of doxorubicin by anti-Fas mAb might be one of the mechanisms responsible for the enhancement of doxorubicin-mediated cytotoxicity in RCC cells.

Background: To explore the relationship between telomerase activity in urine after transurethral resection (TUR) of superficial bladder cancer and early intravesical recurrence. Methods: Urine samples were obtained from 42 patients with superficial bladder cancers prior to TUR and on the postoperative day 1 and day 6. These patients were followed-up prospectively by cystoscopy at 3 and 6 months after TUR in combination with urinary cytology and telomerase activity. Telomerase activity in the urine was assessed by the telomeric repeat amplification protocol assay. Results: Urinary telomerase activity prior to TUR was positive in 24 (57%) of the 42 patients. On the postoperative day 1 and day 6, positive urinary telomerase activity was seen in 13 (31%) and nine (21%) patients, respectively. Postoperative urinary telomerase activity on either day 1 or day 6 was significantly associated with pre-operative urinary telomerase activity status (P = 0.0024). Fifteen patients showed intravesical tumor recurrence at 3 months cystoscopic check-up and an additional nine had recurred at the 6 months check-up. Recurrence rate within 6 months in patients with pre-operative positive urinary telomerase activity was similar to that in those with negative activity (58.3 vs 58.8%). However, recurrence rate at 3 months for patients with positive activity was higher than that of those with negative activity (50 vs 17.7%), in 23 patients treated only by TUR. Conclusions: Presence of cells positive for telomerase activity in urine after TUR of superficial bladder cancer indicates persistently existing cancer cells in the urine. It is, however, not a sole predictor of the early intravesical recurrence.

INTRODUCTION AND OBJECTIVES: Prostate-specific membrane antigen (PSMA) provides an attractive target for monoclonal antibody (mAb) targeted therapies in the treatment of prostate cancer (PC). In this study, we generated an immunotoxin by linking a humanized anti-PSMA mAb (J591) to the ribosome-inactivating protein toxin saporin. The saporinJ591 immunoconjugate was evaluated for antitumor activity against PC cells. METHODS: PSMA-positive cell lines, LNCaP and CWR22Rv1 and a PSMA-negative cell line, PC-3, were used in these experiments. Humanized J591 was biotinylated and mixed in a 1:1 molar ratio with streptavidin-saporin (SAZAP). The binding ability of J591-saporin conjugate was compared to native, unconjugated J591 by enzymelinked immunosorbent assay and the extent of internalization into the cells was tested using immunofluorescence microscopy. The viability of cells treated with J591-SAZAP was examined by MTT assay and apoptotic cells were measured by flow cytometry 72 hours after J591saporin administration. RESULTS: The binding ability of J591-saporin to PSMA was equivalent to that of unconjugated J591. Internalization of J591-saporin was clearly detected in PSMA-positive, but not PSMA-negative, cell lines using fluoresceinisothiocyanateor Cy3-linked secondary antibodies (Fig. 1). The viability of LNCaP cells decreased to 20.16 ± 0.95% at a dose of 6.25 nM compared to the viability of PC-3 cells of 94.90 ± 4.84% at a dose of 100 nM (Fig. 2). In addition, after 72 hours of J591-SAZAP treatment, the percentage of apoptotic cells was 60.29% in LNCaP cells compared to 4.70% in PC-3 cells. CONCLUSIONS: Our findings show that humanized J591saporin conjugate has potent and selective antitumor effects on PSMApositive prostate cancer cells in vitro. This study supports development of conjugates of PSMA antibody to toxin proteins such as saporin for therapy of prostate cancer. Source of Funding: None

Objectives To explore the interrelationship of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors DR4 and DR5 expressions level with patient prognosis and the response to adjuvant therapy in bladder cancer, the synergism function that is between chemotherapy and TRAIL on apoptosis induction in tumor cells. Methods The expression of TRAIL, DR4, and DR5 was studied using immunohistochemistry of paraffin-embedded tumor specimens from 229 bladder cancer patients who had undergone transurethral resection. Results Cytoplasmic TRAIL, DR4, and DR5 expressions were detected in 35%, 75.1%, and 74.2% of bladder cancer patients, respectively. Patients with bladder cancer with either high DR4 or DR5 expression had a significantly longer postoperative recurrence-free rate than those with low expression of both during the 10-year follow-up. Multivariate analysis revealed that the expression of DR4 ( P P P = .034) were independent prognostic indicators of bladder cancer. Furthermore, epirubicin therapy significantly improved recurrence-free rate for the patients with DR4-high ( P = .006) or DR5-high ( P = .042) tumor. Conclusions The results of the present study have shown for the first time that a combination of DR4 and DR5 expression have significant value in predicting the prognosis of bladder cancer. In addition, patients with high expression of both DR4 and DR5 might benefit from epirubicin therapy.

Previous studies indicated that the low molecular weight polysaccharide extracts from Agaricus blazei are potential antitumor agents or adjuvant in tumor treatment. In this study, we investigated the antitumor activity of LMPAB, a low molecular weight polysaccharide isolated from Agaricus blazei, and the molecular mechanisms of its antitumor activity. The antitumor effect of LMPAB was examined using mouse sarcoma 180 (S180) xenograft models. Antiangiogenic effect of LMPAB was determined by chicken embryo chorioallantoic membrane (CAM) angiogenesis and Matrigel-induced neovascularization in vivo models. The mRNA and protein levels of vascular endothelial growth factor (VEGF) were assessed using real-time reverse transcription-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assays. Tumor inhibitory rates in the S180 xenograft models were 9.7, 23.9, and 33.0%, respectively, after administration of LMPAB at dose of 50, 100, and 200 mg/kg/day for 2 weeks. LMPAB also inhibited angiogenesis in the CAM model and Matrigel-induced neovascularization in C57BL/6 mice. The mRNA and protein levels of VEGF in tumor tissues were significantly down-regulated in the BALB/c mice received LMPAB treatment. Furthermore, significant down-regulation of serum VEGF levels was also observed in the mice. Our data suggest that LMPAB might be a promising agent for tumor therapy, and the antitumor and antiangiogenic effects of LMPAB may be related with down-regulation of VEGF.