Apoptotic-associated Proteins Research Articles

Inhibition of S100A9 alleviates neurogenic pulmonary edema after subarachnoid hemorrhage

Background and PurposeNeurogenic pulmonary edema (NPE) frequently arises as a complication subsequent to subarachnoid hemorrhage (SAH). Heterodimers of S100A8 and S100A9 are commonly formed, thereby initiating an inflammatory reaction through receptor binding on the cell surface. Paquinimod serves as a specific inhibitor of S100A9. The objective of this investigation is to assess the impact of Paquinimod administration and S100A9 knockout on NPE following SAH. MethodsIn this study, SAH models of C57BL/6J wild-type (WT) and S100A9 knockout mice were established through intravascular perforation. These models were then divided into several groups, including the WT-sham group, S100A9-KO-sham group, WT-SAH group, WT-SAH + Paquinimod group, and S100A9-KO-SAH group. After 24 h of SAH induction, pulmonary edema was assessed using the lung wet-dry weight method and Hematoxylin and eosin (HE) staining. Additionally, the expression levels of various proteins, such as interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), occludin, claudin-3, Bax, Bcl-2, TLR4, MYD88, and pNF-κB, in lung tissue were analyzed using western blot and immunofluorescence staining. Lung tissue apoptosis was detected by TUNEL staining. ResultsFirstly, our findings indicate that the knockout of S100A9 has a protective effect on early brain injury following subarachnoid hemorrhage (SAH). Additionally, the reduction of brain injury after SAH can also alleviate neurogenic pulmonary edema (NPE). Immunofluorescence staining and western blot analysis revealed that compared to SAH mice with wild-type S100A9 expression (WT-SAH), the lungs of S100A9 knockout SAH mice (S100A9-KO-SAH) and mice treated with Paquinimod exhibited decreased levels of inflammatory molecules (IL-1β and TNF-α) and increased levels of tight junction proteins. Furthermore, the knockout of S100A9 resulted in upregulated expression of the apoptotic-associated protein Bax and down-regulated expression of Bcl-2. Furthermore, a decrease in TLR4, MYD88, and phosphorylated pNF-κB was noted in S100A9-KO-SAH and Paquinimod treated mice, indicating the potential involvement of the TLR4/MYD88/NF-κB signaling pathway in the inhibition of the protective effect of S100A9 on NPE following SAH. ConclusionThe knockout of S100A9 not only ameliorated initial cerebral injury following subarachnoid hemorrhage (SAH), but also mitigated SAH-associated neurogenic pulmonary edema (NPE). Additionally, Paquinimod was found to diminish NPE. These findings imply a correlation between the central nervous system and peripheral organs, highlighting the potential of safeguarding the brain to mitigate harm to peripheral organs.

Umbelliferone Ameliorates Hepatic Steatosis and Lipid-Induced ER Stress in High-Fat Diet-Induced Obese Mice.

Among the characteristics of non-alcoholic fatty liver disease (NAFLD), hepatic steatosis is due to excessive fat accumulation and causes liver damage and lipotoxicity, which are associated with insulin resistance, endoplasmic reticulum (ER) stress, and apoptosis. Umbelliferone (UMB) has various powerful pharmacological properties, such as antioxidant, anti-hyperglycemic, anti-viral, and anti-inflammatory effects. However, the mechanism of action in hepatic steatosis and lipid-induced ER stress is still unclear. Thus, the efficacy of UMB in hepatic steatosis and palmitate (PA)-induced hepatocellular lipotoxicity was evaluated in the present study. Male C57BL/6J mice (n=40) were divided into four groups: regular diet (RD), UMB-supplemented RD, high-fat diet (HFD), and UMB-supplemented HFD. All mice were fed orally for 12 weeks. In addition, the effects of UMB on lipotoxicity were investigated in AML12 cells treated with PA (250 µM) for 24 h; Western blot analysis was used to evaluate the changes in ER stress and apoptotic-associated proteins. Administration with UMB in HFD-fed mice reduced lipid accumulation and hepatic triglyceride (TG) as well as serum insulin and glucose levels. In AML12 cells, UMB treatment reduced lipid accumulation as indicated by decreases in the levels of lipogenesis markers, such as SREBP1, FAS, PPAR-γ, and ADRP. Furthermore, UMB reduced both oxidative stress and ER stress-related cellular apoptosis. UMB supplementation ameliorated hepatic steatosis and improved insulin resistance by inhibiting lipid accumulation and regulating ER stress. These findings strongly suggest that UMB may be a potential therapeutic compound against NAFLD.

α-Mangostin protects lipopolysaccharide-stimulated nucleus pulposus cells against NLRP3 inflammasome-mediated apoptosis via the NF-κB pathway.

Intervertebral disc degeneration (IDD) is a common and chronic inflammatory disorder. α-Mangostin exhibits a novel biological function against inflammation in various inflammatory diseases. Here, we aimed to explore the role of α-mangostin in IDD using an in vitro cell model. Human nucleus pulposus cells (NPCs) were exposed to lipopolysaccharide (LPS) to induce inflammatory injury. Cell viability of NPCs was determined by CCK-8 assay. ELISA was performed to examine the production of interleukin (IL)-1β and IL-18. Apoptotic cell death in NPCs was detected by TUNEL staining. The expression levels of apoptotic-associated proteins were detected by western blotting. Nuclear factor-kappa B (NF-κB) activation was examined by determining the expression levels of p-p65, p65, and nuclear p65. Results showed that treatment with α-mangostin improved the viability of LPS-treated NPCs. α-Mangostin treatment also inhibited the LPS-induced increase in expression levels of NLRP3, ASC and pro-caspase-1, as well as the production of IL-1β and IL-18 in NPCs. Moreover, treatment with α-mangostin or NLRP3 inhibitor (MCC950) significantly decreased apoptotic cell death in NPCs, as compared with treatment with LPS. In addition, the expression levels of cleaved caspase-3 and Bax were decreased, while Bcl-2 expression was increased in α-mangostin- or MCC950-treated NPCs. Treatment with α-mangostin also suppressed LPS-induced increase of p-p65/p65 and nuclear p65 levels. Moreover, inhibition of NF-κB by PDTC aggravated the inhibitory effects of α-mangostin on NLRP3 inflammasome activation and apoptosis in LPS-induced NPCs. These findings suggested that α-mangostin exerted a protective effect on NLRP3 inflammasome-mediated apoptosis in LPS-induced NPCs through regulating NF-κB signaling.

POS-352 MiR-26a-5p PROTECTS PODOCYTE FROM APOPTOSIS VIA DOWNREGULATION OF TRPC6 IN TYPE 2 DIABETIC NEPHROPATHY

Podocyte injury is a major etiological factor in diabetic nephropathy (DN), while transient receptor potential cationic channel 6 protein (TRPC6) channels play a key role in podocyte injury. Our study is designed to investigate the regulatory effect of microRNA-26a-5p (miR-26a-5p) on DN podocyte injury induced by TRPC6 and the underlying mechanisms. 20 SPF, 8-week male Sprague-Dawley (SD) rats were used for establishing the type 2 diabetic mellitus (T2DM) group by feeding them with high fat and high sugar food for 8 weeks and intraperitoneal injection of low-dose streptozotocin (STZ, 30mg/kg). Another 10 normal male SD rats were selected as normal control group (NC). The 24h urinary albumin excretion rate (UAE), urinary creatinine (Ucr), creatinine clearance rate (CCr) and other major biochemical parameters including the fasting insulin level (FINS), serum creatinine (Scr), fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) were measured in both groups after establishing the DM models. Light and electron microscope were used to observe the pathology of kidneys and podocyte ultrastructures, respectively. Immunohistochemical method was used to show the distribution of nephrin and TRPC6 protein. To determine the expression of nephrin, TRPC6, apoptotic-associated proteins (cleaved-caspase-3 and bax/bcl-2), western blot was applied. qRT-PCR was used to detect miR-26a-5p and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2. Podocyte apoptotic rate was measured using TUNEL method. Cultured immortalized mouse podocytes (MPC5) were divided into three groups: normal glucose group (NG), hypertonic group (HM), high glucose group (HG). Before and after miR-26a-5p mimics and inhibitor transfecting, the expression and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2 were detected again using the same methods. Flow cytometry (FCM) was uesd to detect podocyte apoptosis. 1. Compared with NC group, rats in DM group exhibited disrupted biochemical conditions, for uAER, CCr, FINS, FBG, TC, TG and LDL were all significantly increased (all P <0.05) while HDL were decreased (P <0.05), along with significant insulin resistance. 2. Increased glomeruli volume, mesangial cell proliferation, thickened basement membrane and foot process fusion could be observed by light and electron microscope. 3. Immunochemistry results showed a increased expression of TRPC6 and a decreased of nephrin in DM rats than those in NC group (both P <0.05). 4. Both qRT-PCR and Western blot results in vivo and in vitro showed a notable increase in the expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 and decrease in the expression of nephrin and miR-26a-5p (P <0.05). TUNEL and flow cytometry indicated higher podocyte apoptosis in DM rats and HG group. 5. The expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 were down-regulated and nephrin expression was restored after miR-26a-5p mimics transfection (all P <0.05), while miR-26a-5p inhibitor intervention could aggravate all above changes as the effects of HG on podocytes (all P <0.05). High-glucose condition significantly decreased miR-26a-5p expression and increased TRPC6 expression and podocyte apoptosis. In DN, miR-26a-5p can protect podocyte from apoptosis via down-regulating the expression of TRPC6.

Probiotics counteract the expression of hepatic profibrotic genes via the attenuation of TGF-β/SMAD signaling and autophagy in hepatic stellate cells.

Hepatic fibrosis is caused by the increased accumulation and improper degradation of extracellular matrix (ECM) proteins in the liver. Hepatic stellate cells (HSCs) activation is a key process in initiating hepatic fibrosis and can be ameliorated by the administration of probiotic strains. This study hypothesized that LAB strains (Lactiplantibacillus plantarum, Lactobacillus brevis, and Weissella cibaria) might attenuate pro-fibrogenic cytokine TGF-β mediated HSCs activation and induce collagen deposition, expression of other fibrogenic/inflammatory markers, autophagy, and apoptotic processes in vitro. Few studies have evaluated the probiotic effects against fibrogenesis in vitro. In this study, TGF-β exposure increased collagen deposition in LX-2 cells, but this increase was diminished when the cells were pretreated with LAB strains before TGF-β stimulation. TGF-β not only increased collagen deposition, but it also significantly upregulated the mRNA levels of Col1A1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinases-2 (MMP-2), IL-6, CXCL-8, CCL2, and IL-1β in LX-2 cells. Pretreatment of the cells with LAB strains counteracted the TGF-β-induced pro-fibrogenic and inflammatory markers by modulating SMAD-dependent and SMAD-independent TGF-β signaling. In addition, LX-2 cells exposed to TGF-β induced the autophagic and apoptotic associated proteins that were also positively regulated by the LAB strains. These findings suggest that LAB can attenuate TGF-β signaling that is associated with liver fibrogenesis.

Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy.

PurposeTo investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.MethodsCybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.ResultsNineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.ConclusionsOur findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

Resveratrol-induced apoptosis is associated with regulating the miR-492/CD147 pathway in malignant melanoma cells

Resveratrol (RES) as a natural phytoalexin has anti-tumor effects on various cancers through its pro-apoptotic activities. Our aim was to determine that RES induces apoptosis in melanoma cells by regulating miR-492 resulting in decreased CD147 expression. We treated A375 and SK-MEL-28 melanoma cells via RES at different concentrations and time-points. The results have shown that the inhibition rate of A375 and SK-MEL-28 was significantly increased after RES treatment. Subsequently, we investigated cell apoptosis by flow cytometry, as well as detected apoptotic-associated proteins including PARP, Caspase-3, Bcl-2, and Bax by western blotting. Meanwhile, the expression of miR-492 and CD147 was analyzed. We found that RES remarkably induces apoptosis in melanoma cells, along with an upregulation of miR-492 and the inhibition of CD147 expression. Furthermore, the detection of luciferase reporter activity confirmed that miR-492 could target CD147 mRNA, and transfected with mimic miR-492 in cells reduced CD147 expression. We also performed the rescued experiment by using a miR-492 inhibitor in melanoma cells. The results showed that the ability of induced apoptosis by RES in melanoma cells was to be attenuated via inhibiting miR-492 expression resulting in CD147 augment. Finally, we determined that the effect of RES-induced apoptosis in melanoma cells is associated with, at least in part, its ability to regulate the miR-492/CD147 pathway.

Pharmacological evaluation and mechanistic study of compound Xishu Granule in hepatocellular carcinoma

Abstract Objective In this study, we used HepG2 human hepatocellular carcinoma cells to study the effects of Compound Xishu Granule (CXG) on cell proliferation, apoptosis, and the cell cycle in vitro. We also used a xenograft tumor model to study the anti-tumor effects of CXG and related mechanisms in vivo. Methods The effect of CXG on cell viability was measured using Cell Counting Kit-8 and a colony formation assay. The effect of CXG on apoptosis and the cell cycle was analyzed using flow cytometry. The in vivo anti-tumor effect of CXG was assessed by measuring the volume change in xenograft tumors after drug administration. The CXG anti-tumor mechanism was studied using western blotting assay to detect cell cycle and apoptotic associated proteins. Results CXG suppressed HepG2 cell proliferation in a time- and dose-dependent manner in vitro. Colony formation experiments showed that CXG administration for 24 h significantly reduced HepG2 cell formations (P Conclusion CXG has good anti-tumor effects in vitro and in vivo. In vitro, CXG promoted HepG2 cell apoptosis and induced G2/M phase arrest. In vivo, CXG significantly inhibited graft tumor growth. The CXG mechanism in treating hepatocellular carcinoma may be that CXG can induce abnormal apoptotic and cell cycle associated protein expression, leading to mitotic catastrophe and apoptosis.

Sulforaphane protects against skeletal muscle dysfunction in spontaneous type 2 diabetic db/db mice

AimsSkeletal muscle diseases have become to be the most common complication in patients with type 2 diabetic mellitus (T2DM). However, the effective therapies against skeletal muscle diseases are not yet available. Sulforaphane (SFN) is an organic isothiocyanate found in cruciferous plants. Our aim was to explore whether SFN could attenuate the skeletal muscle diseases in spontaneous type 2 diabetic db/db mice. Materials and methodsThe db/m and littermate db/db mice were treated with SFN or dimethyl sulfoxide. The grip strength of mice was measured by a grasping forcing machine. The electron transmission microscopy was used to perform the skeletal muscle. The western blot was used to detect the nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signal pathway related proteins, and inflammatory and apoptotic associated proteins. The mRNA levels of anti-inflammatory and anti-oxidative relative genes were detected by RT-QPCR. Key findingsWe found that SFN could significantly increase the grip strength of the db/db mice. The lean mass and gastrocnemius mass were increased in the db/db mice after administration with SFN. Additionally, the db/db mice restored the skeletal muscle fiber organization after SFN treatment. Mechanistically, SFN could activate the Nrf2/HO-1 signal pathway, and downregulate the expression of inflammatory and apoptotic associated proteins. Furthermore, SFN could also regulate the mRNA levels of anti-inflammatory and anti-oxidative related genes. SignificanceOur results demonstrated that SFN can protect against skeletal muscle diseases in db/db type 2 diabetic mice and provide a potential drug to prevent skeletal muscle dysfunction in T2DM patients.

Sevoflurane attenuates brain damage through inhibiting autophagy and apoptosis in cerebral ischemia‑reperfusion rats.

The present study aimed to investigate the effects of sevoflurane post-conditioning in a rat brain cerebral ischemia-reperfusion (I/R) model and examine its possible mechanism. Rats were randomly divided into six groups: Sham control group (Sham), I/R group, sevoflurane group (Se), Toll-like receptor-4 (TLR4) inhibitor group (Tak-242), nuclear factor (NF)-κB inhibitor group (QNZ) and Sevoflurane post-conditioning combined with TLR4-NF-κB signaling pathway inhibitor group (Se + Tak-242). Morris water maze test and tetrazolium chloride staining were used to investigate the I/R injury. The nerve cell apoptosis and autophagy in cortical tissue were detected by TUNEL and transmission electron microscopy, respectively. The expression of TLR4 protein in cortical tissue was observed by immunohistochemical staining. The expression of autophagy and apoptotic associated proteins in cortical tissues and the activity of TLR4-NF-κB signaling pathway were assayed by western blot analysis. Sevoflurane post-conditioning improved the learning and memory dysfunction caused by cerebral I/R injury. The cerebral infarction area, nerve cell apoptosis and formation of autophagic vacuoles were reduced after sevoflurane administration. The expression of light chain 3II/I, Beclin-1, Bad and Cleaved-Caspase-3 proteins were inhibited and the expression of Bcl-2 protein was upregulated after sevoflurane administration. Sevoflurane post-conditioning also inhibited the TLR4 protein and NF-κB phosphorylation, and increased inhibitor of kBα phosphorylation. The treatment effect of Tak-242 and QNZ groups were not significantly different compared with the Se group (P>0.05), and the Se + Tak-242 group had the best results. The present study demonstrated that sevoflurane post-conditioning could protect middle cerebral artery occlusion-induced brain injury rats by inhibiting autophagy and apoptosis, and that its mechanism is related to the TLR4-NF-κB signaling pathway.

424P - Preclinical study of novel tetracyclic small molecule, CC12, for brain cancer

BackgroundThe development of a novel therapy for malignant glioma is a vigorous area in both chemistry and cancer research. Previously, we reported that the small molecule naphtho [2, 3-f] quinoxaline-7, 12-dione (CC12) effectively inhibits the proliferation of several cancers. In this study, we further investigated the therapeutic effects of CC12 on human glioblastoma (GBM) cells and clarified underlying mechanisms of these effects. MethodsHuman GBM cell lines, U118MG and U87MG, were used for in vitro and in vivo. Analysis of CC12 effects on cell cycle and apoptosis, while nude mice bearing xenograft of the tumor cells were used for in-vivo analysis of CC12 effects on tumor metabolism and size by using animal positron emission tomography (PET). DNA fragmentation, mitochondrial membrane potential change, cell cycle inhibiion and apoptosis were labelled with fluorescent tracers or antibodies, and followed by measured with flow cytometry. Apoptotic-associated proteins were quantified by immunoblot analysis. Tumors in animals were labelled with [18F]-fluorodeoxyglucose ([18F]-FDG), and imaged by animal-PET. Tumor tissue was collected and weighed, and vital organs including the heart, kidneys and liver were extracted for hematoxylin and eosin staining. ResultsWe found that CC12 induced cell cycle arrest as GBM cells were accumulated in the subG1 and G2/M phases in a dose- and time-dependent manner. This effect was caused by DNA damage response in GBM cells. Moreover, the treatment of CC12 reduced the expression of decoy receptor 3 and disrupted the mitochondrial membrane signaling cascade in GBM cells, leading to apoptosis of the cells. In the heterotopic mice model, we found that the size of the GBM cell xenografts was decreased following the treatment of CC12, indicated by [18F]-FDG coupled with PET. ConclusionsThese findings provide evidence from molecular, cellular, and physiological levels that strongly suggest that CC12 is a promising small-molecule agent for human GBM. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways.

Ouabain, a cardiotonic steroid and specific Na+ /K+ -ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca2+ , and mitochondrial membrane potential (ΔΨm ) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca2+ production, but decreased the levels of ΔΨm in DU 145 cells. Ouabain also increased the activities of caspase-3, -8, and -9. Western blotting was used for measuring the alterations of apoptosis-associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATMSer1981 , p-H2A.XSer139 , and p-p53Ser15 ) and ER-stress-associated proteins (Grp78, ATF6β, p-PERKThr981 , PERK, eIF2A, GADD153, CaMKIIβ, and caspase-4) in time-dependently. Furthermore, ouabain increased apoptosis-associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro-apoptotic protein Bax, increased apoptotic-associated proteins, such as cytochrome c, AIF, Endo G, caspase-3, -8, and -9, but reduced anti-apoptotic protein Bcl-2 and Bcl-x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase-dependent and mitochondria-dependent pathways in human prostate cancer DU 145 cells.

The role of chamaejasmine in cellular apoptosis and autophagy in MG-63 cells.

Background: Osteosarcoma (OS) is the most common malignant neoplasm in children and adolescents with a very high propensity for local invasion and poor response to current therapy. Anti-cancer effect of chamaejasmine is newly discovered from Stellera chamaejasmine L. Our study focuses on investigating the effect of chamaejasmine on the cellular apoptosis, proliferation, autophagy, and the underlying mechanisms in MG-63.Methods: Our study investigated the concentration of chamaejasmine in MG-63 cells by MTT and verified that chamaejasmine inhibited cell invasion by transwell. We also used Hoechst staining as well as apoptotic associated-proteins in MG-63 cells. Meanwhile, we also detected the lysophagesome and autophagsome by Lysotracker. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) knockdown was performed with siRNA.Results: Our results show that chamaejasmine exerts cellular growth inhibition, pro-apoptotic and pro-autophagic effect via activating AMPK in MG-63 cells. Furthermore, chamaejasmine significantly increases autophagic cell via the inhibition of mammalian target of rapamycin (mTOR) and activation of AMPK signaling pathways. Administrated with chamaejasmine also induces reactive oxygen species (ROS) generation, indicating cross-talking between these two primary modes of programmed cell death.Conclusion: Our results show that chamaejasmine promotes apoptosis and autophagy by activating AMPK/mTOR signaling pathways with involvement of ROS in MG-63 cells. Chamaejasmine is a promising anti-cancer agent in OS treatment, and further studies are needed to confirm its efficacy and safety in vivo or other cancer cells.

Bufalin Induces Apoptotic Cell Death in Human Nasopharyngeal Carcinoma Cells through Mitochondrial ROS and TRAIL Pathways.

The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca production and decreased the levels of . Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1 , IRE-1 , caspase-4, ATF-6 , Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.

Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway.

PurposeTo reveal the function of miR-134 in myocardial ischemia.MethodsReal-time PCR and western blotting were performed to measure the expression of miR-134, nitric oxide synthase 3 (NOS3) and apoptotic-associated proteins. Lactic dehydrogenase (LDH) assay, cell counting kit-8 (CCK-8), Hoechst 33342/PI double staining and flow cytometry assay were implemented in H9c2 cells, respectively. MiR-134 mimic/inhibitor was used to regulate miR-134 expression. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Rescue experiments exhibited the role of NOS3. The involvement of PI3K/AKT was assessed by western blot analysis.ResultsMiR-134 was high regulated in the myocardial ischemia model, and miR-134 mimic/inhibitor transfection accelerated/impaired the speed of cell apoptosis and attenuated/exerted the cell proliferative prosperity induced by H/R regulating active status of PI3K/AKT signaling. LDH activity was also changed due to the different treatments. Moreover, miR-134 could target NOS3 directly and simultaneously attenuated the expression of NOS3. Co-transfection miR-134 inhibitor and pcDNA3.1-NOS3 highlighted the inhibitory effects of miR-134 on myocardial H/R injury.ConclusionThis present work puts insights into the crucial effects of the miR-134/NOS3 axis in myocardial H/R injury, delivering a potential therapeutic technology in future.

MicroRNA-21 attenuates oxygen and glucose deprivation induced apoptotic death in human neural stem cells with inhibition of JNK and p38 MAPK signaling

Neural stem cells (NSCs) persist in the mammalian brain throughout life and protect against hypoxia-ischemia injury. NSCs are being increasingly recognized as a novel therapeutic target for various neurological disorders. Previous research indicates that miR-21 attenuates hypoxia-ischemia induced apoptotic death in various cell types. However, whether miR-21 plays a role in this protective effect mediated by NSCs is unknown, particularly in human NSCs (hNSCs). The present study investigated whether miR-21 could prevent hNSC injury induced by oxygen and glucose deprivation (OGD). Upon challenge with OGD treatment, loss of cell viability was observed in cultured hNSCs, as shown by CCK-8 assay. Moreover, quantitative real-time PCR (qRT-PCR) analysis indicated that expression of miR-21 increased in a time-dependent manner. TUNEL staining and Western blotting analysis showed that overexpression of miR-21 inhibited excessive hNSCs death induced by OGD treatment. Accordingly, knock down of miR-21 attenuated the neuroprotective effect observed in response to OGD treatment. Furthermore, JNK and p38 MAPKs inhibition was observed after overexpression of miR-21, and knock down of miR-21 had the opposite effect. We suggest that miR-21 prevents OGD-induced hNSCs death and apoptotic-associated protein activities through inhibiting JNK and p38 pathways in cultured hNSCs. Our findings may help to develop strategies for enhancing resident and transplanted NSCs survival after hypoxia-ischemic brain damage.

Hesperetin inhibits PM(2.5)-induced apoptosis in H9c2 cells by attenuating oxidative stress and mitochondrial damage

Objective: To investigate the effects of hesperetin on fine particulate matter (PM(2.5)) induced apoptosis in H9c2 cells and related mechanisms. Methods: H9c2 cells were divided into 4 groups: control group (cells were cultured without intervention), PM(2.5) group (cells were treated with 800 µg/ml PM(2.5)), hesperetin group (H group, cells were treated by 40 µmol/L hesperetin for 1 h at 37 ℃), and hesperetin+PM(2.5) group (H+PM(2.5) group, cells were pretreated with hesperetin before PM(2.5) treatment). Cells were cultured for corresponding interval. Apoptotic cells were detected by Annexin Ⅴ-FITC/PI apoptosis detection kit and Hoechst staining. The intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA Fluorescence Probe and mitochondrial membrane potential (MMP) was detected with JC-1 staining, respectively in these groups. Apoptotic related protein and phosphorylated MAPK expression levels were determined by Western blot. Results: (1) Flow cytometry results showed that the apoptosis rate of PM(2.5) group ((48.94±3.20)%) was significantly higher than that of control group ((8.13±1.40)%, P<0.01), which was significantly reduced in H+PM(2.5) group ((34.80±2.21)%) (P=0.003 2 vs. PM(2.5) group, P<0.01 vs. control group). The number of Hoechst 33258 positive apoptotic cells was distinctly less in H+PM(2.5) group than in PM(2.5) group. (2) The ROS levels was significantly higher in PM(2.5) group ((49.69±5.05)%) than in control group (10.57±1.33)%, P<0.01), which was significantly reduced in H+PM(2.5) group ((35.08±3.90)%) (P=0.000 2 vs. PM(2.5) group, P<0.01 vs. control group). (3) Green fluorescence indicating the JC-1 monomer form, which represented MMP loss of H9c2 cells, was significantly higher in PM(2.5) group ((20.28±4.69)%) than in control group ((10.50±2.72)%, P<0.01), which was significantly decreased in H+PM(2.5) group ((13.41±2.89)%) (P<0.01 vs. PM(2.5) group, P=0.029 4 vs. control group). (4) The expression levels of Bcl-2 protein of H9c2 cells was lower in PM(2.5) group ((76.94±4.52)%) than in control group (100%, P=0.000 9), which was significantly upregulated in H+PM(2.5) group ((92.95±6.82)%) (P=0.027 5 vs. PM(2.5) group, P=0.15 vs. control group). The expression levels of cleaved caspase-3 protein of H9c2 cells was significantly higher in PM(2).5 group ((243.98±17.94)%) than in control group (100%, P=0.000 2), which was significantly downregulated in H+PM(2.5) group ((200.45±4.31)%) (P=0.015 vs. PM(2.5) group, P<0.01 vs. control group). (5) The expression levels of phosphorylated p38 MAPK protein of H9c2 cells was higher in PM(2.5) group ((118.90±4.78)%) than in control group(100%, P=0.002 7), which could be significantly downregulated in H+PM(2.5) group ((103.30±1.27)%) (P=0.01 vs. PM(2.5) group, P=0.05 vs. control group). The expression levels of phosphorylated ERK protein of H9c2 cells was higher in PM(2.5) group ((163.50±4.98)%) than in control group (100%, P<0.01), which was significantly downregulated in H+PM(2.5) group ((139.10±2.72)%) (P=0.001 6 vs. PM(2.5) group, P<0.01 vs. control group). Conclusions: Hesperetin protects H9c2 cells from PM(2.5) stimulation through reducing oxidative stress and protecting mitochondrial function, regulating the expression of apoptotic associated proteins as well as MAPK signal pathway, thus inhibiting H9c2 cells apoptosis.

Inhibition of human prostate cancer (PC-3) cells and targeting of PC-3-derived prostate cancer stem cells with koenimbin, a natural dietary compound from Murraya koenigii (L) Spreng.

BackgroundInhibition of prostate cancer stem cells (PCSCs) is an efficient curative maintenance protocol for the prevention of prostate cancer. The objectives of this study were to assess the efficiency of koenimbin, a major biologically active component of Murraya koenigii (L) Spreng, in the suppression of PC-3 cells and to target PC-3-derived cancer stem cells (CSCs) through apoptotic and CSC signaling pathways in vitro.Materials and methodsThe antiproliferative activity of koenimbin was examined using MTT, and the apoptotic detection was carried out by acridine orange/propidium iodide (AO/PI) double-staining and multiparametric high-content screening (HCS) assays. Caspase bioluminescence assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblotting were conducted to confirm the expression of apoptotic-associated proteins. Cell cycle analysis was investigated using flow cytometry. Involvement of nuclear factor-kappa B (NF-κB) was analyzed using HCS assay. Aldefluor™ and prostasphere formation examinations were used to evaluate the impact of koenimbin on PC-3 CSCs in vitro.ResultsKoenimbin remarkably inhibited cell proliferation in a dose-dependent manner. Koenimbin induced nuclear condensation, formation of apoptotic bodies, and G0/G1 phase arrest of PC-3 cells. Koenimbin triggered the activation of caspase-3/7 and caspase-9 and the release of cytochrome c, decreased anti-apoptotic Bcl-2 and HSP70 proteins, increased pro-apoptotic Bax proteins, and inhibited NF-κB translocation from the cytoplasm to the nucleus, leading to the activation of the intrinsic apoptotic pathway. Koenimbin significantly (P<0.05) reduced the aldehyde dehydrogenase-positive cell population of PC-3 CSCs and the size and number of PC-3 CSCs in primary, secondary, and tertiary prostaspheres in vitro.ConclusionKoenimbin has chemotherapeutic potential that may be employed for future treatment through decreasing the recurrence of cancer, resulting in the improvement of cancer management strategies and patient survival.

The role of aldehyde dehydrogenase 1A1 in B-cell non-Hodgkin's lymphoma.

Previously we showed that aldehyde dehydrogenase1A1 (ALDH1A1) is a new mediator for resistance of DLBCL to CHOP and a facility predictor of clinical prognosis. In the present study, knockdown and inhibitor of ALDH1A1 were applied to identify the role of ALDH1A1 in Raji cells. CCK-8 and clone formation assay were applied to determine the CHOP sensitivity and clone formation ability. Caspase colorimetric assay and AnnexinV/FITC staining was performed to determine the degree of apoptosis. Western blot analysis was used to detect the NF-κB/STAT3 signaling proteins and apoptotic-associated proteins. Real-time quantitative PCR (RT-PCR) was used to identify the differential expression of ALDH1A1 between NHL patients and healthy donors. We demonstrated that inhibition of ALDH1A1 increased the sensitivity of Raji cells to CHOP, as indicated by increased cytotoxicity, reduced clonogenicity, activated caspase-3/-9, decreased NF-κB/STAT3 signaling and increased pro-apoptosis signaling, ad increased apoptosis rate. Moreover, we found high ALDH1A1 expression was associated with poor prognosis in NHL patients. Our data revealed the critical role of ALDH1A1 in NHL and provides a theoretical basis for the use of ALDH1A1 inhibitors in NHL patients.

MiR-133b acts as a tumor suppressor and negatively regulates EMP2 in glioma.

In recent years, the incidence of neuroglioma (glioma) has trended towards a younger age-group. Gene therapy has been widely implemented and a growing number of microRNAs associated with glioma have been identified., Herein, we detected the expression of micro RNA - miR-133b - in glioma by qPCR and also its effect on cell viability, survival and apoptosis of in vitro U87 and A172 cells. The binding effect of miR-133b on epithelial membrane protein-2 (EMP2) was verified and we then investigated the effect of EMP2 on in vitro glioma cells and tested the expression of apoptosis related factors after administration of altered miR-133b and EMP2 expressions. We found that miR-133b was down-regulated in glioma compared to adjacent non-tumorous tissue and also that its over-expression inhibits cell viability and survival and enhances apoptosis in the U87 and A-172 cells. Moreover, miR-133b effectively binds to EMP2, down-regulates its expression and negates its normal function. EMP2 normally promotes cell apoptosis and reduces cell viability and survival while miR-133b over-expression regulates the expression of apoptotic-associated protein and activates the apoptotic pathway, thus counteracting EMP2 regulation of opposite expression effects. Further, miR-133b can be considered a tumor suppressor because of its low expression and effects on cell apoptosis via down-regulating EMP2 expression and activating the apoptotic cell pathway in glioma. EMP2 is a risk factor for glioma, and miR-133b should prove a potential target for glioma clinical prevention and treatment.