Bcl-2 Signaling Pathways Research Articles

Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats.

ObjectivesThis study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra.MethodsFA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA).ResultsOur study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios.ConclusionsOur study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to inhibition of the cytochrome c-mediated caspase-3-dependent apoptotic pathway in the cortical penumbra 7 d after reperfusion.

Valproic acid-mediated myocardial protection of acute hemorrhagic rat via the BCL-2 pathway.

Hemorrhage is a major cause of morbidity and mortality among trauma patients. The pathophysiologic changes following acute severe hemorrhage and tissue hypoxia lead to an imbalance of protein acetylation. Histone deacetylase inhibitors (HDACIs) were reported to restore the acetylation imbalance and serve as potential drugs for treating severe hemorrhage. However, the molecular mechanism of HDACI-mediated cytoprotection remains unclear. In this study, we examined the myocardial protective effects and respective mechanism of the HDACI valproic acid (VPA) administered during hemorrhagic and hypoxic stress in vivo and in vitro. In vivo, the therapeutic effect of VPA was evaluated in acute severe hemorrhagic rats, and the expressions of BCL-2 signal pathway molecules were observed in rat heart tissues. To explore the molecular mechanism of VPA-mediated myocardial protection, a cobalt chloride (CoCl2)-induced hypoxia model of rat H9c2 cardiomyoblasts was applied to mimic hypoxic injury raised by acute hemorrhage. VPA administration significantly improved the 4-hour survival rate of hemorrhagic animals from 55% to 100% and protected H9c2 cells against CoCl2-induced hypoxic injury at a dose of between 12.5 μM and 100 μM. Increased expression of BCL-2 messenger RNA was observed following VPA treatment in the heart tissues of hemorrhagic rats (approximately 4.9-fold) and in H9c2 cells that survived CoCl2-induced hypoxia (approximately 4.9-fold). Western blot analysis showed a concomitant increase in BCL-2 protein expression and Akt phosphorylation following VPA treatment. The cytoprotective activity of VPA was diminished by triciribine-mediated inhibition of Akt activation and by silencing of BCL-2 gene expression. These findings suggest that VPA protects myocardial cells from hemorrhagic and hypoxic stress through the Akt/BCL-2 survival pathway, indicating a potential use of HDACIs for acute severe hemorrhage treatment.

MicroRNA-744 inhibited cervical cancer growth and progression through apoptosis induction by regulating Bcl-2.

Growing evidence suggests that microRNA plays an essential role in the development and metastasis of many tumor progressions, including cervical cancer. Aberrant miR-744 expression has been indicated in many growth of tumor, the mechanism of miR-744 inhibits both the proliferation and metastatic ability for cervical cancer remains unclear. Accumulating evidences reported that Bcl-2 signal pathway plays an important role in the cellular process, such as apoptosis, cell growth and proliferation. The goal of this study was to identify miR-744 that could inhibit the growth, migration, invasion, proliferation and metastasis of gastric cancer through targeting Bcl-2 expression. Real-time PCR (RT-qPCR) was used to quantify miR-744 expression in vitro and vivo experiments. The biological functions of miR-744 were determined via cell proliferation. Our study indicated that miR-744 targeted on Bcl-2, which leads to the inactivation of apoptosis signaling and the cell proliferation of cervical cancer cells, ameliorating cervical cancer growth and progression. In addition, both up-regulation of miR-744 and down-regulation of Bcl-2 could stimulate Caspase-3 expression, promoting apoptosis of cervical cancer cells. Therefore, our research revealed the mechanistic links between miR-744 and Bcl-2 in the pathogenesis of cervical cancer through modulation of Caspase-3, leading to the inhibition of cervical cancer cell growth. And targeting miR-744 could be served as a novel strategy for future cervical cancer therapy clinically.

Involvement of Bcl-2 Signal Pathway in the Protective Effects of Apigenin on Anoxia/Reoxygenation-induced Myocardium Injury.

Apigenin is a type of flavonoids, which has been demonstrated to protect myocardium against ischemia/reperfusion (I/R) injury. However, the mechanism is still unclear. We hypothesized that the mechanism of cardioprotective action of apigenin on the I/R-induced injury might be caused via B-cell lymphoma (Bcl) signaling pathway. In this study, an in vitro I/R model was replicated on Langendorff-perfused heart and H9c2 cardiomyocytes by anoxia/reoxygenation (A/R) treatment. The recovery of cardiac contractile function, infarct size, lactate dehydrogenase (LDH) and creatine kinase (CK) in the perfusate, the expression and activity of Bcl-2 and caspase-3, and cardiomyocyte apoptosis were measured in the Langendorff heart undergoing A/R injury. In addition, the cell viability, LDH release, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), expression of cytochrome c in the cytosol, and cell apoptosis were examined in the culture of H9c2 cardiomyocytes after the A/R. The results showed that apigenin significantly improved rat heart contractile function, reduced LDH release, infarct size and apoptotic rate, upregulated the expression of Bcl-2 and caspase-3, and downregulated the expression of cleaved caspase-3 after the A/R. Moreover, apigenin increased the cell viability and decreased the release of LDH, production of reactive oxygen species, release of mitochondrial cytochrome c into the cytosol, and cell apoptosis in the culture of H9c2 cardiomyocytes after the A/R. In addition, inhibition of Bcl-2 activity by ABT-737 markedly attenuated the protective effect of apigenin on the A/R-induced myocardium injury. Taken together, we firstly demonstrated that the effect of apigenin against A/R injury in cardiomyocytes involves Bcl-2 signal pathway and at least partly depends on its effect of upregulating the expression of Bcl-2.

Interferon-γ Upregulates SOCS3 Expression to Reduce Cisplatin Chemoresistance in Non-Small Cell Lung Cancer A549 Cells

Abstract Background: Cisplatin (DDP)-based chemotherapy is the mainstay of first-line therapeutic strategy for the treatment of advanced non-small cell carcinoma (NSCLC). However, the anticancer efficacy of DDP is often limited by the existence or development of chemoresistance. Thus, we investigated the effect of interferon-γ on DDPresistant A549 cell. Methods: Semi-quantitative RT–PCR was used to compare the differences of SOCS3 mRNA expression in both cisplatin-resistant A549 (A549/DDP) cell and the parental A549 cell. The cellular sensitivity to cisplatin, cell viability and apoptosis were detected by MTT, flow cytometry and Western blotting. Results: Semi-quantitative RT–PCR and western blotting showed that SOCS 3 expression was significantly down-regulated in A549/DDP cell compared to the parental A549 and normal human bronchial epithelial cells BEAS-2B. IFN-γ treatment could restore SOCS3 expression, resulting in an increased sensitivity of these resistant A549 cells to DDP. In addition, p53 and Bcl-2 signaling pathways were also involved in regulating IFN-γ-induced cell death in DDP-resistant A549 cells. Conclusion: Our study indicates that SOCS 3 may play a crucial role in the progress of cisplatin resistance of A549. Moreover, IFN-γ could induce SOCS3 expression and potentiate the re-sensitization of these resistant A549 cells to DDP

Resveratrol Improves Cognitive Impairment by Regulating Apoptosis and Synaptic Plasticity in Streptozotocin-Induced Diabetic Rats

Aims: To investigate the effects of resveratrol on cognitive impairment in streptozotocin (STZ)-induced diabetic rats and to explore the mechanisms of that phenomenon. Methods: Sixty healthy male Sprague Dawley rats were randomly divided into four groups: normal control group (Con group, n = 15), Res group (normal Sprague Dawley rats treated with resveratrol, n = 15), diabetes mellitus group (DM group, n = 15) and DM + Res group (diabetic rats treat with resveratrol, n = 15). Streptozotocin (STZ) was injected intraperitoneally to establish the diabetic model. One week after diabetic model induction, the animals in the Res group and the DM + Res group received resveratrol intraperitoneally once a day for consecutive 4 weeks. The Morris water maze test was applied to assess the effect of resveratrol on learning and memory. To explore the mechanisms of resveratrol on cognition, we detected the protein expression levels of Caspase-3, Bcl-2, Bax, NMDAR1 (N-Methyl-d-Aspartate receptor) and BDNF (Brain Derived Neurotrophic Factor) via western blotting analysis. Results: Resveratrol has no obvious effect on normal SD rats. Compared to Con group, cognitive ability was significantly impaired with increased expression of Caspase-3, Bax and down-regulation of Bcl-2, NMDAR1 and BDNF in diabetic rats. By contrast, resveratrol treatment improved the cognitive decline. Evidently, resveratrol treatment reversed diabetes-induced changes of protein expression. Conclusions: Resveratrol significantly ameliorates cognitive decline in STZ-induced diabetic model rats. The potential mechanism underlying the protective effect could be attributed to the inhibition of hippocampal apoptosis through the Bcl-2, Bax and Caspase-3 signaling pathways and improvement of synaptic dysfunction. BDNF may also play an indispensable role in this mechanism.

Protective Effects of Gastrodin Against Autophagy-Mediated Astrocyte Death.

Gastrodin is an active ingredient derived from the rhizome of Gastrodia elata. This compound is usually used to treat convulsive illness, dizziness, vertigo, and headache. This study aimed to investigate the effect of gastrodin on the autophagy of glial cells exposed to lipopolysaccharides (LPS, 1 µg/mL). Autophagy is a form of programmed cell death, although it also promotes cell survival. In cultured astrocytes, LPS exposure induced excessive autophagy and apoptosis, which were significantly prevented by the pretreatment cells with gastrodin (10 μM). The protective effects of gastrodin via autophagy inhibition were verified by the decreased levels of LC3-II, P62, and Beclin-1, which are classical markers for autophagy. Furthermore, gastrodin protected astrocytes from apoptosis through Bcl-2 and Bax signaling pathway. The treatment of astrocytes with rapamycin (500 nM), wortmannin (100 nM), and LY294002 (10 μM), which are inhibitors of mTOR and PI3K, respectively, eliminated the known effects of gastrodin on the inhibited Beclin-1 expression. Furthermore, gastrodin blocked the down-regulation of glutamine synthetase induced by LPS exposure in astrocytes. Our results suggest that gastrodin can be used as a preventive agent for the excessive autophagy induced by LPS.

Study on effect of lycorine in inducing apoptosis of pulmonary carcinoma cell A549

To explore the effect of lycorine in inducing apoptosis of pulmonary carcinoma cell A549 and its mechanism. In the study, pulmonary carcinoma cell A549 were taken as the experimental subject and processed with different concentrations of lycorine (0, 0.5, 1.0, 2.0, 4.0 and 8.0 μmol x L(-1)). The MTT method was used to observe the cell proliferation. The apoptosis rate of A549 cells was determined by Annexin FITC/PI double staining. The microplate reader was used to detect the activities of Bcl-2, Bax and p53. The changes in mitochondrial membrane potential were measured by the flow cytometry. The expressions of apoptosis-related factors Bcl-2, Bax, p53 and Survivin were determined by Real-time PCR. The results showed that lycorine significantly inhibited the proliferation of A549 cells (P < 0.05), induced the apoptosis on A549 cells (P < 0.05), increased the activities of Bax and p53, reduced Bcl-2 activity and mitochondrial membrane potential, and notably changed the gene expressions of Bcl-2, Bax, p53 and Survivin (P < 0.05). In conclusion, lycorine can induce the apoptosis of A549 cells and be applied to treat pulmonary carcinoma. Its mechanism may be related to the activation of relevant factors in Bcl-2 signaling pathway.

Electromagnetic radiation at 900 MHz induces sperm apoptosis through bcl-2, bax and caspase-3 signaling pathways in rats.

BackgroundThe decreased reproductive capacity of men is an important factor contributing to infertility. Accumulating evidence has shown that Electromagnetic radiation potentially has negative effects on human health. However, whether radio frequency electromagnetic radiation (RF-EMR) affects the human reproductive system still requires further investigation. Therefore, The present study investigates whether RF-EMR at a frequency of 900 MHz can trigger sperm cell apoptosis and affect semen morphology, concentration, and microstructure.MethodsTwenty four rats were exposed to 900 MHz electromagnetic radiation with a special absorption rate of 0.66 ± 0.01 W/kg for 2 h/d. After 50d, the sperm count, morphology, apoptosis, reactive oxygen species (ROS), and total antioxidant capacity (TAC), representing the sum of enzymatic and nonenzymatic antioxidants, were investigated. Western blotting and reverse transcriptase PCR were used to determine the expression levels of apoptosis-related proteins and genes, including bcl-2, bax, cytochrome c, and capase-3.ResultsIn the present study, the percentage of apoptotic sperm cells in the exposure group was significantly increased by 91.42 % compared with the control group. Moreover, the ROS concentration in exposure group was increased by 46.21 %, while the TAC was decreased by 28.01 %. Radiation also dramatically decreased the protein and mRNA expression of bcl-2 and increased that of bax, cytochrome c, and capase-3.ConclusionRF-EMR increases the ROS level and decreases TAC in rat sperm. Excessive oxidative stress alters the expression levels of apoptosis-related genes and triggers sperm apoptosis through bcl-2, bax, cytochrome c and caspase-3 signaling pathways.

Effects of bergamot essential oil and its extractive fractions on SH-SY5Y human neuroblastoma cell growth.

The goals were to investigate the mechanisms underlying the antiproliferative effects of bergamot essential oil (BEO) and to identify the compounds mainly responsible for its SH-SY5Y cells growth rate inhibition. Five BEO extractive fractions (BEOs) differing in their chemical composition were used. Cell proliferation was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell count assays. Trypan blue exclusion test and Annexin V/PI staining were performed to assess their cytotoxic activity. Genotoxicity was detected by comet assay. The cell cycle was checked cytofluorimetrically. Reactive oxygen species (ROS) and Δψm were measured fluorimetrically. Western blotting analyses for some apoptosis-related proteins were carried out. Treatment of SH-SY5Y cells with some types of BEOs decreased cell growth rate by a mechanism correlated to both apoptotic and necrotic cell death. Coloured BEOs act by increasing ROS generation, responsible for the drop in Δψm, and modulate p38 and extracellular signal-regulated kinases (ERK ½) mitogen-activated protein kinases, p53, Bcl-2 and Bax signalling pathways. Finally, we identify bergamottin and 5-geranyloxy-7-methoxycoumarin as the bioactive molecules that could play a pivotal role in the antiproliferative effects exerted by coloured BEOs. Our study provides novel insights into the field of the antiproliferative effects of BEO, which could be exploited in the context of a multitarget pharmacological strategy.

P0107 New insights into the mechanisms of bergamot essential oil and its extractive fractions on SH-SY5Y human neuroblastoma cell growth

<h3>Background</h3> Bergamot essential oil (BEO) is obtained from the fruit peel of <i>Citrus bergamia</i> (bergamot), a small tree cultivated almost exclusively along the southern coast of the Calabria region of Italy. BEO is appreciated for its organoleptic properties and is widely used in the manufacture of perfumes. The goals of our study were to investigate the mechanisms underlying the antiproliferative effects of BEO and to identify the compounds mainly responsible for its inhibition of SH-SY5Y cell growth. <h3>Methods</h3> Five BEO extractive fractions (BEOs) differing in chemical composition were used. Cell proliferation was determined by MTT and cell count assays. The trypan blue exclusion test and annexin V/PI staining were done to assess their cytotoxic activity. Genotoxic effects were detected by the comet assay. The cell cycle was also analysed cytofluorimetrically. Reactive oxygen species and Δ<i>ψ</i>m were measured fluorimetrically. Western blotting analyses for some apoptosis-related proteins were carried out. <h3>Findings</h3> Treatment of SH-SY5Y cells with some types of BEOs decreased rate of cell growth by a mechanism correlated to both apoptotic and necrotic cell death. Coloured BEOs act by increasing generation of reactive oxygen species, responsible for the drop in <mml:math><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>ψ</mml:mi></mml:mrow></mml:math>m, and modulating p38 and ERK1/2 MAPKs, p53, Bcl-2 and Bax signalling pathways. Finally, we identified bergamottin and 5-geranyloxy-7-methoxycoumarin as the bioactive molecules that play a pivotal part in the antiproliferative effects exerted by coloured BEOs. <h3>Interpretation</h3> Our study provides novel insights into the antiproliferative effects of BEO, which could be exploited in the context of multitarget pharmacological strategies.

Cadmium-induced autophagy promotes survival of rat cerebral cortical neurons by activating class III phosphoinositide 3-kinase/beclin-1/B-cell lymphoma 2 signaling pathways.

Autophagy is an evolutionarily conserved response that can be activated in response to heavy metal. Thus, the present study investigated the effect of autophagy on neurotoxic damage caused by cadmium (Cd) in rat cerebral cortical neurons. The results indicated that the viability of cortical neurons treated with Cd was markedly decreased in a dose-and time-dependent manner. The present study provided evidence that cortical neurons treated with Cd underwent autophagy: The conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3) to LC3-II, an increase in the punctate distribution of endogenous LC3-II and the presence of autophagosomes were identified. Combined treatment with Cd and chloroquine, an autophagy inhibitor, reduced the amount of autophagocytosis and cell activity, whereas rapamycin, an autophagy inducer, reduced Cd-mediated cytotoxicity. Furthermore, it was found that beclin-1 and class III phosphoinositide 3 kinase (PI3K) levels were increased, while levels of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) were decreased after Cd treatment. LY294002, a specific inhibitor of PI3K, prevented the decline in Bcl-2 production and the increase in levels of beclin-1, class III PI3K and autophagy following Cd treatment. In conclusion, the results of the present study suggested that Cd can induce cytoprotective autophagy by activating the class III PI3K/beclin-1/Bcl-2 signaling pathway, and that the autophagy pathway can serve as a sensitive biomarker for nervous system injury after exposure to Cd.

Ceramide-induced apoptosis in renal tubular cells: a role of mitochondria and sphingosine-1-phoshate.

Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed.

3B, a novel of photosensitizer, exhibited anti-tumor effects via mitochondrial apoptosis pathway in MCF-7 human breast carcinoma cells.

Photodynamic therapy (PDT) has been recognized as an innovated therapeutic modality for the treatment of various cancers. In this study, we evaluated the anticancer effect of a new photosensitizer 3B in breast cancer, which was considered one of the most common cancers in women worldwide. Here, we determined the effect of 3B not only on the cell growth, apoptosis, and Bcl-2 signal pathway in vitro but also on the anti-cancer effect in nude mice in vivo. Our results showed that 3B was primarily accumulated in mitochondria, increased the level of ROS, induced apoptotic cells death via Bcl-2 family, and its activity could be blocked by the caspase inhibitor (Z-VAD-FMK). In vivo study, 3B made a significant opening inhibition of tumor growth and showed drug toxicity hardly. TUNEL assay indicated that PDT group showed more positive cells (green) than other groups. These data supported that 3B might develop as potential therapeutic drug for the treatment of breast cancer.

Expression and clinical significance of nuclear factor κB/B cell lymphoma-2 signal pathway and glycogen synthase kinase 3β in oral squamous cell carcinoma

This study aims to examine the expression of nuclear factor κB (NF-κB)/B cell lymphoma-2 (Bcl-2) signal pathway and glycogen synthase kinase 3β (GSK-3β) in oral squamous cell carcinoma (OSCC) and provide references for the early diagnosis and prognosis evaluation of OSCC. A total of 55 cases of OSCC and 10 cases of paracan- cerous mucosa were examined in this study. Their expressions of GSK-3β, NF-κB and Bcl-2 were detected using the SP me- thod immunohistochemistry. The correlation between their expression in OSCC and the clinical and pathological peculiarity of OSCC was analyzed. The positive expression of GSK-3β, NF-κB, and Bcl-2 in OSCC were significantly higher than that in paracancerous mucosa (P < 0.01). The expression of GSK-3β, NF-κB, and Bcl-2 had no obvious relationship with patient's age, sex, and clinical stages of the disease (P > 0.05). The expression of Bcl-2 was significantly correlated with the degree of tumor differentiation (P < 0.05), whereas the expression of GSK-3β and NF-κB in OSCC had no obvious relation- ship with the degree of tumor differentiation (P > 0.05). Strong positive correlations were observed among the expressions of GSK-3β, NF-κB, and Bcl-2 (P < 0.05). The positive expression of GSK-3β, NF-κB, and Bcl-2 in OSCC are sig- nificantly higher than that in paracancerous mucosa. Detecting GSK-3β, NF-κB, and Bcl-2 in OSCC may have implications in the early diagnosis and prognosis evaluation of OSCC.

Lactate promotes resistance to glucose starvation via upregulation of Bcl-2 mediated by mTOR activation.

Solid tumors grow faster and need more glucose than normal tissue; however, due to poor angiogenesis and excessive growth, tumors remote from blood vessels are always under glucose starvation. Even so, cancer cells remain alive in vivo. Thus, making cancer cells sensitive to glucose depletion may potentially provide an effective strategy for cancer intervention. Tumors that obtain sufficient glucose generate a large amount of lactate. Therefore, we proposed that lactate, a tumor microenvironment factor, may allow cancer cells to develop resistance to glucose starvation-induced death. We cultured cancer cells in no-glucose medium and added lactate to the medium. During the experiment, lactate helped cancer cells to escape from glucose starvation-induced cell death, without using lactate as an energy substrate, resulting in activation of Akt through PI3K. Akt activation plays a central role in cell growth through the activation of mammalian target of rapamycin (mTOR). Alteration of the PI3K/Akt/mTOR signaling pathway by inhibiting apoptosis induced specific upregulation of B-cell lymphoma 2 (Bcl-2) through translational control. In conclusion, this study showed that lactate rescues cancer cells from glucose starvation-induced cell death through regulation of the PI3K/Akt/mTOR/Bcl-2 signaling pathway. These data suggest that lactate is an important determinant of the sensitivity of tumors to glucose starvation, and reducing lactate or inhibiting the PI3K/Akt/mTOR/Bcl-2 signaling pathway may influence the response of cancers to glucose starvation.

MiR-135a inhibition protects A549 cells from LPS-induced apoptosis by targeting Bcl-2

Acute lung injury (ALI) is a severe clinical condition with high morbidity and mortality. Apoptosis is a key pathologic feature of ALI, and Bcl-2 plays an important role during the pathogenesis of ALI via the regulation of apoptosis. However, the regulation of Bcl-2 during ALI, particularly through microRNAs, remains unclear. We hypothesize that certain miRNAs may play deleterious or protective roles in ALI via the regulation of Bcl-2. The LPS stimulation of A549 cells was used to mimic ALI in vitro. First, we confirmed that Bcl-2 is involved in LPS-induced apoptosis in A549 cells. Then, bioinformatic analyses and quantitative real-time polymerase chain reaction assays were performed to screen for miRNAs targeting Bcl-2. We observed that miR-135a was markedly increased in LPS-challenged A549 cells. miR-135a inhibition markedly restored Bcl-2 expression and protected A549 cells from LPS-induced apoptosis. Furthermore, bioinformatic analysis and luciferase activity assays were conducted to confirm that miR-135a binds directly to the 3′-untranslated region of Bcl-2 and suppresses its expression. Interestingly, the inhibition of miR-135a did not attenuate apoptosis under LPS-treated conditions when Bcl-2 was knocked down. Therefore, we suggest that miR-135a regulation of LPS-induced apoptosis in A549 cells may depend in part on the regulation of Bcl-2. The miR-135a/Bcl-2 signaling pathway may be a novel therapeutic target for the prevention of ALI.

Cannabinoid receptor type 2 agonist attenuates apoptosis by activation of phosphorylated CREB–Bcl-2 pathway after subarachnoid hemorrhage in rats

Early brain injury (EBI) which comprises of vasogenic edema and apoptotic cell death is an important component of subarachnoid hemorrhage (SAH) pathophysiology. This study evaluated whether cannabinoid receptor type 2 (CB2R) agonist, JWH133, attenuates EBI after SAH and whether CB2R stimulation reduces pro-apoptotic caspase-3 via up-regulation of cAMP response element-binding protein (CREB)-Bcl-2 signaling pathway. Male Sprague–Dawley rats (n=123) were subjected to SAH by endovascular perforation. Rats received vehicle or JWH133 at 1h after SAH. Neurological deficits and brain water content were evaluated at 24h after SAH. Western blot was performed to quantify phosphorylated CREB (pCREB), Bcl-2, and cleaved caspase-3 levels. Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling staining. Additionally, CREB siRNA was administered to manipulate the proposed pathway. JWH133 (1.0mg/kg) improved neurological deficits and reduced brain water content in left hemisphere 24h after SAH. JWH133 significantly increased activated CREB (pCREB) and Bcl-2 levels and significantly decreased cleaved caspase-3 levels in left hemisphere 24h after SAH. CREB siRNA reversed the effects of treatment. TUNEL positive neurons in the cortex were reduced with JWH133 treatment. Thus, CB2R stimulation attenuated EBI after SAH possibly through activation of pCREB–Bcl-2 pathway.

MicroRNA-34a regulates high glucose-induced apoptosis in H9c2 cardiomyocytes

Hyperglycemia is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The purpose of the present study was to investigate whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9c2 through microRNA-mediated Bcl-2 signaling pathway. The expression of miR-34a and Bcl-2 mRNA was detected by using real-time PCR. Western blotting was used to examine the changes in apoptosis-associated protein Bcl-2. Apoptosis of H9c2 cells was tested by using flow cytometry. The results showed that the expression of miR-34a was significantly elevated and that of Bcl-2 was strongly reduced, and apoptosis of cardiomyocytes was apparently increased in the high-glucose-treated H9c2 cells as compared with normal-glucose-treated controls. In addition, we identified Bcl-2 gene was the target of miR-34a. miR-34a mimics reduced the expression of Bcl-2 and increased glucose-induced apoptosis, but miR-34a inhibitor acted as the opposite mediator. Our data demonstrate that miR-34a contributes to high glucose-induced decreases in Bcl-2 expression and subsequent cardiomyocyte apoptosis.

Effects of down-regulated TRAF6 gene expression on the proliferation and apoptosis in multiple myeloma cells

To investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells. Detection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot. The levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein. TRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.