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.

Molecular cloning and characterization of cDNAs for the gamma- and epsilon-subunits of mitochondrial F1F0 ATP synthase from the sweet potato.

Mitochondrial F1F0 ATP synthases purified from dicotyledonous plants contain six different subunits named alpha, beta, gamma, delta, delta', and epsilon. Our previous N-terminal amino acid sequence analyses indicated that the gamma- and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma- and epsilon-subunits of animal mitochondrial F1, respectively (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). A cDNA clone for the gamma-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide hybridization selection of a cDNA library, and a cDNA clone for the epsilon-subunit was isolated by reverse polymerase chain reaction and hybridization selection of a cDNA library by the polymerase chain reaction product. The 1.4-kilobase long cDNA for the gamma-subunit contained a 978-base pair open reading frame coding for a precursor for the gamma-subunit. The mature gamma-subunit is composed of 281 amino acids, and its sequence showed significantly higher similarities with the gamma-subunit of animal mitochondrial F1 and bacterial F1 compared with the gamma-subunit of chloroplast CF1 from plants. The precursor for the gamma-subunit contained N-terminal presequence of 45 amino acid residues. By contrast, the 0.46-kilobase long cDNA for the epsilon-subunit contained a coding sequence of 207-base pairs for the mature epsilon-subunit of 69 amino acid residues that is preceded by an ATG codon suggesting that the epsilon-subunit is synthesized without the cleavable presequence for mitochondrial import. The amino acid sequence of the epsilon-subunit of sweet potato mitochondrial F1 showed similarities of 25 and 36% amino acid positional identity with the epsilon-subunits of mitochondrial F1 from yeast and bovine, respectively.

[Clinical studies of varicocele. 1: Clinical statistical analysis of varicocele].

A clinical statistical analysis was conducted of the patients with varicocele who visited the Department of Urology, Osaka City University, and its related hospitals. Regarding the age distribution, patients younger than 50 years old were predominant; this age group included 20 of 24 cases, or 83%. An overwhelming majority of the cases, 75%, had the main complaint of infertility; they had not been able to have children even after 2 to 3 years of marriage. The next most common complaint was swelling and/or an uncomfortable feeling of the contents of the scrotum. In all of the cases, the lesion was seen in the left side of the scrotum. When the patients were classified on the basis of the sperm count, oligospermia was predominant; it was seen in 15 cases (62.5%). A study on the relationship between the sperm count and the grade of varicocele revealed a tendency for the sperm count to show an inverse relationship with the grade. Testicular atrophy showed a direct correlation with the grade. Of the patients with varicocele, 5 patients had one or more children, while 8 patients (61.5%) complained of their inability to have a child. No particular relationship could be identified between the age at the time of visiting our Department as outpatients and the degree of testicular atrophy.