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We investigated the prognostic significance of Death-Associated Protein Kinase 1 (DAPK1) and its role in sunitinib resistance in clear cell renal cell carcinoma (ccRCC). DAPK1 mRNA levels were significantly lower in tumor tissues than normal kidney tissues in TCGA-KIRC dataset (n=428). Both overall survival and disease-free survival were significantly shorter in ccRCC patients with low DAPK1 expression than those with high DAPK1 expression. Receiver operating characteristic curve analysis showed that low DAPK1 expression correlated with poor prognosis in ccRCC patients. Multivariate analysis confirmed that DAPK1 expression was an independent prognostic indicator in ccRCC. Gene set enrichment analysis showed that low DAPK1 expression correlates with upregulation of pathways related to metastasis, drug resistance, hypoxia and invasiveness in ccRCC patients. Sunitinib-resistant ccRCC cells show significantly lower DAPK1 mRNA and protein levels than sunitinib-sensitive ccRCC cells. DAPK1 overexpression enhances apoptosis in sunitinib-resistant ccRCC cells via the ATF6-dependent ER stress pathway. Xenograft tumors derived from DAPK1-overxpressing ccRCC cells were significantly smaller than the controls in nude mice. Our finding demonstrates that low DAPK1 expression is an independent prognostic indicator that correlates with ccRCC progression and sunitinib resistance.

Objective: Prostate cancer (PCa) refers to one of the most common tumors in male’s genitourinary system. Emerging research has confirmed that circRNAs play an important role in the occurrence and development of tumors. However, the correlation between circular RNA circITGA7 and PCa still remains unclear. Here, the role of circITGA7 in PCa was explored and the underlying mechanism was investigated as well.Methods: The circRNA expression profiles in PCa and the paracancerous tissues were established by high-throughput sequencing. The expression levels of circITGA7 in PCa tissues and cells were detected by qRT-PCR. Cell Counting Kit-8, colony formation, EdU and flow cytometry assays were used to detect the effects of circITGA7 on PCa cell proliferation. To further explore the underlying mechanisms, bioinformatics analysis on downstream target genes was carried out. RNA immunoprecipitation and dual-luciferase reporter assays were used to verify the direct relationship between miR-370-3p and circITGA7 or P21CIP1.Results: circITGA7 was downregulated in PCa tissues and cells. Gain- or loss-of-function assays showed that circITGA7 inhibited the proliferation of PCa cells in vivo and in vitro. Mechanically, circITGA7 served as a sponge for miR-370-3p and miR-370-3p could target P21CIP1 in PCa cells. The inhibition of cell proliferation induced by circITGA7 could be reversed by transfecting miR-370-3p mimic. Conclusion: Our data indicated that circITGA7 played an important role in inhibiting tumor proliferation in PCa and might be a potential therapeutic target.

Studies have demonstrated that oxidaive stress-induced apoptosis may be the main pathogenic mechanism of renal ischemia/reperfusion (I/R) injury. Theaflavin, a polyphenolic compound extracted from black tea, has been proven to exert strong antioxidant biological function. The objective of the present study was to investigate the potential role of theaflavin on renal I/R injury and its potential molecular mechanism both in vitro and in vivo. C57/BL6 J mice were used to create a model of I/R injury wherein mice were ligated with bilateral renal pedicles for 45 min, and then reperfused for 24 h. A hypoxia/reoxygenation (H/R) model of TCMK-1 cells was used to simulate I/R in vitro. Theaflavin were administered to the treatment group first and then established the model. Kidney Injury Molecule-1 (KIM-1), serum creatinine, urea nitrogen, and 24-h urinary protein levels were evaluated and changes in mitochondrial membrane potential and the ultrastructure of mitochondria were observed. Cell viability, oxidative stress damage, and apoptosis were assessed. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target genes HO-1 and NQO1 were evaluated. Our results revealed that pretreatment with theaflavin significantly inhibited I/R- and H/R-induced renal injury and cell apoptosis. Theaflavin improved mitochondrial dysfunction by attenuating mitochondrial damage and promoting mitochondrial membrane potential. Theaflavin pretreatment significantly reduced malondialdehyde content, while enhancing superoxide dismutase activity in vivo and in vitro. It also reduced oxidative stress and apoptosis mainly by upregulating Nrf2 and its downstream targets in TCMK-1 cells. Thus, theaflavin exerted a protective effect against renal I/R injury by inhibiting oxidative stress and apoptosis via activation of the Nrf2-NQO1/HO-1 pathway as well as correcting mitochondrial dysfunction, thereby presenting its potential as a clinical therapeutic in cases of acute kidney injury.

Prostate cancer (PCa) refers to one of the most common tumors in male’s genitourinary system. Emerging research has confirmed that circRNAs play an important role in the occurrence and development of tumors. However, the correlation between circular RNA circITGA7 and PCa still remains unclear. Here, the role of circITGA7 in PCa was explored and the underlying mechanism was investigated as well. The circRNA expression profiles in PCa and the paracancerous tissues were established by high-throughput sequencing. The expression levels of circITGA7 in PCa tissues and cells were detected by qRT-PCR. Cell Counting Kit-8, colony formation, EdU, and flow cytometry assays were used to detect the effects of circITGA7 on PCa cell proliferation. To further explore the underlying mechanisms, bioinformatics analysis on downstream target genes was carried out. RNA immunoprecipitation and dual-luciferase reporter assays were used to verify the direct relationship between miR-370-3p and circITGA7 or P21CIP1. The present results demonstrated that circITGA7 was downregulated in PCa tissues and cells. Gain- or loss-of-function assays showed that circITGA7 inhibited the proliferation of PCa cells in vivo and in vitro. Mechanically, circITGA7 served as a sponge for miR-370-3p, and miR-370-3p could target P21CIP1 in PCa cells. The inhibition of cell proliferation induced by circITGA7 could be reversed by transfecting miR-370-3p mimic. Collectively, our data indicated that circITGA7 played an important role in inhibiting tumor proliferation in PCa and might be a potential therapeutic target.

Objective: This study aimed to investigate the correlation of A-kinase interacting protein 1 (AKIP1) with chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2, as well as their associations with clinical characteristics and prognosis in prostate cancer patients. Methods: A total of 248 eligible prostate cancer patients who underwent surgery were consecutively recruited, and tumor tissues were collected during the surgery. AKIP1, CXCL1, and CXCL2 expression in tumor tissues were assessed by immunohistochemistry. Disease-free survival and overall survival were recorded, and the median follow-up time was 27 months. Results: The proportion of patients with AKIP1, CXCL1, and CXCL2 high expression was 56.5%, 63.7%, and 56.9%, respectively. Additionally, AKIP1 expression positively correlated with CXCL1 expression ( PConclusion: AKIP1 positively associates with CXCL1/2 and is a potential biomarker for disease monitoring as well as prognosis in prostate cancer.

The primary purpose of the current study was to investigate the antitumor activity of limonene which is a plant monoterpene along with evaluating its effects on cell apoptosis, cell cycle phase distribution, cell migration and invasion.The cell proliferation of T24 bladder cancer cells was examined by WTS-1 assay. The apoptotic effects induced by limonene were investigated by a combination of fluorescence microscopy and flow cytometry and then confirmed by western blot assay. The effects of limonene on cell cycle in T24 bladder cancer cells were studied by flow cytometry. The effects on cell migration and invasion were examined by wound healing assay and transwell assay using Matrigel.The results showed that limonene induced cytotoxic effects and reduced cell viability of T24 human bladder cancer cells showing an IC50 value of 9 μM. Limonene also induced significant apoptosis in bladder cancer cells since it induced significant nuclear fragmentation, chromatin condensation, and splitting of the nucleus, representative of the apoptotic cascade. The apoptotic cell percentage was 1.95, 5.35, 15.61 and 34.71% at limonene concentrations of 0, 9, 18 and 36 µM. Further, the apoptotic effects of limonene were also confirmed by Western blot analysis and the results showed increase in the expression of Bax and caspase-3 and decrease of Bcl-2 expression. Limonene also caused G2/M phase cell cycle arrest as well as suppression of cell migration and invasion.These results indicate that limonene might be used as a potent anticancer agent against human bladder cancer for which further in depth studies are needed, especially over its toxicological studies.

Supplemental material, Supplementary_material for A-kinase interacting protein 1, a potential biomarker associated with advanced tumor features and CXCL1/2 in prostate cancer by Danlan Wang, Yuanfang Luo, Yonglian Guo, Guohao Li and Fan Li in The International Journal of Biological Markers

Renal cell carcinoma (RCC), which was one of the most common malignant tumors in urinary system, had gradually increased incidence and mortality in recent years. Although significant advances had been made in molecular and biology research on the pathogenesis of RCC, effective treatments and prognostic indicators were still lacking. In order to predict the prognosis of RCC better, we identified 17 genes that were associated with the overall survival (OS) of RCC patients from The Cancer Genome Atlas (TCGA) dataset and a 17-gene signature was developed. Through SurvExpress, we analyzed the expression differences of the 17 genes and their correlation with the survival of RCC patients in five datasets (ZHAO, TCGA, KIPAN, KIRC, and KIRP), and then evaluated the survival prognostic significance of the 17-gene signature for RCC. Our results showed that the 17-gene signature had a predictive prognostic value not only in single pathologic RCC, but also in multiple pathologic types of RCC. In conclusion, the 17-gene signature model was related to the survival of RCC patients and could help predict the prognosis with significant clinical implications.

Type 2 diabetes mellitus (T2DM) is a common cause of erectile dysfunction (ED). It has been demonstrated that G protein-coupled receptor kinase 2 (GRK2) overexpression contributes to diabetic endothelial dysfunction and oxidative stress, which also underlies ED in T2DM. We hypothesized that GRK2 overexpressed and attenuated endothelial function of the cavernosal tissue in a rat model of T2DM. T2DM rats were established by feeding with a high-fat diet (HFD) for 2 weeks and then administering two intraperitoneal (IP) injections of a low dose of streptozotocin (STZ), followed by continuous feeding with a HFD for 6 weeks. GRK2 was inhibited by IP injection of paroxetine, a selective GRK2 inhibitor, after STZ injection. Insulin challenge tests, intracavernous pressure (ICP), GRK2 expression, the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox, nitric oxide (NO), reactive oxygen species (ROS) production, and apoptosis in cavernosal tissue were examined. Less response to insulin injection was observed in T2DM rats 2 weeks after HFD. Markedly increased GRK2 expression, along with impaired Akt/eNOS pathway, reduced NO production, increased gp91phox expression and ROS generation, increased apoptosis and impaired erectile function were found in T2DM rats. Inhibition of GRK2 with paroxetine ameliorated Akt/eNOS signaling, restored NO production, downregulated NADPH oxidase, subsequently inhibited ROS generation and apoptosis, and ultimately preserved erectile function. These results indicated that GRK2 upregulation may be an important mechanism underlying T2DM ED, and GRK2 inhibition may be a potential therapeutic strategy for T2DM ED.

Figure S1. The Effect of PCM on T Cells and Jurkat cells, Related to Fig. 1. Figure S2. ROS Scavenger and Inhibitor of CIII Could Weaken the Effect of PCM on T cells. Figure S3. ROS Scavenger and Inhibitor of CIII Could Weaken the Effect of PCM on T cells. Figure S4. The Media of RM-1 Have No Effect on SHP1 Knockdown T Cells. Figure S5. Quality Control of the RNAseq, Rlated to Fig. 3. Figure S6. Quality Control of the Metabonomics, Rlated to Fig. 5. Figure S7. The Efficiency of PRODH Knockdown and the Effect to Cell Number, and the Effect of Additional P5C on Human CD3+ T Cells, Related to Fig. 6. Figure S8. The Change of PRODH Expression Affect CD4+ and CD8+ T cells Infiltration in vivo Which Have no Influence on Nude Mice Xenograft. Table S1. The clinical information on the patients. (DOCX 3226 kb)

Background Cavernous nerve injury (CNI) causes fibrosis and loss of smooth muscle cells (SMCs) in the corpus cavernosum and leads to erectile dysfunction, and lysyl oxidase (LOX) activation has been found to play an important role in fibrotic diseases. Aim To evaluate the role of LOX in penile fibrosis after bilateral CNI (BCNI). Methods Rats underwent BCNI or a sham operation and were treated with vehicle or β-aminopropionitrile, a specific LOX activity inhibitor. 30 days after BCNI, rats were tested for erectile function before penile tissue harvest. LOX and extracellular matrix component expression levels in the corpus cavernosum, including matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), fibronectin (FN), collagen (COL) I, and COL IV, were evaluated by real-time quantitative polymerase chain reaction and western blot. Corporal fibrosis was evaluated by Masson trichrome staining. Localization of LOX and SMC content in the corpus cavernosum were assessed by immunohistochemistry. Outcomes Ratio of intracavernous pressure to mean arterial blood pressure; LOX, MMPs, TIMPs, COL I, COL IV, and FN expression; penile fibrosis; penile SMC content. Results After BCNI, there was an increase in penile LOX expression and activity, increased penile fibrosis, decreased SMC content, and impaired erectile function. TIMP1, TIMP2, COL I, COL IV, and FN expression was markedly upregulated, whereas the enzyme activity of MMPs was decreased after BCNI. β-Aminopropionitrile treatment, at least in part, prevented a decrease in the ratio of intracavernous pressure to mean arterial blood pressure, decreased penile expression of TIMP1, TIMP2, COL I, COL IV, and FN, increased MMP activity, prevented corporal fibrosis, and preserved SMC content. Clinical Translation LOX over-activation contributes to penile fibrosis and LOX inhibition could be a promising strategy in preventing the progression of CNI-induced erectile dysfunction. Strengths and Limitations This is the 1st study to demonstrate the role of LOX activation in penile fibrosis. However, the exact mechanism of how LOX influences extracellular matrix protein synthesis and SMC content preservation awaits further investigation. Conclusion CNI induced LOX over-activation in cavernous tissue, and inhibition of LOX preserved penile morphology and improved erectile function in a rat model of BCNI.

Background Huachansu (HCS), a class of toxic steroids extracted from toad venom, which has been shown to be a valuable anticancer drug in many kinds of cancers. However, the effect of HCS on bladder cancer has not been elucidated. In this study, we focused on the antitumor activities and related mechanisms of HCS on bladder cancer in vitro and in vivo. Methods Cell viability of T24, EJ, RT-4, SV-HUC-1 cells after HCS treatment was measured by MTS, whereas the changes of cell morphology were observed by transmission electron microscopy. The early apoptosis induced by HCS was evaluated by flow cytometry, and the expression level of apoptosis-related molecules (Bax, Bcl-2, XIAP, PARP, cleaved-Caspases 3, 8, 9) was detected using Western blot. We then evaluated the impact of HCS on the expression of Fas/Fasl, TNF- alpha/TNFR1, and the activation of NF-Kappa B pathway, and furthermore the effect of these pathways in HCS induced-apoptosis were also detected. At last, xenograft tumor in nude mice was used to further investigate the antitumor effect of HCS in vivo. Results Our results showed that HCS could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cell lines. The expression of Fas, Fasl, TNF- alpha were all elevated at both mRNA and protein level after HCS treatment. Furthermore, down regulation of TNF- alpha, TNFR1, Fas or inhibition of Fas/Fasl interaction decreased the relative number of death cells induced by HCS. In vivo, HCS treatment significantly suppressed tumor growth and induced apoptosis in xenografts tumor in nude mice. Conclusions HCS could efficiently inhibit proliferation and induce apoptosis in human bladder cancer cells in vitro and in vivo, which is largely mediated by Fas/Fasl and TNF- alpha/TNFR1 pathway. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0134-9) contains supplementary material, which is available to authorized users.

Introduction: Erectile dysfunction (ED) after cavernous nerve (CN) injury remains difficult to treat. Calpain plays a critical role in causing neurodegenerative diseases. This study aimed to evaluate whether calpain inhibition preserves erectile function in a rat model of CN injury. Materials and Methods: Rats underwent sham surgery or CN crush injury. The CN-crushed rats were treated with vehicle or MDL-28170, a specific calpain inhibitor. At 1, 2, 3, and 7 days post-surgery, major pelvic ganglia (MPG) were harvested, followed by the measurement of erectile function, respectively. At 28 days, penile tissue and distal CN were harvested, followed by the measurement of erectile function in rats. Calpain activity in MPG and corpus cavernosum, as well as TGF-β1/Smad2 and collagen content in corpus cavernosum, were measured by western blot. Neuronal nitric oxide synthase (nNOS) was observed by immunohistochemistry. Results: Increased calpain activity was observed in MPG and corpus cavernosum. CN crush markedly attenuated the erectile responses and nNOS expression in CN, and these were improved by MDL-28170 treatment. Furthermore, treatment prevented increased TGF-β1/Smad2 and collagen expression in corpus cavernosum. Conclusions: Our results suggested that calpain activation plays a role in pathogenesis of CN injury-associated ED. Calpain inhibition could be a novel approach for preventing the development of ED following CN injury.

Background Recently, leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a negative regulator of EGFR, was discovered is a novel agent for suppressing bladder cancer. The aim of this study was to investigate the impact of LRIG1 on the biological features of aggressive bladder cancer cells and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1. Methods In this study, we examined the mRNA and protein expression of LRIG1 and EGFR in bladder cancers and normal bladder. Meanwhile, we overexpressed LRIG1 with adenovirus vector in T24/5637 bladder cancer cell lines, and we used real time-PCR, western blot, and co-immunoprecipitation analysis in order to examine the effects of LRIG1 gene on EGFR. Furthermore, we evaluate the impact of LRIG1 gene on the function of human bladder cancer cells and EGFR signaling. Results The expression of LRIG1 was decreased, while the expression of EGFR was increased in the majority of bladder cancer, and the ratio of EGFR/LRIG1 was increased in tumors versus normal tissue. We found that upregulation of LRIG1 induced cell apoptosis and cell growth inhibition, and further reversed invasion in bladder cancer cell lines in vitro by inhibiting phosphorylation of downstream MAPK and AKT signaling pathway. Conclusion Taken together, our findings provide us with an insight into LRIG1 function, and we conclude that LRIG1 evolved in bladder cancer as a rare feedback negative attenuator of EGFR, thus could offer a novel therapeutic target to treat patients with bladder cancer.