Junichi Asakuma

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.

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.

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.

Chemosensitization of Androgen-Independent Prostate Cancer with Neutral Endopeptidase

Purpose: We investigated whether neutral endopeptidase (NEP) could augment chemosensitivity to anticancer drugs by promoting protein kinase C (PKC)δ-mediated mitochondrial apoptosis in prostate cancer (PC) cells. Experimental Design: Human PC cell lines LNCaP and PC-3, and a normal prostate epithelial cell line (PrEC) were used. The protein expression was detected by Western blot analysis, and the protein turnover was determined by pulse-chase assay. Apoptotic ratio was measured by annexin V staining. Results: Western blot analyses and pulse-chase assays showed that the specific NEP inhibitor CGS24592 decreased PKCδ protein expression by promoting PKCδ protein degradation in NEP-expressing LNCaP cells. Conversely, recombinant NEP (rNEP) increased PKCδ protein expression by delaying PKCδ protein degradation in NEP-negative PC-3 cells. Apoptosis assays showed that rNEP promoted anticancer drug-induced apoptosis in PC-3 cells specifically through PKCδ activity that mediated anticancer drug-induced mitochondrial change such as cytochrome-c release and caspase-9 activation. Of note, rNEP was able to increase PKCδ protein expression predominantly in PC-3 cells rather than in PrEC cells. Treatment with rNEP before subtoxic concentrations of etoposide (0.1 μm) significantly promoted mitochondrial apoptosis compared with only etoposide in PC-3 cells (P < 0.01) but not in PrEC cells. Conclusions: These results suggest that NEP enzyme activity contributes to anticancer drug-induced PC cell apoptosis dependent on PKCδ-mediated mitochondrial events. More importantly, the combination of NEP with anticancer drugs may be a promising therapeutic modality because rNEP is able to augment chemosensitivity in androgen-independent PC with minimal toxicity in normal tissues.

Impact of Postoperative C-Reactive Protein Level on Recurrence and Prognosis in Patients With N0M0 Clear Cell Renal Cell Carcinoma

PURPOSE Preoperative C-reactive protein is a strong predictor of recurrence and prognosis in patients with renal cell carcinoma while postoperative C-reactive protein reportedly predicts survival in patients with metastatic renal cell carcinoma. We evaluated the impact of postoperative C-reactive protein on recurrence and prognosis in patients with N0M0 clear cell renal cell carcinoma. MATERIALS AND METHODS We defined increased preoperative C-reactive protein as 1 mg/dl or greater and postoperative C-reactive protein normalization as at least 1 postoperative measurement of less than 0.3 mg/dl. We reviewed the records of 263 patients with N0M0 clear cell renal cell carcinoma who underwent nephrectomy, and in whom preoperative and postoperative C-reactive protein values were available. We used multivariate analysis to identify independent factors predicting recurrence and prognosis. We also evaluated C-reactive protein at recurrence and its impact on survival. RESULTS Increased preoperative C-reactive protein and nonnormalization of postoperative C-reactive protein were associated with worse clinicopathological factors. Postoperative C-reactive protein nonnormalization, increased preoperative C-reactive protein, microvascular invasion and histological tumor necrosis were independent predictors for recurrence. Risk stratification using these factors effectively predicted the possibility of recurrence. Anemia, thrombocytosis and postoperative C-reactive protein nonnormalization were independent predictors of overall survival. Postoperative followup revealed recurrence in 50 patients. The 3-year survival rate in patients with C-reactive protein 0.3 mg/dl or greater at recurrence was significantly lower than that in patients with less than 0.3 mg/dl at recurrence (47.3% vs 81.6%). CONCLUSIONS Nonnormalization of postoperative C-reactive protein is a strong predictor of recurrence and prognosis. Patients with C-reactive protein 0.3 mg/dl or greater at recurrence might not survive as long as those with C-reactive protein less than 0.3 mg/dl at recurrence.

Glucose-Regulated Protein 78 Positivity as a Predictor of Poor Survival in Patients with Renal Cell Carcinoma

Introduction: Glucose-regulated protein 78 (GRP78), a chaperone for newly formed proteins during folding and glycosylation, is associated with resistance to apoptosis in some forms of cancer. We assessed GRP78 expression and its correlation with clinicopathological parameters and survival. Patients and Methods: Immunohistochemistry was performed using formalin-fixed, paraffin-embedded specimens: 128 primary renal cell carcinoma (RCC) specimens (120 conventional and 8 other cell types) and 9 metastatic specimens. GRP78 positivity was determined based on intensity of staining and percentage of cells stained. Correlation of GRP78 positivity with clinicopathological parameters including patients’ survival was evaluated. Results: A statistically significant association was found between GRP78 positivity and higher tumor grade (G3; p <0.0001), advanced T stage (≧pT3; p = 0.0002), lymphovascular invasion (positive; p <0.0001), regional nodal involvement (≧N1; p = 0.0086), and distant metastases at presentation (M1; p = 0.001). Positivity of GRP78 expression was significantly associated with shorter disease-specific survival and shorter progression-free survival. Cox proportional hazard model showed that strong GRP78 positivity was an independent predictor of shortened progression-free survival in N0M0 RCC patients. Conclusions: There was a significant relationship between GRP78 expression levels and aggressiveness of RCC. Increased expression of GRP78 might be a useful parameter to predict shortened survival in patients with RCC.

Anterior perirectal fat tissue thickness is a strong predictor of recurrence after high‐intensity focused ultrasound for prostate cancer

Objective:  To evaluate if and why obesity affects the clinical outcome in patients undergoing high‐intensity focused ultrasound (HIFU) treatment for prostate cancer (CaP).

Transurethral resection of the prostate immediately after high-intensity focused ultrasound treatment for prostate cancer

Objectives:  To evaluate the long‐term outcomes of transurethral resection of the prostate (TURP) immediately after high‐intensity focused ultrasound (HIFU) treatment for prostate cancer (CaP).

Prognostic factors for upper urinary tract urothelial carcinoma after nephroureterectomy.

Introduction: The purpose of this study was to evaluate prognostic factors for patients with upper urinary tract urothelial carcinoma (UUT-UC) after nephroureterectomy and to seek a better way of finding more favorable clinical results for these patients. Patients and Methods: We retrospectively reviewed the medical records of 121 UUT-UC patients who underwent a nephroureterectomy at our institution, and analyzed the prognostic significance of various clinicopathological parameters for progression-free and disease-specific survival rates by using univariate and multivariate analysis. Results: A Cox proportional hazards model showed that extravesical tumor recurrence after surgery was an independent prognostic factor for disease-specific survival (p < 0.0001). An additional model showed that lymphovascular invasion (LVI) was one of the independent predictors of lower extravesical-recurrence-free survival rates (p = 0.0004). Our final finding was that pathological tumor stage and positive surgical margin were significantly associated with the presence of LVI (p < 0.0001 and p = 0.0029, respectively). Conclusions: We conclude that there is a high possibility of LVI in patients with large tumors. Our findings should be helpful in terms of determining whether or not to perform neoadjuvant chemotherapy for patients with large tumors, given the fact that we frequently find a severe reduction in renal function after nephroureterectomy.