Apigenin inhibits renal cell carcinoma cell proliferation through G2/M phase cell cycle arrest.

Apigenin is a flavonoid widely presented in fruits and vegetables, and is known to possess anti­inflammatory, antioxidant, and anticancer properties. The present study was designed to investigate the effects of apigenin on renal cell carcinoma (RCC) cells. These effects on cell growth were evaluated using a cell counting kit, while cell cycle distribution was investigated by flow cytometry following propidium iodide DNA staining. The human RCC cell lines, Caki­1, ACHN, and NC65, were each treated with 1­100 µM apigenin for 24 h, which resulted in concentration­dependent cell growth inhibition, with the effects confirmed by trypan blue staining. Furthermore, even when the apigenin treatment period was shortened to 3 h, the same cytostatic effect on RCC cells was noted. Similarly, a concentration­dependent cell growth inhibitory effect was also observed in primary RCC cells, as apigenin induced G2/M phase cell cycle arrest and reduced the expression levels of cyclin A, B1, D3, and E in RCC cells in both dose­ and time­dependent manners. These findings suggest the possibility of the use of apigenin as a novel therapeutic strategy for treatment of RCC due to its anticancer activity and ability to function as a cell cycle modulating agent.

[SERTOLI CELL TUMOR OF TESTIS: CASE REPORT].

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 case of Endocrine Active Retroperitoneal Ganglioneuroma, Difficult to Differentiate From Adrenal Pheochromocytoma].

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.

Enhancement of death receptor 4-mediated apoptosis and cytotoxicity in renal cell carcinoma cells by anisomycin.

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.

Synergistic induction of apoptosis by mapatumumab and anthracyclines in human bladder cancer cells.

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.

Expression of TRAIL, DR4, and DR5 in bladder cancer: correlation with response to adjuvant therapy and implications of prognosis.

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 < .001), DR5 (P < .001) and epirubicin therapy (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.

Delineation of apoptotic genes for synergistic apoptosis of lexatumumab and anthracyclines in human renal cell carcinoma cells by polymerase chain reaction array.

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.

Expression of autotaxin and acylglycerol kinase in prostate cancer: association with cancer development and progression.

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 < 0.0001) and capsular invasion (P = 0.03 and 0.003 respectively). ATX expression was significantly correlated with probability of prostate specific antigen (PSA)-failure after surgery (P < 0.0001). In conclusion, LPA-producing enzymes (ATX and AGK) were frequently expressed in prostate cancer cells and precancerous HG-PIN. In particular, high expression levels of ATX were associated with both malignant potentials and poor outcomes.

Effect of verapamil on the expression of EGFR and NM23 in A549 human lung cancer cells.

Despite the advances in the detection and treatment of lung cancer, the overall 5-year survival is only 10-20%. Accumulating evidence suggests that verapamil, a calcium channel antagonist, is a potential anticancer agent. Epidermal growth factor receptor (EGFR) is a key therapeutic target in many types of cancer, whereas nm23 is a putative metastasis suppressor gene. In this study, the effect of verapamil on the expression of nm23 and EGFR in A549 human lung cancer cells was investigated by quantitative real-time reverse transcription-polymerase reaction and immunohistochemical assays. The expression of EGFR and nm23 was also determined in lung cancer patients. Verapamil significantly reduced EGFR expression at both the mRNA and protein levels in A549 cells (p < 0.01). Verapamil also significantly increased the protein levels of nm23 in these cells (p < 0.01), although the mRNA levels of nm23 were not changed after verapamil treatment. Furthermore, the expression of EGFR in human lung cancer tissues was significantly higher than in normal lung tissues (p < 0.001). However, the expression of nm23 was not different between lung cancer and normal tissues. Our data suggest that verapamil may regulate the expression of EGFR and nw23 in lung cancer cells by transcriptional and post-transcriptional levels, respectively. EGFR may be a promising therapeutic molecular target for lung cancer treatment using verapamil and/or chemotherapeutic agents.

Enhancement of lexatumumab-induced apoptosis in human solid cancer cells by Cisplatin in caspase-dependent manner.

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.

Thymidylate synthase was associated with patient prognosis and the response to adjuvant therapy in bladder cancer.

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 < 0.001). CONCLUSIONS: High TS expression might be a marker of poor prognosis for patients with bladder cancer. In addition, patients with high TS expression might also be benefit from adjuvant therapy.

Gene expression profiles of lysophosphatidic acid-related molecules in the prostate: relevance to prostate cancer and benign hyperplasia.

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.

Induction of apoptosis in human renal cell carcinoma cells by vitamin E succinate in caspase-independent manner.

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.

[Assessment of the urinary status by self-administered questionnaires in men after radical prostatectomy-- comparison with the urination status of elderly women].

The lower urinary tract anatomy in men after radical prostatectomy (RP) resembles that in women. Out of 112 male patients who had undergone RP for localized prostate cancer, 102 (91%) of them responded to a questionnaire survey. The mean age of the responders at the time of RP was 65.9 +/- 5.3 years. The time of response after RP ranged from 2 months to 6 years (median: 44 months). The instruments used for the assessment of urinary status were the International Prostate Symptom Score (IPSS) and QOL score, the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and Overactive Bladder Symptom Score (OABSS). Urinary status of 40 elderly female patients aged 59.5 +/- 6.9 years who consulted our outpatient clinic due to conditions (microhematuria, simple renal cyst, etc.) unrelated to lower urinary tract disorders were assessed with the IPSS. In the male patients, total IPSS and QOL score showed significant improvement over time after RP (P = 0.0004, P = 0.0015, respectively). In particular, the voiding symptom score of IPSS showed significant improvement (P < 0.0001). The improvement of incontinence within 1 year after RP was confirmed with ICIQ-SF (p = 0.06). In contrast, the storage symptom score of IPSS after RP was not different with time after RP. Furthermore, the OABSS rose with time after RP (p = 0.08). On the other hand, in the elderly female controls, the storage symptom score of IPSS was significantly higher than voiding symptom score (P = 0.0019). Men who underwent RP showed significant improvement in their voiding symptoms and continence status, but the storage symptoms, progressively worsened following RP. Consequently, careful follow-up and appropriate medical intervention are needed in men after RP as in aging women.

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells.

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.

Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2-mediated apoptosis by inducing TRAIL-R2 expression.

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.

Uroplakin III-delta4 messenger RNA as a promising marker to identify nonulcerative interstitial cystitis.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 (p <0.001). 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. CONCLUSIONS: 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.

Enhancement of death receptor 4 mediated apoptosis and cytotoxicity in renal cell carcinoma cells by subtoxic concentrations of doxorubicin.

PURPOSE: 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. MATERIALS AND METHODS: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. RESULTS: 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 (p<0.05). 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. CONCLUSIONS: 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.

Human agonistic antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 induces cytotoxicity and apoptosis in prostate cancer and bladder cancer cells.

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.

Uroplakin II as a promising marker for molecular diagnosis of nodal metastases from bladder cancer: comparison with cytokeratin 20.

PURPOSE: Current methods used to determine pathological examination of the lymphatics after radical cystectomy are tedious and costly. We performed a systemic study of uroplakin II (UP II) and cytokeratin 20 (CK 20) expression in pelvic lymph nodes on multiple sides in patients with bladder cancer. MATERIALS AND METHODS: A total of 82 pelvic lymph node and 19 bladder tumor samples were obtained from 21 patients with bladder cancer by radical cystectomy with pelvic lymphadenectomy for reverse transcriptase-polymerase chain reaction assay. RESULTS: Of the 19 bladder tumor tissue specimens 19 (100%) and 13 (68.4%) were positive for UP II and CK 20 mRNA expression, respectively. UP II mRNA was detected in 15 of 16 pelvic lymph node samples (93.8%) with pathologically proven metastases, whereas 9 (56.6%) were positive for CK 20 mRNA. The reverse transcriptase-polymerase chain reaction assay for UP II was statistically more sensitive than that for CK 20 in detecting not only primary tumors, but also metastatic pelvic lymph nodes (p = 0.0179 and 0.0373, respectively). Of 66 pelvic lymph node samples without metastasis UP II was detected in 6 (10%), while CK 20 was not. In addition, UP II and CK 20 mRNA could be detected in at least 50 and 500 bladder cancer HT1197 cells, respectively. CONCLUSIONS: These results indicate that UP II might be a more useful marker than CK 20 for detecting micrometastases of bladder cancer in the pelvic lymph nodes, although a greater number of patients and longer followup are needed to come to a definitive conclusion.