Induction Of Bcl-2 Expression Research Articles

Plasmodium yoelii Infection Enhances the Expansion of Myeloid-Derived Suppressor Cells via JAK/STAT3 Pathway.

Myeloid-derived suppressor cells (MDSCs), the negative immune regulators, have been demonstrated to be involved in immune responses to a variety of pathological conditions, such as tumors, chronic inflammation, and infectious diseases. However, the roles and mechanisms underlying the expansion of MDSCs in malaria remain unclear. In this study, the phenotypic and functional characteristics of splenic MDSCs during Plasmodium yoelii NSM infection are described. Furthermore, we provide compelling evidence that the sera from P. yoelii-infected C57BL/6 mice containing excess IL-6 and granulocyte-macrophage colony-stimulating factor promote the accumulation of MDSCs by inducing Bcl2 expression. Serum-induced MDSCs exert more potent suppressive effects on T cell responses than control MDSCs within both invivo P. yoelii infection and invitro serum-treated bone marrow cells experiments. Serum treatment increases the MDSC inhibitory effect, which is dependent on Arg1 expression. Moreover, mechanistic studies reveal that the serum effects are mediated by JAK/STAT3 signaling. By inhibiting STAT3 phosphorylation with the JAK inhibitor JSI-124, effects of serum on MDSCs are almost eliminated. In vivo depletion of MDSCs with anti-Gr-1 or 5-fluorouracil significantly reduces the parasitemia and promotes Th1 immune response in P. yoelii-infected C57BL/6 mice by upregulating IFN-γ expression. In summary, this study indicates that P. yoelii infection facilitates the accumulation and function of MDSCs by upregulating the expression of Bcl2 and Arg1 via JAK/STAT3 signaling pathway invivo and invitro. Manipulating the JAK/STAT3 signaling pathway or depleting MDSCs could be promising therapeutic interventions to treat malaria.

Neuroprotective effect of piracetam-loaded magnetic chitosan nanoparticles against thiacloprid-induced neurotoxicity in albino rats

Thiacloprid (TH) is a neurotoxic agricultural insecticide and potential food contaminant. The purpose of this study was to investigate the relationship between TH exposure and memory dysfunction in rats, as well as the potential protective effect of piracetam and piracetam-loaded magnetic chitosan nanoparticles (PMC NPs). Rats were divided into five equal groups (six rats/group). The control group received saline. Group II was treated with PMC NPs at a dose level of 200 mg/kg body weight (Bwt); Group III was treated with 1/10 LD50 of TH (65 mg/kg Bwt); Group IV was treated with TH (65 mg/kg Bwt) and piracetam (200 mg/kg Bwt); Group V was co-treated with TH (65 mg/kg Bwt) and PMC NPs (200 mg/kg Bwt). All animal groups were dosed daily for 6 weeks by oral gavage. Footprint analysis, hanging wire test, open field test, and Y-maze test were employed to assess behavioral deficits. Animals were euthanized, and brain tissues were analyzed for oxidative stress biomarkers, proinflammatory cytokines, and gene expression levels of glial fibrillary acidic protein (GFAP), amyloid-beta precursor protein (APP), B-cell lymphoma 2 (Bcl-2), and caspase-3. Brain and sciatic nerve tissues were used for the evaluation of histopathological changes and immunohistochemical expression of tau protein and nuclear factor kappa B (NF-κB), respectively. The results revealed that TH-treated rats suffered from oxidative damage and inflammatory effect on the central and peripheral nerves. The administration of PMC NPs considerably protected against TH-induced neuronal damage, increased antioxidant enzyme activity, decreased inflammatory markers, and improved behavioral performance than the group treated with piracetam. The neuroprotective effect of PMC NPs was mediated through the inhibition of GFAP, APP, caspase-3, Tau, and NF-κB gene expression with induction of Bcl-2 expression. In conclusion, TH could induce oxidative stress, inflammatory and neurobehavior impairment in rats. However, PMC NPs administration markedly mitigated TH-induced brain toxicity, possibly via oxidative and inflammatory modulation rather than using piracetam alone.

β2-AR Signaling Enhances MDSC Survival through Metabolic Reprograming and Impairs Therapeutic Efficacy in Hematologic Malignancies

Nerve-driven chronic adrenergic stress can suppress anti-tumor immunity. However, the mechanistic details of how this occurs are still poorly understood. Catecholamines (epinephrine, norepinephrine) released by the autonomic nervous system (ANS) can activate adrenergic receptors (AR) present on nearly every cell type, including myeloid derived suppressor cells (MDSCs). MDSCs are important regulatory cells that hinder anti-tumor immune responses in hematological malignancies by inhibiting numerous cell types, including effector NK cells, CD8+ T cells, CD4+ T cells, and processes, such as blocking cytokine secretion. Because first line treatment for most hematologic malignancies includes chemotherapy, and MDSCs are known to be detrimental to its efficacy, understanding their role, including through chronic stress pathways, is relevant. Recently, using preclinical models of solid tumors, we have shown β2 adrenergic receptor (β2-AR) signaling, activated by chronic stress, increases the accumulation of MDSCs in the tumor microenvironment (TME). However, the role of β2-AR in MDSC survival and accumulation in hematological malignancies after chemotherapy, and the precise mechanisms by which β2-AR increases MDSC accumulation, remain unresolved. We have discovered β2-AR signaling drives MDSC survival in tumor bearing mice treated with chemotherapy. We have also shown the combination of propranolol, a non-selective β-AR blocker, with doxorubicin prolongs survival in mice bearing EL4 and C1498 tumors. Examination of immune cell compartments demonstrated a significant reduction of both M-MDSC and PMN-MDSC abundance in mice receiving the combination therapy, indicating the key effect of stress induced β2-AR signaling on MDSC accumulation. Further, we implanted EL4 tumors into β2-AR-/- and β2ARfl/fl LysmCre mice and confirmed doxorubicin increases survival in both β2-AR-/- and β2ARfl/fl LysmCre mice compared to WT control mice, suggesting β2-AR signaling in MDSCs is the main receptor driving the effects of propranolol in combination therapy. Using both in vitro and in vivo systems, we showed β2-AR signaling increased MDSC survival by the induction of BCL-2 expression and the suppression of caspase-3 and caspase-8. Seahorse metabolic studies further showed β2-AR signaling induced a metabolic shift to increased mitochondrial respiration (OCR) and ATP production in MDSCs. We then applied single cell RNA sequencing (scRNAseq) pathway analysis and multiplexed D7-Glc plus 13C5-Gln tracing in Stable Isotope-Resolved Metabolomics (SIRM). We found β2-AR signaling induces a metabolic shift toward higher ATP generation and decreased mitochondrial reactive oxygen species (mROS) that serves as a survival signal for MDSCs. We also found β2-AR signaling suppressed the expression of ATPase Inhibitory Factor 1 (ATPIF1) in MDSCs, which is responsible for increased ATP generation. These results show β2-AR signaling induces MDSC survival through metabolic reprogramming in mitochondria by enhancing ATP generation and modulating key survival signals to increase the ratio of ATP/mROS. Altogether, these studies reveal β2-AR signaling plays an important role in mitochondrial metabolic fitness in MDSCs, priming them to survive chemotherapy-induced cytotoxicity. The combination of the stress blocker propranolol, with chemotherapy regimens, could potentially improve the current therapeutic regimens used for hematological malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TPPU Downregulates Oxidative Stress Damage and Induces BDNF Expression in PC-12 Cells.

Objective Ischemia-reperfusion is an ongoing clinical challenge that can lead to a series of pathological changes including oxidative stress. The inhibition of soluble epoxide hydrolase inhibitor (sEH) by 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU) results in an anti-inflammatory, cardioprotective, and blood vessel growth-promoting effects. Therefore, this study focused on the protective effect of TPPU on a rat pheochromocytoma (PC-12) cell oxidative stress model induced by H2O2. Methods CCK-8 and Hoechst 33342 were used to evaluate cell apoptosis and western blot to detect the apoptotic proteins and brain-derived neurotrophic factor (BDNF) expression. Result The incubation with 100 μM, 50 μM, and 25 μM TPPU significantly increased PC-12 cell viability. Epoxyeicosatrienoic acid (EET) pretreatment also protected PC-12 cells from oxidative stress. In addition, TPPU reduced caspase-3 and Bax expression and induced Bcl-2 expression, and EETs exerted the same effect on caspase-3 expression as TPPU. A positive relationship was found between TPPU or EET incubation and BDNF expression. Conclusion These results revealed that TPPU reduced PC-12 cell oxidative stress injury induced by H2O2 and promoted BDNF expression.

Effect of Hypoxia Inducible Factor-1α on Chemosensitivity of B-ALL Cells to Vincristine and Its Mechanism

To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia （B-ALL） cells to Vincristine (VCR) and the mechanisms. B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels. CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups. Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.

Ginsenoside Rb3 Alleviates the Toxic Effect of Cisplatin on the Kidney during Its Treatment to Oral Cancer via TGF-β-Mediated Mitochondrial Apoptosis.

Objective The research aimed to confirm the role of the transforming growth factor-β (TGF-β) in cisplatin- (CPT-) evoked kidney toxicity and elucidate the mechanism that ginsenoside Rb3 (Rb3) could alleviate the kidney toxicity by CPT during its treatment to oral cancer via TGF-β-mediated mitochondrial apoptosis. Methods The model of xenograft nude mice bearing oral carcinoma cells ACC83 was established and treated with CPT and/or Rb3, respectively. Bodyweights of the treated mice were weighed, and the kidney tissues were collected; following, the histopathology and the expression of TGF-β were examined using H&E staining and immunohistochemistry. Afterward, the renal cells GP-293 were treated with CPT and/or Rb3. The expression and phosphoration of TGF-β, Smad2, Smad3, Bcl-2, and Bax in GP-293 cells were detected by Western blotting. The cellular apoptosis and mitochondrial membrane potential were analyzed using flow cytometry. Results The xenograft nude mice exposure to CPT presented the bodyweight loss, necrotic areas, and the increased expression of TGF in kidney tissue, and Rb3 pretreatment relieved these changes evoked by CPT. In GP-293 cells, CPT administration induced the phosphorylation of Smad2 and Smad3, and Rb3 pretreatment suppressed the induced phosphorylation by CPT. Besides, flow cytometry analysis showed that Rb3 inhibited the CPT-evoked cellular apoptosis ratio and mitochondrial membrane depolarization. The Western blotting test indicated that Rb3 alleviated the cleavage of PARP, caspase 3, caspase 8, and caspase 9, the induction of Bax expression, and inhibition of Bcl-2 expression. Additionally, after treating with the TGF inhibitor of disitertide, Rb3 exhibited no alleviation effects on CPT-evoked cellular apoptosis ratio, inhibition of Bax expression, and induction of Bcl-2 expression in GP-293 cells. Conclusion Rb3 could alleviate CPT-evoked toxic effects on kidney cells during its treatment to oral cancer via TGF-β-mediated mitochondrial apoptosis.

Combination of Bortezomib and Venetoclax Induces Synergistic Killing of Epstein-Barr Virus-Driven Lymphoproliferative Diseases By Targeting the Pro-Survival Function of Latent Membrane Protein-1 and Epstein-Barr Nuclear Antigen-3C

Epstein-Barr virus (EBV) is strongly associated with lymphoproliferative diseases (LPDs), in particular B cell, in both immunocompetent and immunocompromised hosts. The virus is able to manipulate host cell machineries such as the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and the survival of the host cells through expression of various viral proteins in distinct latency patterns. Latent membrane protein-1 (LMP-1) is a constitutively active CD40 receptor homolog and activates NF-κB signaling pathways to induce Bcl-2 expression whilst Epstein-Barr nuclear antigen-3C (EBNA-3C) interacts with and mediates proteasomal degradation of Bcl-6 protein which, in turn, increases Bcl-2 expression. We hypothesize that combining a proteasome inhibitor, bortezomib, with a Bcl-2 inhibitor, venetoclax, will induce synergistic killing of EBV-driven LPDs expressing both LMP-1 and EBNA-3C in the latency III pattern. Isobologram showed that combination of bortezomib and venetoclax could synergistically induce potent apoptosis of spontaneous lymphoblastoid cell lines (sLCL), derived from patients with post-transplant lymphoproliferative disorder (PTLD), expressing LMP-1 and EBNA-3C proteins. The mechanism of killing was related to the suppression of NF-κB signaling pathway induced by LMP-1. The phosphorylation of serine 70 of Bcl-2, which enhances the anti-apoptotic activity of Bcl-2 through stabilization of its interaction with other pro-apoptotic proteins such as Bak and Bim, was decreased in the sLCL, but not in the LMP-1 or EBNA-3C knockdown LCL, upon treatment with the drug combination. Activation of DNA damage response and production of reactive oxygen species were observed in the sLCL, contributing to the synergistic cell death. Bortezomib induced the expression of pro-apoptotic initiator, NOXA, to enhance the susceptibility of the sLCL to apoptosis upon treatment with venetoclax whilst knockdown of NOXA in the sLCL led to the resistance of the cells to apoptosis induced by the drug combination. In-vivo study demonstrated that the drug combination significantly inhibited the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, we conclude that the combination of bortezomib and venetoclax induces synergistic killing of EBV-driven LPDs such as PTLD by targeting the pro-survival function of LMP-1 and EBNA-3C. Disclosures No relevant conflicts of interest to declare.

Trigonelline protects hippocampal neurons from oxygen-glucose deprivation-induced injury through activating the PI3K/Akt pathway

Trigonelline is a plant alkaloid that has generated interest for its neuroprotective roles in brain pathology. However, the protective effect of trigonelline on cerebral ischemia/reperfusion (I/R) injury and the potential mechanism have not been fully evaluated. Our results showed that trigonelline pretreatment ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced hippocampal neurons injury. The OGD/R-caused reactive oxygen species (ROS) generation and decreased concentrations of superoxide dismutases (SOD) and glutathione peroxidase (GPx) were markedly attenuated by trigonelline. In addition, the increased levels of TNF-α, IL-6 and IL-1β in OGD/R-induced hippocampal neurons were significantly decreased by trigonelline pretreatment. Trigonelline also suppressed caspase-3 activity and bax expression, and induced bcl-2 expression in OGD/R-induced hippocampal neurons. Furthermore, trigonelline induced the activation of PI3K/Akt pathway in hippocampal neurons exposed to OGD/R condition. Inhibition of PI3K/Akt signaling reversed the protective effects of trigonelline on OGD/R-induced hippocampal neurons injury. Taken together, these findings indicated that trigonelline protected hippocampal neurons from OGD/R-induced injury, which was mediated by the activation of PI3K/Akt signaling pathway.

HOXA10 induces BCL2 expression, inhibits apoptosis, and promotes cell proliferation in gastric cancer.

Homeobox A10 (HOXA10) has been implicated critical for the promotion of carcinogenesis, but the underlying mechanism between HOXA10 and malignant gastric cancer (GC) phenotype remains elusive. In the present study, we analyzed and validated that HOXA10 and BCL2 expressions were elevated both at the mRNA and protein levels in GC tissues. Upregulated HOXA10 promoted GC cell proliferation with reduced apoptosis in vitro and accelerated GC tumor growth in vivo. Bioinformatics analysis and quantitative real‐time polymerase chain reaction (qRT‐PCR) experiment inferred that HOXA10 might upregulate the expression of BCL2. By performing western blot, chromatin immunoprecipitation and quantitative PCR (ChIP‐qPCR), and rescue experiment, we found that HOXA10 might bind to BCL2 promoter region, induce its expression, and thus inhibit intrinsic apoptosis pathway. Moreover, higher expression of HOXA10 and BCL2 predicted poor overall survival (OS) in GC patients. In summary, our study indicated that HOXA10 was upregulated in GC, and that HOXA10 might promote cell proliferation by elevating BCL2 expression and inhibiting apoptosis.

Paeoniflorin protects pancreatic β cells from STZ-induced damage through inhibition of the p38 MAPK and JNK signaling pathways

Pancreatic β-cells are responsible for insulin secretion and control of plasma glucose levels. Accumulating evidences indicate a relationship between β-cell dysfunction/death and diabetes onset. Paeoniflorin (PF), a natural glycoside, has antihyperglycemic effect. However, the role of PF in pancreatic β-cells has not been examined. The aim of this study was to evaluate the protective effect of PF on streptozotocin (STZ)-induced β-cell damage. Our results showed that PF improved STZ-caused inhibitory effect on cell viability and insulin secretion ability in INS-1 cells. PF reduced caspase-3 activity and bax expression, and induced bcl-2 expression in STZ-treated INS-1 cells. PF resulted in a decrease in production of reactive oxygen species and MDA, and an increase in SOD activity in STZ-treated INS-1 cells. Furthermore, PF inhibited the phosphorylation of p38 and JNK, which is induced by STZ in INS-1 cells. The results suggested that PF protected INS-1 cells from STZ-induced cell damage. Meanwhile, PF suppressed the activation of p38 MAPK and JNK pathways in STZ-treated INS-1 cells. These results indicated that PF might be a natural anti-diabetic agent by improving pancreatic β-cells injury through inhibition of the p38 MAPK and JNK signaling pathways.

A Small Molecule BH3-mimetic Suppresses Cigarette Smoke-Induced Mucous Expression in Airway Epithelial Cells

Cigarette smoke (CS) exposure is one of the primary risk factors associated with the chronic mucous hypersecretion (CMH). The antiapoptotic protein, Bcl-2 sustains hyperplastic mucous cells, and the airway epithelium of ex-smokers with CMH as well as mice exposed to chronic CS showed increased Bcl-2 expression. Therefore, we investigated whether Bcl-2 plays a role in CS-induced mucous expression. Primary airway epithelial cells (AECs) of murine and human origin were treated with CS extract (CSE), and there was a concentration- and time-dependent increase in secretory mucin (MUC5AC), mucous regulator (SPDEF) and Bcl-2 expression. Using differentiated human AECs cultured on air-liquid interface, EGFR and ERK1/2 pathways were interrogated. Bcl-2 activity was blocked using a small molecule BH3 mimetic ABT-263 that disrupts the Bcl-2 interaction with pro-apoptotic proteins. The ABT-263 treatment resulted in the downregulation of CSE-induced mucus expression and disrupted the EGFR-signaling while inducing the apoptosis and the pro-apoptotic protein, Bik expression. This strategy significantly suppressed the mainstream CS-induced mucous phenotype in a 3-D human airway epithelium model. Therefore, the present study suggests that CS induces Bcl-2 expression to help promote mucous cell survival; and small molecule BH3 mimetics targeting Bcl-2 could be useful in suppressing the CS-induced mucous response.

Platycodin D inhibits oxidative stress and apoptosis in H9c2 cardiomyocytes following hypoxia/reoxygenation injury

Myocardial ischemia/reperfusion (I/R) injury is a complex pathophysiological process related to the occurrence of myocardial infarction (MI). Oxidative stress is known to play a crucial role in the pathogenesis of I/R injury. Platycodin D (PD) is an active natural saponin that possesses strong anti-oxidant activity. The aim of the present study was to investigate the effect of PD on myocardial I/R injury. An in vitro hypoxia/reoxygenation (H/R) model was established in cardiomyocyte H9c2 cells. The results showed that PD improved the cell viability in H/R-stimulated H9c2 cells. The H/R-induced increase in the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and decrease in the activities of superoxide dismutase (SOD) and catalase (CAT) were reversed by PD pretreatment. The histone-associated DNA fragment was increased by H/R stimulation, while decreased after PD treatment. Besides, PD pretreatment reduced the expressions of Bax and cleaved caspase-3, while induced Bcl-2 expression in H/R-induced H9c2 cells. Furthermore, PD was found to induce the activation of Akt/Nrf2/HO-1 pathway. The inhibitor of Akt, LY294002, attenuated the effects of PD on H/R-induced H9c2 cells. These findings indicated that PD exerted its protective effect via inducing the activation of Akt/Nrf2/HO-1 pathway. Our work provided new insights into the potential therapeutic role of PD in myocardial I/R injury.

Phytonutrient genistein is a survival factor for pancreatic β-cells via GPR30-mediated mechanism

We previously discovered that phytonutrient genistein rapidly activates cAMP signaling in β-cells and improves islet mass in diabetic mice. However, the mechanism underlying these actions of genistein is still unclear. Here, we show that pharmacological or molecular inhibition of Gαs blocked genistein-stimulated adenylate cyclase activity in plasma membrane and intracellular cAMP production in INS1 cells and islets. Further, genistein stimulation of cAMP generation was abolished in islets exposed to a specific GPR30 inhibitor G15 or islets from GPR30 deficient (GPR30−/−) mice. In vivo, dietary provision of genistein (0.5 g/kg diet) significantly mitigated streptozotocin-induced hyperglycemia in male WT mice, which was associated with improved blood insulin levels and pancreatic islet mass and survival, whereas these effects were absent in Gpr30−/− mice. Genistein treatment promoted survival of INS1 cells and human islets chronically exposed to palmitate and high glucose. At molecular level, genistein activated CREB phosphorylation and subsequently induced Bcl-2 expression, and knockdown of CREB diminished the protective effect of genistein on β-cells induced by lipoglucotoxicity. Finally, deletion of GPR30 in β-cells or islets ablated genistein-induced CREB phosphorylation and its cytoprotective effect. These findings demonstrate that genistein is a survival factor for β-cells via GPR30-initiated, Gαs-mediated activation of CREB.

MicroRNA-21 abrogates palmitate-induced cardiomyocyte apoptosis through caspase-3/NF-κB signal pathways.

Objective:The aim of the study was to investigate the role of microRNA-21 (miR-21) in cardiomyocyte apoptosis and to determine a possible mechanism.Methods:H9c2 embryonic rat heart-derived cells were used in the study. Cell viability was determined using the 3-(4.5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and flow cytometry was used to evaluate cell apoptosis. Reverse transcription-polymerase chain reaction and western blot assays were used to detect mRNA and protein expression of the apoptosis-related proteins and miR-21. ELISA was used to detect reactive oxygen species (ROS).Results:Palmitate exposure greatly reduced miR-21 expression in cardiomyocytes. Apoptosis increased when miR-21 was inhibited with or without palmitate exposure. Consistently, reduced apoptosis was observed when miR-21 was overexpressed in cardiomyocytes. Caspase-3 activity was reduced after palmitate exposure. Bcl-2 protein expression was increased in H9c2 cells when transfected with the miR-21 mimic. MiR-21 overexpression alone did not induce ROS or DNA fragmentation; however, in conjunction with palmitate exposure, miR-21 mimic reduced ROS and DNA fragmentation. Moreover, palmitate administration overcame the antioxidant effect of 3 mM N-acetylcysteine to significantly inhibit apoptosis, DNA fragmentation, and caspase-3 activity. The exposure to palmitate greatly reduced p65 and p-p38 expression in the nucleus. A p38 inhibitor had no effect on the expression of Bcl-2 and cleaved caspase-3 in H9c2 cells alone; however, when combined with exposure to palmitate the p38 inhibitor induced Bcl-2 expression and inhibited caspase-3 activity. The p38 inhibitor by itself did not induce apoptosis, ROS production, or DNA fragmentation in H9c2 cells, but when palmitate was included with the p38 inhibitor, apoptosis, ROS production, and DNA fragmentation were reduced.Conclusion:miR-21 protects cardiomyocytes from apoptosis that is induced by palmitate through the caspase-3/NF-κB signal pathways.

Tao-Hong-Si-Wu Decoction ameliorates steroid-induced avascular necrosis of the femoral head by regulating the HIF-1α pathway and cell apoptosis.

The aim of this study was to corroborate the hypothesis that Tao-Hong-Si-Wu Decoction (THSWD) affects steroid-induced avascular necrosis of the femoral head (SANFH) by regulating the hypoxia-inducible factor 1α (HIF-1α) pathway. Forty-eight New Zealand rabbits were randomly divided into a normal control group (NC group), a model group (SANFH group), a THSWD group, and a dimethyloxalylglycine group (DMOG group). Rabbits in the SANFH group were injected with both horse serum and methylprednisolone. Rabbits in the THSWD group were gavaged with THSWD in addition to receiving the same treatment as the SANFH group. Rabbits in the DMOG group were injected with extra DMOG in conjunction with the same treatment as the SANFH group. Rabbits in the NC group received the same amount of normal saline. Eight weeks after steroid treatment, the femoral heads of rabbits were removed to examine HIF-1α, vascular endothelial growth factor (VEGF), caspase-3, and bcl-2. Results indicated that THSWD significantly promoted the expression of HIF-1α and VEGF in the femoral head tissue of rabbits and markedly inhibit the apoptosis of osteocytes, chondrocytes, and bone marrow cells. In addition, THSWD suppressed caspase-3 expression and induced bcl-2 expression in femoral head tissues. In conclusion, THSWD can suppress SANFH by regulating the HIF-1α pathway and cell apoptosis.

Halofuginone promotes satellite cell activation and survival in muscular dystrophies

Halofuginone is a leading agent in preventing fibrosis and inflammation in various muscular dystrophies. We hypothesized that in addition to these actions, halofuginone directly promotes the cell-cycle events of satellite cells in the mdx and dysf−/− mouse models of early-onset Duchenne muscular dystrophy and late-onset dysferlinopathy, respectively. In both models, addition of halofuginone to freshly prepared single gastrocnemius myofibers derived from 6-week-old mice increased BrdU incorporation at as early as 18h of incubation, as well as phospho-histone H3 (PHH3) and MyoD protein expression in the attached satellite cells, while having no apparent effect on myofibers derived from wild-type mice. BrdU incorporation was abolished by an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated protein kinase, suggesting involvement of this pathway in mediating halofuginone's effects on cell-cycle events. In cultures of myofibers and myoblasts isolated from dysf−/− mice, halofuginone reduced Bax and induced Bcl2 expression levels and induced Akt phosphorylation in a time-dependent manner. Addition of an inhibitor of the phosphinositide-3-kinase/Akt pathway reversed the halofuginone-induced cell survival, suggesting this pathway's involvement in mediating halofuginone's effects on survival. Thus, in addition to its known role in inhibiting fibrosis and inflammation, halofuginone plays a direct role in satellite cell activity and survival in muscular dystrophies, regardless of the mutation. These actions are of the utmost importance for improving muscle pathology and function in muscular dystrophies.

Neuroprotective effect of Citrus unshiu immature peel and nobiletin inhibiting hydrogen peroxide-induced oxidative stress in HT22 murine hippocampal neuronal cells.

Background:Oxidative stress-induced cell damage is common in the etiology of several neurobiological disorders, including Alzheimer's disease and Parkinson's disease. In a case study, nobiletin-rich Citrus reticulata peels could prevent the progression of cognitive impairment in donepezil-preadministered Alzheimer's disease patients.Objective:In this study, we investigated the effects and underlying mechanism of nobiletin and Citrus unshiu immature peel (CUIP) water extract, which contains nobiletin as a major compound, on hydrogen peroxide-induced oxidative stress in HT22 cells, a murine hippocampal neuronal model.Materials and Methods:HT22 cells were treated with hydrogen peroxide in the presence or absence of various concentrations of CUIP and nobiletin. Cytotoxicity and apoptotic protein levels were measured by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Western blotting.Results:Pretreatment with CUIP and nobiletin inhibited cell death due to hydrogen peroxide. Hydrogen peroxide-induced the expression of phospho-Jun N-terminal kinases (p-JNK) and p-p38 proteins in HT22 cells; however CUIP and nobiletin suppressed p-JNK and p-p38 without changing JNK or p38. Regarding apoptosis, caspase 3, B-cell lymphoma 2 (Bcl-2), and Bax protein expression was determined. CUIP and nobiletin suppressed caspase 3 and Bax expression, but they induced Bcl-2 expression in HT22 cells.Conclusion:These results show that CUIP and nobiletin can protect against hydrogen peroxide-induced cell death in HT22 neurons via mitogen-activated protein kinases and apoptotic pathways.

Epidermal growth factor-induced prostate cancer (PC3) cell survival and proliferation is inhibited by quercetin, a plant flavonoid through apoptotic machinery

Epidermal growth factor (EGF) plays a key role in epithelial malignancies by enhancing cancer cell proliferation, survival, invasion, and metastasis. The aberrant expression of epidermal growth factor receptor (EGFR) by tumors typically confers a more aggressive phenotype and is often predictive of poor prognosis. Quercetin is an anti-oxidative flavonoid widely distributed in fruits and vegetables and have attracted much attention as potential anti-carcinogens. Prostate cancer is the most common cause of cancer related deaths in men. In the present study, we examined the effects of quercetin on EGF induced signaling molecules involved in proliferation, survival and apoptosis in PC-3 cells. EGF-stimulated EGFR, Akt, PI3K, PDK1 and ERK1/2 protein levels were inhibited by quercetin. The inhibitory effects of quercetin on EGF induced signaling were compared with PI3K inhibitor (LY294002) and MAPK inhibitor (PD98059). Quercetin down-regulated EGF induced Bcl-2 expression and upregulated Bax protein levels. Caspase-3 activity was significantly increased by quercetin treatment. Acridine orange and ethidium bromide staining showed that quercetin was able to induce apoptosis even in the presence of EGF. To conclude, the present study showed that quercetin inhibits EGF induced cell survival, proliferation and induced apoptosis in PC-3 cells.

The Transcription Factor GLI1 Interacts with SMAD Proteins to Modulate Transforming Growth Factor β-Induced Gene Expression in a p300/CREB-binding Protein-associated Factor (PCAF)-dependent Manner

The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFβ signaling in the regulation of gene expression in cancer cells. TGFβ stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFβ-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFβ pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFβ- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFβ targets. We found that two additional TGFβ-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells.

Stable over‐expression of the 2‐oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl‐2‐dependent mitochondrial GSH transport

Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron-like cell lines over-expressing the mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up-regulation of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl-2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co-immunoprecipitated with Bcl-2 from rat brain lysates in a GSH-dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl-2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels. Stable over-expression of the 2-oxoglutarate carrier (OGC) in a motor neuronal cell line induced a specific increase in mitochondrial GSH and markedly enhanced resistance to oxidative stress. Over-expression of OGC also induced Bcl-2 expression which was owing to the specific increase in mitochondrial GSH. Intriguingly, enhanced expression of Bcl-2 was required to sustain OGC-dependent GSH transport into the mitochondria. Thus, OGC and Bcl-2 work in a concerted manner to maintain the mitochondrial GSH pool which is crucial for neuronal survival.