Mitochondrial dysfunction of peripheral blood mononuclear cells is associated with lung carcinogenesis.

The composition, quantity, and function of peripheral blood mononuclear cells (PBMCs) are closely correlated with tumorigenesis. However, the mechanisms of PBMCs in lung cancer are not clear. Mitochondria are energy factories of cells, and almost all cellular functions rely on their energy metabolism level. The present study aimed to test whether the mitochondrial function of PBMCs directly determines their tumor immune monitoring function. We recruited 211 subjects, including 105 healthy controls and 106 patients with recently diagnosed with lung cancer. The model of lung carcinogenesis induced by BaP was used in animal experiment, and the Bap carcinogenic metabolite, Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), was used in cell experiment. We found that mitochondrial function of PBMCs decreased significantly in patients with new lung cancer, regardless of age. In vivo, BaP caused PBMC mitochondrial dysfunction in mice before the appearance of visible malignant tissue. Moreover, mitochondrial function decreased significantly in mice with lung cancers induced by BaP compared to those without lung cancer after BaP intervention. In vitro, BPDE also induced mitochondrial dysfunction and reduced the aggressiveness of PBMCs toward cancer cells. Furthermore, the changes in mitochondrial energy metabolism gene expression caused by BPDE are involved in this process. Thus, the mitochondrial function of PBMCs is a potential prognostic biomarker or therapeutic target to improve clinical outcomes in patients with lung cancer.

A human-specific insertion promotes cell proliferation and migration by enhancing TBC1D8B expression.

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC. Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo. Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

Association between oxidative stress, mitochondrial function of peripheral blood mononuclear cells and gastrointestinal cancers.

BACKGROUND: The incidence and mortality rate of gastrointestinal cancers are high worldwide. Increasing studies have illustrated that the occurrence, progression, metastasis and prognosis of cancers are intimately linked to the immune system. Mitochondria, as the main source of cellular energy, play an important role in maintaining the physiological function of immune cells. However, the relationship between mitochondrial function of immune cells and tumorigenesis has not yet been systematically investigated. METHODS: A total of 150 cases, including 60 healthy donors and 90 primary gastrointestinal cancer patients without anti-tumor treatments (30 with gastric cancer, 30 with liver cancer and 30 with colorectal cancer) were involved in our study. The oxidant/antioxidant and cytokine levels in plasma, the ROS level, mitochondrial function and apoptosis ratio of peripheral blood mononuclear cells (PBMCs) were evaluated. RESULTS: The imbalance between oxidant and antioxidant in plasma was discovered in the primary gastrointestinal cancer patients. The levels of cell reactive oxygen species (ROS) and mitochondrial ROS in PBMCs of primary gastrointestinal cancers were significantly increased compared with that in healthy donors. Meanwhile, the ATP content, the mtDNA copy number and the mitochondrial membrane potential (MMP) in PBMCs of patients with primary gastrointestinal cancers were lower than those in control group. The decreased MMP also occurred in immune cells of gastrointestinal cancers, including T cell, B cell, NK cell and monocyte. Furthermore, the PBMCs apoptosis ratio of primary gastrointestinal cancer patients was significantly higher than that of control group. Importantly, an increase of IL-2 and IL-6 and a decrease of IgG in plasma were found in the patients with primary gastrointestinal cancers. These changes of mitochondrial function in immune cells were consistent among primary gastrointestinal cancers without anti-tumor treatments, such as liver cancer, gastric cancer and colorectal cancer. CONCLUSION: Our study demonstrated that the imbalance of oxidation/antioxidation in primary gastrointestinal cancer patients without anti-tumor treatments results in excessive ROS. The oxidative stress was associated to the mitochondrial dysfunction, the apoptosis of immune cells and eventually the abnormal immune function in primary gastrointestinal cancers. The application of immune cell mitochondrial dysfunction into clinical evaluation is anticipated.

The regulatory roles of DDIT4 in TDCIPP-induced autophagy and apoptosis in PC12 cells.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is a commonly used organophosphate-based flame retardant and can bio-accumulate in human tissues and organs. As its structure is similar to that of neurotoxic organophosphate pesticides, the neurotoxicity of TDCIPP has raised widespread concerns. TDCIPP can increase neuronal apoptosis and induce autophagy. However, its regulatory mechanism remains unclear. In this study, we found that the expression upregulation of the DNA Damage-Inducible Transcript 4 (DDIT4) protein, which might play essential roles in TDCIPP-induced neuronal autophagy and apoptosis, was observed in TDCIPP-treated differentiated rat PC12 cells. Furthermore, we determined the protective effect of the DDIT4 suppression on the autophagy and apoptosis induced by TDCIPP using Western blot (WB) and Flow cytometry (FACS) analysis. We observed that TDCIPP treatment increased the DDIT4, the autophagy marker Beclin-1, and the microtubule-associated protein light chain 3-II (LC3II) expressions and decreased the mTOR phosphorylation levels. Conversely, the suppression of DDIT4 expression increased the p-mTOR expression and decreased cell autophagy and apoptosis. Collectively, our results revealed the function of DDIT4 in cell death mechanisms triggered by TDCIPP through the mTOR signaling axis in differentiated PC12 cells. Thus, this study provided vital evidence necessary to explain the mechanism of TDCIPP-induced neurotoxicity in differentiated PC12 cells.

[Construction and evaluation of a gradient stress model of PC12 cells induced by corticosterone].

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 µmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 µmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 µmol/L, 25 µmol/L, 50 µmol/L, 100 µmol/L, 150 µmol/L and 200 µmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.

Resveratrol Ameliorates High Altitude Hypoxia-Induced Osteoporosis by Suppressing the ROS/HIF Signaling Pathway.

Hypoxia at high-altitude leads to osteoporosis. Resveratrol (RES), as an antioxidant, has been reported to promote osteoblastogenesis and suppress osteoclastogenesis. However, the therapeutic effect of RES against osteoporosis induced by high-altitude hypoxia remains unclear. Thus, this study was intended to investigate the potential effects of RES on high-altitude hypoxia-induced osteoporosis both in vivo and in vitro. Male Wistar rats were given RES (400 mg/kg) once daily for nine weeks under hypoxia, while the control was allowed to grow under normoxia. Bone mineral density (BMD), the levels of bone metabolism-related markers, and the changes on a histological level were measured. Bone marrow-derived mesenchymal stem cells (BMSCs) and RAW264.7 were incubated with RES under hypoxia, with a control growing under normoxia, followed by the evaluation of proliferation and differentiation. The results showed that RES inhibited high-altitude hypoxia-induced reduction in BMD, enhanced alkaline phosphatase (ALP), osteocalcin (OCN), calcitonin (CT) and runt-related transcription factor 2 (RUNX2) levels, whereas it reduced cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I) levels and tartrate-resistant acid phosphatase (TRAP) activity in vivo. In addition, RES attenuated histological deteriorations in the femurs. In vitro, RES promoted osteoblastogenesis and mineralization in hypoxia-exposed BMSCs, along with promotion in RUNX2, ALP, OCN and osteopontin (OPN) levels, and inhibited the proliferation and osteoclastogenesis of RAW264.7. The promotion effects of RES on osteoblastogenesis were accompanied by the down-regulation of reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) induced by hypoxia. These results demonstrate that RES can alleviate high-altitude hypoxia-induced osteoporosis via promoting osteoblastogenesis by suppressing the ROS/HIF-1α signaling pathway. Thus, we suggest that RES might be a potential treatment with minimal side effects to protect against high-altitude hypoxia-induced osteoporosis.

[Effects of three Polyphenolic compounds on the intestinal flora of mice exposed simulated intermittent plateau hypoxia].

OBJECTIVE: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. METHODS: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. RESULTS: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (P＜0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(P＜0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(P＜0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(P＜ 0.05). CONCLUSION: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.

Expression and significance of SOX B1 genes in glioblastoma multiforme patients.

The overall survival of glioblastoma multiforme (GBM) patients remains poor. To improve patient outcomes, effective diagnostic and prognostic biomarkers for GBM are needed. In this study, we first applied bioinformatic analyses to identify biomarkers for GBM, focusing on SOX (sex-determining region on the Y chromosome (SRY)-related high mobility group (HMG) box) B1 family members. The ONCOMINE, GEPIA, LinkedOmics and CCLE databases were used to assess mRNA expression levels of the SOX B1 family members in different cancers and normal tissue. Further bioinformatic analysis was performed using the ONCOMINE database in combination with the LinkedOmics data set to identify the prognostic value of SOX B1 family members for GBM. We found mRNA expression levels of all tested SOX B1 genes were significantly increased in GBM. In the LinkedOmics database, increased expression of SOX3 indicated a better overall survival. In GEPIA databases, increased expression of all SOX B1 family members suggested an improved overall survival, but none of them were statistically different. Then, Transwell assays and wound healing were employed to evaluate the motility and invasive captivity of U251 cells when silencing SOX2 and SOX3. We found exogenous inhibition of SOX2 appeared to reduce the migration and invasion of U251 cells in vitro. Collectively, our research suggested that SOX2 might serve as a cancer-promoting gene to identify high-risk GBM patients, and SOX3 had the potential to be a prognostic biomarker for GBM patients.

Sestrin2 as a gatekeeper of cellular homeostasis: Physiological effects for the regulation of hypoxia-related diseases.

Sestrin2 (SESN2) is a conserved stress-inducible protein (also known as hypoxia-inducible gene 95 (HI95)) that is induced under hypoxic conditions. SESN2 represses the production of reactive oxygen species (ROS) and provides cytoprotection against various noxious stimuli, including hypoxia, oxidative stress, endoplasmic reticulum (ER) stress and DNA damage. In recent years, the determination of the regulation and signalling mechanisms of SESN2 has increased our understanding of its role in the hypoxic response. SESN2 has well-documented roles in hypoxia-related diseases, making it a potential target for diagnosis and treatment. This review discusses the regulatory mechanisms of SESN2 and highlights the significance of SESN2 as a biomarker and therapeutic target in hypoxia-related diseases, such as cancer, respiratory-related diseases, cardiovascular diseases and cerebrovascular diseases.

Intermittent hypobaric hypoxia causes deleterious effects on the reproductive system in female rats.

BACKGROUNDS: Numerous studies have revealed that hypobaric hypoxia exposure elicited imbalance of homeostasis. However, the effects of intermittent hypobaric hypoxia (IHH) exposure on the female reproductive system have been rarely reported. This study aims to assess the effects of IHH on rat female reproductive system and explore the corresponding mechanism at the histological, endocrine and molecular levels. METHODS: The female rats were randomly divided into control and IHH groups. Multiple pathophysiological parameters, including body weight gain, organ coefficients, estrus cycle, and health signs were measured. Also, the reproductive hormones, hormone receptor mRNA expression and oxidant/antioxidant level were evaluated. RESULTS: Significant increases of the heart, liver and lung coefficients were observed after IHH exposure. There were no statistically significant differences in ovarian and uterine coefficients, but changes were found in the morphology of the ovary and uterus. Additionally, the diestrus phase duration was significantly increased during IHH exposure. Furthermore, estrogen increased and the Luteinizing hormone and progesterone decreased after IHH exposure. Altered expression of ER, PR and LHR were also found in the IHH exposed rats. Importantly, IHH exposure significantly repressed the activities of GSH-Px and T-SOD and improved the contents of MDA. CONCLUSIONS: Our results evince that IHH exposure caused estrus cycle irregularity. IHH induced oxidative stress along with ovarian and uterine structure damages, reproductive hormone disturbances and unusual expression of hormone receptors, thus suggesting a potential mechanism underlying IHH-induced reproductive system dysfunction.

Downregulation of miR-541 induced by heat stress contributes to malignant transformation of human bronchial epithelial cells via HSP27.

Environmental factors are one of the important factors affecting the occurrence of lung cancer. However, few studies have been done on the relationship between hot environment and lung cancer. In the present study, we demonstrated that heat stress leads to anchorage-independent proliferation, mitochondrial apoptosis, and autophagy of Beas-2B cells, which are normal lung bronchial epithelial cells. Heat shock protein 27 (HSP27) promoted heat stress-induced anchorage-independent proliferation and autophagy, but suppressed mitochondrial apoptosis, indicating that HSP27 might act as an oncogene in the malignant transformation of lung epithelial cells. We also showed that HSP27 promoted autophagy of these cells under heat stress via autophagy related 7 (ATG7) and ETS Transcription Factor ELK1 (ELK1), a transcription factor of ATG7, under heat stress. In addition, we showed that HSP27 translation could be repressed by microRNA miR-541, and the biological effects of miR-541 were the opposite to HSP27, suggesting that HSP27 is a downstream target of miR-541. In this study, we characterized a new mechanism whereby HSP27 promotes cell transformation during the onset of lung cancer. Our studies provide new insights into the molecular mechanisms underlying the lung carcinogenic effect of heat exposure. Specifically, heat stress promotes translation of HSP27 by inhibiting miR-541 accumulation, ultimately resulting in activation of autophagy, inhibition of mitochondrial apoptotic pathway and malignant transformation of Human Bronchial Epithelial Cells. This study identifies miR-541 as a potential prognostic biomarker or therapeutic target to improve theory of environmental carcinogenesis.

[The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism].

OBJECTIVE: To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. METHODS: Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. RESULTS: With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. CONCLUSION: Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

[Effects of soybean isoflavones on the energy metabolism of swimming mice].

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(P<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(P<0.05). CONCLUSIONS: Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.

Novel long noncoding RNA NMR promotes tumor progression via NSUN2 and BPTF in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNA) have been implicated in cancer but most of them remain largely unstudied. Here, we identified a novel NSUN2 methylated lncRNA (NMR), which was significantly upregulated in esophageal squamous cell carcinoma (ESCC), functioned as a key regulator of ESCC tumor metastasis and drug resistance. Upregulation of NMR correlated with tumor metastasis and indicated poor overall survival in ESCC patients. Functionally, NMR could promote tumor cell migration and invasion, inhibit cisplatin-induced apoptosis and increase drug resistance in ESCC cells. Mechanistically, transcription of NMR could be upregulated by NF-κB activation after IL-1ß and TNF-α treatment. NMR was methylated by NSUN2 and might competitively inhibit methylation of potential mRNAs. NMR could directly bind to chromatin regulator BPTF, and potentially promote MMP3 and MMP10 expression by ERK1/2 pathway through recruiting BPTF to chromatin. Taken together, NMR functions as an oncogenic gene and may serve as new biomarker and therapeutic target in ESCC.

Prediction of Chemotherapeutic Efficacy in Non-Small Cell Lung Cancer by Serum Metabolomic Profiling.

Purpose: No validated biomarkers that could identify the subset of patients with lung adenocarcinoma who might benefit from chemotherapy have yet been well established. This study aimed to explore potential biomarker model predictive of efficacy and survival outcomes after first-line pemetrexed plus platinum doublet based on metabolomics profiling.Experimental Design: In total, 354 consecutive eligible patients were assigned to receive first-line chemotherapy of pemetrexed in combination with either cisplatin or carboplatin. Prospectively collected serum samples before initial treatment were utilized to perform metabolomics profiling analyses under the application of LC/MS-MS. Binary logistic regression analysis was carried out to establish discrimination models.Results: There were 251 cases randomly sorted into discovery set, the rest of 103 cases into validation set. Seven metabolites including hypotaurine, uridine, dodecanoylcarnitine, choline, dimethylglycine, niacinamide, and l-palmitoylcarnitine were identified associated with chemo response. On the basis of the seven-metabolite panel, a discriminant model according to logistic regression values g(z) was established with the receiver operating characteristic curve (AUC) of 0.912 (Discovery set) and 0.909 (Validation set) in differentiating progressive disease (PD) groups from disease control (DC) groups. The median progression-free survival (PFS) after chemotherapy in patients with g(z) ≤0.155 was significantly longer than that in those with g(z) > 0.155 (10.3 vs.4.5 months, P < 0.001).Conclusions: This study developed an effective and convenient discriminant model that can accurately predict the efficacy and survival outcomes of pemetrexed plus platinum doublet chemotherapy prior to treatment delivery. Clin Cancer Res; 24(9); 2100-9. ©2018 AACR.

TGF-ß-induced NKILA inhibits ESCC cell migration and invasion through NF-κB/MMP14 signaling.

The transforming growth factor ß (TGF-ß) signaling pathway plays anti- and pro-tumoral roles in the vast majority of cancers, and long noncoding RNAs have been reported to play key roles in the highly contextual response process. However, the roles of long noncoding RNAs (lncRNAs) in TGF-ß signaling in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we performed RNA-seq to compare lncRNAs expression levels between TGF-ß1-treated and untreated ESCC cells and observed that NF-kappaB-interacting lncRNA (NKILA) was remarkably upregulated by the classical TGF-ß signaling pathway. RNA profiling of 39 pairs ESCC tumor and adjacent nontumor samples using RT-qPCR demonstrated that NKILA is significantly downregulated in ESCC tumor tissues, and NKILA expression levels were significantly decreased in advanced tumor tissues (III and IV) compared to early stages (I and II) (p < 0.01). Gain- and loss-of-function assays showed that NKILA inhibited ESCC cell metastasis in vitro and in vivo, and mechanism studies showed that NKILA repressed MMP14 expression by inhibiting IκBα phosphorylation and NF-κB activation. Collectively, these findings suggest that the TGF-ß-induced lncRNA NKILA has potential as an antimetastasis therapy. KEY MESSAGES: Long noncoding RNA NKILA could be remarkably upregulated by classical TGF-ß signal pathway in ESCC. NKILA was significantly downregulated in esophageal squamous cell carcinoma and negatively correlated with TNM stage. NKILA inhibits ESCC cell metastasis via repressing MMP14 expression by suppressing the phosphorylation of IκBα and NF-κB activation.

Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and is generally resistant to chemotherapy. In the present study, the cytotoxic activity of Rabdocoestin B (Rabd-B) against ESCC and the underlying mechanisms were investigated. METHODS: The inhibitory effect of Rabd-B on KYSE30 and KYSE450 was evaluated by Cell Counting Kit-8 (CCK8) and colony formation assays in vitro. The cell cycle distribution and apoptosis of cells treated with Rabd-B were determined by flow cytometry. The mechanisms underlying the effects of Rabd-B were systematically examined by Western blot. The in vivo anti-tumor ability of Rabd-B was measured in mouse xenograft models and cisplatin (DDP) was used as positive control. RESULTS: Rabd-B efficiently induced G2/M phase arrest in ESCC cells by upregulating the Chk1/Chk2-Cdc25C axis to inhibit the G2→M transition facilitated by Cdc2/Cyclin B1. Furthermore, Rabd-B suppressed ATM/ATR phosphorylation, thereby inhibiting BRCA1-mediated DNA repair, which resulted in mitotic catastrophe and induced cell apoptosis. Rabd-B also decreased the activity of the Akt and NF-κB survival signaling pathways and ultimately initiated the caspase-9-dependent intrinsic apoptotic pathway in ESCC cells. The apoptosis induced by Rabd-B could be partially reversed by a caspase-9-specific inhibitor (Z-LEHD-FMK) and a pan-caspase inhibitor (Z-VAD-FMK). Moreover, Rabd-B effectively suppressed tumor growth in mouse xenografts which was comparable to that of DDP without significant injuries to the mice. CONCLUSION: Taken together, these findings indicate that Rabd-B is a promising precursor compound that may be useful as a treatment for ESCC and thus warrants further investigation.

Therapeutic Efficacy of Methazolamide Against Intermittent Hypoxia-Induced Excessive Erythrocytosis in Rats.

Zhang, Zhiqing, Zhonghai Xiao, Bingnan Deng, Xiaohua Liu, Wei Liu, Hongjing Nie, Xi Li, Zhaoli Chen, Danfeng Yang, and Ruifeng Duan. Therapeutic efficacy of methazolamide against intermittent hypoxia-induced excessive erythrocytosis in rats. High Alt Med Biol 19:69-80, 2018.-This study aimed to determine whether methazolamide is effective for the treatment of chronic mountain sickness. Forty-eight male Wistar rats were randomly divided into eight groups: normoxia control, hypoxia control, hypoxia + acetazolamide (30 mg·kg-1·d-1), and five hypoxia + methazolamide groups (5, 10, 30, 90, and 120 mg·kg-1·d-1). Excessive erythrocytosis was induced through 4 weeks of hypobaric hypoxia (8 hours O2 10%/16 hours O2 21%). Rats were then treated for 4 weeks, and their body weight was measured. Hematological, hemorheological, and biochemical parameters were analyzed. Renal hypoxia-inducible factor-1alpha (HIF-1α) and vascular endothelial growth factor (VEGF) levels were detected by immunohistochemistry. Proteomic analysis of plasma was conducted to determine the most differentially expressed proteins. Methazolamide with doses lower than 30 mg·kg-1·d-1 had no significant effects on body weight compared with the hypoxia control group (p > 0.05). Methazolamide dose-dependently reduced the hemoglobin concentration, hematocrit (Hct), and blood viscosity. Hct/blood viscosity, an oxygen delivery index, dose-dependently increased after methazolamide treatment. A methazolamide dose of 10 mg·kg-1·d-1 showed similar efficacy to an acetazolamide dose of 30 mg·kg-1·d-1 for all the above parameters. Plasma levels of low-density lipoprotein cholesterol, total cholesterol, creatinine, and hemoglobin increased substantially after long-term hypoxia, but decreased after methazolamide treatment. HIF-1α and VEGF both increased substantially after long-term hypoxia and decreased in the kidney after methazolamide treatment. The most differentially expressed protein was haptoglobin, an endogenous protective factor, which was depleted in rats with excessive erythrocytosis and increased substantially after methazolamide treatment. In summary, methazolamide exhibits dose-dependent efficacy for the treatment of excessive erythrocytosis induced by long-term hypoxia. It also has beneficial effects on oxygen transport and lipid metabolism, which are encouraging with regard to the development of methazolamide-based chronic mountain sickness therapies.

Immune signature profiling identified predictive and prognostic factors for esophageal squamous cell carcinoma.

Understanding interactions between tumor and the host immune system holds great promise to uncover biomarkers for targeted therapies and clinical outcomes. However, systematical analysis of immune signatures in esophageal squamous cell carcinoma (ESCC) remains largely unstudied. In this study, immune signatures containing 708 immune related genes were curated from mRNA microarray data with tumor and paired normal tissues from 119 ESCC patients. Differential expression and survival analysis were performed with validations from Human Protein Atlas and an independent cohort of 110 ESCC patients by immunohistochemistry staining. We identified a total of 186 significantly dysregulated genes in ESCC, including downregulated genes SPINK5, IL1RN and upregulated genes SPP1 and PLAU, which were further confirmed in Human Protein Atlas data. Moreover, nine immune related genes (ABL1, ATF2, ATG5, C6, CD38, HMGB1, ICOSLG, IL12RB2 and PLAU) were significantly associated with patients' overall survival, among which, prognostic model was built including three independent factors ABL1, CD38 and ICOSLG. Validation by immunohistochemistry staining suggested that combination with tumor infiltrated CD4+ and CD8+ T lymphocytes would yield higher performance in distinguishing cases as high or low risk of unfavorable prognosis. In summary, we profiled the immune status in ESCC and established predictive and prognostic factors for ESCC, which could reflect immune disorders within tumor microenvironments and independently distinguish patients with a high risk of reduced survival, providing novel predictive and therapeutic targets for ESCC patients in the future.