Mototsugu Muramaki

Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer. [Mol Cancer Ther 2007;6(1):299–308]

Risk factors for intravesical recurrence after surgical management of transitional cell carcinoma of the upper urinary tract.

OBJECTIVES To identify risk factors for developing subsequent bladder cancer in patients undergoing surgical management of transitional cell carcinoma (TCC) of the upper urinary tract. METHODS This study included 177 patients who were diagnosed as having clinically localized upper urinary tract TCC and thereafter underwent nephroureterectomy after exclusion of those with a previous and/or concurrent history of bladder cancer. Univariate and multivariate analyses using both the logistic regression model and the Cox proportional hazards model were carried out in these 177 patients to determine the risk factors for intravesical recurrence after nephroureterectomy. RESULTS Of the 177 patients, 63 (35.6%) developed recurrent bladder cancer after a median interval of 7.5 months. Intravesical recurrence-free survival rates for these 177 patients at 1, 3, and 5 years were 75.7%, 63.7%, and 54.1%, respectively. Univariate analyses showed that patients with low-stage tumors and those with multifocal tumors were likely to undergo subsequent intravesical recurrence; however, there was no significant impact of other factors on subsequent intravesical recurrence, including age, tumor side, tumor location, surgical modality, operation time, management of the distal ureter, tumor grade, lymph node metastasis, microvascular invasion, lymphatic invasion, and margin status. Furthermore, pathologic stage and tumor multifocality were identified as independent predictors for the development of recurrent bladder cancer by multivariate analyses. CONCLUSIONS The incidence of intravesical recurrence after nephroureterectomy for upper urinary tract TCC is comparatively high. It could be important to perform careful follow-up targeting intravesical recurrence for such patients after nephroureterectomy, particularly those with low-stage tumors and/or multifocal tumors.

Expression of the secreted form of clusterin protein in renal cell carcinoma as a predictor of disease extension

To evaluate the significance of clusterin expression in surgically resected renal cell carcinoma (RCC) specimens.

Expression of potential molecular markers in prostate cancer: correlation with clinicopathological outcomes in patients undergoing radical prostatectomy.

The objective of this study was to evaluate the expression levels of multiple potential molecular markers in prostate cancer to clarify the significance of these markers as prognostic indicators in patients undergoing radical prostatectomy (RP). This study included a total of 193 patients with clinically organ-confined prostate cancer who underwent RP without any neoadjuvant therapies. Expression levels of 12 proteins, including Ki-67, p53, androgen receptor (AR), matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor, Aurora-A, Bcl-2, clusterin, heat shock protein 27 (HSP27), HSP70, and HSP90, in RP specimens obtained from these 193 patients were measured by immunohistochemical staining. Of the 12 molecules, Ki-67, p53, AR, MMP-2, MMP-9, and HSP27 expression were significantly associated with several conventional prognostic factors. Univariate analysis identified these 6 markers as significant predictors for biochemical recurrence as well, while prostate-specific antigen, Gleason score, seminal vesicle invasion (SVI), surgical margin status (SMS), lymph node metastasis, and tumor volume were also significant. Of these significant factors, Ki-67 expression, SVI, and SMS appeared to be independently related to biochemical recurrence by multivariate analysis. Furthermore, there were significant differences in biochemical recurrence-free survival according to positive numbers of these three independent risk factors. These findings suggest that consideration of expression levels of potential molecular markers in RP specimens, in addition to conventional prognostic parameters, would contribute to accurate prediction of biochemical recurrence following RP in patients with clinically localized prostate cancer, and that combined evaluation of Ki-67 expression, SVI, and SMS would be particularly useful for further refinement of the system in predicting biochemical outcome.

GLI2 Knockdown Using an Antisense Oligonucleotide Induces Apoptosis and Chemosensitizes Cells to Paclitaxel in Androgen-Independent Prostate Cancer

Purpose: GLI transcription factors mediate hedgehog signaling and have been implicated in several human malignancies, including prostate cancer. The objectives of this study were to characterize GLI2 expression levels in human prostate cancer cell lines and tissues to test the effect of antisense oligonucleotide (ASO) targeting GLI2 on androgen-independent (AI) prostate cancer cell lines. Experimental Design: A tissue microarray was used to characterize differences in GLI2 expression in benign prostate hyperplasia, prostate cancer treated by neoadjuvant hormonal therapy and AI prostate cancer. The effects of GLI2 ASO on PC-3 cell growth and paclitaxel chemosensitivity were assessed in vitro and in vivo. Oligonucleotide spotted microarray analysis was used to determine alteration in GLI2 coregulated genes after ASO treatment. Results: The expression of GLI2 was significantly higher in prostate cancer than in benign prostate hyperplasia, decreased after androgen ablation in a time-dependent fashion, but became highly expressed again in AI prostate cancer. GLI2 ASO treatment of PC-3 cells reduced GLI2 mRNA and protein levels in a dose-dependent manner. GLI2 knockdown increased PC-3 cell apoptotic rates and significantly decreased cell growth and modulated levels of apoptosis-related genes, such as Bcl2, Bcl-xL, and clusterin. GLI2 knockdown also changed levels of several cell cycle regulators, such as cyclin D1, p27, and PKC-η. Systematic administration of GLI2 ASO in athymic mice significantly delayed PC-3 tumor progression and enhanced paclitaxel chemosensitivity. Conclusions: These findings suggest that increased levels of GLI2 correlates with AI progression and that GLI2 may be a therapeutic target in castrate-resistant prostate cancer.

Abnormalities of thyroid function in Japanese patients with metastatic renal cell carcinoma treated with sorafenib: a prospective evaluation.

The objective of this study was to characterize features of thyroid dysfunction in Japanese patients with metastatic renal cell carcinoma (RCC) who were treated with sorafenib. We performed a prospective observational study including 69 Japanese patients who were diagnosed as having metastatic RCC refractory to cytokine therapy and subsequently treated with sorafenib for at least 12 weeks. Thyroid function was assessed before and every 4 weeks after the initiation of sorafenib treatment. Of the 69 patients, 23 (33.3%) did not show any biochemical thyroid abnormality, while the remaining 46 (67.7%) developed hypothyroidism. However, 11 (23.9%) of these 46 hypothyroid patients initially had a suppressed thyroid-stimulating hormone (TSH) value accompanying the increase in free triiodothyronine (T3) and/or free thyroxine (T4) before developing hypothyroidism, suggesting sorafenib-induced thyroiditis. During the observation period of this study, 4 patients (5.8%) demonstrated severe clinical symptoms caused by hypothyroidism and received thyroid hormone replacement. Among several factors examined, only age was significantly associated with the risk for hypothyroidism. These findings suggest that although the incidence of clinically significant hypothyroidism requiring thyroid hormone replacement therapy was not very high, biochemical thyroid abnormality was frequently observed in Japanese RCC patients treated with sorafenib. Accordingly, regular surveillance of thyroid function by the measurement of TSH, free T3, and T4 is warranted during sorafenib treatment in Japanese RCC patients.

Expression of urokinase-type plasminogen activator system in prostate cancer: Correlation with clinicopathological outcomes in patients undergoing radical prostatectomy

The objective of this study was to evaluate the expression levels of urokinase-type plasminogen activator (uPA) system in radical prostatectomy (RP) specimens in order to clarify the significance of the uPA system in prostate cancer. Expression levels of uPA, uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and PAI-2 in RP specimens obtained from 153 patients with clinically organ-confined prostate cancer who had not received any neoadjuvant therapies were evaluated by immunohistochemical staining. Various expression levels of uPA, uPAR, PAI-1, and PAI-2 were noted in the majority of prostate cancer specimens. Expression levels of uPA and uPAR were significantly associated with major prognostic indicators, including pathological stage, Gleason score, lymphatic invasion, surgical margin status and lymph node metastasis. However, PAI-1 expression was related to only pathological stage and surgical margin status, and there was no significant association between the expression level of PAI-2 and several parameters examined. Despite the lack of prognostic significance in PAI-2 expression, biochemical recurrence-free survival of patients with strong uPA, uPAR, and PAI-1 expression was significantly lower than that of those with weak uPA, uPAR, and PAI-1 expression, respectively. Furthermore, strong expression of uPA in addition to a Gleason score, positive surgical margin, and lymph node metastasis could be independent predictors for biochemical recurrence after RP. These findings suggest that the uPA system may be involved in the progression of prostate cancer, and that the expression level of uPA in prostate cancer tissue could be used as a useful predictor of biochemical recurrence in patients undergoing RP.

Expression profile of E-cadherin and N-cadherin in non-muscle-invasive bladder cancer as a novel predictor of intravesical recurrence following transurethral resection

The objective of this study was to investigate the impact of the expression profile of E-cadherin and N-cadherin in newly diagnosed non-muscle-invasive bladder cancer (NMIBC) on the probability of intravesical recurrence in patients undergoing transurethral resection (TUR). This study included 115 consecutive patients diagnosed as having NMIBC following TUR. Expression levels of E-cadherin and N-cadherin in TUR specimens from these patients were measured by immunohistochemical staining. In this series, intravesical recurrence occurred in 35 of 115 patients (30.4%). Immunohistochemical study showed that positive expression of E-cadherin and N-cadherin were noted in 62 (53.9%) and 48 (41.7%) specimens, respectively. Intravesical recurrence was detected in only 7 of 62 patients (11.3%) with positive E-cadherin expression, while 33 of 48 patients (68.8%) with positive N-cadherin expression developed intravesical recurrence. When patients were divided into 4 groups according to the positivities of E-cadherin and N-cadherin expression, intravesical recurrence was detected in 27 of 30 patients (90.0%) with negative E-cadherin as well as positive N-cadherin expression, and the intravesical recurrence-free survival of this group was significantly poorer than those of the remaining 3 groups. Furthermore, negative E-cadherin as well as positive N-cadherin expression was identified as the most powerful independent predictor for intravesical recurrence following TUR on multivariate analysis. These findings suggest that the loss of E-cadherin and gain of N-cadherin expression in on NMIBC appeared to be significantly associated with postoperative recurrence; therefore, the switch from E-cadherin to N-cadherin expression might be involved in the mechanism underlying intravesical recurrence of on NMIBC.

Comparison of the Transperitoneal and Retroperitoneal Approach in Robot-Assisted Partial Nephrectomy in an Initial Case Series in Japan

PURPOSE To compare the results from the transperitoneal and retroperitoneal approaches in our initial case series of robot-assisted partial nephrectomy (RAPN) in terms of surgical time, renal artery clamping time, postoperative renal function, adverse events, and surgical margin status. PATIENTS AND METHODS The initial 26 consecutive RAPNs performed for solid renal tumors in our hospital were categorized by the approach used, transperitoneal or retroperitoneal, and compared for body mass index, tumor size, R.E.N.A.L. nephrometry score, PADUA score, tumor location, surgical time, renal artery clamping time, renal function change after surgery, operative blood loss, surgical margin status, and adverse events (AEs). RESULTS The median tumor size was 25 mm (range 15-50). A transperitoneal approach was used in 16 patients and a retroperitoneal approach was used in 10 patients. There was no significant difference in renal tumor and patient characteristics between the two groups except tumor location (anterior tumor was significantly more in the transperitoneal approach and posterior tumor was significantly more in retroperitoneal approach (P=0.0144 and P=0.0100, respectively)). Operative time (239 ± 63.0 minutes in the transperitoneal group vs. 193 ± 40.6 minutes in the retroperitoneal group), warm ischemic time (24.3 ± 9.07 minutes in the transperitoneal group vs. 24.7 ± 8.35 minutes in the retroperitoneal group) and AEs (1/16 in the transperitoneal group vs. 1/10 in the retroperitoneal group; both cases were Clavien-Dindo grade I) did not show any significant difference between the two approaches (P=0.0792, 0.5485, and 0.7270, respectively). CONCLUSIONS The retroperitoneal approach in RAPN appears to be a safe and technically feasible minimally invasive option for nephron-sparing surgery, based on our initial case series, and showed equivalent outcomes to those of the transperitoneal approach even though it was an initial robotic renal surgery series. Future studies, including a larger number of cases, are planned to draw more definitive conclusions.

Serum Level of Clusterin and Its Density in Men with Prostate Cancer as Novel Biomarkers Reflecting Disease Extension

OBJECTIVES To assess whether the serum level of clusterin and its density could be used as novel biomarkers of prostate cancer. METHODS Sera were obtained from 380 patients with prostate cancer and 120 with benign prostatic hyperplasia. Serum clusterin level was measured by a sandwich enzyme immunoassay, and clusterin density, which was determined by dividing the serum clusterin level by the prostate volume, was also calculated. These findings were analyzed with respect to several clinicopathologic factors. RESULTS The mean serum level of clusterin in prostate cancer patients was significantly higher than that in the benign prostatic hyperplasia group. Both the serum clusterin level and clusterin density in prostate cancer patients were significantly associated with major prognostic factors other than biopsy Gleason score. Of the 380 prostate cancer patients, 162 underwent radical prostatectomy and pelvic lymphadenectomy, and 104 and 58 were diagnosed as having organ-confined and extraprostatic diseases, respectively. The clusterin density in patients with organ-confined disease was significantly higher than that in patients with extraprostatic disease; however, there was no significant difference in the serum clusterin level between these 2 groups. Furthermore, biochemical recurrence-free survival in patients with elevated clusterin density was significantly lower than that in patients with normal density. CONCLUSIONS These findings suggest that serum clusterin level and its density could serve as a useful practical adjuncts to conventional parameters for estimating the extension of prostate cancer.