SEPT9, H4C6, and RASSF1A methylation in nasopharyngeal swabs: A reflection of potential minimally invasive biomarkers for early screening of nasopharyngeal cancer.

The potential value of epigenetic DNA methylation in early cancer screening has been demonstrated. Therefore, in this study, we performed QMS-PCR and quantitative reverse transcription PCR on the genes RASSF1A, H4C6, SEPT9, GSTP1, PAX1, SHOX2, and SOX2, which are common in epithelial cancers. We found hypermethylation in RASSF1A, H4C6 and SEPT9. The mRNA expressions of RASSF1A, H4C6 and SEPT9 in tumor group were significantly different from those in the inflammatory group and healthy group (P < .05). Receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of RASSF1A, H4C6 and SEPT9 genes were 0.831, 0.856 and 0.767, respectively. The areas under the AUC curve of SEPT9 + H4C6, SEPT9 + RASSF1A and H4C6 + RASSF1A are 0.946, 0.912 and 0.851, respectively. The diagnostic ability of dual gene combination is better than that of single gene combination, among which SEPT9 and H4C6 combination has the best diagnostic effect. In conclusion, our findings suggest that H4C6, RASSF1A, and SEPT9 methylation occur more frequently in nasopharyngeal carcinoma, and their detection in nasopharyngeal swabs may be a minimally invasive tool for diagnosis of nasopharyngeal carcinoma.

Morphine stimulates angiogenesis through Akt/mTOR/eIF4E activation under serum deprivation or H2 O2 -induced oxidative stress condition.

Morphine is an opioid analgesic drug routinely used to treat pain in several medical conditions including cancer. Increasing evidence has shown that morphine can directly modulate cancer growth via regulating angiogenesis. In this work, we investigated the effect of morphine on angiogenesis under pathological conditions. We showed that morphine, in a concentration typical of that observed in patient's blood, stimulates tumour angiogenesis under serum deprivation and H2 O2 -induced oxidative stress conditions. We found that morphine protected human lung tumour associated-endothelial cell (HLT-EC) against serum deprivation or H2 O2 -induced inhibition of capillary network formation. Furthermore, morphine stimulated other biological functions of HLT-EC under serum deprivation and H2 O2 -induced pathological conditions, such as growth, migration and survival, without affecting HLT-EC adhesion. Interestingly, morphine at the same concentration did not affect lung tumour cell growth and survival, suggesting the specific protective role of morphine at low micromolar concentrations on tumour angiogenesis. Using in vivo Matrigel angiogenesis assay, it was found that morphine stimulated in vivo angiogenesis under H2 O2 -induced pathological condition. The opioid receptor antagonist, naloxone, did not inhibit the protective activity of morphine in in vivo angiogenesis, indicating that the effect was less likely to be mediated by the typical opioid receptors. Mechanism analysis indicated that morphine alleviated serum deprivation and H2 O2 -induced angiogenesis inhibition via reducing oxidative stress and damage, and activating Akt/mTOR/eIF4E signalling. We demonstrate the protective role of morphine on tumour angiogenesis under pathological conditions. Our work suggests that clinical use of morphine may be harmful in patients with angiogenesis-dependent cancers.

The Roles of Autophagy in Acute Lung Injury Induced by Myocardial Ischemia Reperfusion in Diabetic Rats.

Patients with diabetes are vulnerable to myocardial ischemia reperfusion (IR) injury, which may also induce acute lung injury (ALI) due to overaccumulation of reactive oxygen species (ROS) and inflammation cytokine in circulation. Despite autophagy plays a significant role in diabetes and pulmonary IR injury, the role of autophagy in ALI secondary to myocardial IR in diabetes remains largely elusive. We aimed to investigate pulmonary autophagy status and its roles in oxidative stress and inflammation reaction in lung tissues from diabetic rats subjected to myocardial IR. Control or diabetic rats were either treated with or without autophagy inducer rapamycin (Rap) or autophagy inhibitor 3-methyladenine (3-MA) before myocardial IR, which was achieved by occluding the left anterior descending coronary artery for 30 min and followed by reperfusion for 120 min. Diabetic rats subjected to myocardial IR showed more serious ALI with higher lung injury score and WET/DRY ratio and lower PaO2 as compared with control rats, accompanied with impaired autophagy indicated by reduced LC-3II/LC-3I ratio and Beclin-1 expression, decreased superoxide dismutase (SOD) activity, and increased 15-F2t-Isoprostane formation in lung tissues, as well as increased levels of leukocyte count and proinflammatory cytokines in BAL fluid. Improving autophagy with Rap significantly attenuated all these changes, but the autophagy inhibitor 3-MA exhibited adverse or opposite effects as Rap. In conclusion, diabetic lungs are more vulnerable to myocardial IR, which are involved in impaired autophagy. Improving autophagy could attenuate ALI induced by myocardial IR in diabetic rats, possibly through inhibiting inflammatory reaction and oxidative stress.

Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCß2 Activation and Improving Mitochondrial Quality.

BACKGROUND: Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKCß2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKCß2 activation and improving mitochondrial quality in cultured H9C2 cells. METHODS: H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKCß2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKCß2 activation were detected by Western blot. RESULTS: HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKCß2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKCß2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation. CONCLUSIONS: Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKCß2 activation and improving mitochondria quality.

Cell cycle protein Bora serves as a novel poor prognostic factor in multiple adenocarcinomas.

Cell cycle protein Bora has been identified to integrate the functions of three major mitotic kinases: Cyclin-dependent kinase-1, Polo-like kinase-1, and Aurora A kinase. Overexpression of Bora disrupts spindle assembly and causes genomic instability. However, the clinical relevance of Bora in cancer remains unclear. In this study, we examined the expression of Bora and its association with clinical characteristics in breast (n = 538), lung (n = 144) and gastric (n = 77) adenocarcinomas. We found that Bora was overexpressed in primary breast cancer tissues compared to paired non-cancerous tissues. Bora overexpression was observed at a higher proportion in triple-negative breast cancer (TNBC, 77.63%) compared with non-TNBC subtypes (42.76%, P < 0.0001). Kaplan-Meier survival analysis indicated that Bora overexpression was associated with unfavourable overall survival (OS, P < 0.0001) and disease-free survival (DFS, P = 0.007) in breast cancer. In addition, Bora subclassified patients with distinct clinical outcomes in both stages (II/III) and subtypes (HR+, HER2+) of breast cancer. Consistently, Bora was associated with adverse prognosis in lung (P = 0.005 for OS and DFS P = 0.001 for DFS) and gastric adenocarcinomas (P < 0.0001 for OS, and P < 0.0001 for DFS). Moreover, Bora was positively correlated with proliferation index Ki67 in breast and gastric cancer (P < 0.001, P = 0.005, respectively). Multivariate analyses further revealed that Bora was an independent prognostic parameter for OS and DFS in all three types of adenocarcinomas. In conclusion, our findings demonstrated that Bora was overexpressed and served as an independent biomarker for poor prognosis in multiple adenocarcinomas.

Upregulation of long noncoding RNA HOTTIP promotes metastasis of esophageal squamous cell carcinoma via induction of EMT.

Esophageal squamous cell carcinoma (ESCC) is one of the prevalent and deadly cancers worldwide, especially in Eastern Asia. The prognosis of ESCC remains poor; thus, it is still necessary to further dissect the underlying mechanisms and explore therapeutic targets of ESCC. Recent studies show that lncRNAs involve in the initiation and progression of various cancers including ESCC. HOTTIP has been recently revealed as oncogenic regulator in different cancers, however, whether HOTTIP is involved in ESCC remains poorly understood. To investigate the role of HOTTIP in ESCC, we evaluated the HOTTIP expression levels in a series of ESCC tissues and a panel of ESCC cell line using qRT-PCR. Moreover, we investigated the effect of HOTTIP on cell proliferation, migration and invasion of ESCC cells. Here, we reported that HOTTIP was upregulated in ESCC. Further experiments revealed that HOTTIP knockdown significantly inhibited ESCC cells proliferation by causing G1 arrest. Furthermore, inhibitory effects of HOTTIP on cell migration and invasion were partly associated with EMT process. In conclusion, these data suggest that HOTTIP could be an oncogene for ESCC, and may be served as a candidate target for new therapies in human ESCC.

N-acetylcysteine attenuates myocardial dysfunction and postischemic injury by restoring caveolin-3/eNOS signaling in diabetic rats.

BACKGROUND: Patients with diabetes are prone to develop cardiac hypertrophy and more susceptible to myocardial ischemia-reperfusion (I/R) injury, which are concomitant with hyperglycemia-induced oxidative stress and impaired endothelial nitric oxide (NO) synthase (eNOS)/NO signaling. Caveolae are critical in the transduction of eNOS/NO signaling in cardiovascular system. Caveolin (Cav)-3, the cardiomyocytes-specific caveolae structural protein, is decreased in the diabetic heart in which production of reactive oxygen species are increased. We hypothesized that treatment with antioxidant N-acetylcysteine (NAC) could enhance cardiac Cav-3 expression and attenuate caveolae dysfunction and the accompanying eNOS/NO signaling abnormalities in diabetes. METHODS: Control or streptozotocin-induced diabetic rats were either untreated or treated with NAC (1.5 g/kg/day, NAC) by oral gavage for 4 weeks. Rats in subgroup were randomly assigned to receive 30 min of left anterior descending artery ligation followed by 2 h of reperfusion. Isolated rat cardiomyocytes or H9C2 cells were exposed to low glucose (LG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) for 36 h before being subjected to 4 h of hypoxia followed by 4 h of reoxygenation (H/R). RESULTS: NAC treatment ameliorated myocardial dysfunction and cardiac hypertrophy, and attenuated myocardial I/R injury and post-ischemic cardiac dysfunction in diabetic rats. NAC attenuated the reductions of NO, Cav-3 and phosphorylated eNOS and mitigated the augmentation of O2-, nitrotyrosine and 15-F2t-isoprostane in diabetic myocardium. Immunofluorescence analysis demonstrated the colocalization of Cav-3 and eNOS in isolated cardiomyocytes. Immunoprecipitation analysis revealed that diabetic conditions decreased the association of Cav-3 and eNOS in isolated cardiomyocytes, which was enhanced by treatment with NAC. Disruption of caveolae by methyl-ß-cyclodextrin or Cav-3 siRNA transfection reduced eNOS phosphorylation. NAC treatment attenuated the reductions of Cav-3 expression and eNOS phosphorylation in HG-treated cardiomyocytes or H9C2 cells. NAC treatment attenuated HG and H/R induced cell injury, which was abolished during concomitant treatment with Cav-3 siRNA or eNOS siRNA. CONCLUSIONS: Hyperglycemia-induced inhibition of eNOS activity might be consequences of caveolae dysfunction and reduced Cav-3 expression. Antioxidant NAC attenuated myocardial dysfunction and myocardial I/R injury by improving Cav-3/eNOS signaling.

Aurora kinase A inhibition-induced autophagy triggers drug resistance in breast cancer cells.

We have previously shown that elevated expression of mitotic kinase aurora kinase A (AURKA) in cancer cells promotes the development of metastatic phenotypes and is associated clinically with adverse prognosis. Here, we first revealed a clinically positive correlation between AURKA and autophagy-associated protein SQSTM1 in breast cancer and further demonstrated that AURKA regulated SQSTM1 through autophagy. Indeed, depletion by siRNA or chemical inhibition of AURKA by the small molecule VX-680 increased both the level of microtubule-associated protein 1 light chain 3-II (LC3-II) and the number of autophagosomes, along with decreased SQSTM1. Conversely, overexpression of AURKA inhibited autophagy, as assessed by decreased LC3-II and increased SQSTM1 either upon nutrient deprivation or normal conditions. In addition, phosphorylated forms of both RPS6KB1 and mechanistic target of rapamycin (MTOR) were elevated by overexpression of AURKA whereas they were suppressed by depletion or inhibition of AURKA. Moreover, inhibition of MTOR by PP242, an inhibitor of MTOR complex1/2, abrogated the changes in both LC3-II and SQSTM1 in AURKA-overexpressing BT-549 cells, suggesting that AURKA-suppressed autophagy might be associated with MTOR activation. Lastly, repression of autophagy by depletion of either LC3 or ATG5, sensitized breast cancer cells to VX-680-induced apoptosis. Similar findings were observed in cells treated with the autophagy inhibitors chloroquine (CQ) and bafilomycin A 1 (BAF). Our data thus revealed a novel role of AURKA as a negative regulator of autophagy, showing that AURKA inhibition induced autophagy, which may represent a novel mechanism of drug resistance in apoptosis-aimed therapy for breast cancer.