Tomohiko Asano

Cyclosporine induces cancer progression by a cell-autonomous mechanism

Malignancy is a common and dreaded complication following organ transplantation,,,. The high incidence of neoplasm and its aggressive progression, which are associated with immunosuppressive therapy, are thought to be due to the resulting impairment of the organ recipients immune-surveillance system,,,,. Here we report a mechanism for the heightened malignancy that is independent of host immunity. We show that cyclosporine (cyclosporin A), an immunosuppressant that has had a major impact on improving patient outcome following organ transplantation,, induces phenotypic changes, including invasiveness of non-transformed cells, by a cell-autonomous mechanism. Our studies show that cyclosporine treatment of adenocarcinoma cells results in striking morphological alterations, including membrane ruffling and numerous pseudopodial protrusions, increased cell motility, and anchorage-independent (invasive) growth. These changes are prevented by treatment with monoclonal antibodies directed at transforming growth factor-β (TGF-β). In vivo, cyclosporine enhances tumour growth in immunodeficient SCID–beige mice; anti-TGF-β monoclonal antibodies but not control antibodies prevent the cyclosporine-induced increase in the number of metastases. Our findings suggest that immunosuppressants like cyclosporine can promote cancer progression by a direct cellular effect that is independent of its effect on the hosts immune cells, and that cyclosporine-induced TGF-β production is involved in this.

Protein kinase Cδ amplifies ceramide formation via mitochondrial signaling in prostate cancer cells

We studied the role of protein kinase C isoform PKCδ in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCδ-positive LNCaP and DU145 cells but not in PKCδ-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCδ inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative–type PKCδ. Overexpression of wild-type PKCδ had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCδ and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCδ mitochondrial translocation. These results indicate that PKCδ plays a crucial role in activating anticancer drug–induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.

3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitor, Fluvastatin, as a Novel Agent for Prophylaxis of Renal Cancer Metastasis

Purpose: Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also called statins, are currently used widely as a safe, effective therapeutic in the treatment of hypercholesterolemia. Recently, statins have been recognized for their activity against cancer. In the present study, we examined the effect of a synthetic statin, fluvastatin, on the development of renal cancer. Experimental Design: The effects of fluvastatin on cell viability, cell cycle, in vitro angiogenesis, and invasive properties were examined in murine renal cancer cell Renca. The changes in cell cycle-associated proteins, p21Waf1/Cip1 and p53, and rac1 phosphorylation were analyzed by Western blotting. The prophylactic efficacy of fluvastatin to murine pulmonary metastasis of Renca was examined. Results: Fluvastatin inhibited in vitro growth of Renca cells in a time- and dose-dependent manner, with up to 70% inhibition at a concentration of 10 μmol/L. This inhibitory effect was due to cell cycle arrest at the G1 phase and induction of apoptosis accompanied by up-regulation of p21Waf1/Cip1 and p53. The invasive properties of Renca cells through Matrigel were inhibited by fluvastatin, with decreased phosphorylation of rac1. In vitro angiogenesis was also inhibited by fluvastatin. Furthermore, oral administration at doses of 1 to 10 mg/kg/d, for 12 days after inoculation of Renca cells via the tail vein, significantly decreased the amount of pulmonary metastasis. Conclusions: Because our results suggest that fluvastatin may effectively inhibit in vitro tumor growth, invasion, angiogenesis, and metastasis of Renca cells, oral administration of fluvastatin could be a novel, safe, and effective agent for preventing metastasis of renal cancer.

Impact of thrombocytosis and C-reactive protein elevation on the prognosis for patients with renal cell carcinoma.

Aim: C‐reactive protein (CRP) elevation is reportedly a prognostic factor in patients with renal cell carcinoma (RCC). Thrombocytosis has recently been reported also to be a prognostic factor in RCC and, like CRP, to be related to inflammatory cytokines such as interleukin‐6. The aim of this study was to evaluate the importance of both thrombocytosis and CRP elevation in tumor recurrence and prognosis for patients with RCC.

STAT3 inhibitor WP1066 as a novel therapeutic agent for renal cell carcinoma

Background:Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes that mediate cell survival, proliferation, and angiogenesis and is aberrantly activated in various types of malignancies, including renal cell carcinoma (RCC). We examined whether it could be a novel therapeutic target for RCC by using the STAT3 inhibitor WP1066.Methods:The antitumour activities and related mechanisms of WP1066 were investigated in vitro on renal cancer cell lines and in vivo on murine xenografts.Results:In Caki-1 and 786-O renal cancer cells, 5 μM WP1066 prevented the phosphorylation of STAT3, and 2.5 μM WP1066 significantly (P<0.01) inhibited cell survival and proliferation. WP1066 suppressed the expression of Bcl-2, induced apoptosis, and inhibited the basal and hypoxia-induced expression of HIF1α and HIF2α, as well as vascular endothelial growth factor secretion into cell culture medium. Human umbilical vascular endothelial cells cocultured with media from WP1066-treated cells showed significantly reduced tubulogenesis (P<0.05). Systemic oral administration of WP1066 to mice for 19 days significantly inhibited the growth of Caki-1 xenograft tumours (P<0.05), and pathological analysis of xenografts of WP1066-treated mice showed decreased immunostaining of phosphorylated STAT3 and reduced length of CD34-positive vessels (P<0.05).Conclusion:Our results suggest that using WP1066 to inhibit the STAT3 signalling pathway could be a novel therapeutic strategy against RCC.

Novel SN-38–Incorporated Polymeric Micelle, NK012, Strongly Suppresses Renal Cancer Progression

It has been recently reported that NK012, a 7-ethyl-10-hydroxy-camptothecin (SN-38)-releasing nanodevice, markedly enhances the antitumor activity of SN-38, especially in hypervascular tumors through the enhanced permeability and retention effect. Renal cell carcinoma (RCC) is a typical hypervascular tumor with an irregular vascular architecture. We therefore investigated the antitumor activity of NK012 in a hypervascular tumor model from RCC. Immunohistochemical examination revealed that Renca tumors contained much more CD34-positive neovessels than SKRC-49 tumors. Compared with CPT-11, NK012 had significant antitumor activity against both bulky Renca and SKRC-49 tumors. Notably, NK012 eradicated rapid-growing Renca tumors in 6 of 10 mice, whereas it failed to eradicate SKRC-49 tumors. In the pulmonary metastasis treatment model, an enhanced and prolonged distribution of free SN-38 was observed in metastatic lung tissues but not in nonmetastatic lung tissues after NK012 administration. NK012 treatment resulted in a significant decrease in metastatic nodule number and was of benefit to survival. Our study shows the outstanding advantage of polymeric micelle-based drug carriers and suggests that NK012 would be effective in treating disseminated RCCs with irregular vascular architectures.

ZD1839 Modulates Paclitaxel Response in Renal Cancer by Blocking Paclitaxel-Induced Activation of the Epidermal Growth Factor Receptor–Extracellular Signal-Regulated Kinase Pathway

Purpose: We evaluated the antitumor activity of ZD1839, a selective epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, in combination with paclitaxel in human renal cell carcinomas (RCCs). Experimental Design: Eight human RCC lines and the surgical specimens obtained from 10 RCC patients were used. The protein expression was detected by Western blotting, immunohistochemistry and/or flow cytometry. Apoptosis was evaluated by flow cytometry and fragmented DNA ELISA. SKRC-49 tumor xenografts in athymic nude mice were treated with ZD1839 and/or paclitaxel, and tumor volume was determined Results: EGFR protein was expressed and phosphorylated in eight RCC lines and EGFR expression was markedly increased in RCC specimens compared with adjacent normal renal tissues. Treatment of SKRC-49 with 1 μm ZD1839 resulted in a marked decrease in the phosphorylation of EGFR but not of HER-2. Treatment of SKRC-49 with ZD1839 in combination with 5 nm paclitaxel resulted in a significant increase in apoptotic cell number compared with paclitaxel alone, whereas ZD1839 alone failed to induce apoptosis. Although administration of ZD1839 or paclitaxel resulted in a transient growth inhibition in SKRC-49 xenografts, significant tumor regrowth delay was observed when paclitaxel was combined with ZD1839. Paclitaxel phosphorylated extracellular signal-regulated kinase through EGFR activation predominantly in cancer cells. ZD1839 promoted paclitaxel-induced Bcl-2 down-regulation resulting in promoting apoptosis by blocking paclitaxel-induced activation of the EGFR—extracellular signal-regulated kinase antiapoptotic pathway independent of Akt activity in SKRC-49. Conclusions: Our findings support the idea that the significant clinical benefit is obtained from ZD1839 in combination with paclitaxel for the treatment of RCC.

Fatty Acid Synthase Over Expression is an Indicator of Tumor Aggressiveness and Poor Prognosis in Renal Cell Carcinoma

PURPOSE Fatty acid synthase is a key enzyme in the de novo biosynthesis of fatty acids. Increased fatty acid synthase expression and its association with tumor aggressiveness and poor prognosis have been demonstrated in various human malignant tumors. We investigated fatty acid synthase expression in patients with renal cell carcinoma and its impact on clinicopathological parameters. MATERIALS AND METHODS Fatty acid synthase expression in 120 patients with renal cancer was examined by immunohistochemistry. The relationship between fatty acid synthase expression status and various clinicopathological parameters was analyzed. Survival analysis was performed using the log rank test and a Cox multivariate hazard model. RESULTS Of 120 tumors 18 (15%) showed positive fatty acid synthase expression, which was significantly associated with advanced pathological T stage (pT3-4, p = 0.0009), regional lymph node metastasis (p = 0.0429), distant metastasis (p = 0.0042), higher histological grade (G3, p = 0.0017) and microvascular invasion (p = 0.0357). Patients with positive fatty acid synthase expression had significantly shorter cancer specific survival than those with negative FAS expression (p <0.0001). Multivariate Cox proportional hazards model analysis demonstrated that positive fatty acid synthase expression was an independent predictor of shortened cancer specific survival (p = 0.0363, HR 3.736). CONCLUSIONS Increased FAS expression could be an indicator of tumor aggressiveness and poor prognosis of renal cell carcinoma. Patients with fatty acid synthase positive tumors should be followed closely and carefully, and adjuvant therapy should be considered.

Pharmacological Inhibitor of Fatty Acid Synthase Suppresses Growth and Invasiveness of Renal Cancer Cells

PURPOSE Fatty acid synthase has been shown to be over expressed in a wide range of cancers and it has emerged as a therapeutic target. We examined whether fatty acid synthase could be a novel therapeutic target for renal cell carcinoma using the pharmacological fatty acid synthase inhibitor C75 (Cayman Chemical, Ann Arbor, Michigan). MATERIALS AND METHODS The effects of C75 on cell viability and proliferation in human renal cancer 769P (ATCC(R)), Caki-1 and KU20-01 cells were examined by MTS assay and cell counts. Cell cycle distribution was analyzed by flow cytometry and cell invasiveness was assessed by wound healing and Matrigel(trade mark) invasion assays. Fatty acid synthase expression and the effects of C75 on intracellular signaling pathways were analyzed by Western blotting. The antitumor efficacy of C75 was examined using Caki-1 cell xenografts. RESULTS All renal cancer cell lines expressed detectable fatty acid synthase. C75 (10 mug/ml) significantly inhibited cell viability and growth by arresting the cell cycle at the G2/M phase and inducing apoptosis (p <0.01). The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with C75 than they were for control cells treated with vehicle (p <0.001). C75 suppressed Her2 and epidermal growth factor receptor expression as well as STAT3 phosphorylation, while increasing p53 and p21(Waf1/Cip1) expression. Intraperitoneal administration of C75 at doses of 20 mg/kg per week for 28 days significantly reduced the tumor volume of Caki-1 xenografts (p <0.05). CONCLUSIONS Pharmacological inhibition of fatty acid synthase could be an effective strategy for treating renal cell carcinoma.

Decreased Serum Adiponectin Levels in Patients with Metastatic Renal Cell Carcinoma

BACKGROUND Low levels of serum adiponectin are associated with increased risk and aggressiveness of obesity-related cancer. The purpose of the study reported here was to investigate the association between serum adiponectin levels and clinicopathological parameters of renal cell carcinoma. METHODS Preoperative serum total and high-molecular-weight (HMW) adiponectin levels were measured in 118 patients with renal cell carcinoma, and their association with clinicopathological parameters was analysed. RESULTS There were no statistically significant associations between total adiponectin and HMW adiponectin and pathological stage, regional lymph node involvement, histological grade, histological type (clear cell carcinoma versus other types) or presence of venous invasion. Total and HMW adiponectin levels in patients with metastasis, however, were significantly lower than in patients without metastasis (P = 0.044 for total adiponectin and P = 0.041 for HMW adiponectin). Low total and HMW adiponectin levels were significantly associated with metastasis in patients with a normal BMI (<25 kg/m(2)) (P = 0.034 for total adiponectin and P = 0.028 for HMW adiponectin) but not in overweight and obese patients (P = 0.652 for total adiponectin and P = 0.489 for HMW adiponectin). Multivariate logistic regression analysis showed that total adiponectin level was an independent predictor of metastasis of renal cell carcinoma in all patients (P = 0.024, 95% CI = 1.031-1.560) and in patients with a normal BMI (P = 0.040, 95% CI = 1.043-6.534). CONCLUSIONS Serum total and HMW adiponectin levels were decreased in patients with metastatic renal cell carcinoma. Adiponectin might be a molecular link between obesity and the progression of renal cell carcinoma.