Purpose: This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. Experimental Design: We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. Results: HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6. The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G₂-M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. Conclusions: Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G₂-M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

The polycomb group gene, EZH2, is highly expressed in advanced bladder cancer. Here we demonstrated that down-regulation of EZH2 in tumor tissues after neo-adjuvant chemotherapy correlated with good therapeutic response in advanced bladder cancer. We next developed a small molecule, NSC745885, derived from natural anthraquinone emodin, which down-regulated EZH2 via proteasome-mediated degradation. NSC745885 showed potent selective toxicity against multiple cancer cell lines but not normal cells. NSC745885 treatment overcame multiple-drug resistance and inhibited growth of resistant cancer cells. Over-expression of EZH2 in cancer cells attenuated effects of NSC745885, suggesting that down-regulation of EZH2 was responsible for growth inhibition of NSC745885. NSC745885 also suppressed tumor growth and down-regulated EZH2 in vivo. These results indicate that NSC7455889 suppresses bladder cancer by targeting EZH2.

The aim of this study was to investigate the effects of metformin supplementation on metabolic dysfunction, testicular antioxidant capacity, apoptosis, inflammation and spermatogenesis in male mice with high-fat and high-cholesterol diet-induced obesity. Forty male C57BL/6 mice were fed a normal diet (NC group, n = 10) or a high-fat and high-cholesterol diet (HFC group, n = 30) for 24 weeks, and mice randomly chosen from the HFC group were later treated with metformin for the final 8 weeks of HFC feeding (HFC + Met group, n = 15). Compared with the HFC group, the obese mice supplemented with metformin exhibited improved blood cholesterol, glucose and insulin resistance. The HFC group diminishes in the sperm motility and normal sperm morphology, while the poorer maturity of testicular spermatogenesis was improved by metformin treatment. The HFC group exhibited a higher estradiol level and a lower 17β-HSD protein expression. Further analyses showed that metformin treatment increased the activities of superoxide dismutase, catalase and glutathione peroxidase and reduced lipid peroxidation. Nevertheless, both the HFC and HFC + Met groups exhibited increased expressions of apoptosis and inflammation proteins in the testis. Metformin treatment ameliorated obesity-induced poor testicular spermatogenesis and semen quality through increasing the testosterone level and antioxidant capacity.

Histone deacetylase inhibitors (HDACIs) have potent anti-cancer activity in a variety of cancer models. Understanding the molecular mechanisms involved in the therapeutic responsiveness of HDACI is needed before its clinical application. This study aimed to determine if a potent HDACI, LBH589 (Panobinostat), had differential therapeutic responsiveness towards LNCaP and PC-3 prostate cancer (PCa) cells. The former showed prometaphase arrest with subsequent apoptosis upon LBH589 treatment, while the latter was less sensitive and had late G2 arrest. The LBH589 treatment down-regulated HDAC6 and sustained ERK activation, and contributed to prometaphase arrest. Mechanistically, LBH589 inhibited HDAC6 activity, caused its dissociation from protein phosphatase PP1α, and increased 14-3-3ζ acetylation. Acetylated 14-3-3ζ released its mask effect on serine 259 of c-Raf and serine 216 of Cdc25C subsequent to de-phosphorylation by PP1α, which contributed to ERK activation. Enhanced ERK activity by LBH589 further down-regulated HDAC6 protein levels and sustained ERK activation by free-forward regulation. The sustained Cdc25C and ERK activation resulted in early M-phase (prometaphase) arrest and subsequent apoptosis in the most sensitive LNCaP cells but not in PC-3 cells. This study provides pre-clinical evidence that HDAC6 may serve as a sensitive therapeutic target in the treatment of prostate cancer with HDACI LBH589 for clinical translation. This study also posits a novel mechanism of HDAC6 participation in regulating the c-Raf-PP1-ERK signaling pathway and contributing to M phase cell-cycle transition.

To determine whether a relationship between obesity and varicocele occurrence exists, the prevalence and severity of varicoceles related to obesity were investigated in a general population of young males. A total of 1050 young males attending the Navy Recruit Training Center were evaluated from their physical screening examinations. All subjects underwent history taking and physical examinations to evaluate for the presence and severity of varicocele. The anthropometric indexes including body mass index (BMI), waist circumference (WC) and waist‐to‐hip ratio (WHR) were recorded. All subjects were categorized by quartiles according to each anthropometric index. Means were compared with the Student’s t‐test. Severity was compared by analysis of variance testing and frequency was analysed using the chi‐square method. Statistical significance was considered at p <0.05. A total of 490 (46.67%) subjects had varicoceles. The means of BMI, WC and WHR of those without varicoceles was 23.99 ± 3.82 kg/m², 83.20 ± 9.97 cm and 0.85 ± 0.05, respectively. These judged values were greater than those with varicoceles (22.02 ± 3.18 kg/m², 79.19 ± 9.01 cm and 0.83 ± 0.05) (p < 0.001). In the univariate regression analysis, BMI, WC and WHR all had a significantly negative correlation with severity of varicocele (all p < 0.001). Analysis comparing varicocele frequency based on each grade per anthropometric index group was performed. The logistic regression revealed that the prevalence of grade II and III varicoceles showed a statistically inverse association with all three anthropometric indexes. The prevalence and severity of varicoceles inversely correlated with obesity. The present data support the explanation that obesity may result in a decreased nutcracker effect, which accounts for prevention of the renal vein compression by the adipose tissue.

Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf^S338) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK^S308), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF^C-Raf−/− or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK^S308, this drug–target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf^S338 and pDAPK^S308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK^S308 to DAPK. PP2A then dissociated from the C-Raf–DAPK complex and induced profound cancer cell death. Increased pDAPK^S308 modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK^S308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. Cancer Res; 75(17); 3568–82. ©2015 AACR.