Erk phosphorylation reduces the thymoquinone toxicity in human hepatocarcinoma.

Although enormous achievements have been made in targeted molecular therapies against hepatocellular carcinoma (HCC), the treatments can only prolong the life of patients with extrahepatic metastases. We evaluated thymoquinone (TQ), a compound from Nigella sativa Linn., for its anti-cancer effect on SK-Hep1 cells and HCC-xenograft nude mice. TQ effectively triggered cell death    and activated p38 and extracellular signal-regulated kinases (Erk) pathways up to 24 h after treatment in cells. TQ-induced cell death was reversed by p38 inhibitor; however, it was enhanced by si-Erk. The caspase3 activation and TUNEL assay revealed a stronger toxic effect upon co-treatment with TQ and si-Erk. Our study suggested that phosphorylation of p38 in SK-Hep1 cells constituted the major factor leading to cell apoptosis, whereas phosphorylation of Erk led to drug resistance. Furthermore, TQ therapeutic effect was improved upon Erk inhibition in HCC-xenograft nude mice. TQ could present excellent anti-HCC potential under suitable p-Erk inhibiting conditions.

Estrogen and/or Estrogen Receptor α Inhibits BNIP3-Induced Apoptosis and Autophagy in H9c2 Cardiomyoblast Cells.

The process of autophagy in heart cells maintains homeostasis during cellular stress such as hypoxia by removing aggregated proteins and damaged organelles and thereby protects the heart during the times of starvation and ischemia. However, autophagy can lead to substantial cell death under certain circumstances. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a hypoxia-induced marker, has been shown to induce both autophagy and apoptosis. A BNIP3-docked organelle, e.g., mitochondria, also determines whether autophagy or apoptosis will take place. Estrogen (E2) and estrogen receptor (ER) alpha (ERα) have been shown to protect the heart against mitochondria-dependent apoptosis. The aim of the present study is to investigate the mechanisms by which ERα regulates BNIP3-induced apoptosis and autophagy, which is associated with hypoxic injury, in cardiomyoblast cells. An in vitro model to mimic hypoxic injury in the heart by engineering H9c2 cardiomyoblast cells to overexpress BNIP3 was established. Further, the effects of E2 and ERα in BNIP3-induced apoptosis and autophagy were determined in BNIP3 expressing H9c2 cells. Results from TUNEL assay and Immunoflourecense assay for LC3 puncta formation, respectively, revealed that ERα/E2 suppresses BNIP3-induced apoptosis and autophagy. The Western blot analysis showed ERα/E2 decreases the protein levels of caspase 3 (apoptotic marker), Atg5, and LC3-II (autophagic markers). Co-immunoprecipitation of BNIP3 and immunoblotting of Bcl-2 and Rheb showed that ERα reduced the interaction between BNIP3 and Bcl-2 or Rheb. The results confirm that ERα binds to BNIP3 causing a reduction in the levels of functional BNIP3 and thereby inhibits cellular apoptosis and autophagy. In addition, ERα attenuated the activity of the BNIP3 promoter by binding to SP-1 or NFκB sites.

BNIP3 induces IL6 and calcineurin/NFAT3 hypertrophic-related pathways in H9c2 cardiomyoblast cells.

Ischemia/reperfusion injury causes cardiomyocyte apoptosis, ventricular remodeling, leading to a dilated heart. Hypoxia is one of the causes involved in ischemia damage, and BNIP3 is a hypoxia-inducible marker and also a sensor to induce mitochondria-dependent apoptosis. Recent reports discussed ablating BNIP3 can restrain cardiomyocytes apoptosis and post-infarction remodeling. BNIP3 is a crucial therapeutic target. However, the BNIP3-induced hypertrophy aspect is rarely investigated. Here, we transiently transfected BNIP3 plasmids into H9c2 cardiomyoblast cells to evaluate the molecular signaling and hypertrophy markers using Western blot. We measured the cell size change using actin staining. We disclose that BNIP3 overexpression induced an increase in cell size, activated the pathological-related hypertrophy signaling pathways, such as IL6-MEK5-ERK5, IL6-JAK2-STAT1/3, calcineurin/NFAT3 and p38ß MAPK resulting in the fetal genes, ANP and BNP expressing. Concluding above, BNIP3 acts as a pathological hypertrophy inducer, which might be a potential therapeutic target for heart damage prevention.

E4BP4 is a cardiac survival factor and essential for embryonic heart development.

The bZIP transcription factor E4BP4, has been demonstrated to be a survival factor in pro-B lymphocytes. GATA factors play important roles in transducing the IL-3 survival signal and transactivating the downstream survival gene, E4BP4. In heart, GATA sites are essential for proper transcription of several cardiac genes, and GATA-4 is a mediator of cardiomyocyte survival. However, the role E4BP4 plays in heart is still poorly understood. In this study, Dot-blot hybridization assays using Dig-labeled RNA probes revealed that the E4BP4 gene was expressed in cardiac tissue from several species including, monkey, dog, rabbit, and human. Western blot analysis showed that the E4BP4 protein was consistently present in all of these four species. Furthermore, immunohistochemistry revealed that the E4BP4 protein was overexpressed in diseased heart tissue in comparison with normal heart tissue. In addition, the overexpression of E4BP4 in vitro activated cell survival signaling pathway of cardiomyocytes. At last, siRNA-mediated knock down of E4BP4 in zebrafish resulted in malformed looping of the embryonic heart tube and decreased heart beating. Based on these results, we conclude that E4BP4 plays as a survival factor in heart and E4BP4 is essential for proper embryonic heart development.

Taohe Chengqi Tang ameliorates acute liver injury induced by carbon tetrachloride in rats.

OBJECTIVE: To clarify the efficacy of Taohe Chengqi Tang (THCQT), a compound traditional Chinese herbal medicine, in protecting liver damage induced by carbon tetrachloride (CCl(4)) in rats. METHODS: Thirty male Wistar rats were divided into normal control group, untreated group, low-dose THCQT group (receiving 0.3 g/kg of THCQT), high-dose THCQT group (receiving 0.5 g/kg of THCQT), and positive control group (receiving silymarin 25 mg/kg). All testing substances were orally administered 1 hour before the intraperitoneal injection of CCl(4) (1.5 mL/kg). Twenty-four hours after CCl(4) injection, the rats were sacrificed to observe liver histopathological changes, and to evaluate activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and lipid peroxidation (LPO) and glutathione (GSH) levels in liver tissues. RESULTS: CCl(4) injection elevated the serum AST and ALT activities, but THCQT significantly reversed this effect. The increase of hepatic LPO by CCl(4) was markedly reduced by THCQT. Also, this herbal mixture increased hepatic GSH in the rats. In histopathology analysis, THCQT decreased the fatty accumulation, necrosis and lymphocyte infiltration. The in vitro study in rat brain showed that LPO induced by Fe(2+)/ascorbic acid was dose-dependently reduced by THCQT. According to the biochemical and morphological data, THCQT could protect the liver from CCl(4-)induced injuries. CONCLUSION: THCQT seems helpful for protection of liver damage induced by chemicals depending on its anti-oxidant-like function, and THCQT is more effective than silymarin.

Applying WCACG modified process is beneficial on reduced door-to-balloon time of acute STEMI patients.

BACKGROUND: Various systems have employed with the objective to reduce the time from emergency medical services contact to balloon inflammation for ST-elevation myocardial infraction (STEMI) patients. The WCACG message system was used to an alternative communication platform to improve confirmation of the diagnosis and movement to treatment, resulted in shorten the door-to-balloon (D-to-B) time for STEMI patients. METHODS: We collected 366 STEMI patients admitted at the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Department of Cardiology, during the period from June 2013 to October 2015. The patients were divided into two groups one underwent the current GC processes and the other group was handled using WCACG system. We compared between two groups with several indicators including D-to-B time, duration of hospitalization, associated costs, and incidence of adverse cardiovascular events. RESULTS: The results show that the new method with WCACG system significantly reduced the average D-to-B time (from 100.42 ± 25.14 mins to 79.81 ± 20.51 mins, P < 0.05) compared to the GC processes, and also reduced the duration, costs and undesirable cardiac incidence during hospitalization. CONCLUSIONS: The modified WCACG process is an applicable system to save pieces of time and efficiently integrate the opinions of experts in emergency.

Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner.

Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estradiol (E2) have been reported to downregulate the expression of E2 receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E2 replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. These data strongly suggest the protective roles of E2 and ER against colorectal cancer. However, the mechanisms remain unexplored. LoVo cells were transient transfected to overexpress ER-beta, DNA fragmentation and caspase activity assay were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the TNF-alpha promoter activity. Our data clearly demonstrated that E2 and ER-beta alone could upregulate p21 and p27 proteins, which further activate caspase-8 and caspase-9 to induce apoptosis in LoVo cell, and the ER-beta. effects were enhanced by E2. However, overexpressed ER-beta did not influence the expression and promoter activity of TNF-alpha. In addition, E2 and overexpressed ER-beta downregulated the beta-catenin proteins which cause the downregulation of its target genes, cyclin D1 and Rb, to inhibit the cell cycle and cell proliferation. The results indicate that overexpressed ER-beta may induce LoVo cell apoptosis and anti-proliferation by increasing p53 signaling in a ligand-dependent manner, and without hTNF-alpha involvement. Efforts aiming at enhancing ER-beta expression and/or activity may prove to be an attractive alternative therapy against colorectal cancer.

ß-catenin plays a key role in metastasis of human hepatocellular carcinoma.

Currently, there are no diagnostic or metastatic markers that can be used in early diagnosis and treatment of human hepatocellular carcinoma (HCC). The aim of this study was to find a molecular marker that regulated migration and metastasis in HCC. We analyzed the gene expression of ß-catenin, c-Myc and IL-8 in human HCC tissue by RT-PCR and immunohistochemistry and analyzed five variously differentiated HCC cell lines by Western blotting and migration and invasion assays to find markers for HCC diagnosis and HCC metastasis. mRNA expression of ß-catenin was significantly higher in the tumor area compared to the non-tumor area and was more abundant in specimens of late-stage HCC. Immunohistochemistry revealed that the translocation of ß-catenin into the nucleus was closely correlated with IL-8 protein levels and tumor stage. Similarly, the level of expression and nuclear translocation of ß-catenin was greater in HA22T cells with high proliferative activity than in HCC cell lines with low proliferative activity (PLC, Hep3B, HepG2). Knockdown of the ß-catenin gene with ß-catenin antisense oligonucleotides resulted in inhibition of cell migration and invasion of HA22T cells. Taken together, these results suggest that ß-catenin may be a suitable diagnostic marker of metastasis in human HCC.

Proliferative effects of chishao on injured peripheral neurons.

The aim of the study was to evaluate the proliferative effects of chishao on neuron regeneration. A silicone rubber nerve guide across a 15-mm gap was filled with different concentrations of chishao (0-125 mg/ml) in the dissected sciatic nerve of the right leg in SD rats. The left legs were used as control. After eight weeks, the regenerated nerves showed dose-dependently activated fibroblast growth factor-2 (FGF-2) signaling with increased urokinase plasminogen activator (uPA), decreased plasminogen activator inhibitor-1 (PAI-1) and enhanced proliferative proteins, extracellular signal regulated kinase (ERK)- and c-Jun N-terminal kinase (JNK)-signalings. The results imply that applying an appropriate dose of chishao would be a potential approach for enhancing neuron regeneration.

Hypoxia-induced compensatory effect as related to Shh and HIF-1alpha in ischemia embryo rat heart.

Chronic cardiac ischemia/hypoxia induces coronary collateral formation and cardiomyocyte proliferation. Hypoxia can induce cellular adaptive responses, such as synthesis of VEGF for angiogenesis and IGF-2 for proliferation. Both reduce apoptotic effects to minimize injury or damage. To investigate the mechanism of neoangiogenesis and proliferation of fetal heart under umbilical cord compression situation, we used H9c2 cardiomyoblast cell culture, and in vivo embryonic hearts as our study models. Results showed hypoxia induced not only the increase of IGF-2 and VEGF expression but also the activation of their upstream regulatory genes, HIF-1alpha and Shh. The relationship between HIF-1alpha and Shh was further studied by using cyclopamine and 2-ME2, inhibitor of Shh and HIF-1alpha signaling, respectively, in the cardiomyoblast cell culture under hypoxia. We found that the two inhibitors not only blocked their own signal pathway, but also inhibited each other. The observations revealed when fetal heart under hypoxia that HIF-1alpha and Shh pathways maybe involve in cell proliferation and neoangiogenesis to minimize injury or damage, whereas the complex cross-talk between the two pathways remains unknown.

Over-expressed estrogen receptor-alpha up-regulates hTNF-alpha gene expression and down-regulates beta-catenin signaling activity to induce the apoptosis and inhibit proliferation of LoVo colon cancer cells.

Epidemiologic studies reported that the prevalence of hereditary non-polyposis colon cancer (HNPCC) in male is about 1.5-fold higher than that in female. Decreases in circulatory estrogen (E(2)) have been reported to downregulate the expression of E(2) receptor (ER) and significantly increase the risk of colorectal cancer. Patients that received E(2) replacement therapy were found to have a reduction in the incidence of colon adenoma and carcinoma. Furthermore, significant decreases in the expression of ER have been found in colorectal cancer specimens. Evidences strongly suggest the protective roles of E(2) and ER against colorectal cancer. However, the mechanisms of ERalpha effects on colorectal cancer cells remained un-clear. LoVo cells were transient transfected to overexpress ERalpha, DNA fragmentation and the activated caspases measurements were performed to evaluate apoptotic effects. Western blotting was used to evaluate protein levels, and luciferase activity assay to measure the Htnf-a promoter activity. The results clearly demonstrated that overexpressed ERalpha with or without E(2) (10(-8) M) treatment could activate caspase -8, -9, and 3 and induce DNA fragmentation in LoVo cell. At the same time, overexpressed ERalpha plus E(2) significantly increases the expression and promoter activity of hTNF-alpha, and the DNA fragmentation effect induced by E(2) plus ERalpha were reduced by the addition of hTNF antibody (0.1 ng(ml). In addition, E(2) plus ERalpha significantly upregulated p21 and p27 levels and downregulated the beta-catenin and its target genes, cyclin D1 and Rb, which regulate the cell cycle and cell proliferation. The results indicate that E(2) plus overexpressed ERalpha induce LoVo cell apoptosis might mediate through the increase of hTNF-alpha gene expression, which in turn activate caspase-8, -9 and caspase-3 and lead to the DNA fragmentation and apoptosis. E(2) plus ERalpha also showed the downregulation of beta-catenin signalings implicating the suppression of proliferation and metastasis of colorectal cells. Efforts aiming at enhancing ERalpha expression and(or activity may be proved to be an alternative therapy against colorectal cancer.