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Recent evidence revealed an inhibitory effect of circ-ITCH on the progression of papillary thyroid cancer via affecting the circ-ITCH/miR-22-3p/CBL axis. Rs4911154, an SNP located in circ-ITHC, was previously reported to be significantly associated with an increased risk of hepatocellular carcinoma. Ultrasound testing was used to evaluate the doubling time of thyroid nodules. 202 patients diagnosed with thyroid nodule disorders were divided into three groups according to their genotypes at rs4911154. We found that the A allele was correlated with a shortening doubling time of thyroid nodules. Moreover, the A allele contributed to reduced expression of circ-ITCH/CBL and increased expression of miR-22-3p. Besides, decreased tissue apoptosis was linked to the A allele. Luciferase assays indicated that miR-22-3p could effectively suppress the luciferase activities of CBL and circ-ITCH. Furthermore, manual up-regulation of miR-22-3p effectively suppressed the expression of CBL, while CBL siRNA apparently abolished circ-ITCH induced CBL upregulation, reduced proliferation and increased apoptosis of K1 and TPC-1 cells. A signaling pathway of circ-ITCH/miR-22-3p/CBL axis was established to explain the effect of SNP of circ-ITCH in thyroid tumor malignancy. Compared with the G allele, the A allele in rs4911154 contributed to the malignancy of thyroid nodules with decreased doubling time and down-regulated CBL expression.

Hepatocellular carcinoma (HCC) remains the most common malignant tumor worldwide. Long noncoding RNAs can modulate various tumorigenic processes. In addition, growing evidence has indicated tha the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is activated in multiple cancers, including HCC. Recently, it was found that LINC00346 can participate in several cancers. Nevertheless, the biological roles of LINC00346 in HCC have been barely investigated. In this study, the function of LINC00346 was specifically concentrated upon. We observed that LINC00346 was obviously elevated in HCC cells (Bel7404, Huh-6, HepG2, and QGY-7703 cells). Then, Bel7404 and HepG2 cells were overexpressed with LINC00346. Overexpression of LINC00346 repressed HCC cell survival and cell proliferation. In addition, apoptosis of Bel7404 and HepG2 cells was triggered by LINC00346 upregulation. Bel7404 and HepG2 cell cycle was arrested in the G1 phase by LINC00346. Meanwhile, we conducted wound-healing assay and Transwell invasion assays. As shown, we observed that the migratory and invasive capacities of Bel7404 and HepG2 cells were remarkably restrained by the increase of LINC00346. Moreover, we showed that LINC00346 overexpression activated the JAK-STAT3 pathway, which is involved in many cancers. Afterward, in vivo experiments were utilized and we proved that LINC00346 was able to induce HCC tumor growth via activating the JAK-STAT3 pathway. To conclude, we revealed the potential possibility of developing LINC00346 as an indicator for HCC.

BACKGROUND: Caspase-cleaved K18 (cK18) may accurately reflect hepatocyte apoptosis in patients with non-alcoholic steatohepatitis (NASH). However, NASH can also exist within the normal range of cK18. The aim of this study was to investigate the risk factors and characteristics of NASH within the normal serum levels of cK18. METHODS: In the study, 227 histopathologically confirmed non-alcoholic fatty liver disease (NAFLD) patients with normal cK18 levels (≤200 U/L), measured in serum using ELISA kits, were enrolled. The Rs738409 allele, coding patatin-like phospholipase domain-containing protein 3 (PNPLA3), was detected by MALDI-TOF mass spectrometry. Non-alcoholic steatohepatitis was defined as an NAFLD activity score (NAS) ≥5 with each part >0. RESULTS: The prevalence of NASH was 31.7% among NAFLD patients with normal serum cK18 levels. Compared with non-NASH, NASH had a higher possibility of occurrence with central obesity, insulin resistance, and the G allele of PNPLA3. The mean serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were higher in NASH patients. Moreover, ALT, AST, TC, LDL-C, central obesity, and the PNPLA3 G allele were risk factors for NASH in NAFLD patients with normal serum cK18 levels, with odds ratios of 1.01 (95% CI: 1.00, 1.02), 1.03 (95% CI: 1.01, 1.05), 1.33 (95% CI: 1.04, 1.68), 1.41 (95% CI: 1.03, 1.92), 2.19 (95% CI: 1.15, 4.18), and 2.48 (95% CI: 1.15, 5.36), respectively; all P < .05. CONCLUSIONS: The major risk factors for NASH were central obesity, AST, and the PNPLA3 G allele, in NAFLD with low hepatocyte apoptosis.

With the increasing application of tetracycline (TC) in medical treatment, animal husbandry and aquaculture in recent decades, high quantities of TC have been frequently detected in the aquatic environment, and accordingly TC-related toxicity and environmental pollution have become a global concern. The present study was performed to explore the toxicological influences of TC exposure at its environmentally relevant concentrations on the gills of tilapia Oreochromis niloticus, based on the alteration in histopathology, oxidative stress, inflammatory response, cell cycle, mitochondrial function, apoptosis, and transcriptomic analysis. Our findings revealed that TC exposure damaged the structure and function, induced oxidative stress, affected inflammatory responses, and reduced Na+/K+-ATPase (NKA) activity in the gills. TC also caused the inhibition in cell cycle, resulted in mitochondrial dysfunction and activated apoptosis. Further transcriptomic analysis indicated the extensive influences of TC exposure on the gill function, and immune system was the main target to waterborne TC exposure. These results elucidated that environmental TC had more complex toxicological effects on gills of fish than previously assessed, and provided novel insight into molecular toxicology of TC on fish and good basis for assessing the environmental risk of TC.

Natrin, a new member of the cysteine-rich secretory protein (CRISP) family purified from the snake venom of Naja naja atra, has been demonstrated to have anticancer activity. However, the underlying molecular mechanisms need further elucidation. In this study, MTT was used to evaluate cell viability. Apoptotic cells were analyzed by employing a transmission electron microscope (TEM). Metabolomic study of the metabolic perturbations caused by natrin-induced apoptosis in differentiated SMMC-7721 cells was performed for the first time by using integrative ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). To investigate the possible mechanism in the mitochondrial pathway of natrin-induced apoptosis, we measured apoptosis-related mRNA changes using real-time fluorescent quantitative PCR (FQ-PCR). Cell proliferation was significantly inhibited after treatment with natrin in a dose-dependent manner. Principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) clearly demonstrated that metabolic profiles were affected by natrin. The results of multivariate statistical analysis showed that a total of 13 metabolites were characterized as potential biomarkers highly implicated in natrin-induced apoptosis, which corresponded to fluctuations of five pathways, including sphingolipid metabolism, fatty acid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism and glycosphingolipid biosynthesis. Furthermore, natrin-induced apoptosis showed an increase in the Bax/Bcl-2 ratio in the mitochondrial pathway compared with controls. This study illustrated that rapid and holistic cell metabolomics combining molecular biological approaches might be a powerful tool for evaluating the underlying mechanisms of natrin-induced apoptosis, which would help to deepen specific insights into the anti-hepatoma mechanisms of natrin and facilitate the clinical application of natrin in the future.

This study aimed to investigate whether inhibition of glucagon-like peptide-1 (GLP-1) on pressure overload induced cardiac hypertrophy and apoptosis is related to activation of ATP sensitive potassium (KATP) channels. Male SD rats were randomly divided into five groups: sham, control (abdominal aortic constriction), GLP-1 analog liraglutide (0.3 mg/kg/twice day), KATP channel blocker glibenclamide (5 mg/kg/day), and liraglutide plus glibenclamide. Relative to the control on week 16, liraglutide upregulated protein and mRNA levels of KATP channel subunits Kir6.2/SUR2 and their expression in the myocardium, vascular smooth muscle, aortic endothelium, and cardiac microvasculature. Consistent with a reduction in aortic wall thickness (61.4 ± 7.6 vs. 75.0 ± 7.6 μm, p

Enrofloxacin (ENR), one representative of fluoroquinolones widely used in husbandry, poses potential threats to aquatic organisms. However, its toxicological effect remained largely unknown. Here, we explored the influences of environmentally relevant concentration of ENR exposure on the gills of yellow catfish Pelteobagrus fulvidraco, a well-known freshwater teleost. After 10-weeks of waterborne ENR exposure at environmentally relevant concentrations, the gill tissues were collected and subsequently used for histopathology, enzyme activity, protein and mRNA expression analysis in yellow catfish. Our study revealed that waterborne ENR exposure reduced Na+/K+-ATPase activity, tight junction genes' expression and cytokines secretion, activated oxidative stress and induced apoptosis in gills. ENR exposure also activated MAPK signaling in a concentration-dependent manner. Thus, we demonstrated that environmentally relevant concentration of ENR exposure activated oxidative stress and MAPK pathway, resulted in immune dysfunction and induced intrinsic apoptosis. These results elucidated that ENR, as an environmental pollutant, had more complex toxicological effects on gills of fish than previously assessed, which provided novel insight into ENR toxicology and the assessment of its environmental risks.

Hepatitis B virus × protein (HBx) serves an important role in the pathogenesis of the hepatitis B virus infection. Previous studies have reported that the interaction between HBx and hepatocyte mitochondria is an important factor leading to liver cell injury and apoptosis, ultimately inducing the formation of liver cancer. In the present study, a mouse model expressing HBx was constructed using hydrodynamic in vivo transfection based on the interaction between HBx and cytochrome c oxidase (COX) subunit III. The specific mechanism of HBx-induced oxidative stress in mouse hepatocytes and the subsequent effect on mitochondrial function and inflammatory injury was assessed. The results demonstrated that HBx reduced the activity of COX and the expression of superoxide dismutase and upregulated the expression of malondialdehyde, NF-κB and phospho-AKT, thus increasing oxidative stress. In addition, HBx induced an increase in interleukin (IL)-6, IL-1β and IL-18 expression levels, which created an inflammatory microenvironment in the liver, further promoting hepatocyte inflammatory injury. Therefore, it was proposed that HBx may affect hepatocyte mitochondrial respiration by reducing the activity of cytochrome c oxidase, leading to mitochondrial dysfunction and inducing hepatocyte inflammation and injury.

Background: Multiple myeloma (MM) is the second most common hematological neoplasm. Wide administration of bortezomib significantly improves the survival of MM patients compared with conventional chemotherapy. Bromodomain-containing protein 4 (BRD4) inhibitors also have been demonstrated to retard cell proliferation and induce cellular apoptosis in various cancers. However, it is unclear whether the BRD4 inhibitor nitroxoline plus bortezomib has a synergistic anti-tumor effect on MM. Methods: Cell viability was determined via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and cell apoptosis were assessed via flow cytometry. Protein expression levels were determined via western blotting. The expression of apoptosis-related proteins in xenograft tissue were detected by means of immunohistochemistry. Results: Treatment with nitroxoline or bortezomib suppressed cell proliferation, and caused G0/G1 phase arrest and apoptosis in H929 and RPMI8226 cells. Furthermore, nitroxoline intensified the retardation of cell proliferation, as well as further enhanced the G0/G1 phase arrest and apoptosis induced by bortezomib in H929 and RPMI8226 cells. The western blot analysis revealed that nitroxoline or bortezomib treatment markedly diminished the levels of Bcl-2 and cyclin D1, and increased the levels of p21, Bax, cleaved PARP and cleaved caspase-3. Combination of these two agents was observed to result in further marked changes on these levels compared with nitroxoline or bortezomib treatment alone. What is more, in the xenograft tumor model, combinative treatment markedly inhibited tumor growth compared with the single drug treatment. Conclusion: Combination of bortezomib with nitroxoline has a synergistic anti-tumor activity in MM cells and may be a novel treatment method for MM.

We induced changes in the tumor microenvironment (TME) through the synergistic actions of two drugs used in breast cancer therapy. The anti-fibrotic drug silibinin (SLB) targets tumor-associated fibroblasts and exerts immune-mediated anti-cancer effects. IPI-549, an efficient and highly selective phosphoinositide-3-kinase-gamma (PI3Kγ) inhibitor, was applied to alter the balance of immunosuppressive cells by inhibiting PI3Kγ molecules; it also promotes anti-tumor immunity. We developed nanoparticle formulations to encapsulate both drugs into the targeting carrier aminoethyl anisamide-polyethylene glycol-polycaprolactone (AEAA-PEG-PCL) respectively. The drugs were intravenously delivered in mice and resulted in an increase in anti-tumor efficacy and apoptotic tumor tissue compared with either IPI-549 or SLB alone in 4T1 breast cancer cell-derived tumors. Furthermore, a significant reduction in regulatory T (Treg) cells and myeloid suppressor cells (MDSCs) was observed. A normalized TME structure was also observed, including angiogenesis suppression, antifibrotic effects and the inhibition of collagen formation in the tumor tissue, significantly enhancing the anti-tumor effects. In summary, this combination strategy may offer an alternative treatment for breast cancer.

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem‑like cells (GSCs), by culturing patient‑derived primary GSCs as well as a GSC cell line in glucose‑restricted, β‑hydroxybutyrate‑containing medium (BHB‑Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB‑Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self‑renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB‑Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB‑Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia‑inducible factor 1 and B‑cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB‑Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.

Extracellular vesicles (EVs) including exosomes can serve as mediators of cell–cell communication under physiological and pathological conditions. However, cargo molecules carried by EVs to exert their functions, as well as mechanisms for their regulated release and intake, have been poorly understood. In this study, we examined the effects of endothelial cells-derived EVs on neurons suffering from oxygen-glucose deprivation (OGD), which mimics neuronal ischemia-reperfusion injury in human diseases. In a human umbilical endothelial cell (HUVEC)–neuron coculture assay, we found that HUVECs reduced apoptosis of neurons under OGD, and this effect was compromised by GW4869, a blocker of exosome release. Purified EVs could be internalized by neurons and alleviate neuronal apoptosis under OGD. A miRNA, miR-1290, was highly enriched in HUVECs-derived EVs and was responsible for EV-mediated neuronal protection under OGD. Interestingly, we found that OGD enhanced intake of EVs by neurons cultured in vitro. We examined the expression of several potential receptors for EV intake and found that caveolin-1 (Cav-1) was upregulated in OGD-treated neurons and mice suffering from middle cerebral artery occlusion (MCAO). Knock-down of Cav-1 in neurons reduced EV intake, and canceled EV-mediated neuronal protection under OGD. HUVEC-derived EVs alleviated MCAO-induced neuronal apoptosis in vivo. These findings suggested that ischemia likely upregulates Cav-1 expression in neurons to increase EV intake, which protects neurons by attenuating apoptosis via miR-1290.

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 μg shrimp−1) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 μg shrimp−1), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.

Morphology and immune functions of Penaeus monodon haemocytes were analysed by flow cytometry (FCM). Basing on the relative size and granularity, there are three types of haemocyte identified by FCM, including hyaline cells (HC), semigranular cells (SGC) and granular cells (GC). GC and SGC were significantly larger than HC. GC contained more granules in the cytoplasm than the two other types. SGC were the most abundant cells, while HC were the least. The mean total apoptotic cell ratio was 5.09 % in the fresh shrimp haemocytes. SGC contributed most to the phagocytic activity, and HC contributed least, probably due to the great difference of percentage. GC had most mass of mitochondria and lysosome, and HC contained least. Defined by different characteristic of lysosomal mass, three subsets of haemocytes were observed. GC were most active in non-induced and zymosan-induced reactive oxygen species (ROS) production, while HC showed lowest oxidative activity. Both non-induced and zymosan-induced esterase activity (EA) in GC were higher than those in SGC and HC. The present flow cytometric analysis clearly demonstrated the variation in morphologic and immunological characterisation among the three haemocyte subpopulations. The facts that SGC account for most of the number of circulating haemocyte, while GC contain most organelles in relation to energy supply and immune defence, and have highest immune activites, suggesting that GC and SGC are the main haemocyte types involved in the cellular defence of P. monodon.

// Shaojun Chen 1 , Longsheng Wang 1 , Xudong Yao 1 , Hui Chen 2 , Chen Xu 1 , Lu Tong 2 , Abdussaboor Shah 2 , Tingmei Huang 2 , Geng Chen 2 , Jiwei Chen 2 , Tie-Long Liu 3 , Xiao-Tao Li 2, 4 , Jun-Hua Zheng 1 and Lei Li 2 1 Department of Urology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China 2 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai 200241, China 3 Department of Orthopedic Oncology, Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China 4 Department of Molecular and Cellular Biology, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas 77030, USA Correspondence to: Tie-Long Liu, email: czyyltl@163.com Xiao-Tao Li, email: xiaotaol@bcm.edu Jun-Hua Zheng, email: renalzjh@163.com Lei Li, email: lllkzj@163.com Keywords: miR-195-5p, REGγ, wnt/β-catenin, renal cell carcinoma Received: March 21, 2017 Accepted: June 12, 2017 Published: July 15, 2017 ABSTRACT Renal cell carcinoma (RCC) is the most prevalent malignancy of kidney and accounts for approximately 4% of all cancer diagnoses in adults. Previous studies demonstrated microRNA-195-5p (miR-195-5p) as a tumor suppressor which is deregulated in many human cancers. However, the role of miR-195-5p in RCC is largely unknown. In the present study, we demonstrated that miR-195-5p was downregulated and negatively correlated with advanced clinical stage in RCC. Overexpression of miR-195-5p significantly suppressed RCC cells growth in vitro and in vivo , induced apoptosis and enhanced chemosensitivity to sorafenib. Conversely, suppression of miR-195-5p exhibited a reverse effect. REGγ, a proteasome activator, was identified as a novel downstream target of miR-195-5p in RCC. Knockdown of REGγ inhibited proliferation, induced apoptosis, increased sorafenib chemosensitivity and suppressed the wnt/β-catenin pathway in RCC cells. Moreover, restoration of REGγ markedly abolished the effects of miR-195-5p in RCC, and the wnt/β-catenin pathway was suppressed by miR-195-5p overexpression while activated by miR-195-5p inhibition in RCC cells. Our findings suggest that miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in RCC development and may serve as a novel target for RCC treatment.

The effect of miR-146a-dependent regulation of STAT1 on apoptosis in acute lymphoblastic leukemia (ALL) Jurkat cells was investigated. The miR-146a mimic and miR-146a inhibitor vectors were constructed in vitro, and experimental grouping was as follows: Control group (untreated Jurkat cells), empty vector group (Jurkat cells transfected with empty vector), agonist group (Jurkat cells transfected with miR-146a mimic) and the inhibitor group (Jurkat cells transfected with miR-146a inhibitor). Western blot analysis was used to observe the expression, respectively, of STAT1, p-STAT1 and Bcl-xL, and flow cytometry was used to test apoptosis in Jurkat cells. STAT1 and p-STAT1 expression in the agonist group was higher than that in the control and empty vector groups, but lower in the inhibitor group, and differences were statistically significant (P

Objective We aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression. Methods qRT‐PCR was used for the assessment of BIRC5, miRNA‐203 and MALAT1 expression. Furthermore, the targeted relationships between miR‐203 and BIRC5, as well as MALAT1 and miR‐203, were predicted by the miRanda/starBase database and verified by dual‐luciferase reporter gene assay. The effects of MALAT1, miRNA‐203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK‐8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis. Results The expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA‐203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase. Conclusions Our study demonstrated that MALAT1 functions as a miR‐203 decoy to increase BIRC5 expression in RCC.

Diabetes is one of the most severe chronic diseases worldwide. It is widely accepted that apoptosis of the pancreatic beta cell is an important cause for the induction of hyperglycemia and high levels of free fatty acids (FFAs), also called lipotoxicity associated with pancreatic beta cell dysfunction. Lipotoxicity has been proven to be an important pathogenic factor of diabetes. However, until now, the mechanism of FFA-induced lipotoxicity in INS-1 cells has not been fully understood. Current anti-diabetic drugs that protect islet cells are often toxic to healthy cells, resulting in negative side effects. Thus, there is an urgent need to identify more effective and safer anti-diabetic agents to protect pancreatic islet cells. Rubusoside (RUB) is a major ingredient in the leaves of Rubus suavissimus S. Lee, which decreases blood glucose levels by protecting pancreatic islet cells. However, the exact mechanism of this effect is unknown. In this study, metabolomics experiments based on UPLC-Q/TOF MS characterized a total of 15 metabolites as potential biomarkers associated with lipotoxicity induced by palmitic acid in INS-1 cells. According to the metabolic pathway analysis, pentose and glucuronate interconversions, and glycerophospholipid metabolism were recognized as the most influenced metabolic pathways associated with lipotoxicity. Unexpectedly, deviations of 14 metabolites in lipotoxic INS-1 cells were regulated by RUB, suggesting synergistic mediation of the abnormal metabolic pathways. The metabolomics strategy based on UPLC/Q-TOF MS analysis provides a new insight into the mechanisms of lipotoxicity induced by palmitic acid and the anti-lipotoxic activity of RUB in INS-1 cells.

Periodontitis is a common inflammatory disorder affecting the tissues surrounding the teeth, which can lead to the destruction of periodontal tissue and tooth loss. Resveratrol, a natural phytoalexin, exerts multiple biological effects. For example, its anti‑inflammatory activity has been widely studied for the treatment of inflammatory bowel disease for a number of years. However, its effect on bone repair and new bone formation in an inflammatory microenvironment is not well understood. Accordingly, the effect of resveratrol on inflammation‑affected human periodontal ligament stem cells (hPDLSCs) requires further investigation. In the present study, the effect of tumor necrosis factor‑α (TNF‑α), resveratrol, or the combination of both on the osteogenic differentiation of hPDLSCs, as well as the underlying mechanisms involved, were investigated. Cell Counting Kit‑8 assay, alkaline phosphatase staining, Alizarin red staining, Oil Red O staining, reverse transcription‑quantitative PCR and western blotting were used in the present study. It was demonstrated that resveratrol enhanced hPDLSC osteogenesis and reversed the inhibitory effects of TNF‑α on this process. Further mechanistic studies indicated that resveratrol exerted anti‑inflammatory activity by activating the ERK1/2 pathway, decreasing the secretion of interleukin (IL)‑6 and IL‑8 induced by TNF‑α, and enhancing hPDLSCs osteogenesis. The present study suggested that resveratrol may be a novel and promising therapeutic choice for periodontitis.

Background BRAF-activated long non-coding RNA (BANCR) has been associated with various types of cancer. Nevertheless, the role of BANCR in clear cell renal cell carcinoma (ccRCC) is still not fully understood. This study aims to investigate the relationship between ccRCC and BANCR. Methods Expression of BANCR in TCGA renal cancer data sets was analyzed. The expression pattern of BANCR in Immortalized normal human proximal tubule epithelial cell line HK-2 and ccRCC cell lines (ACHN, CAKI-1, A498 and 786-O) was detected by real-time quantitative RT-PCR (qRT-PCR). ccRCC tissues with adjacent normal renal tissues diagnosed by pathological methods from 62 patients were used to detect the expression of BANCR, and its correlation with prognosis of ccRCC patients was assessed by Kaplan-Meier method. The LV-BANCR vector was used to examine the influence of BANCR on the proliferation, migration, invasion, apoptosis and cell cycle distribution of ccRCC cells in vitro. Results BANCR was downregulated in renal cancer according to TCGA data sets. Compared with adjacent normal renal tissues and normal human proximal tubule epithelial cell line HK-2, BANCR expression was significantly decreased in ccRCC tissues and ccRCC cell lines, and its low expression was associated with poor prognosis. Moreover, in the condition of BANCR overexpression by LV-BANCR vector, the proliferation, migration, invasion capacity of ccRCC cells was inhibited, while the apoptosis was increased and the G1 cell cycle arrest was induced in vitro. Conclusions BANCR is downregulated in ccRCC tissues and cell lines, and is associated with ccRCC progression. Thus, BANCR may represent a novel prognostic biomarker and a potential therapeutic target for ccRCC patients.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL‑60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose‑ and time‑dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT‑22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6‑induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase‑3, ‑8 and ‑9, and decreases in the levels of B‑cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.

Neuregulin receptor degradation protein-1 (Nrdp1) is involved in a plethora of cellular processes and plays an essential role in the development and progression of human cancers. However, its role in renal cell carcinoma (RCC) remains unclear. Therefore, the present study aimed to explore the biological significance of Nrdp1 in RCC. Western blot analyses of tissue samples from 24 patients with primary RCC revealed lower Nrdp1 and higher baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme (BRUCE) protein levels in RCC tissues compared with adjacent normal tissues. In addition, MTT and apoptosis assays demonstrated that Nrdp1 overexpression resulted in decreased cell viability and enhanced apoptosis in RCC 786-O cells; conversely, Nrdp1 knockdown increased 786-O cell viability and inhibited apoptosis. Further analysis showed that BRUCE downregulation partially attenuated the effects of Nrdp1 knockdown on RCC cell viability and apoptosis. Moreover, an inverse association was obtained between BRUCE and Nrdp1 protein levels. These findings suggest that Nrdp1-mediated degradation of BRUCE decreases cell viability and induces apoptosis in RCC cells, highlighting Nrdp1 as a potential target for RCC treatment.

Background: Multiple myeloma (MM) is the second most common hematological neoplasm. Wide administration of bortezomib significantly improves the survival of MM patients compared with conventional chemotherapy. Bromodomain-containing protein 4 (BRD4) inhibitors also have been demonstrated to retard cell proliferation and induce cellular apoptosis in various cancers. However, it is unclear whether the BRD4 inhibitor nitroxoline plus bortezomib has a synergistic anti-tumor effect on MM. Methods: Cell viability was determined via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and cell apoptosis were assessed via flow cytometry. Protein expression levels were determined via western blotting. The expression of apoptosis-related proteins in xenograft tissue were detected by means of immunohistochemistry. Results: Treatment with nitroxoline or bortezomib suppressed cell proliferation, and caused G0/G1 phase arrest and apoptosis in H929 and RPMI8226 cells. Furthermore, nitroxoline intensified the retardation of cell proliferation, as well as further enhanced the G0/G1 phase arrest and apoptosis induced by bortezomib in H929 and RPMI8226 cells. The western blot analysis revealed that nitroxoline or bortezomib treatment markedly diminished the levels of Bcl-2 and cyclin D1, and increased the levels of p21, Bax, cleaved PARP and cleaved caspase-3. Combination of these two agents was observed to result in further marked changes on these levels compared with nitroxoline or bortezomib treatment alone. What is more, in the xenograft tumor model, combinative treatment markedly inhibited tumor growth compared with the single drug treatment. Conclusion: Combination of bortezomib with nitroxoline has a synergistic anti-tumor activity in MM cells and may be a novel treatment method for MM.

Ferulic acid (FA), a naturally derived phenolic compound, has antioxidant and antidepressant-like effects. It is still a challenge to study its mechanism due to the complexity of the pathophysiology of depression. In this study, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to perform metabolomics studies based on biochemical changes in differentiated rat pheochromocytoma (PC12) cells treated with corticosterone-induced neurological damage after FA treatment. A total of 31 metabolites were identified as potential biomarkers for corticosterone-induced PC12 cells injury. Among them, 24 metabolites were regulated after FA treatment. Pathway analysis revealed that these metabolites were mainly involved in the amino acid metabolism, energy metabolism and glycerophospholipid metabolism. In addition, based on the results of metabolomics, three cell signaling pathways related to glutamate were discovered. To further study the interactions between FA and major targets in three signaling pathways, a molecular docking method was employed. The results showed that FA had the strongest binding power with protein kinase B (AKT). Furthermore, the result of mRNA changes analyzed by quantitative real time RT-PCR indicated that AKT and protein kinase A (PKA) in the signaling pathway were up regulated after treatment with FA compared with model group. This study shows that strategies based on cell metabolomics associated with molecular docking and molecular biology is a helpful tool to elucidate the neuroprotective mechanism of FA.

Previous studies have reported that hyperoside and quercetin in combination (QH; 1:1) inhibited the growth of human leukemia cells. The aim of the present study was to investigate the anti‑cancer effect of QH on prostate cancer cells. The results demonstrated that QH decreased the production of reactive oxygen species (ROS) and increased antioxidant capacity in PC3 cells at various concentrations (2.5‑60 µg/ml) with peak inhibition and augmentation changes of 3.22‑ and 3.00‑fold, respectively. Following treatment with QH for 48 and 72 h, the IC50-values on PC3 cells were 19.7 and 12.4 µg/ml, respectively. Western blot analysis revealed that QH induced apoptosis in human prostate cancer cells via activation of caspase‑3 and cleavage of poly(adenosine diphosphate ribose) polymerase. In addition, QH significantly inhibited the invasion and migration of PC3 cells as well as reduced the expression of numerous prostate tumor‑associated microRNAs (miRs), including miR‑21, compared to that of untreated human prostate cancer cells. QH was also found to enhance the expression of tumor suppressor programmed cell death protein 4, which was negatively regulated by miR‑21. Furthermore, induced overexpression of miR‑21 using pre‑miR‑21 oligonucleotides attenuated the beneficial effect of QH on prostate cancer cells. In conclusion, the results of the present study indicated that QH exerted an anti‑cancer effect on human prostate cancer cells, the mechanism of which proceeded, at least in part, via the inhibition of the miR‑21 signaling pathway.

Low-intensity ultrasound (LIUS) combined with chemotherapy is an innovative modality for cancer treatment, but its effect on orthotopic carcinoma remains unknown. Our previous study revealed that LIUS enhanced the growth inhibitory effects of several chemotherapeutic drugs in nude mice with transplanted tumors. In the present study, we used 7,12-dimethylbenz(alpha)anthracene to induce orthotopic tongue carcinogenesis in hamsters. We used the first-line chemotherapy drug for tongue cancer, carboplatin (CBP) in combination with LIUS to investigate the synergistic effect. The results revealed that LIUS combined with low-dose CBP enhanced the inhibitory effects of CBP on tumor growth, prolonged survival, and did not increase the incidence of side-effects. It also enhanced the inherent DNA damage caused by CBP, suppressed the expression of the DNA repair proteins O6-methylguanine DNA methyltransferase (MGMT) and Chk1, and increased the expression of DNA damage-inducible protein GADD45α. Furthermore, compared with CBP alone, LIUS combined with CBP reduced the expression of cyclin D1 and cyclin B1, induced the expression of caspase-3, cleaved caspase-3, caspase-8, Bax, and Bak, and inhibited the expression of Bcl-2. Examination of clinical samples revealed that MGMT, Chk1, and Gadd45α were higher in OTSCC than in adjacent normal tissue. Hence, our results indicated that LIUS enhanced the ability of low-dose CBP to damage DNA in an orthotopic hamster model of tongue cancer, induced apoptosis, inhibited tumor growth and progression, while it did not increase the toxic side-effects of the drug, suggesting additional clinical benefits for patients treated with the combination of CBP with LIUS.

This study aimed to investigate the effects of microRNA-184 (miR-184) on the proliferation and apoptosis of human colon cancer cells through the regulation of C-MYC and BCL-2. Human colon cancer tissues were selected as case group, and adjacent normal tissues were as control group. Human colon cancer SW480 and HCT116 cells were allocated into blank, miR-184 mimic negative control (mimic-NC), miR-184 inhibitor NC (inhibitor-NC), miR-184 mimic, and miR-184 inhibitor groups. Flow cytometry, Annexin V/PI and MTT assay were used to examine the cell cycle, apoptosis and viability. The expressions of C-MYC, BCL-2 and miR-184 were detected via immunohistochemistry, Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR). C-MYC and BCL-2 were direct targets to miR-184. The growth of colon cancer cells in the miR-184 mimic group was inhibited and exhibited an increase in apoptosis. Cell growth in the miR-184 mimic group was increased in addition to the inhibition of apoptosis. Compared with miR-184 mimic group, the expressions of C-MYC and BCL-2 in miR-184 inhibitor group were increased. The expressions of C-MYC and BCL-2 in colon cancer tissues exhibited high levels of expression, while miR-184 displayed relatively low levels in comparison to the adjacent normal tissues. An association was detected regarding the expressions of miR-184, C-MYC and BCL-2 with the differentiation, invasion depth and lymph node metastasis. MiR-184 expression was negatively related to C-MYC and BCL-2 expressions. Our study suggested that miR-184 could inhibit proliferation and promote apoptosis of colon cancer cells by down-regulating expressions of C-MYC and BCL-2.

// Zhiguo Chen 1 , Rujian Zhu 2, 3 , Jiayi Zheng 4 , Chen Chen 2 , Chi Huang 2 , Junjie Ma 2 , Chen Xu 1 , Wei Zhai 1, 5 and Junhua Zheng 1 1 Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China 2 Department of Urology, The Affiliated Shanghai Tenth People’s Hospital, Nanjing Medical University, Shanghai, China 3 Department of Urology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China 4 Department of Pathology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China 5 Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Correspondence to: Wei Zhai, email: jacky_zw2002@hotmail.com Junhua Zheng, email: zhengjh0471@sina.com.cn Keywords: cryptotanshinone, renal cell carcinoma, cell proliferation, cell apoptosis, STAT3 Abbreviations: CPT: cryptotanshinone; RCC: renal cell carcinoma; STAT3: signal transducer and activator of transcription 3; DMSO: dimethyl sulfoxide Received: January 23, 2017 Accepted: May 04, 2017 Published: June 15, 2017 ABSTRACT It has been established that signal transducer and activator of transcription 3 serves as an oncoprotein in various human cancers; targeting it is therefore a reasonable approach for emerging cancer therapies. Cryptotanshinone, a natural compound extracted from the root of Salvia miltiorrhiza Bunge, has been identified as a potential STAT3 inhibitor. However, its functional role in renal cell carcinomas remains largely unknown. Therefore, we investigated the mode of action for cryptotanshinone. We found that cryptotanshinone substantially suppressed cancer cell growth while it promoted cell apoptosis by inhibiting the phosphorylation of STAT3 at Tyr705 and its blocking nuclear translocation. Coordinately, P-AKT, CyclinD1, C-MYC, MEKK2, and HGF were down-regulated and cell cycle progression was arrested at the G0/G1 phase, thereby attenuating cell proliferation. Moreover, the level of Cleaved-Caspase-3 was elevated while Bcl-2 and Survivin were down-regulated, accounting for the increased apoptosis. Furthermore, in vivo results revealed that cryptotanshinone effectively inhibits tumorigenesis in an A498-xenografted mouse model. Taken together, our data gives a more comprehensive understanding of how cryptotanshinone functions in renal cell carcinomas and demonstrates its potential as a powerful therapeutic approach to treat renal cell carcinomas.

Background: Nin one binding protein (NOB1) was identified as a potential oncogene in human glioma and miR-646 plays an important role in human growth and development. However, the underlying molecular mechanisms of NOB1 in tumorigenicity and its correlation with miR-646 in renal cell carcinoma (RCC) have not been investigated. Methods: We performed bioinformatic analysis to explore miRNA targeting NOB1. The expression of NOB1 and miR-646 from 100 cases of clear cell RCC (ccRCC) and 30 cases of adjacent non-tumour tissues were detected by quantitative real-time PCR. The expression of miR-646 was correlated with NOB1 expression, tumour features and patient metastasis-free survival. The effect of overexpression of mir-646 on renal cancer cell proliferation was detected by colony formation in soft agar. Using a xenograft tumour model, we observed the in vivo tumorigenesis effect of miR-646 and NOB1. Results: miR-646 negatively regulated NOB1 and inhibited the proliferation and migration of renal cancer cells. There was a significant upregulation of NOB1 in ccRCC and it was further increased in metastatic cases, while miR-646 was downregulated in tumour tissues and further decreased in metastatic ccRCC. Additionally, expression of miR-646 was inversely correlated with the expression of NOB1. The downregulation of miR-646 also indicated a higher probability of developing metastasis. Most importantly, miR-646 expression was an independent predictor of ccRCC metastasis by the univariate analysis and binary logistic regression model (both P

The aim of the present study was to investigate the effects of methylthiouracil (MTU) on the proliferation and apoptosis of rat bone marrow stromal cells (BMSCs). Rat BMSCs were isolated, cultured in vitro and treated with various concentrations of MTU. Cell growth curves were determined using the Cell Counting Kit-8 method and the effect of MTU on BMSCs in a logarithmic growth phase was observed. BMSC apoptosis following MTU treatment was detected by flow cytometry. The experimental results demonstrated that the proliferation-inhibition effect was gradually enhanced with increasing MTU concentrations and the extension of treatment time. Statistically significant differences were observed between the treatment and the control groups (P

MicroRNAs (miRNAs) have been reported to be critical players in osteosarcoma (OS). Among numerous cancer-related miRNAs, the expression level of miR-92a and its potential role in OS has not been investigated. Here, We showed that overexpression of miR-92a was identified in OS specimens and cells compared to normal bone tissues. The high level of miR-92a was correlated with high T classification and advanced clinical stages of OS patients. Notably, miR-92a highly expressing OS patients showed a notably reduced survival rate. In vitro experiments showed that loss of miR-92a inhibited U2OS cell proliferation and cell-cycle progression while induced apoptosis. In turn, its restoration facilitated MG-63 cell growth and suppressed apoptosis. Experimental nude mice showed that miR-92a silencing prohibited the in vivo growth of OS cells. Furthermore, bioinformatics software predicted that F-box and WD repeat-containing protein 7 (FBXW7) was a direct target of miR-92a. We then observed the negative regulation of miR-92a on FBXW7 expression and the direct binding between them was further verified by dual-luciferase assays in OS cells. Forced expression of FBXW7 resulted in reduced proliferation, cell cycle arrest at G1 phase and increased apoptosis in miR-92a overexpressing MG-63 cells. In summary, this study demonstrates miR-92a probably functions as a driver of tumor progression by targeting FBXW7, and highlights the potential effects of miR-92a on prognosis and treatment of OS.

Cancer induced bone pain (CIBP) remains one of the most intractable clinical problems due to poor understanding of its underlying mechanisms. Recent studies demonstrate the decline of inhibitory interneurons, especially GABAergic interneurons in the spinal cord, can evoke generation of chronic pain. It has also been reported that neuronal MHC-I expression renders neurons vulnerable to cytotoxic CD8+ T cells and finally lead to neurons apoptosis in a variety neurological disorders. However, whether MHC-I could induce the apoptosis of GABAergic interneurons in spinal cord and contribute to the development of CIBP remains unknown. In this study, we investigated roles of MHC-I and underlying mechanisms in CIBP on a rat model. Our results showed that increased MHC-I expression on GABAergic interneurons could deplete GABAergic interneurons by inducing their apoptosis in the spinal dorsal horn of tumor-bearing rats. Pretreatment of MHC-I RNAi-lentivirus could prevent the apoptosis of GABAergic interneurons and therefore alleviated mechanical allodynia induced by tumor cells intratibial injection. Additionally, we also found that CD8+ T cells were colocalized with MHC-I and GABAergic neurons and presented a significant and persistent increase in the spinal cord of tumor-bearing rats. Taken together, these findings indicated that MHC-I could evoke CIBP by promoting apoptosis of GABAergic interneurons in the dorsal horn, and this apoptosis was closely related to local CD8+ T cells.

This study investigated the function of HOTAIR in the growth and apoptosis of OS MG-63 cell linein vitroand further clarified its mechanism. The expression levels of HOTAIR in OS MG-63 cell line and normal osteoblast hFOB1.19 cell line were determined by RT-PCR, respectively. The growth and apoptosis of MG-63 cellsin vitrowere investigated by MTT assay and flow cytometry assay after HOTAIR was knocked down with retroviral vector construction. And the expression levels of cell growth and apoptosis related factors TGF-β, p53, Bcl-2, and TNF-αwere determined to clarify the mechanism. We found that HOTAIR was highly expressed in osteosarcoma MG-63 cell line compared with normal osteoblast hFOB1.19 cell line. The proliferation rate was lower and the apoptosis rate was higher significantly in shHOTAIR MG-63 cells than those in EV MG-63 cells. TGF-βand Bcl-2 were downregulated significantly when HOTAIR was knocked down. p53 and TNF-αwere upregulated significantly when HOTAIR was knocked down. These results indicated that HOTAIR functioned as a carcinogenic lncRNA, which could promote the proliferation and inhibit the apoptosis of MG-63 cellsin vitro. HOTAIR could be a potential target for the treatment of osteosarcoma.

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Previous studies have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The purpose of this investigation was to examine the protective effect of remifentanil preconditioning in cerebral ischemia/reperfusion injury (CIR) and its possible molecular mechanism. Results showed that Remifentanil pretreatment significantly decreased the CD4(+) and increased the CD8(+) in cerebral tissues. Additionally, CD4(+)/CD8(+) in CIR + Remifentanil group was markedly lower than that in CIR group. TNF-α and TNFR1 in CIR + Remifentanil group rats was found to be significant lower than that in CIR group rats. The expression levels of Cyt-c, caspase-3, caspase-9 and pJNK proteins in brain of CIR + Remifentanil group rats were found to significantly decreased compared to CIR group rats. In addition, decreased ROS level indirectly inhibit JNK activation and cell death in CIR rat receiving Remifentanil preconditioning. From current experiment results, at least two signal pathways involve into the process of Remifentanil preconditioning inhibiting cerebral damage induced by ischemia reperfusion. The inhibitory effects of Remifentanil preconditioning on the brain damage are achieved probably through blocking the activation of TNF-α/TNFR1, JNK signal transduction pathways, which implies that Remifentanil preconditioning may be a potential and effective way for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TNF-α/TNFR1, JNK signal pathways. Taken together, this study indicated that regulation of the TNF-α/TNFR1 and JNK signal pathways may provide a new therapy for cerebral damage induced by ischemia and reperfusion.

Glucocorticoids (GCs) have been proved to be an important pathogenic factor of some neuropsychiatric disorders. Usually, a classical injury model based on corticosterone-induced cytotoxicity of differentiated rat pheochromocytoma (PC12) cells was used to stimulate the state of GC damage of hippocampal neurons and investigate its potential mechanisms involved. However, up to now, the mechanism of corticosterone-induced cytotoxicity in PC12 cells was still looking forward to further elucidation. In this work, the metabolomic study of the biochemical changes caused by corticosterone-induced cytotoxicity in differentiated PC12 cells with different corticosterone concentrations was performed for the first time, using the ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). Partial least squares-discriminate analysis (PLS-DA) indicated that metabolic profiles of different corticosterone treatment groups deviated from the control group. A total of fifteen metabolites were characterized as potential biomarkers involved in corticosterone-induced cytotoxicity, which were corresponding to the dysfunctions of five pathways including glycerophospholipid metabolism, sphingolipid metabolism, oxidation of fatty acids, glycerolipid metabolism and sterol lipid metabolism. This study indicated that the rapid and holistic cell metabolomics approach might be a powerful tool to further study the pathogenesis mechanism of corticosterone-induced cytotoxicity in PC12 cells.

AIM: To investigate the role of Aquaporin-1 (AQP-1) in lens epithelial cells (LECs) and its potential target genes. AQP-1 is specifically expressed in LECs of eyes and is significant for lens homeostasis and transparency maintenance. Herein, AQP-1 expression in LECs was investigated to evaluate its influence on cell survival in association with its potential role in cataract formation. METHODS: LECs were transfected with lentivirus carrying AQP-1 small interfering RNA (siRNA). Real-time polymerase chain reaction (PCR) and Western blotting were conducted to detect AQP-1 expression in LECs from different groups. Meanwhile, cell counting kit-8 (CCK-8) assay and flow cytometry were performed to measure LEC proliferation and apoptosis, respectively. RESULTS: AQP-1 expression was significantly reduced in LECs, both at mRNA and protein levels (P

Summary Background Polo-like kinase 1 (PLK1) is an important molecule in proliferation of many human cancers. The aim of study is to clarify the expression patterns and potential function of PLK1 in colorectal cancers. Material/Methods Fifty-six colorectal cancers samples were collected and arranged onto a tissue array and the expression of PLK1 were detected by immunohistochemistry and correlated with clinico-pathological characteristics and expression of PCNA. Expression of PLK1 in 9 colorectal cancer cells lines was investigated by RT-PCR and Western blot, then SW1116 cells lines were treated with PLK1 siRNA and the efficiency was examined by Western blot. Transwell test was applied to detect the migration and invasion capability of cancer cells by counting the number of cells passing through the membranes. Cell proliferation and apoptosis were examined by Cell Counting Kit-8 (CCK-8) and Annexin-V Kit. Results PLK1 was positively expressed in 73.2% (41/56) of colorectal cancers tissues, but in only 3.6% (2/56) of normal tissues, and was associated with Duke’s stage (P

Zearalenone (ZEN) is commonly found in many food commodities and is known to cause reproductive disorders and genotoxic effects. However, the mode of ZEN-induced cell death of macrophages and the mechanisms by which ZEN causes cytotoxicity remain unclear. The present study shows that ZEN treatment reduces viability of RAW264.7 cells in a dose-dependent manner. ZEN causes predominantly necrotic and late apoptotic cell death. ZEN treatment also results in the loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Pre-treatment of the cells with either z-VAD-fmk or z-IETD-fmk does not attenuate ZEN-mediated cell death, whereas catalase suppresses the ZEN-induced decrease in viability in RAW264.7 cells. Treating the cells with c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or p53 inhibitor prevented ZEN-mediated changes, such as MMP loss, cellular reactive oxygen species (ROS) increase, and cell death. JNK or p38 MAPK inhibitor inhibited mitochondrial alterations of Bcl-2 and Bax proteins with attendant decreases in cellular ROS levels. Knockdown of AIF via siRNA transfection also diminished ZEN-induced cell death. Further, adenosine triphosphate was markedly depleted in the ZEN-exposed cells. Collectively, these results suggest that ZEN induces cytotoxicity in RAW264.7 cells via AIF- and ROS-mediated signaling, in which the activations of p53 and JNK/p38 play a key role.

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel-3 (ClC-3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin-induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl-2, decreased the protein expressions of cytochrome C and caspase-3, and increased the mRNA expressions of cyclin D1 and ClC-3 in cells treated with cisplatin. The caspase-3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC-3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC-3. NPPB-induced CIC-3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin-induced apoptosis in human erythroleukemia K562 and RK562 cells.

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na + , K + -ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.

Studies have shown that statins may induce vascular smooth muscle cells (VSMCs) apoptosis. But its mechanisms are incompletely understood. In this study, we investigate the effects of atorvastatin and survivin antisense oligonucleotides (ASODN) on VSMCs apoptosis and the relation between survivin and VSMCs apoptosis.Cultured VSMCs were treated with atorvastatin and vascular endothelial grow factor (VEGF). Apoptosis of VSMCs at 6-72 h after treatment with atorvastatin was detected by means of Hoechst33258 staining. Survivin and Fas factors expression were detected by means of immunohistochemistry. Survivin and Fas mRNA expression were detected by means of RT-PCR. In order to determine the relations between survivin and VSMCs apoptosis, we also performed transfection of VSMCs with survivin ASODN using GenePORTER Transfection Reagent and studied the survivin protein expression by means of western blotting.VSMCs apoptosis after treatment with atorvastatin was increased in a dose- and time-dependent manner. The expression of survivin and survivin mRNA in VSMCs was significantly down-regulated at 24 h and disappeared at 48-72 h after treatment with atorvastatin. Fas and Fas mRNA in VSMCs could only be detected at 72 h and not at 6-48 h after treatment with atorvastatin. We did not observe any effects of VEGF on VSMCs apoptosis, on survivin and survivin mRNA expression, and on Fas and Fas mRNA expression in VSMCs after treatment with atorvastatin. At 48 hours after the start of transfection, survivin protein expression was significantly reduced after transfection with 0.5, 1.0 and 2.0 microg/ml of survivin ASODN and there was no survivin protein expression after transfection with 3.0 and 4.0 microg/ml of survivin ASODN. In contrast, in the GenePORTER only and SODN studies, survivin protein expression was observed with western blotting. Hoechst33258 staining showed that treatment of VSMCs with survivin ASODN resulted in VSMCs apoptosis.Atorvastatin induces VSMCs apoptosis in a dose- and time-dependent manner. Transfection of survivin ASODN can directly induce VSMCs apoptosis. The mechanisms of VSMCs apoptosis induced by atorvastatin may be mainly associated with down-regulation of survivin expression in VSMCs. Up-regulation of Fas in VSMCs may play a role in later stages following atorvastatin treatment.

Background Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC). Methods miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines. Results miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506. Conclusions miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.

The molecular mechanisms of aluminum (Al) toxicity and tolerance in plants have been the focus of ongoing research in the area of stress phytophysiology. Recent studies have described Al-induced apoptosis-like cell death in plant and animal cells. In this study, we show that yeast (Saccharomyces cerevisiae) exposed to low effective concentrations of Al for short times undergoes enhanced cell division in a manner that is dose and cell density dependent. At higher concentrations of Al or longer exposure times, Al induces cell death and growth inhibition. Several apoptotic features appear during Al treatment, including cell shrinkage, vacuolation, chromatin marginalization, nuclear fragmentation, DNA degradation, and DNA strand breaks, as well as concomitant cell aggregation. Yeast strains expressing Ced-9, Bcl-2, and PpBI-1 (a plant Bax inhibitor-1 isolated from Phyllostachys praecox), respectively, display more resistance to Al toxicity compared with control cells. Data from flow cytometric studies show these three antiapoptotic members do not affect reactive oxygen species levels, but decrease calcium ion (Ca2+) signals in response to Al stress, although both intracellular reactive oxygen species and Ca2+ levels were increased. The data presented suggest that manipulation of the negative regulation process of programmed cell death may provide a novel mechanism for conferring Al tolerance.

To study the protective effect and possible mechanisms of Dihydromyricetin(DMY)on PC12 cells injury in- duced by sodium nitroprusside( SNP).SNP toxicity cellular model was established using PC12 cells treated with SNP. Cell via- bility was determined by MTT assay. The apoptosis of treated cells was detected by Hoechst Staining. Effect of DMY on accumulation of ROS in PC12 cells induced by SNP was detected by fluorometric analysis. The pathways involved were studied by kinase specific inhibi- tors; The level of phosphorylated Akt and ERK1/2 was detected by Western blot with specific phosphor-antibodies.SNP in- duced the apoptosis of PC12 cells in a dose-dependent manner. DMY dose-dependently protected PC12 cells from injury induced by SNP. Hoechst staining indicated that SNP decreased the number of viable cells and induced shrinkage and aggregation of the nucleus, whereas DMY attenuiated the toxic effects of SNP. The level of ROS induced by SNP in PC12 cells was decreased gradually by DMY. PI3K specific inhibitor LY294002 and the MAPK pathway specific inhibitor PD98059 attenuated the protective effect of DMY on SNP-induced injury of PC12 cells. However, the effect of DMY could be blocked by LY294002 and PD98059 respectively.DMY possesses protective effect against apoptosis induced by SNP in PC12 cells,and its mechanisms may be partially related with Akt and ERK1/2 signaling.

The anticancer properties of epigallocatechin-3-gallate (EGCG) are documented in the treatment of several types of cancer; however, there is no relevant evidence for its efficacy in the treatment of renal cell carcinoma (RCC). In the present study, the therapeutic effects of EGCG in vitro were investigated, with particular attention to the metastatic behavior of human RCC cells. MTT assays and flow cytometry were performed to detect the effects of EGCG on the proliferation and apoptosis of RCC cells. The migration and invasion abilities of RCC cells following treatment with EGCG were assessed by wound-healing and Transwell assays, respectively. Gelatin zymography and western blot analysis were performed to analyze the effect of EGCG on matrix metalloproteinase-2 (MMP-2) and MMP-9 expression levels. The results suggested that EGCG was able to inhibit the proliferation of RCC cells, induce apoptosis and effectively suppressed the migration and invasion of RCC cells. In addition, EGCG treatment resulted in the downregulation of MMP-2 and MMP-9 in RCC cells. We hypothesize that the anticancer effect associated with EGCG may involve the downregulation of MMP-2 and MMP-9. The present results suggest the potential of EGCG as a novel therapeutic agent against RCC.

To observe the protective effect of rutin (RUT) on neuronal cells against sodium nitroprusside (SNP) induced neurotoxicity.PC12 cells were treated with different concentration of SNP for 24 h and MTT assay was applied to analyze the survival rate; PC12 cells were pretreated with rutin for 1 h, and then incubated for 24 h with SNP. MTT assay, morphological observation, as well as immunofluorescence were performed to evaluate both the SNP neurotoxicity and the protective effects of RUT, Western blot was used to analyzed the level of phosphorylated extra cellular regulated protein kinases (ERK1/2) after treatment with RUT, the results were also testified in primary cultured neurons.Results from MTT assay showed that SNP caused cell death in a concentration-dependent manner in PC12 cells. The effect of SNP was observed at 200 - 1 000 micromol/L and was significant at 800 micromol/L. 25 micromol/L rutin partly blocked the neurotoxicity of SNP by preventing PC12 cells from apoptosis. Hoechst and PI staining indicated that SNP treatment decreased the number of viable cells and induced shrinkage and aggregation of the nucleus, whereas RUT pretreatment attenuated the toxic effects of SNP, after treatment with RUT in PC12 cells, the phosphorylation of ERK1/2 was increased and peaked at 20 min. Most importantly, the protective effect of RUT on PC12 cells was confirmed on cultured neurons.RUT possesses protective effect against neuronal apoptosis induced by SNP and this effect may be partially related with ERK1/2 signaling.

Long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has recently been identified to be involved in tumorigenesis of several cancers such as lung cancer, pancreatic cancer, and cervical cancer. However, the role of lncRNA MALAT1 in clear cell renal cell carcinoma (ccRCC) remains unclear. Expression levels of lncRNA MALAT1 in ccRCC tissues and renal cancer cell lines were evaluated by quantitative real-time PCR (qRT-PCR), and its association with overall survival of patients was analyzed by statistical analysis. Small interfering RNA (siRNA) was used to suppress MALAT1 expression in renal cancer cells. In vitro assays were conducted to further explore its role in tumor progression. The expression level of MALAT1 was higher in ccRCC tissues and renal cancer cells compared to adjacent non-tumor tissues and normal human proximal tubule epithelial cells HK-2. The ccRCC patients with higher MALAT1 expression had an advanced clinical features and a shorter overall survival time than those with lower MALAT1 expression. And multivariate analysis showed that the status of MALAT1 expression was an independent predictor of overall survival in ccRCC. Additionally, our data indicated that knockdown expression of MALAT1 decreased renal cancer cell proliferation, migration, and invasion. Our data suggested that lncRNA MALAT1 was a novel molecule involved in ccRCC progression, which provided a potential prognostic biomarker and therapeutic target.