Myc upregulates Ggct, γ-glutamylcyclotransferase to promote development of p53-deficient osteosarcoma.

Osteosarcoma (OS) in humans is characterized by alterations in the TP53 gene. In mice, loss of p53 triggers OS development, for which c-Myc (Myc) oncogenicity is indispensable. However, little is known about which genes are targeted by Myc to promote tumorigenesis. Here, we examined the role of γ-glutamylcyclotransferase (Ggct) which is a component enzyme of the γ-glutamyl cycle essential for glutathione homeostasis, in human and mouse OS development. We found that GGCT is a poor prognostic factor for human OS, and that deletion of Ggct suppresses p53-deficient osteosarcomagenesis in mice. Myc upregulates Ggct directly by binding to the Ggct promoter, and deletion of a Myc binding site therein by genome editing attenuated the tumorigenic potential of p53-deficient OS cells. Taken together, these results show a rationale that GGCT is widely upregulated in cancer cells and solidify its suitability as a target for anticancer drugs.

Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

Clinical, pathological, and therapeutic features of newly diagnosed prostate cancer predominantly detected by opportunistic PSA screening: A survey of Shiga Prefecture, Japan.

BACKGROUND: In all the prefectures of Japan, with the exception of Shiga Prefecture, more than half of local governments have an organized prostate-specific antigen (PSA) screening system in place. However, in the Shiga Prefecture, only a single city performed PSA screening over the time period of this survey. The purpose of the present study was to determine the clinical, pathological, and therapeutic features of newly diagnosed prostate cancer in localities where a formally organized screening system was almost entirely absent. METHODS: A multicenter observational study was conducted in the Shiga Prefecture, which has the lowest rate of population-based PSA-screening in Japan. Patients' age, initial PSA, reasons for PSA testing, Gleason score, clinical stage, and primary treatments were surveyed. We stratified patients according to the reasons for PSA measurement, and compared the differences between groups subject to organized versus opportunistic screening. RESULTS: In the 2 years 2012 and 2017, 984 newly diagnosed prostate cancer patients were analyzed. Of these, 954 (97%) were opportunistically tested (i.e., not as part of an organized screening system), with the remaining 29 (3%) measured as part of an organized screening program. Patients in the former group exhibited a higher initial PSA value than in the organized screening group (median: 11.49 vs. 5.67 ng/ml). They also had worse clinical features, including higher Gleason score and TNM stage. More patients in the organized screening group were treated curatively than in the nonorganized screening group in terms of the primary treatment. The results were similar in a subanalysis of the patients of age 50-69 years. CONCLUSIONS: Organized PSA screening contributes to increasing the number of patients diagnosed with early-stage cancer who can be treated curatively.

Differences in the Central Energy Metabolism of Cancer Cells between Conventional 2D and Novel 3D Culture Systems.

The conventional two-dimensional (2D) culture is available as an in vitro experimental model. However, the culture system reportedly does not recapitulate the in vivo cancer microenvironment. We recently developed a tissueoid cell culture system using Cellbed, which resembles the loose connective tissue in living organisms. The present study performed 2D and three-dimensional (3D) culture using prostate and bladder cancer cell lines and a comprehensive metabolome analysis. Compared to 3D, the 2D culture had significantly lower levels of most metabolites. The 3D culture system did not impair mitochondrial function in the cancer cells and produce energy through the mitochondria simultaneously with aerobic glycolysis. Conversely, ATP production, biomass (nucleotides, amino acids, lipids and NADPH) synthesis and redox balance maintenance were conducted in 3D culture. In contrast, in 2D culture, biomass production was delayed due to the suppression of metabolic activity. The 3D metabolome analysis using the tissueoid cell culture system capable of in vivo cancer cell culture yielded results consistent with previously reported cancer metabolism theories. This system is expected to be an essential experimental tool in a wide range of cancer research fields, especially in preclinical stages while transitioning from in vitro to in vivo.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

A risk stratification model based on four novel biomarkers predicts prognosis for patients with renal cell carcinoma.

BACKGROUND: Accurate prediction of the prognosis of RCC using a single biomarker is challenging due to the genetic heterogeneity of the disease. However, it is essential to develop an accurate system to allow better patient selection for optimal treatment strategies. ARL4C, ECT2, SOD2, and STEAP3 are novel molecular biomarkers identified in earlier studies as survival-related genes by comprehensive analyses of 43 primary RCC tissues and RCC cell lines. METHODS: To develop a prognostic model based on these multiple biomarkers, the expression of four biomarkers ARL4C, ECT2, SOD2, and STEAP3 in primary RCC tissue were semi-quantitatively investigated by immunohistochemical analysis in an independent cohort of 97 patients who underwent nephrectomy, and the clinical significance of these biomarkers were analyzed by survival analysis using Kaplan-Meier curves. The prognostic model was constructed by calculation of the contribution score to prognosis of each biomarker on Cox regression analysis, and its prognostic performance was validated. RESULTS: Patients whose tumors had high expression of the individual biomarkers had shorter cancer-specific survival (CSS) from the time of primary nephrectomy. The prognostic model based on four biomarkers segregated the patients into a high- and low-risk scored group according to defined cut-off value. This approach was more robust in predicting CSS compared to each single biomarker alone in the total of 97 patients with RCC. Especially in the 36 metastatic RCC patients, our prognostic model could more accurately predict early events within 2 years of diagnosis of metastasis. In addition, high risk-scored patients with particular strong SOD2 expression had a much worse prognosis in 25 patients with metastatic RCC who were treated with molecular targeting agents. CONCLUSIONS: Our findings indicate that a prognostic model based on four novel biomarkers provides valuable data for prediction of clinical prognosis and useful information for considering the follow-up conditions and therapeutic strategies for patients with primary and metastatic RCC.

Depletion of gamma-glutamylcyclotransferase inhibits cancer cell growth by activating the AMPK-FOXO3a-p21 axis.

Inhibition of gamma-glutamylcyclotransferase (GGCT), which is highly expressed in various cancer tissues, exerts anticancer effects both in vitro and in vivo. Previous studies have shown that depletion of GGCT blocks the growth of MCF7 breast cancer cells via upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21); in addition, induction of autophagy plays a role in the upregulation of p21 upon GGCT knockdown. However, the mechanisms underlying induction of p21 in cancer cells are not fully understood. Here, we show that GGCT knockdown in PC3 human prostate cancer and A172 glioblastoma cells upregulates the mRNA and nuclear protein levels of Forkhead box O transcription factor 3a (FOXO3a), a transcriptional factor involved in tumor suppression. Simultaneous knockdown of FOXO3a and GGCT in PC3 and A172 cells attenuated upregulation of p21, followed by growth inhibition and cell death. Furthermore, simultaneous knockdown of GGCT and AMP-activated protein kinase (AMPK) α, a metabolic stress sensor, in PC3 and A172 cells led to marked attenuation of cellular responses induced by GGCT knockdown, including an increase in FOXO3a phosphorylation at Ser413, upregulation of p21, growth inhibition, and cell death. These results indicate that the AMPK-FOXO3a-p21 axis plays an important role in inhibition of cancer cell growth by depletion of GGCT.

Pediatric voiding cystourethrography: An essential examination for urologists but a terrible experience for children.

Voiding cystourethrography is the most important fluoroscopic examination in pediatric urology for the investigation of lower urogenital tract diseases, such as vesicoureteral reflux or urethral stricture. However, this invasive procedure imposes a significant burden on children and their parents, and recently there has been a paradigm shift in the diagnosis and treatment of vesicoureteral reflux. In the 2011 revision, the American Academy of Pediatrics guidelines on urinary tract infection recommended abandoning routine voiding cystourethrography after the first febrile urinary tract infection. In 2014, the randomized intervention for children with vesicoureteral reflux study recommended discontinuation of routine continuous antibiotic prophylaxis for vesicoureteral reflux. The time is now ripe to radically reconsider indications for voiding cystourethrography and the procedure itself.

Prohibitin-2 is a novel regulator of p21WAF1/CIP1 induced by depletion of γ-glutamylcyclotransferase.

Previous studies show that gamma-glutamylcyclotransferase (GGCT) is expressed at high levels in various cancer tissues and that its knockdown inhibits MCF7 cancer cell growth via upregulation of p21WAF1/CIP1 (p21). However, the detailed underlying mechanism is unclear. Here, we used yeast two-hybrid screening and co-immunoprecipitation to identify Prohibitin-2 (PHB2) as a novel protein that interacts with GGCT. We also show that nuclear expression of PHB2 in MCF7 cells falls upon GGCT knockdown, and that overexpression of PHB2 inhibits p21 upregulation. A chromatin immunoprecipitation assay revealed that nuclear PHB2 proteins bind to the p21 promoter, and that this interaction is abrogated by GGCT knockdown. Moreover, knockdown of PHB2 alone led to significant upregulation of p21 and mimicked the cellular events induced by GGCT depletion, including G0/G1 arrest, cellular senescence, and growth inhibition, in a p21 induction-dependent manner. Taken together, the results indicate that PHB2 plays a central role in p21 upregulation following GGCT knockdown and as such may promote deregulated proliferation of cancer cells by suppressing p21.

A comparison of nephrotoxicity between patients with a solitary-functioning kidney and those with bilateral-functioning kidneys in cisplatin-based chemotherapy for advanced urothelial carcinoma: a Japanese retrospective multi-institutional study.

BACKGROUND: To compare the prevalence of nephrotoxicity between patients with a solitary-functioning kidney versus those with bilateral-functioning kidneys during the administration of cisplatin-based chemotherapy for advanced urothelial carcinoma. METHODS: We retrospectively analyzed 244 advanced urothelial carcinoma patients treated with cisplatin-based chemotherapy between 2004 and 2010 at 17 institutes in Japan. The 24 h creatinine clearance, Cockcroft-Gault formula, and estimated glomerular filtration rate equation (eGFR), were compared before all chemotherapies. The urinary tract function status was determined based on the data of nephroureterectomy, hydronephrosis, and relief of upper urinary tract obstruction. A total of 244 patients were divided into four groups according to their urinary tract functioning status and eGFR results, including bilateral-functioning kidneys with pretreatment eGFR ≥60 mL/min/1.73 m2 group (n = 83, 34.0%); a solitary-functioning kidney with pretreatment eGFR ≥60 mL/min/1.73 m2 group (n = 36, 14.8%); bilateral-functioning kidneys with pretreatment eGFR < 60 mL/min/1.73 m2 group (n = 45, 18.4%); and a solitary-functioning kidney with pretreatment eGFR < 60 mL/min/1.73 m2 group (n = 80, 32.8%). RESULTS: The prevalence of nephrotoxicity with impaired eGFR of > 10% and 30% from baseline in the post-third-course of chemotherapy was significantly higher in patients with bilateral-functioning kidneys than in those with a solitary-functioning kidney, among patients with pretreatment eGFR < 60 mL/min/1.73 m2 (p = 0.023 and p = 0.026). During all courses of chemotherapy, the prevalence of nephrotoxicity with impaired eGFR of > 20% from baseline were significantly higher in patients with bilateral-functioning kidneys than those with a solitary-functioning kidney among patients with pretreatment eGFR < 60 mL/min/1.73 m2 (p = 0.034), whereas no significant difference was observed among patients with pretreatment eGFR ≥60 mL/min/1.73 m2. CONCLUSIONS: The results suggest that cisplatin-based chemotherapy may have more nephrotoxicity in patients with bilateral-functioning kidneys than in those with a solitary-functioning kidney.

The location of the bladder neck in postoperative cystography predicts continence convalescence after radical prostatectomy.

BACKGROUND: This study was conducted to determine whether the location of the bladder neck in postoperative cystography predicts recovery of continence after radical prostatectomy. METHODS: Between 2008 and 2015, 203 patients who underwent laparoscopic radical prostatectomy (LRP, n = 99) and robot assisted radical prostatectomy (RARP, n = 104) were analyzed. The location of the bladder neck was visualized by postoperative routine cystography, and quantitative evaluation of the bladder neck position was performed according to the bladder neck to pubic symphysis (BNPS) ratio proposed by Olgin et al. (J Endourol, 2014). Recovery of continence was defined as no pad use or one security pad per day. To determine the predictive factors for recovery of continence at 1, 3, 6 and 12 months, several parameters were analyzed using logistic regression analysis, including age (≤68 vs. > 68, BMI (≤23.4 vs. > 23.4 kg/m2), surgical procedure (LRP vs. RARP), prostate volume (≤38 vs. > 38 mL), nerve-sparing technique, vesico-urethral anastomosis leakage, and BNPS ratio (≤0.59 vs. > 0.59). RESULTS: The mean postoperative follow-up was 1131 days (79-2880). At 1, 3, 6 and 12 months after surgery, continence recovery rates were 25, 53, 68 and 81%, respectively. Although older age (> 68) and RARP were significant risk factors for incontinence within 3 months, neither was significant after 6 months. A high BNPS ratio (> 0.59) was the only significant risk factor for the persistence of incontinence at all observation points, up to 12 months. CONCLUSIONS: A lower bladder neck position after prostatectomy predicts prolonged incontinence.

Intravesical bacillus Calmette-Guerin therapy after second transurethral resection for primary T1 bladder cancer.

BACKGROUND: To evaluate the effect of intravesical bacillus Calmette-Guerin (BCG) instillation therapy after second transurethral resection (TUR) on primary T1 bladder cancer. METHODS: The subjects were 180 patients diagnosed with T1 bladder cancer at our university and at affiliated hospitals between January 1990 and December 2015. Tumor residual rate, intravesical recurrence rate, and risk factors for intravesical recurrence were investigated. RESULTS: The median follow-up period was 26 (1-175) months. Of the 180 patients, 78 (43%) underwent a second TUR. Residual tumors were detected in 42 patients (53.8%), and no up-staging cases were observed. Within the whole group, 42 patients were treated with intravesical BCG therapy following a second TUR (group 1), 36 were treated with second TUR alone (group 2), 28 were treated with intravesical BCG therapy alone (group 3), and 74 were treated without second TUR or intravesical BCG therapy (group 4). The 1- and 5-year recurrence-free survival rates of the four groups were 80.7 and 59.7% (group 1), 69.0 and 26.3% (group 2), 76.3 and 56.6% (group 3), 64.6 and 48.6% (group 4), respectively. There was no significant difference between group 1 and group 3 (p = 0.401). Intravesical BCG therapy was the only factor preventing intravesical recurrence (p = 0.013). CONCLUSIONS: Intravesical BCG therapy alone showed a significant preventive effect with regard to intravesical recurrence. In our cohort, however, second TUR did not improve recurrence-free survival in those individuals who underwent BCG instillation.

Urological laparoendoscopic single-site and reduced port surgery: A nationwide survey in Japan.

OBJECTIVES: To evaluate the current status of urological laparoendoscopic single-site and reduced port surgery in Japan. METHODS: Of the 152 institutions to which councilors of the Japanese Society of Endourology belong, 42 (28%) have carried out laparoendoscopic single-site and reduced port surgery. A total of 32 of these institutions agreed to participate in this survey. Patients who had undergone surgery between January 2008 and March 2014 were included in the present study. RESULTS: Overall, 1145 cases of laparoendoscopic single-site and reduced port surgery were recorded during the study period. The most frequent procedures were adrenalectomy and radical nephrectomy. Laparoendoscopic single-site and reduced port surgery represented 12% (872/7311) of all laparoscopic procedures carried out at participating institutions. The number of patients who underwent pyeloplasty, donor nephrectomy and simple nephrectomy tended to increase, whereas those who underwent adrenalectomy, radical nephrectomy and nephroureterectomy peaked in 2012, and then tended to decrease in 2013. The rates of conversion, perioperative and postoperative complications, were 2.7%, 2.2% and 4.5%, respectively. CONCLUSIONS: The number of laparoendoscopic single-site and reduced port urological surgeries in Japan has increased for benign indications, such as pyeloplasty, donor nephrectomy and simple nephrectomy. In contrast, procedures such as adrenalectomy and radical nephrectomy are trending down after reaching a peak in 2012. Overall, laparoendoscopic single-site and reduced port urological surgery in Japan is being safely carried out when compared with other reported series of laparoendoscopic single-site surgery and conventional laparoscopic surgery.

Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy.

γ-Glutamylcyclotransferase (GGCT), which is one of the major enzymes involved in glutathione metabolism, is upregulated in a wide range of cancers-glioma, breast, lung, esophageal, gastric, colorectal, urinary bladder, prostate, cervical, ovarian cancers and osteosarcoma-and promotes cancer progression; its depletion leads to the suppression of proliferation, invasion, and migration of cancer cells. It has been demonstrated that the suppression or inhibition of GGCT has an antitumor effect in cancer-bearing xenograft mice. Based on these observations, GGCT is now recognized as a promising therapeutic target in various cancers. This review summarizes recent advances on the mechanisms of the antitumor activity of GGCT inhibition.

Impact of acute kidney injury defined by CTCAE v4.0 during first course of cisplatin-based chemotherapy on treatment outcomes in advanced urothelial cancer patients.

BACKGROUND: The Kidney Disease: Improving Global Outcomes group (KDIGO) defined acute kidney injury (AKI) as an elevation of serum creatinine (sCR) exceeding 0.3 mg/dl within 48 h. The widely used adverse events criteria for chemotherapy, Common Toxicity Criteria for Adverse Events Version 4.0 (CTCAE v4.0), also defined AKI as sCR exceeding 0.3 mg/dl, but with no provision of a time course. Here, we attempted to clarify the impact of AKI (CTCAE v4.0) during cisplatin-based chemotherapy on clinical outcome of patients with advanced urothelial cancer (UC). METHODS: This multicenter retrospective study included 230 UC patients who received cisplatin-based chemotherapy. RESULTS: During the first chemotherapy course, AKI (CTCAE v4.0) episodes were observed in 61 patients (26.5 %), whereas only four patients (1.5 %) experienced AKI (KDIGO) episodes. Both the pretreatment estimated glomerular filtration rate (eGFR) and creatinine clearance by Cockcroft-Gault formula were not efficient predictors for the development of AKI (CTCAE v4.0). AKI (CTCAE v4.0) impacted renal function: at the start of second-course chemotherapy, the average eGFR of the patients with AKI (CTCAE v4.0) was 54.1 ml/min/1.73 m2, significantly lower than that of patients without AKI (CTCAE v4.0) (63.4 ml/min/1.73 m2). As a result, only 57.4 % of patients with AKI (CTCAE v4.0) received the planned treatment at the second course. The survival of the patients who developed AKI (CTCAE v4.0) was significantly worse than that of the patients who did not. The 3-year OSs were 10.3 and 21.4 %, respectively (P = 0.02). CONCLUSION: The present study demonstrated that AKI (CTCAE v4.0) during chemotherapy had a negative impact on both the intensity of subsequent chemotherapy and oncological outcomes.

[Locally Advanced Ureteral Cancer Seen in an Ectopic Kidney : A Case Report].

An ectopic kidney is a common congenital anomaly of the urogenital system, but malignant tumor in an ectopic kidney has been rarely reported. We report a case of ureteral carcinoma arising from an ectopic kidney in an 83-year-old male. He visited a hospital complaining of gross hematuria. Computed tomography revealed right ectopic kidney, right ureteral tumor and bladder tumor around the right ureteral orifice. Transurethral resection of the bladder tumor was performed and histopathological diagnosis was urothelial carcinoma. He was referred to our clinic for surgery of the right ureteral tumor. We performed open right nephroureterectomy and partial cystectomy. The histopathological diagnosis was a high grade urothelial carcinoma of the right ureter, pT3N0. Four months postoperatively, there was no evidence of recurrence. We discuss the clinical and pathological features of the malignancy in an ectopic kidney.

The Effect of D-Aspartate on Spermatogenesis in Mouse Testis.

Spermatogenesis is controlled by hormonal secretions from the hypothalamus and pituitary gland, by factors produced locally in the testis, and by direct interaction between germ cells and Sertoli cells in seminiferous tubules. Although the mammalian testis contains high levels of D-aspartate (D-Asp), and D-Asp is known to stimulate the secretion of testosterone in cultured Leydig cells, its role in testis is unclear. We describe here biochemical, immunohistochemical, and flow cytometric studies designed to elucidate developmental changes in testicular D-Asp levels and the direct effect of D-Asp on germ cells. We found that the concentration of D-Asp in mouse testis increased with growth and that fluctuations in D-Asp levels were controlled in part by its degradative enzyme, D-aspartate oxidase expressed in Sertoli cells. In vitro sperm production studies showed that mitosis in premeiotic germ cells was strongly inhibited by the addition of D-Asp to the culture medium. Moreover, immunohistochemical analysis demonstrated that d-Asp accumulated in the differentiated spermatids, indicating either transport of D-Asp to spermatids or its de novo synthesis in these cells. Such compartmentation seems to prevent premeiotic germ cells in mouse testis from being exposed to the excess amount of D-Asp. In concert, our results indicate that in mouse testis, levels of D-Asp are regulated in a spatiotemporal manner and that D-Asp functions as a modulator of spermatogenesis.

Depletion of γ-glutamylcyclotransferase inhibits breast cancer cell growth via cellular senescence induction mediated by CDK inhibitor upregulation.

BACKGROUND: Chromosome 7 open reading frame 24 (C7orf24) was originally identified as a highly expressed protein in various types of cancer, and later shown to be a γ-glutamylcyclotransferase (GGCT). GGCT depletion in cancer cells has anti-proliferative effects in vitro and in vivo, and it is therefore considered a promising candidate as a therapeutic target. However, the cellular events induced by GGCT depletion remain unclear. METHODS: GGCT was depleted by siRNA in MCF7, MDA-MB-231, PC3, A172, Hela, and LNCaP cells. Induction of cellular senescence was evaluated with senescence-associated ß-galactosidase (SA-ß-Gal) staining. Expression levels of p21WAF1/CIP1 and p16INK4A were assessed by qRT-PCR and Western blotting. Effects of simultaneous double knockdown of p21WAF1/CIP1 and p16INK4A together with GGCT on cell cycle regulation and cell growth was measured by flow cytometry, and trypan blue dye exclusion test. RESULTS: We found that GGCT knockdown induces significant cellular senescence in various cancer cells. Cyclin dependent kinase inhibitor p21WAF1/CIP1 and/or p16INK4A were upregulated in all cell lines tested. Simultaneous knockdown of p21WAF1/CIP1 recovered the cell cycle arrest, attenuated cellular senescence induction, and rescued the subsequent growth inhibition in GGCT-silenced MCF7 breast cancer cells. In contrast, in GGCT silenced MDA-MB-231 breast cancer cells, GGCT depletion upregulated p16INK4A, which played a regulatory role in senescence induction, instead of p21WAF1/CIP1. CONCLUSIONS: Our findings demonstrate that induction of cellular senescence mediated by the upregulation of cyclin-dependent kinase inhibitors is a major event underlying the anti-proliferative effect of GGCT depletion in breast cancer cells, highlighting the potential of GGCT blockade as a therapeutic strategy to induce cellular senescence.

Do metastatic upper tract urothelial carcinoma and bladder carcinoma have similar clinical responses to systemic chemotherapy? A Japanese multi-institutional experience.

OBJECTIVE: There has been no clear evidence supporting similar chemo-responses for upper and lower urothelial carcinomas. METHODS: We conducted a multicenter retrospective cohort study to analyze urothelial carcinoma patients who underwent systemic chemotherapy at 17 centers from 2004 to 2010. A total of 298 patients with either urothelial carcinoma of the bladder (N = 151) or upper tract urothelial carcinoma (N = 147) were included. Differences in tumor location (urothelial carcinoma of the bladder vs. upper tract urothelial carcinoma) were evaluated in relation to the patient backgrounds and clinical responses to systemic chemotherapy. RESULTS: Overall 216 patients were treated with cisplatin-based chemo-regimens (gemcitabine and cisplatin in 92, or methotrexate, vinblastine, adriamycin and cisplatin/methotrexate, epirubicin and cisplatin in 124). Among 186 initially metastatic patients, the incidences of lung metastasis and liver metastasis were 39.2 and 34.1%, respectively, in upper tract urothelial carcinoma patients, and were significantly higher than those with urothelial carcinoma of the bladder (22.4% for lung; 8.4% for liver metastasis). Among 112 post-surgical recurrent/metastatic patients, age was significantly higher and estimated glomerular filtration rate at baseline was significantly lower in upper tract urothelial carcinoma patients than those with urothelial carcinoma of the bladder. No significant differences were observed in overall clinical response rates for systemic chemotherapy between urothelial carcinoma of the bladder (45.8%) and upper tract urothelial carcinoma (38%) in initially metastatic patients or between urothelial carcinoma of the bladder (43.2%) and upper tract urothelial carcinoma (44.1%) in post-surgical recurrent/metastatic patients. Tumor location was not independently associated with cancer-specific survival in either initially metastatic or post-surgical recurrent/metastatic urothelial carcinoma patients. CONCLUSIONS: No significant difference was observed in response rates of urothelial carcinoma of the bladder and upper tract urothelial carcinoma to systemic chemotherapy, suggesting that a similar chemo-regimen can be applied to metastatic urothelial carcinoma patients regardless of tumor location (upper vs. lower).