Construction of an immune-related prognostic model and potential drugs screening for esophageal cancer based on bioinformatics analyses and network pharmacology.

BACKGROUND: Esophageal cancer (ESCA) is a highly invasive malignant tumor with poor prognosis. This study aimed to discover a generalized and high-sensitivity immune prognostic signature that could stratify ESCA patients and predict their overall survival, and to discover potential therapeutic drugs by the connectivity map. METHODS: The key gene modules significantly related to clinical traits (survival time and state) of ESCA patients were selected by weighted gene coexpression network analysis (WCGNA), then the univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to construct a 15-immune-related gene prognostic signature. RESULTS: The immune-related risk model was related to clinical and pathologic factors and remained an effective independent prognostic factor. Enrichment analyses revealed that the differentially expressed genes (DEGs) of the high- and low-risk groups were associated with tumor cell proliferation and immune mechanisms. Based on the gathered data, a small molecule drug named perphenazine (PPZ) was elected. The pharmacological analysis indicates that PPZ could help in adjuvant therapy of ESCA through regulation of metabolic process and cellular proliferation, enhancement of immunologic functions, and inhibition of inflammatory reactions. Furthermore, molecular docking was performed to explore and verify the PPZ-core target interactions. CONCLUSION: We succeed in structuring the immune-related prognostic model, which could be used to distinguish and predict patients' survival outcome, and screening a small molecule drug named PPZ. Prospective studies also are needed to further validate its analytical accuracy for estimating prognoses and confirm the potential use of PPZ for treating ESCA.

Xiasangju alleviate metabolic syndrome by enhancing noradrenaline biosynthesis and activating brown adipose tissue.

Metabolic syndrome (MetS) is a clinical condition associated with multiple metabolic risk factors leading to type 2 diabetes mellitus and other metabolic diseases. Recent evidence suggests that modulating adipose tissue to adaptive thermogenesis may offer therapeutic potential for MetS. Xiasangju (XSJ) is a marketed drug and dietary supplement used for the treatment of metabolic disease with anti-inflammatory activity. This study investigated the therapeutic effects of XSJ and the underlying mechanisms affecting the activation of brown adipose tissue (BAT) in MetS. The results revealed that XSJ ameliorated MetS by enhancing glucose and lipid metabolism, leading to reduced body weight and abdominal circumference, decreased adipose tissue and liver index, and improved blood glucose tolerance. XSJ administration stimulated catecholamine biosynthesis, increasing noradrenaline (NA) levels and activating NA-mediated proteins in BAT. Thus, BAT enhanced thermogenesis and oxidative phosphorylation (OXPHOS). Moreover, XSJ induced changes in gut microbiota composition, with an increase in Oscillibacter abundance and a decrease in Bilophila, Candidatus Stoquefichus, Holdemania, Parasutterella and Rothia. XSJ upregulated the proteins associated with intestinal tight junctions corresponding with lower serum lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α) monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) levels to maintain NA signaling transport. In summary, XSJ may alleviate MetS by promoting thermogenesis in BAT to ultimately boost energy metabolism through increasing NA biosynthesis, strengthening intestinal barrier integrity and reducing low-grade inflammation. These findings suggest XSJ has potential as a natural therapeutic agent for the treatment of MetS.

Amlodipine inhibits the proliferation and migration of esophageal carcinoma cells through the induction of endoplasmic reticulum stress.

BACKGROUND: L-type calcium channels are the only protein channels sensitive to calcium channel blockers, and are expressed in various cancer types. The Cancer Genome Atlas database shows that the mRNA levels of multiple L-type calcium channel subunits in esophageal squamous cell carcinoma tumor tissue are significantly higher than those in normal esophageal epithelial tissue. Therefore, we hypothesized that amlodipine, a long-acting dihydropyridine L-type calcium channel blocker, may inhibit the occurrence and development of esophageal cancer (EC). AIM: To investigate the inhibitory effects of amlodipine on EC through endoplasmic reticulum (ER) stress. METHODS: Cav1.3 protein expression levels in 50 pairs of EC tissues and corresponding paracancerous tissues were examined. Subsequently, the inhibitory effects of amlodipine on proliferation and migration of EC cells in vitro were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and Transwell assays. In vivo experiments were performed using murine xenograft model. To elucidate the underlying mechanisms, in vitro cell studies were performed to confirm that ER stress plays a role in inhibition proliferation and migration of EC cells treated with amlodipine. RESULTS: The expression level of Cav1.3 in esophageal carcinoma was 1.6 times higher than that in paracancerous tissues. Amlodipine treatment decreased the viability of esophageal carcinoma cells in a dose- and time-dependent manner. In vivo animal experiments also clearly indicated that amlodipine inhibited the growth of EC tumors in mice. Additionally, amlodipine reduces the migration of tumor cells by inhibiting epithelial-mesenchymal transition (EMT). Mechanistic studies have demonstrated that amlodipine induces ER stress-mediated apoptosis and suppresses EMT. Moreover, amlodipine-induced autophagy was characterized by an increase in autophagy lysosomes and the accumulation of light chain 3B protein. The combination of amlodipine with the ER stress inhibitor 4-phenylbutyric acid further confirmed the role of the ER stress response in amlodipine-induced apoptosis, EMT, and autophagy. Furthermore, blocking autophagy increases the ratio of apoptosis and migration. CONCLUSION: Collectively, we demonstrate for the first time that amlodipine promotes apoptosis, induces autophagy, and inhibits migration through ER stress, thereby exerting anti-tumor effects in EC.

NLRP3-GSDMD-dependent IL-1ß Secretion from Microglia Mediates Learning and Memory Impairment in a Chronic Intermittent Hypoxia-induced Mouse Model.

Hypoxia/reoxygenation caused by chronic intermittent hypoxia (CIH) plays an important role in cognitive deficits in patients with obstructive sleep apnea. However, the precise underlying mechanism remains unclear. This study investigated whether the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is involved in CIH-induced spatial learning and memory impairment in mice, and the possible underlying upstream and downstream mechanisms. The C57BL/6 male mice were exposed to CIH (21% O2-6% O2, 4 min/cycle, 8 h/day) for 9 weeks to investigate the role of NLRP3 in CIH-induced spatial learning and memory impairment in mice. BV2 cells were exposed to intermittent hypoxia (21% O2-1% O2, 90 min/cycle) for 48 h to investigate the possible mechanisms in vitro. We found that: 1) inhibition of NLRP3 inflammasome activation improved CIH-induced spatial learning and memory impairment in mice. 2) CIH damaged hippocampal neurons but increased the number of microglia in mice hippocampi; CIH activated microglia-specific NLRP3 inflammasome, leading to upregulation of matured IL-1ß and N-GSDMD. 3) intermittent hypoxia activated NLRP3 inflammasome via the ROS-NF-κB signaling pathway to promote the release of matured IL-1ß from microglia in a GSDMD-dependent manner without pyroptosis. 4) The IL-1ß released from microglia might impair the synaptic plasticity of hippocampal CA3-CA1 synapses by acting on IL-1 receptors in hippocampal neurons. Our findings reveal that ROS-NF-κB-NLRP3 inflammasome-GSDMD dependent IL-1ß release from microglia may participate in CIH-induced spatial learning and memory impairment by acting on hippocampal neuronal IL-1 receptor, leading to synaptic plasticity impairment.

Long-term outcomes of esophageal and gastric cancer patients with cardiovascular and metabolic diseases: A two-center propensity score-matched cohort study.

Background and Objectives: An increased risk of cardiovascular and metabolic diseases (CVMDs) among patients with cancer suggests a potential link between CVMD and cancer. The impact of CVMD on the survival time of patients with esophageal and gastric cancer remains unknown. We aimed to determine the incidence of CVMD and its impact on the longterm outcomes in esophageal and gastric cancer patients. Methods: A total of 2074 cancer patients were enrolled from January 1, 2007 to December 31, 2017 in two hospitals, including 1205 cases of esophageal cancer and 869 cases of gastric cancer, who were followed up for a median of 79.8 and 79.3 months, respectively. Survival time was analyzed using the Kaplan-Meier method before and after propensity score matching. Results: The incidence of CVMD in patients with esophageal and gastric cancer was 34.1% (411/1205) and 34.3% (298/869), respectively. The effects of hypertension, diabetes, and stroke on the long-term survival of esophageal and gastric cancer patients were not significant (all P > 0.05). The survival time was significantly longer in esophageal cancer patients without ischemic heart disease than in patients with ischemic heart disease, both before matching (36.5 vs. 29.1 months, P = 0.027) and after matching (37.4 vs. 27.9 months, P = 0.011). The survival time in gastric cancer patients without ischemic heart disease was significantly longer than in patients with ischemic heart disease, both before (28.4 vs.17.5 months, P = 0.032) and after matching (29.5 vs.17.5 months, P = 0.02). Conclusion: The survival time of esophageal and gastric cancer patients with ischemic heart disease was significantly reduced compared to that of esophageal and gastric cancer patients without ischemic heart disease.

Implications of obstructive sleep apnea in lung adenocarcinoma: A valuable omission in cancer prognosis and immunotherapy.

Lung adenocarcinoma (LUAD) is a highly invasive malignant tumor with poor prognosis, and there is growing evidence that obstructive sleep apnea (OSA) could significantly promotes the risk of LUAD. In order to improve the treatment outcomes of patients with LUAD and OSA, we aim to screen OSA-related genes that may potentially affect LUAD and to discover a high sensitivity prognostic signature that can stratify LUAD/OSA patients and to further accurately identify LUAD patients who might respond to immunotherapy. Molecular subtypes classified by the prognostic signature did not belong to any previously reported subtypes of LUAD. The tumor microenvironment (TME), mutation, and so on, were significantly distinct between patients within different risk groups or clusters. Combined with gene set variation analysis (GSVA) and drug susceptibility analysis, patients in the low-risk group (The vast majority of patients belonging to cluster2 by molecular subtyping) were not suitable for immunotherapy due to T-cell exhaustion caused by long-term inflammatory response; the question of how to reverse T-cell exhaustion may be a primary consideration. Cluster3 patients had the highest benefit from immunotherapy, and although cluster1 patients had the worst prognosis, they were more sensitive to traditional chemotherapeutic drugs. Animal experiments showed that chronic intermittent hypoxia (CIH) could not only significantly promote the tumor growth of LUAD, but also increase the expression levels of risk genes. This risk model may contribute greatly to the evaluation of prognosis, molecular characteristics, and treatment modalities of LUAD/OSA, and could be further translated into clinical applications to ameliorate the treatment dilemmas.

Decision-making experiences of breast cancer patients related to contralateral prophylactic mastectomy-a systematic meta-synthesis of qualitative studies.

PURPOSE: Currently, the choice of contralateral prophylactic mastectomy (CPM) for breast cancer patients is variable and controversial. Breast cancer patients must make complex and rapid decisions based on the benefits and risks of CPM. Although there are many qualitative studies on the decision-making experiences of breast cancer patients, there is a lack of synthesis of these qualitative studies. Our study goals were to conduct a meta-synthesis of qualitative studies on the decision-making experiences, real-life experiences, psychological feelings and needs of breast cancer patients in CPM decision-making, with the aim of providing information to support the development of CPM practice decisions. METHODS: Using a meta-ethnographic approach, qualitative research studies were analysed and synthesised using the method of "reciprocal translational analysis", and themes related to the decision-making experiences of breast cancer patients with respect to CPM were identified. RESULTS: Five hundred ninety-three documents were retrieved. This meta-synthesis ultimately collected 8 studies. Four themes were identified: (1) decision motivations for survival and body intention; (2) negative and vacillating decision emotions; (3) diverse but weak decision support; (4) short-term satisfaction but long-term unknown and differentiated decision effects. CONCLUSIONS: We found that although patients had different feelings about the effects of CPM in detail, most patients were satisfied with the short-term effects of CPM, but the long-term effects of CPM were still unknown. The study protocol was registered with PROSPERO (International prospective register of systematic reviews) in May 2022 (Registration number: CRD42022334260).

Inhibition of bromodomain-containing protein 4 enhances the migration of esophageal squamous cell carcinoma cells by inducing cell autophagy.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC), the predominant type of esophageal cancer, has a 5-year survival rate less than 20%. Although the cause of poor prognosis is the high incidence and mortality of ESCC, the high rate of metastasis after esophageal cancer surgery is the main cause of death after the surgery. Bromodomain-containing protein 4 (BRD4), an epigenetic reader of chromatin-acetylated histones in tumorigenesis and development, plays an essential role in regulating oncogene expression. BRD4 inhibition and BRD4 inhibition-based treatment can potentially suppress ESCC growth. However, the effects and mechanisms of action of BRD4 on ESCC cell migration remain unclear. AIM: To explore the effect of BRD4 on cell migration of ESCC in vitro and its possible molecular mechanism. METHODS: Human ESCC cell lines KYSE-450 and KYSE-150 were used. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was performed to examine cell proliferation, and the transwell migration assay was conducted to test ESCC cell migration. JQ1, a BRD4 inhibitor, was applied to cells, and BRD4 siRNA was transfected into ESCC cells to knockdown endogenous BRD4. GFP-RFP-LC3 adenovirus was infected into ESCC cells to evaluate the effect of JQ1 on autophagy. Western blotting was performed to determine the protein levels of BRD4, E-cadherin, vimentin, AMP-activated protein kinase (AMPK), and p-AMPK. RESULTS: BRD4 was either downregulated by small interfering RNA or pretreated with JQ1 in ESCC cells, leading to increased tumor migration in ESCC cells in a dose- and time-dependent manner. Inhibition of BRD4 not only significantly suppressed cell proliferation but also strongly increased cell migration by inducing epithelial-mesenchymal transition (EMT). The protein expression of vimentin was increased and E-cadherin decreased in a dose-dependent manner, subsequently promoting autophagy in KYSE-450 and KYSE-150 cells. Pretreatment with JQ1, a BRD4 inhibitor, inhibited BRD4-induced LC3-II activation and upregulated AMPK phosphorylation in a dose-dependent manner. Additionally, an increased number of autophagosomes and autolysosomes were observed in JQ1-treated ESCC cells. The autophagy inhibitor 3-methyladenine (3-MA) reversed the effects of BRD4 knockdown on ESCC cell migration and blocked JQ1-induced cell migration. 3-MA also downregulated the expression of vimentin and upregulation E-cadherin. CONCLUSION: BRD4 inhibition enhances cell migration by inducing EMT and autophagy in ESCC cells via the AMPK-modified pathway. Thus, the facilitating role on ESCC cell migration should be considered for BRD4 inhibitor clinical application to ESCC patients.

[Effects of TOFA on growth of Eca109 and KYSE-450 cells in human esophageal squamous cell carcinoma].

Objective: To investigate the effects of 5-tetradecanoxy 2-furanic acid (TOFA) on cell proliferation, cell cycle and apoptosis of esophageal squamous cell carcinoma (ESCC) cells. Methods: Eca-109 cells and KYSE-450 cells were divided into control group (DMSO) and experimental group (TOFA), respectively. The cells (4×103 cells/100 µl) were inoculated into 96-well plates with 5 multiple wells at each concentration. After 24 h culture, cells were treated with DMSO or different concentrations (1, 3, 5, 10 µg/ ml) of TOFA for 24, 48 and 72 h. Cell proliferation was detected by MTT, cell cycle and apoptosis were detected by flow cytometry, the expression levels of p21 and Cleaved caspase-3 and modification levels of p-Akt, p-mTOR and p-4EBP1 were detected by Western blot, and intracellular free fatty acids were detected by special kits. Results: MTT results showed that TOFA inhibited the proliferation of Eca109 and KYSE-450 cells in a concentration and time dependent manner (all P<0.05), with IC50 of 4.65 µg/ml and 3.93 µg/ml for 48 h, respectively. Flow cytometry results showed that compared with DMSO group, the percentage of cells in G2/M phase was increased and the apoptosis rate was increased in the experimental group. Western blotting results showed that compared with DMSO group, p21 and Cleaved caspase-3 protein expression levels were up-regulated, and p-AKT, p-mTOR and p-4EBP1 protein expression levels were down-regulated (all P<0.05). Conclusion: TOFA inhibits the proliferation, blocks the cycle progression and promotes apoptosis of ESCC, the mechanism may be related to the AKT/mTOR/4EBP1 signaling pathway.

Therapeutic mechanisms of mulberry leaves in type 2 diabetes based on metabolomics.

Background: Type 2 diabetes (T2D) is considered as one of the most significant metabolic syndromes worldwide, and the long-term use of the drugs already on the market for T2D often gives rise to some side effects. The mulberry leaf (ML), Morus alba L., has advantages in terms of its comprehensive therapeutic efficacy, which are characterized as multicomponent, multitarget, multipathway, and matching with the complex pathological mechanisms of diabetes. Methods: T2D rats were established by a high-fat diet combined with an intraperitoneal injection of streptozotocin; an evaluation of the hypoglycemic effects of the ML in combination with fasting blood glucose and other indicators, in addition to the utilization of metabolomics technology, was performed to analysis the metabolite changes in serum of rats. Results: MLs significantly reduced the fasting blood glucose of T2D rats, while improving the symptoms of polyphagia and polyuria. ML treatment altered the levels of various metabolites in the serum of T2D rats, which are involved in multiple metabolic pathways (amino acid metabolism, carbohydrate metabolism, and lipid metabolism), played a role in antioxidative stress and anti-inflammation, modulated immune and gluconeogenesis processes, and improved obesity as well as insulin resistance (IR). Conclusion: The ML contains a variety of chemical components, and metabolomic results have shown that MLs regulate multiple metabolic pathways to exert hypoglycemic effects, suggesting that MLs may have great promise in the development of new hypoglycemic drugs.

[Research on the mechanism of hypoxia promoting the migration of lung adenocarcinoma A549 cells].

Objective: To investigate the mechanism that hypoxia promotes the migration of lung adenocarcinoma A549 cells. Methods: A549 cells were cultured and cells that knockdown of acetyl-CoA carboxylase 1 (ACC1) were obtained by transfection with lentivirus, and cells that knockdown of sterol regulatory element-binding proteins-1 (SREBP-1) were obtained by treated with si-RNA. A549 cells were treated with hypoxia combined with hypoxia inducible factor-1α (HIF-1α) inhibitor PX-478 (25 µmol); Hypoxia combined with linoleic acid (LA) (20 µmol) treated A549 cells with ACC1 knockdown, and A549 cells with SREBP-1 knockdown were treated by hypoxia. Transwell migration assay was used to detect cell migration. Western blot was conducted to detect HIF-1α, ACC1 and epithelial mesenchymal transition (EMT) related proteins, Vimentin, E-Cadherin and SREBP-1; Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed to detect the changes of ACC1 and SREBP-1 mRNA in A549 cells after hypoxia and HIF-1α inhibitor PX-478 (25 µmol) treatment. Each experiment was repeated three times. Results: Compared with the normoxic control group, hypoxia promoted the migration of A549 cells (P＜0.01), and up-regulated the expressions of ACC1, HIF-1α (all P＜0.01) and SREBP-1 (P＜0.05). PX-478 (25 µmol) inhibited the migration of A549 cells induced by hypoxia and down-regulated the expression of SREBP-1 (all P＜0.05). ACC1 mRNA and SREBP-1 mRNA levels were increased after hypoxia treatment of A549 cells (all P＜0.05). The levels of ACC1 mRNA and SREBP-1 mRNA were decreased after A549 cells treated with hypoxia combined with PX-478 (25 µmol) for 24 h (P＜0.05, P＜0.01). Knockdown of SREBP-1 in A549 cells was obtained by transfection with si-RNA. Transwell migration assay showed the number of cell migration in si-SREBP-1 group was less than that in normoxia control group (P＜0.01). The si-SREBP-1 group and the si-NC group were treated with hypoxia. Compared with the control group, the number of cell migration in the si-SREBP-1 group was decreased (P＜0.01), however, the difference was not statistically significant compared with the normoxia si-SREBP-1 group (P＞0.05). Western blot showed that the expression of ACC1 in the si-SREBP-1 group was lower than that in the control group (P＜0.01). Compared with the control group, the expression of ACC1 was decreased after si-SREBP-1 group treated with hypoxia (P＜0.01). Knockdown of ACC1 inhibited the migration of A549 cells (P＜0.05). After knockdown of ACC1, the migration number of A549 cells under normoxia and 5% O2 conditions had no significant difference (P＞0.05). Application of LA under hypoxia condition rescued ACC1-knockdown induced inhibitory effect on hypoxia-promoted A549 cell migration (P＜0.05). Conclusion: Hypoxia promotes migration of lung adenocarcinoma A549 cells by regulating fatty acid metabolism through HIF-1α/SREBP-1/ACC1 pathway.

Qingwei San treats oral ulcer subjected to stomach heat syndrome in db/db mice by targeting TLR4/MyD88/NF-κB pathway.

BACKGROUND: Qingwei San (QWS), one of classic Chinese Medicine prescripts, has been widely used to treat stomach heat syndrome which manifests oral ulcer (OU), periodontitis and upper gastrointestinal bleeding for seven hundred years. However, the therapeutic effects of QWS on diabetic OU subjected to stomach heat syndrome are still ambiguous. In the study, we investigated the pharmacological mechanisms. METHODS: The main components of QWS aqueous extract were analyzed by LC-MS, and potential pathways of QWS targeting OU were predicted by network pharmacology. The db/db mice were administered with the decoction of dried Zingiber officinale Rosc. rhizome combined with NaOH cauterization to establish the model of diabetic OU subjected to stomach heat syndrome. Subsequently, the model mice were treated with QWS, and OU wound healing status were recorded. The pathological changes of gastric tissue and oral mucosa were evaluated using hematoxylin-eosin staining, and the morphology of collagen fibers in oral mucosa was assessed by Masson staining. The levels of thromboxane B2 (TXB2), 6-Keto-prostaglandin F1α (6-keto-PGF1α), interleukin-1 ß (IL-1ß), IL-2, IL-6, tumor necrosis factor-α (TNF-α), ß-endorphin (ß-EP) and 5-Hydroxytryptamine (5-HT) were determined by ELISA assay. The protein expressions of Toll-like receptor 4 (TLR4), TNF receptor associated factor 6 (TRAF6), myeloid differentiation factor 88 (MyD88), inhibitor of NF-κB alpha (IκΒα), p-IκΒα and nuclear factor kappa-B (NF-κB) p65 were measured by Western Blotting. RESULTS: A total of 183 compounds in QWS were identified by LC-MS, and identified 79 bioactive compounds corresponded to 269 targets and 59 pathways. QWS high-dose treatment significantly reduced the level of TXB2 and the ratio of TXB2/6-keto-PGF1α. Meanwhile, it improved mucosal pathological morphology, and reduced the area of OU and local edema. Simultaneously, the levels of TNF-α, IL-1ß, IL-6, IL-2 and 5-HT, and the expressions of TLR4, TRAF6, MyD88, p-IκΒα and NF-κB p65 were decreased. CONCLUSION: QWS treatment facilitates the healing of OU, ameliorates pathological morphologies of gastric and oral mucosa and decreases the levels of pro-inflammatory cytokines in db/db mice subjected to stomach heat syndrome, whose mechanism may be associated with the inhibition of TLR4/MyD88/NF-κB signaling pathway to exert anti-inflammatory effects.

[Effects of knockdown ACC1 on glioma U251 cell migration and its mechanisms].

Objective: To investigate the effects of ACC1 knockdown on human glioma U251 cell migration and its molecular mechanisms. Methods: Human glioma U251 cell line was used. The experiment was carried out in three steps. Experiment 1: knockdown of ACC1 in U251 cells (shACC1) and its control (NC) U251 cells were established by transfection of shACC1 lentivirus and negative control virus. The cell migration was detected by Transwell migration assay and scratch test. Western blot (WB) was performed to detect the levels of ACC1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Experiment 2: RT-qPCR and WB were performed to verify the RNA-seq result, upregulation effect of ACC1 knockdown on PAI-1 in U251 cells. The cells then were treated with PAI-1 inhibitor PAI-039, and the cell migration was detected by Transwell migration assay and scratch assay. The protein levels of ACC1, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug were examined by WB. Experiment 3: the molecular mechanisms of knocking down ACC1 to increase PAI-1 were explored. The cells were treated with acetyltransferase inhibitor C646, and cell migration was examined by Transwell migration assay and scratch assay. WB was conducted to test the levels of ACC1, H3K9ac, PAI-1, Vimentin, Fibronectin, N-cadherin, E-cadherin and Slug proteins. Each experiment was repeated three times. Results: Experiment 1: lentivirus transfection was performed on glioma U251 cells. Compared with NC group, the expression level of ACC1 in shACC1 group was decreased significantly, indicating that lentivirus transfection was successful (P＜0.01), and the number of migrated cells in shACC1 group was increased significantly (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Experiment 2: Compared with NC group, PAI-1 mRNA level in shACC1 group was up-regulated. Compared with control group, cell migration in shACC1+PAI-039 group was decreased (P＜0.01), and migration-related proteins Vimentin, Fibronectin, N-cadherin, and Slug were up-regulated. E-cadherin expression was down-regulated (P＜0.01). Experiment 3: Compared with NC group, the concentration of acetyl-coA and the expression level of H3K9ac in shACC1 group were increased significantly (P＜0.01); After further treatment with histone acetyl transferase inhibitor C646, PAI-1 mRNA level was decreased, cell migration number and H3K9ac expression level were decreased in shACC1+C646 group compared with control group (P＜0.01). Migration-related proteins Vimentin, Fibronectin, N-cadherin and Slug were up-regulated, while E-cadherin was down-regulated (P＜0.01). Conclusion: Knockdown of ACC1 promotes the migration of human glioma U251 cells by increasing histone acetylation which elevates the level of PAI-1.

[Effects of propranolol on biological function of human esophageal squamous cell carcinoma cells].

Objective: To investigate the effects of propranolol on the subcutaneous tumorigenesis of esophageal squamous cell carcinoma (ESCC) cells and the proliferation, migration, cell cycle, apoptosis and autophagy of ESCC cells and its possible molecular mechanisms. Methods: The cell proliferation was detected by MTT (methyl thiazol tetrazolium) assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS (Phosphate buffer saline) group (without propranolol) and treated groups (40, 60, 80, 100 µmol/L propranolol) were set up with 5 wells in each group. After treatment for 0, 24, 48, 72 h, 10 µl (5 mg/ml) of MTT was added to each well, and the absorbance was measured at 490 nm. The cell migration was tested by Transwell assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured, and PBS group (without propranolol) and treated groups (40, 60 µmol/L) were set up with 2 wells in each group. Photos were taken 40 h later, and the experiment was repeated for three times before statistical analysis. The cell cycle and apoptosis were detected by flow cytometry assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS group (without propranolol) and treated group (80 µmol/L) were set up, fixed, stained, and fluorescence at 488 nm was detected. The protein levels were detected by Western blot: ESCC Eca109 and KYSE-450 cells were routinely cultured. PBS group (without propranolol) and treated groups (60, 80 µmol/L) were set up followed by gel electrophoresis, wet membrane transfer, and ECL imaging. The experiment was repeated for three times and then analyzed statistically. Subcutaneous tumor formation experiment in nude mice: 10 nude mice were assigned PBS group (without propranolol) and treated group (with propranolol). Five mice in each group were inoculated with 5×106 cells/100 µl (Eca109) into the right underarm. The treated group was given a gavage of 0.4 ml/kg (6 mg/kg) every other day, and the tumor size was measured every other day for 3 weeks. After 20 days, the nude mice were dislocated and sacrificed to take tumor tissue. Result: The results showed that propranolol inhibited the proliferation of Eca109, KYSE-450 and TE-1 cells with IC50 of around 70 µmol/L for 48 h. Eca109, KYSE-450 and TE-1 cell migration was inhibited by propranolol in a dose-dependent manner (P＜0.05); Propranolol blocked the cell cycle of Eca109 in G2/M phase, blocked the cell cycle of KYSE-450 and TE-1 in G0/G1 phase, and promoted apoptosis of three kinds of cells (P＜0.05). The results of cell fluorescence showed that LC3 fluorescence intensity of TE-1 was increased after 12 h, 24 h and 36 h treatment with propranolol (P＜0.05). Western blot results showed that compared with PBS group, the protein expressions of p-mTOR, p-Akt and cyclin D1 were down-regulated, while cleaved caspase 9 level was up-regulated (P＜0.05). The results of subcutaneous tumor formation in nude mice showed that the tumor weight of PBS group was (0.91±0.05)g, and that of the experimental group was(0.65±0.12)g, the difference was statistically significant (P＜0.05). Conclusion: Propranolol inhibits the proliferation, migration and cell cycle,promotes apoptosis and autophagy of ESCC cells, and inhibits subcutaneous tumor growth in nude mice. The mechanism might be related to the inhibition of PI3K/AKT/mTOR signaling pathway.

Trichostatin A promotes esophageal squamous cell carcinoma cell migration and EMT through BRD4/ERK1/2-dependent pathway.

BACKGROUND: Histone deacetylases (HDACs) have been demonstrated to be aberrantly activated in tumorigenesis and cancer development. Thus, HDAC inhibitors (HDACIs) are considered to be promising anti-cancer therapeutics. However, recent studies have shown that HDACIs promote the migration of many cancer cells. Therefore, there is a need to elucidate the underlying mechanisms of HDACIs on cancer cell migration to establish a combination therapy that overcomes HDACI-induced cell migration. METHODS: KYSE-150 and EC9706 cells were treated differently. Effects of drugs and siRNA treatment on tumor cell migration and cell signaling pathways were investigated by transwell migration assy. Gene expression for SNAI2 was tested by RT-qPCR. Western blot analysis was employed to detect the level of E-cadherin, ß-catenin, vimentin,Slug，ERK1/2, H3, PAI-1 and BRD4. The effect of drugs on cell morphology was evaluated through phase-contrast microscopic images. RESULTS: TSA promotes epithelial-mesenchymal transition (EMT) in ESCC cells by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers ß-catenin, vimentin, Slug, and PAI-1. Knockdown of Slug by siRNA or inhibition of PAI-1 clearly suppressed TSA-induced ESCC cell migration and resulted in the reversal of TSA-triggered E-cadherin, ß-catenin, and vimentin expression. However, no crosstalk between Slug and PAI-1 was observed in TSA-treated ESCC cells. Blocking ERK1/2 activation also inhibited TSA-induced ESCC cell migration, EMT, and upregulation of Slug and PAI-1 levels in ESCC cells. Interestingly, inhibition of BRD4 suppressed TSA-induced ESCC cell migration and attenuated TSA-induced ERK1/2 activation and upregulation of Slug and PAI-1 levels. CONCLUSIONS: Our data indicate the existence of at least two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration: an ERK1/2-Slug branch and an ERK1/2-PAI-1 branch. Both branches of TSA-induced ESCC cell migration appear to favor the EMT process, while BRD4 is responsible for two separable ERK1/2-dependent signaling pathways in TSA-mediated ESCC cell migration.

Drug-Metabolizing Cytochrome P450 Enzymes Have Multifarious Influences on Treatment Outcomes.

Drug metabolism is a critical process for the removal of unwanted substances from the body. In humans, approximately 80% of oxidative metabolism and almost 50% of the overall elimination of commonly used drugs can be attributed to one or more of various cytochrome P450 (CYP) enzymes from CYP families 1-3. In addition to the basic metabolic effects for elimination, CYP enzymes in vivo are capable of affecting the treatment outcomes in many cases. Drug-metabolizing CYP enzymes are mainly expressed in the liver and intestine, the two principal drug oxidation and elimination organs, where they can significantly influence the drug action, safety, and bioavailability by mediating phase I metabolism and first-pass metabolism. Furthermore, CYP-mediated local drug metabolism in the sites of action may also have the potential to impact drug response, according to the literature in recent years. This article underlines the ability of CYP enzymes to influence treatment outcomes by discussing CYP-mediated diversified drug metabolism in primary metabolic sites (liver and intestine) and typical action sites (brain and tumors) according to their expression levels and metabolic activity. Moreover, intrinsic and extrinsic factors of personal differential CYP phenotypes that contribute to interindividual variation of treatment outcomes are also reviewed to introduce the multifarious pivotal role of CYP-mediated metabolism and clearance in drug therapy.

Therapeutic effects and mechanisms study of Hanchuan Zupa Granule in a Guinea pig model of cough variant asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Hanchuan Zupa Granule (HCZP), a traditional Chinese ethnodrug, has the functions of supressing a cough, resolving phlegm, warming the lungs, and relieving asthma. In clinical practice employing traditional Chinese medicine (TCM), HCZP is commonly used to treat acute colds, cough and abnormal mucous asthma caused by a cold, or "Nai-Zi-Lai" in the Uygur language. Studies have confirmed the use of HCZP to treat cough variant asthma (CVA) and other respiratory diseases. However, the pharmacological mechanisms of HCZP remain unrevealed. AIM OF THE STUDY: To investigate the anti-tussive and anti-asthmatic effects and the possible pharmacological mechanisms of HCZP in the treatment of CVA. MATERIALS AND METHODS: A guinea pig CVA animal model was established by intraperitoneal injection of ovalbumin (OVA) combined with intraperitoneal injection of aluminium hydroxide adjuvant and atomized OVA. Meanwhile, guinea pigs with CVA received oral HCZP (at dosages of 0.571, 0.285 and 0.143 g/kg bodyweight). The number of coughs induced by aerosol capsaicin was recorded, and the airway hyperresponsiveness (AHR) of CVA guinea pigs was detected with the FinePointe series RC system. H&E staining of lung tissues was performed to observe pathological changes. ELISA was used to detect inflammatory cytokines. qRT-PCR and western blotting analyses were used to detect the expression of Th1-specific transcription factor (T-bet), Th2-specific transcription factor (GATA3), and Toll-like receptor 4 (TLR4) signal transduction elements. These methods were performed to assess the protective effects and the potential mechanisms of HCZP on CVA. RESULTS: Great changes were found in the CVA guinea pig model after HCZP treatment. The number of coughs induced by capsaicin in guinea pigs decreased, the body weights of guinea pigs increased, and inflammation of the eosinophilic airway and AHR were reduced simultaneously. These results indicate that HCZP has a significant protective effect on CVA. A pharmacological study of HCZP showed that the levels of interleukin-4 (IL-4) and IL-5 and tumour necrosis factor-α (TNF-α) in serum decreased. The amount of interferon-γ (IFN-γ) increased, mRNA and protein expression of TLR4 and GATA3 weakened, and mRNA and protein expression of T-bet increased. CONCLUSIONS: HCZP ameliorated the symptoms of guinea pigs with CVA induced by OVA by regulating the Th1/Th2 imbalance and TLR4 receptors.

Anti-tumor and Phenotypic Regulation Effect of Matrine on Dendritic Cells through Regulating TLRs Pathway.

OBJECTIVE: To investigate the effects of matrine on antigen presentation of dendritic cells (DCs), and to explore the pharmacological mechanism of matrine on anti-tumor effect. METHODS: Different concentrations (0, 1, 2, 4, 8 and 16 µ g/mL) of matrine were co-cultured with DCs, the harvested DCs were co-cultured with antigens of Lewis lung cancer (LLC) cells, and then DCs and T cells were co-cultured to produce DCs-activated killer (DAK) cells, which have significant tumor-killing activity. The expression of cytokines, mRNA and protein of toll-like receptors (TLRs) in DCs were detected by enzyme linked immunosobent assay, polymerase chain reaction and Western blot, respectively. And the killing effect of DAK were measured by MTT assay. RESULTS: Matrine significantly increased the mRNA expression of TLR7, TLR8, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and I κ B kinase (IKK), as well as the protein expression of TLR7 and TLR8, and up-regulated the levels of interleukin-12 (IL-12), IL-6 and tumor necrosis factor-α (TNF-α), meanwhile, it also increased the expressions of MHC-II, CD54, CD80 and CD86 in DCs. DCs-activated effector T cells had significant tumor-killing activity. When the concentration of matrine was more than 4 µg/mL, all indices had significant difference (P<0.01 or P<0.05). CONCLUSION: Matrine plays an anti-tumor role by regulating TLRs signal transduction pathway, promoting the secretion of inflammatory cytokines and enhancing immune function.

Trichostatin A augments esophageal squamous cell carcinoma cells migration by inducing acetylation of RelA at K310 leading epithelia-mesenchymal transition.

Protein acetylation modification controlled by acetyltransferases (HATs) and histone deacetylases (HDACs) regulates multiple biologic processes including cell proliferation and migration. HDAC inhibitors (HDACi) are currently used as a promising epigenetic-based therapy for cancer treatment. Of the anticancer activity, accumulating evidence has shown that HDACi can enhance cell migration in subset of cancer cells. Thus, there is a critical need to identify such counter anticancer activity to HDACi in different cancer cell types and elucidate the rational in order to develop appropriate combination therapies in cancer treatment. In seeking to address the effect of HDACi on esophageal squamous cell carcinoma (ESCC) cells migration, trichostatin A (TSA), a canonical HDACi targeting class I and class II HDACs, was used. Here, we report the discovery that TSA augmented ESCC cells migration by increasing the acetylation of nuclear factor-κB/RelA at lysine 310 (K310). To elucidate the mechanism by which TSA promotes the migration of ESCC cells, plasmid of RelA K310R, a mutant precluding acetylation at K310, was transfected into ESCC cells. Blocking acetylation of RelA at K310 significantly arrogated TSA-induced cell migration. Mechanistic investigations revealed that TSA increased the level of acetylated RelA at K310 (RelA K310ac), thereby increasing the level of epithelia-mesenchymal transition (EMT) transcription factor slug mRNA, which in turn induced EMT. Overall, this study indicates that TSA promotes ESCC cells migration by RelA K310ac-slug-EMT pathway. Our findings provide a strategy to eradicate HDACi-induced ESCC cells migration by targeting RelA as a combination therapy with nonspecific HDACi in ESCC treatment.

In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of Herba Epimedii extracts.

Herba Epimedii is a famous Chinese edible herb, and due to its potential hepatotoxic effects, the safety associated with this herb has attracted a great deal of attention. In this study, the components of four types of the Herba Epimedii extracts were identified by HPLC-MS/MS. Among these components, 11 components that were present in all four extracts and could be obtained as reference substances were evaluated for their ability of cytotoxicity in HL-7702 and HepG2 cells, resulting in the identification of icarisid I and sagittatoside A as the most relevant with respect to the toxicity of the extracts. The targeted toxicological effects were further investigated using a series of correlated biological indicators to elucidate potentially hepatotoxic mechanisms. The results showed that the extracts and the selected compounds had varying degrees of influence on the leakage of ALT, AST and LDH; the activity of SOD, GSH and MDA; the increase in intercellular ROS; and the decrease in MMP. Among the tested substances, the ethanol extracts exhibited stronger hepatotoxicity, with icarisid I and sagittatoside A correlating with this toxic effect, and the hepatoxic mechanisms of which may be associated with damaged cell structure, increased oxidative stress and induction of apoptosis.