Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3beta positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kappaB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3beta and to assess the anti-cancer effect of GSK-3beta inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3beta in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT-PCR, BrDU incorporation and MTS assays to study the effect of GSK-3beta inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3beta in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kappaB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3beta as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Molecular targeting of Bcl-2 overcomes prostate cancer cell adaptation to XIAP gene downregulation.

X-linked inhibitor of apoptosis (XIAP) is a suppressor of apoptosis that supports an increased survival and resistance to chemotherapy of human prostate cancer (PCa) cells. Effects of transient (24 h) and chronic (beyond 1 month) downregulation of XIAP in DU145 hormone refractory prostate cancer (HRPC) cells were studied. We found that transient downregulation of XIAP by siRNAs resulted in an increase of apoptosis and a decrease in Bcl-2 levels and sensitized PCa cells to cisplatin. XIAP downregulation by shRNA vector stable transfection led to upregulation of Bcl-2 protein. Our results identify the adaptability of PCa cells to chronic loss of XIAP in part through upregulation of Bcl-2 and indicate that multitargeting approach is the most effective application in the chemotherapy of human HRPC.

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis.

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.

Absence of Bcl-2 and Fas/CD95/APO-1 predicts the response to immunotherapy in metastatic renal cell carcinoma.

Immunotherapy is the only available treatment for metastatic renal cell cancer (RCC), but the response rate is only about 20% and the treatment is occasionally associated with severe adverse effects. Thus, the selection of patients with a high susceptibility to immunotherapy is needed; however, there is no promising molecular marker that can predict the response to immunotherapy for RCC. This study was carried out to elucidate the potential role of apoptosis-related molecules Bcl-2 and Fas, as well as apoptotic and proliferating indexes (AI, PI) as predictors of the susceptibility of metastatic RCC to immunotherapy. Immunohistochemical examination of tumour tissues from 40 patients with metastatic RCC undergoing postoperative immunotherapy after radical nephrectomy was performed. Patients with progressive disease (PD) after immunotherapy presented with decreased survival (P=0.006). Progressive disease correlated with higher PI in the primary lesion (P=0.0087). All primary tumours of CR or PR patients were negative for Bcl-2, whereas among NC+PD patients, 40.6% were positive for Bcl-2 (P=0.0373). Patients in whom the primary tumours were both Bcl-2- and Fas-negative showed significantly better responses to immunotherapy in comparison with the remaining group (P=0.0022). The Bcl-2 and Fas status of the primary lesion may become useful criteria for the selection of patients with metastatic RCC for immunotherapy.

Prognostic impact of FAS/CD95/APO-1 in urothelial cancers: decreased expression of Fas is associated with disease progression.

The death receptor Fas (Apo1/CD95) and Fas ligand (FasL) system is recognised as a major pathway for the induction of apoptosis in vivo, and antiapoptosis via its blockade plays a critical role in carcinogenesis and progression in several malignancies. However, the function of Fas-FasL system in urothelial cancer (UC) has not been elucidated. We therefore investigated the expression of Fas, FasL and Decoy receptor 3 for FasL (DcR3) in UC specimens and cell lines, and examined the cytotoxic effect of an anti-Fas-activating monoclonal antibody (mAb) in vitro. Immunohistochemical examinations of Fas-related molecules were performed on 123 UC and 30 normal urothelium surgical specimens. Normal urothelium showed Fas staining in the cell membrane and cytoplasm. In UC, less frequent Fas expression was significantly associated with a higher pathological grade (P < 0.0001), a more advanced stage (P = 0.023) and poorer prognosis (P = 0.010). Fas and the absence thereof were suggested to be crucial factors with which to select patients requiring more aggressive treatment. Moreover, low-dose anti-Fas-activating mAb sensitised resistant cells to adriamycin, and this synergistic effect could be applied in the development of new treatment strategy for UC patients with multidrug-resistant tumours.

Impact of frequent Bcl-2 expression on better prognosis in renal cell carcinoma patients.

Previously, we reported that Bcl-2 was frequently expressed in renal cell carcinoma (RCC) specimens, but p53 mutation was a rare event. However, it was unclear whether Bcl-2 positivity was associated with the clinicopathological characteristics and prognosis in RCC. Therefore, we investigated the expression of Bcl-2 protein and its roles in 101 RCC specimens. In addition, the proliferation index (PI), apoptotic index (AI), caspase-3 and p53 expression were examined. The immunohistochemical method was applied for Bcl-2, caspase-3 and p53 protein expression. To investigate the proliferation activity and apoptosis of tumour cells, PI and AI were calculated based on Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL)-positive cells, respectively. Bcl-2 expression was detected in 72 out of 101 (71.3%) specimens. Bcl-2 positivity was inversely correlated with PI (P<0.0001) and AI (P=0.0074). Furthermore, Bcl-2 positivity was significantly correlated with better survival (P=0.0014), and was associated with lower stage (P=0.0301) and grade (P=0.0020). In RCC, frequent Bcl-2 expression was correlated with favourable character without higher PI and AI. Thus, Bcl-2 expression might be applied as a novel predictor of better prognosis in RCC patients.

Sequential detection of alphafetoprotein-bearing cells in blood stem cell fraction of germ cell tumour patients.

High-dose chemotherapy with peripheral blood stem cell (PBSC) transplantation in advanced germ cell tumour (GCT) patients is widely applied. The aims of this study were: (1) To examine the presence of alphafetoprotein (AFP) bearing tumour cells in PBSC harvests from advanced GCT patients obtained after multiple cycles of induction chemotherapy. (2) To determine whether induction chemotherapy contributed to in vivo purging of the tumour. We evaluated cryopreserved PBSC samples from 5 patients with advanced stage II/III AFP producing GCT. PBSC were separated after the first, second and third cycles of induction chemotherapy. Those samples were analysed using the nested reverse transcription polymerase chain reaction (RT-PCR) method to detect AFP mRNA. Although, in all patients, AFP mRNA was detected in PBSC samples after the first or second cycle of induction chemotherapy, but was not detected in 3 of 4 samples after the third cycle of chemotherapy. Although it is not clear whether tumour cells contaminating PBSC fraction contribute to disease relapse, PBSC harvested after at least 3 cycles of induction chemotherapy might be recommended to avoid such a possibility.

Frequency of PSA-mRNA-bearing cells in the peripheral blood of patients after prostate biopsy.

Transrectal ultrasound (TRUS) guided prostate biopsy is standard diagnostic procedure for prostate cancer (PCa). However, possibility of dissemination of cancer cells by biopsy is not negligible. To investigate this possibility, we examined prostate specific antigen (PSA)-bearing cells in peripheral blood of the 108 patients before and after prostate biopsy. Peripheral blood samples were obtained from 108 patients with elevated serum PSA (sPSA) levels, who had undergone sextant prostate biopsy using TRUS. The presence of PSA-mRNA bearing cells was examined using the nested RT-PCR method enabling detection of one LNCaP cell diluted in 1 ml of whole blood. Among 108 patients, 62 and 46 were diagnosed with benign prostatic hyperplasia (BPH) and PCa, respectively. PSA-mRNA was detected in 3 PCa cases but in no BPH patients before and after biopsy, and in 16 BPH (25.8%) and in 21 PCa (45.7%) patients only after biopsy (P< 0.01). The patients with positive mRNA before biopsy had higher sPSA (P< 0.001), and those after biopsy had higher sPSA and PSA density (PSAD) levels (P< 0.05). Positive PSA-mRNA cases had more cancer involved biopsy cores than the negative PSA-mRNA cases (P< 0.001). Although further investigations are needed, the present findings suggest that prostate biopsy might scatter prostate cells in the blood stream especially in cases with high sPSA and, thus, might contribute to tumour spreading in the cases of prostate cancer.

Caspase involved synergistic cytotoxicity of bcl-2 antisense oligonucleotides and adriamycin on transitional cell cancer cells.

We have previously shown that Bcl-2 expression was negative prognostic factor in transitional cell cancer (TCC), and that TCC cell lines expressing high levels of Bcl-2 are resistant to Adriamycin triggered apoptosis. Here we examined antisense oligonucleotide-mediated downregulation of Bcl-2 expression and its effect on sensitivity to Adriamycin (ADM) treatment in T24 cells. Treatment of T24 cells with 20 microM of bcl-2 antisense phosphorothioate oligodeoxynucleotide (PODN) reduced the Bcl-2 protein level. Combined administration with Adriamycin resulted in synergistic cytotoxicity, accompanied with a 2.4-fold increase in DEVD-specific caspase activity. The finding provides evidence that Bcl-2 expression may be critical for maintaining the drug resistance of TCC. bcl-2 antisense PODN might be useful means for overcoming drug resistance in highly malignant TCC.

Altered expression of beta-catenin in renal cell cancer and transitional cell cancer with the absence of beta-catenin gene mutations.

Loss of normal beta-catenin expression and the beta-catenin gene mutations have been shown to contribute to the malignant character of various cancers. Using PCR-single-strand conformation polymorphism and DNA direct sequencing, we examined the presence of genetic alterations within the third exon of beta-catenin, which are frequently observed in other tumors, in transitional cell cancer (TCC) and renal cell cancer (RCC) cell lines, and in tumor specimens. The degrees of expression and intracellular distribution of beta-catenin were detected by immunohistochemical staining in 77 primary and 12 metastatic RCCs and in 81 primary TCCs. Western blot analysis was also applied to confirm the degree of beta-catenin expression in the cell lines and some tumor samples. We failed to reveal any genetic alterations, at least in the third exon of the beta-catenin gene, in RCC and TCC. Reduced membranous immunoreactivity of beta-catenin was observed in portions of RCC (15.5%) and TCC (24.7%) and was correlated with advanced stages and nodal involvement in RCC and with advanced stages and multiple tumors in TCC. Within the power limitations of this small study, beta-catenin abnormal expression was not correlated with recurrence or survival in either RCC or TCC. Interstitial deletions and mutations in the third exon of beta-catenin do not play a significant role in RCC or TCC tumorigenesis. Down-regulation of normal beta-catenin expression might contribute to the malignant character of RCC and TCC and result in tumor progression. However, this event is not an independent prognostic factor for recurrence or tumor specific survival.

Epidermoid cyst of the penis: a case report and review of the literature.

Penile cysts are uncommon. A case of a 7-year-old boy with an epidermoid cyst of the penis is reported. He had an asymptomatic, slowly growing soft mass in the frenulum of the penis. Excision of the mass was performed, and the diagnosis was epidermoid cyst of the penis. No recurrence has been noted within the year since excision. To our knowledge, there have been no previous reports of malignancy developing in cystic disease of the penis. In such cases, clinicians should attempt more appropriate management, involving either watchful observation or complete excision of the cyst, by considering the embryogenesis and nature of the disease.

Altered expression of beta-catenin and c-erbB-2 in early gastric cancer.

To investigate the possible relationship between altered expression (loss of membranous staining or nuclear accumulation) of beta-catenin and invasion/metastasis in early gastric cancer (EGC), beta-catenin was detected immunohistochemically in 116 cases of EGC, including 86 differentiated and 30 undifferentiated carcinomas. In parallel, immunohistochemical expression of c-erbB-2 was analyzed in all EGC cases. Regardless of histological type, altered expression of beta-catenin was found in 47% of mucosal carcinomas and 89% of carcinomas with submucosal invasion (p<0.001). Of particular interest is that beta-catenin alteration was found in almost all EGCs with lymph node metastasis, even though no significant statistical comparison could be made. These results suggest that molecular changes resulting in abnormal beta-catenin expression participate in the process of submucosal invasion and metastasis. While loss of expression was preferentially observed in undifferentiated EGCs, nuclear accumulation was found exclusively in 24% of differentiated EGCs. c-erbB-2 was overexpressed in only 16% of differentiated EGCs but there was no correlation between this overexpression and invasion or metastasis. However, it is intriguing that 12 out of 14 cases with c-erbB-2 overexpression also showed altered beta-catenin expression, suggesting that both molecules are involved in the development of a certain set of differentiated EGCs.

Adriamycin induced G2/M cell cycle arrest in transitional cell cancer cells with wt p53 and p21(WAF1/CIP1) genes.

Adriamycin (ADM), widely used for systemic and local treatment of bladder tumors, triggers apoptosis in bladder cancer cells. Here we investigated the effect of ADM on cell cycle progression and expression of cell cycle regulating proteins in bladder cancer cell lines with various p53 and p21(WAF1/CIP1) status. Flowcytometric analysis was used to estimate the cell cycle distribution of T24, HT-1376, RT4, and SCaBER bladder cancer cell lines. Cell cycle regulating proteins were analyzed by Immunoblot. Treatment of RT4 cells, bearing wild type p53 and p21(WAF1/CIP1), with ADM induced expression of both proteins and cell cycle arrest, not in G1, as was anticipated, but in the G2 phase. Simultaneously, Retinoblastoma (Rb) protein expression was decreased. Expression of PCNA, which is a target gene of E2F, was not changed. The results suggest that even if the tumor cells bear wild type (wt) p53 and wt p21(WAF1/CIP1) and both proteins accumulate due to genotoxic stimuli, the cell cycle arrest might happen not in the G1 but in the G2 phase.

Infrequent alteration of p53 pathway in metastatic renal cell carcinoma.

Renal-cell carcinoma (RCC) is known to be highly resistant to conventional chemotherapy and irradiation suggesting that RCC cells do not easily undergo apoptosis, though p53 mutation is an infrequent event in RCC. p53 is responsible for the expression of p21WAF1 and bax genes, and these expressions are involved in the G1 arrest or apoptosis when cells are exposed to genotoxic stimuli. These gene mutations have been detected and their dysfunction may lead to accumulation of genomic alterations. We investigated the p53, p21WAF1 and bax gene mutations in 5 patients who had primary RCC and metastatic tumors. In one case only the metastatic tumor had non-sense transversion in the p53 gene, whereas the primary tumor showed no p53 gene mutation. no p21WAF1 and bax mutations were detected in any primary RCC or metastatic tumors. These findings suggest that, in RCC, inactivation of p53 might contribute to progression of the disease but inactivation of p21WAF1 and bax are not likely to play significant roles in the defective p53 pathway.

Recurrence of IgA nephropathy 17 months after renal transplantation in the allograft transmitted thin basement membrane disease (TBMD) from donor.

Recurrence of IgA nephropathy (IgAN) following renal transplantation has been described in 40-50% of such patients and it usually has a good outcome. We present the case of a 20-yr-old woman with IgAN who developed end-stage renal failure in 1995. In November 1996, she received a kidney from a living-related donor and was treated with tacrolimus, azathioprine and steroids. Zero- and one-hour biopsies were performed, which revealed minor glomerular abnormalities in light microscopy, thin basement membrane disease (TBMD) in electron microscopy. Eight months later she developed microscopic hematuria and proteinuria; however, the graft function was normal. Renal biopsy revealed an IgAN that is thought to be due to recurrence of the original disease.

Absence of frameshift mutations in the bax gene in renal cell cancer (RCC) and transitional cell cancer (TCC).

BACKGROUND: Bax protein, a member of the Bcl-2 family, regulates the apoptotic pathway that involves both Bcl-2 and p53. bax mutations are found in hematological malignancies, colon cancer with microsatellite mutator phenotype, and some other cancers. We evaluated the possibility that Bax functions as a TCC or RCC tumor suppressor gene. MATERIALS AND METHODS: Using Western blot and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) we looked for somatic mutations in a tract of eight deoxyguanosines (G)8 within third exon of bax in TCC and RCC cell lines and tumor specimens. RESULTS: We failed to reveal genetic alterations, at least in this specific region of bax gene in urological cancers. CONCLUSION: Alterations of bax, at least at investigated specific region, do not play considerable role in RCC or TCC tumorigenesis.

Variable Bcl-2 phenotype in benign and malignant lesions of urothelium.

We examined Bcl-2, Bax and p53 expression in transitional epithelium, benign lesions derived from transitional epithelium (Brunn's nests and inverted papillomas) and transitional cell cancer (TCC) of the upper urinary tract and urinary bladder using immunostaining of cryostat sections. We also performed Western blot analysis of normal urothelium and TCCs for Bcl-2 and p53. Immunohistochemical staining showed that Bcl-2 was expressed only on basal layer cells, whereas Bax expression was restricted to superficial layers in normal transitional epithelium. Benign lesions of the urinary bladder (Brunn's nests and inverted papillomas) showed strong immunoreactivity to Bcl-2. Taken together, 16 (17%) TCCs were positive for Bcl-2, 62 (64.6%) TCCs were positive for Bax and 28 (29%) TCCs were positive for p53. The expression of Bcl-2 on TCC had a statistical correlation with tumor stage, histopathologic grade and p53 protein accumulation. The results suggest that although Bcl-2 overexpression is detected in normal urothelium and benign lesions of the urinary bladder, it might also contribute to the high grade tumor malignancy of TCC.

Adriamycin (ADM) induced apoptosis in transitional cell cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction.

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1, at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1, Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.

Abrogation of apoptosis induced by DNA-damaging agents in human bladder-cancer cell lines with p21/WAF1/CIP1 and/or p53 gene alterations.

The p53-inducible cyclin-dependent kinase inhibitor, p21/WAF1/CIP1 (p21), plays a pivotal role in the G1 arrest or apoptosis of cells exposed to genotoxic stimuli. To determine whether p21 is a putative tumor-suppressor gene, p21 status was investigated in 4 human bladder-cancer cell lines of known p53 status. A p21-gene mutation, one base-pair insertion at codon 20 resulting in a chain-termination change at codon 35, was observed in one cell line, HT1376, suggesting structural or functional alteration of the p21 protein. When exposed to DNA-damaging agents, cisplatin or mitomycin C, apoptosis was induced in RT4 with the wild-type (wt) p53/wt p21, whereas T24 with the p53 non-sense mutation/wt p21 was resistant. Of the other 2 cell lines with the p53 mis-sense mutation, apoptosis was induced in SCaBER with the wt p21, but HT1376 with the p21 frame-shift mutation was fairly resistant. These findings suggest that not only p53 alteration, but also p21 alteration, is important to prevent apoptosis induced by DNA-damaging agents. When exposed to these agents, p53 and p21 expression was increased in RT4, and not induced in T24. p53 was not induced, but p21 expression was increased in SCaBER, whereas p53 expression was increased but p21 expression was absent in HT1376. Thus, p21 expression itself may have an important role in the induction of apoptosis by DNA-damaging agents.

Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1(CD95)-mediated apoptosis in renal-cell-cancer cells.

The apoptotic machinery has been intensively investigated, and interleukin-1-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apo1 (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethyl-ketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family.