Bcl-2 Signaling Pathways Research Articles

ENT1 inhibition attenuates apoptosis by activation of cAMP/pCREB/Bcl2 pathway after MCAO in rats

Background and purposeThe present study was designed to investigate the potential role and the mechanism of equilibrative nucleoside transporter 1 (ENT1) on neuronal apoptosis and neurological deficits after middle cerebral artery occlusion (MCAO) in rats. MethodsOne hundred and thirty-four male Sprague-Dawley rats were subjected to two hours of MCAO followed by reperfusion. The time course of the expression level of ENT1 and phosphorylation of CREB were detected by western blot and immunofluorescence staining. Another set of animals were administrated with NBTI, the ENT1 inhibitor, by daily intraperitoneal injection starting at 0.5 h post-MCAO, infarction volume and neurological deficits were measured both at 24 h and 72 h post MCAO. We further explored the neuroprotection machenism by using H89, cAMP dependent protein kinase inhibitor, the expression of Bcl-2, Bax, phosphorylated CREB and Cleaved caspase-3 were quantified by Western blot, neuronal apoptosis were analyed by TUNEL staining. ResultsThe endogenous expression of ENT1 were significantly increased and peaked at 12 h after MCAO. High-dose of NBTI (15 mg/kg) reduced brain infarction volume and improved neurologic deficits both at 24 h and 72 h post MCAO. Moreover, NBTI significantly increased the level of CREB phosphorylation and extracellular adenosine concentration, and decreased the neuronal apoptosis 24 h after MCAO. NBTI treatment reduced the expression of Bax and cleaved caspase-3, while up-regulated Bcl-2 compared with vehicle group. These effects were abolished by H89 pretreatment. ConclusionsENT1 inhibition prevented neuronal apoptosis and improves neurological deficits through cAMP/PKA/CREB/Bcl-2 signaling pathway after MCAO in rats. ENT1 might be an effective target in the treatment strategy for ischemic stroke.

Delayed recanalization after MCAO ameliorates ischemic stroke by inhibiting apoptosis via HGF/c-Met/STAT3/Bcl-2 pathway in rats

The activation of tyrosine kinase receptor c-Met by hepatocyte growth factor (HGF) showed an anti-apoptotic effect in numerous disease models. This study aimed to investigate the neuroprotective mechanism of the HGF/c-Met axis-mediated anti-apoptosis underlying the delayed recanalization in a rat model of middle cerebral artery occlusion (MCAO). Permanent MCAO model (pMCAO) was induced by intravascular filament insertion. Recanalization was induced by withdrawing the filament at 3 days after MCAO (rMCAO). HGF levels in the blood serum and brain tissue expressions of HGF, c-Met, phosphorylated-STAT3 (p-STAT3), STAT3, Bcl-2, Bax, cleaved caspase-3(CC3) were assessed using ELISA and western blot, respectively. To study the mechanism, HGF small interfering ribonucleic acid (siRNA) and c-Met inhibitor, su11274, were administered intracerebroventricularly (i.c.v.) or intranasally, respectively. The concentration of HGF in the serum was increased significantly after MCAO. Brain expression of HGF was increased after MCAO and peaked at 3 days after recanalization. HGF and c-Met were both co-localized with neurons. Compared to rats received permanent MCAO, delayed recanalization after MCAO decreased the infarction volume, inhibited neuronal apoptosis, and improved neurobehavioral function, increased expressions of p-STAT3 and its downstream Bcl-2. Mechanistic studies indicated that HGF siRNA and su11274 reversed the neuroprotection including anti-apoptotic effects provided by delayed recanalization. In conclusion, the delayed recanalization after MCAO increased the expression of HGF in the brain, and reduced the infarction and neuronal apoptosis after MCAO, partly via the activation of the HGF/c-Met/STAT3/Bcl-2 signaling pathway. The delayed recanalization may serve as a therapeutic alternative for a subset of ischemic stroke patients.

Impact of Age-Related Genetic Differences on the Therapeutic Outcome of Papillary Thyroid Cancer.

The incidence of papillary thyroid carcinoma (PTC) has been increasing worldwide. PTC is the most common type of differentiated thyroid cancer and usually shows good prognosis. However, some PTC is driven to advanced stage by epithelial-mesenchymal transition (EMT)-mediated drug resistance, which is particularly noticeable in pediatric patients. There are limited options for systemic treatment, necessitating development of new clinical approaches. Here, we aimed to clarify genetic differences due to age of patients with PTC, and thereby aid in developing novel therapeutics. Patients with biochemically and histologically confirmed PTC were included in this study. PTC cells were acquired from young and older patients showing drug resistance, and were compared via microarray analysis. Cellular proliferation and other properties were determined after treatments with lenvatinib and sorafenib. In vivo, tumor volume and other properties were examined using a mouse xenograft model. Lenvatinib-treated group showed obvious suppression of markers of anti-apoptosis, EMT, and the FGFR signaling pathway, compared with control and Sorafenib-treated group. In the xenograft models, lenvatinib treatment induced significant tumor shrinkage and blocked the proto-oncogene Bcl-2 (B cell lymphoma/leukemia gene-2) and FGFR signaling pathway, along with reduced levels of EMT markers, compared with control and Sorafenib-treated group. Our findings clarify the age-dependent characteristics of pediatric PTC, giving insights into the relationship between young age and poor prognosis. Furthermore, it provides a basis for developing novel therapeutics tailored to the age at diagnosis.

Purified Tetrastigma hemsleyanum vines polysaccharide attenuates EC-induced toxicity in Caco-2 cells and Caenorhabditis elegans via DAF-16/FOXO pathway

Ethyl Carbamate (EC), as a carcinogen widely found in fermented foods, was verified that its cytotoxicity was associated with oxidative stress. Polysaccharides from natural sources due to their antioxidative capacity have attracted great attention in the past time. In this study, purified polysaccharide from Tetrastigma hemsleyanum vines (TVP) with 64.89 kDA was extracted and conducted multiple analysis to identify its structural information. It could be discovered that TVP was composed of mannose, rhamnose, glucuronic acid, glucose, galactose, and arabinose. In vitro, TVP could inhibit cytotoxicity and genotoxicity, attenuate oxidative damage and mitochondrial dysfunction induced by EC in Caco-2 cells. Meanwhile, TVP could suppress apoptosis by mTOR and Bcl-2 signaling pathways, ameliorate oxidative via Sirt1-FoxO1 and Nrf2-Keap1 signaling pathways. In vivo, EC as well triggered the decline of survival and athletic ability in Caenorhabditis elegans (C. elegans) and TVP could reverse the decline. In the meantime, TVP could ameliorate oxidative damage in N2 and daf-2 (-) mutant but fail in daf-16 (-) mutant, which suggested that DAF-16 (FOXO) might affect the antioxidative protection of TVP in C. elegans. In brief, our results manifested that TVP could attenuate EC-induced cytotoxicity both in vitro and in vivo.

Novel Antrodia cinnamomea Extract Reduced Cancer Stem-Like Phenotype Changes and Resensitized KRAS-Mutant Colorectal Cancer via a MicroRNA-27a Pathway.

Colorectal cancer (CRC) is one of the most common causes of death in Taiwan. Previous studies showed that Antrodia cinnamomea (AC) can treat poisoning, diarrhea, and various types of cancer. Therefore, we purified a novel ubiquinone derivative, AC009, and investigated its antitumor effects. Cell viability assays revealed that AC009 reduced the viability of several human CRC cell lines. AC009 treatment resulted in cell-cycle arrest/apoptosis, and these effects may occur via caspase and Bcl-2 signaling pathways. We demonstrated that AC009 could significantly inhibit in vivo tumor growth in xenograft mouse models. Using messenger RNA (mRNA) and microRNA (miRNA) microarrays, we found that KRAS gene expression was also regulated by AC009, possibly through specific miRNAs. AC009 also reduced cancer stem-cell marker CD44+/CD24+ expression and restored the tumor inhibition effect of cetuximab in KRAS-mutant CRC. Moreover, we found that miRNA-27a could restore the tumor inhibition effect of cetuximab in KRAS-mutant CRC cells. Taken together, our results suggest that AC009 has therapeutic potential against human wild-type and KRAS-mutant CRC.

Doxorubicin-loaded functionalized selenium nanoparticles for enhanced antitumor efficacy in cervical carcinoma therapy.

Development of novel tumor-targeted drug vehicles for cancer therapy is very important and has become one of major topics for designing nanoscale chemotherapeutics delivery systems. In the present study, selenium nanoparticles (SeNPs) was decorated with hyaluronic acid (HA) to prepare HA-SeNPs nanoparticles which were used to load doxorubicin (DOX) to fabricate tumor-targeted functionalized selenium nanoparticles HA-Se@DOX. In vitro and in vivo antitumor activities of HA-Se@DOX in human cervical carcinoma treatment were investigated. HA-Se@DOX showed selective cellular uptakes between cervical cancer HeLa cells and human umbilical vein endothelial cells (HUVEC). In vitro release result indicated that DOX was released from HA-SeNPs faster in acidic environment in comparison with normal physiological environment and 76.9% DOX was released in pH 5.4 during initial 30 h. HA-Se@DOX showed high activity to inhibit HeLa cell proliferation and triggered HeLa cell apoptosis via activating Bcl-2 signaling pathway. In vivo antitumor study showed that HA-Se@DOX inhibited tumor growth through suppressing cancer cells proliferation and inducing cancer cells apoptosis. Interestingly, HA-Se@DOX exhibited stronger anticancer activity than free DOX and Se@DOX in vitro and in vivo. Additionally, HA-Se@DOX did not cause damage to major organs at the used dose. HA-Se@DOX is a promising antitumor agent for human cervical carcinoma treatment and this research provides a novel therapeutic strategy for cancer therapy.

Effects of combined 5-Fluorouracil and ZnO NPs on human breast cancer MCF-7 Cells: P53 gene expression, Bcl-2 signaling pathway, and invasion activity

Objective(s): The significant contribution of nanoparticles to cancer treatment has attracted therapeutic attention. The present study aimed to evaluate the synergistic effects of 5-fluorouracil (5-FU) and zinc oxide nanoparticles (ZnO NPs) as multimodal drug delivery on human breast cancer MCF-7 cells.Materials and Methods: In this in-vitro study, the impact of 5-FU and ZnO NPs in the single or combined forms was evaluated on cell viability, colony formation, apoptosis, p53 gene expression, and Bcl-2 signaling protein in MCF-7 breast cancer cell line using several techniques, such as MTT, clonogenic assay, flow cytometry, real-time quantitative polymerase chain reaction, and Western blot.Results: In this study, 5-FU combined with ZnO NPs showed synergistic effects against MCF-7 within 48 hours. In addition, the combination of 5-FU and ZnO NPs at the respective concentrations of 1 µM and 45 µg/ml exhibited significant apoptosis (79.53), p53 gene expression (3.6 folds), reduction of cell invasion (9.82), and plating efficiency (5), thereby leading to the significant reduction of cell viability (40±0.9) and decreased Bcl-2 anti-apoptotic protein relative to untreated control cells. Conclusion: According to the results, the synergistic effects of combined ZnO NPs and 5-FU on MCF-7 human breast cancer cells were exerted via Bcl-2 inhibition and the up-regulation of p53 expression.

Combination of metformin and luteolin synergistically protects carbon tetrachloride-induced hepatotoxicity: Mechanism involves antioxidant, anti-inflammatory, antiapoptotic, and Nrf2/HO-1 signaling pathway.

Liver diseases are one of the fatal disorders due to the vital role of the liver. Carbon tetrachloride (CCl4 ) is the most perceived chemical substance utilized in developing models of hepatic damage. Metformin (Met) is a potent antidiabetic and redox modulatory agent that has shown anticancer and protective effects on various organs. Therefore, addition of therapy with natural antioxidative agents or herbal extracts shows defensive impacts against different injuries inside the body. Luteolin (Lut) can be found in several customary Chinese remedies. It has been reported for various pharmacological actions such as antitumor, antioxidative, and anti-inflammatory impacts. Here, the liver injury rat model was established using CCl4 (1.00 mL/kg body weight) in vivo. The protective roles of Met and Lut separately or in combination were observed in hepatotoxicity induced by CCl4 . The result was shown that both Met and Lut, while individually used, were normally active in diminishing CCl4 -caused hepatotoxicity. The combination of two drugs performed synergistically to improve liver damage caused by CCl4 , as shown by the considerably improved liver dysfunction. Met and Lut showed highly antioxidative effects on CCl4 -treated rats moderately by increasing the activities and expression of the antioxidant enzymes. Along with this, a combination of Met and Lut significantly suppressed inflammatory responses, which is evidenced by the reduced level of inflammatory cytokines together with interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6). Additionally, CCl4 -agitated apoptosis was intensely reduced by Met and Lut through reducing cleaved caspase-3 and Bax (pro-apoptotic factor) while increasing Bcl-2 (antiapoptotic factor) signaling pathways. Cotreatments of Met and Lut upregulated nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) expression in the CCl4 -intoxicated rat's liver. The above result recommended that combination of Met and Lut may have a substantial potential and synergizing impact against CCl4 -induced hepatotoxicity.

Curcumin inhibits proliferation,migration and invasion of gastric cancer cells via Wnt3a/β-catenin/EMT signaling pathway

The aim of this paper was to investigate the effects of curcumin on the proliferation,migration,invasion and apoptosis of human gastric cancer cells and to explore the potential mechanisms. SGC7901,MKN45 and NCI N87 cells lines were cultured under different concentrations of curcumin( 2. 5,5,10,20,40,80 and 160 μmol·L~(-1)) at different time points( 12,24,48 and 72 h),and the effect of curcumin on cell proliferation was detected by CCK-8 assay. The migration and invasiveness of cells were determined by wound healing and Transwell assays,the apoptosis rate was assessed by flow cytometry,the expression of N-cadherin,E-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6,Bcl-2 and Bax were detected by Western blot,and the enzymatic activity of caspase-3,caspase-8 and caspase-9 was evaluated via caspase kit. RESULTS:: indicated that the proliferation of MKN45 cells was significantly inhibited by curcumin in a dose-and time-dependent manner( IC50= 21. 93 μmol·L~(-1)). Moreover,curcumin could inhibit the migration and invasion of MKN45 cells,downregulate the expression of N-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6 and Bcl-2,and upregulate the expression of E-cadherin and Bax,it could increase the activity of caspase-3,caspase-8,caspase-9 and induce apoptosis as well. The potential mechanism is through inhibiting the Wnt3 a/β-catenin/EMT pathway,regulating Bcl-2 signaling and caspase pathway,which might provide new potential strategies for gastric cancer treatment.

Melatonin Prevents Oxidative Stress-Induced Mitochondrial Dysfunction and Apoptosis in High Glucose-Treated Schwann Cells via Upregulation of Bcl2, NF-κB, mTOR, Wnt Signalling Pathways.

Neuropathy is a complication that affects more than 50% of long-standing diabetic patients. One of the causes of diabetes neuropathy (DN) is the apoptosis of Schwann cells due to prolonged exposure to high glucose and build-up of oxidative stress. Melatonin is a hormone that has a known antioxidant property. In this study, we investigated the protective effect of melatonin on high glucose-induced Schwann cells’ apoptosis. Our results revealed that high glucose promoted apoptosis via mitochondrial-related oxidative stress and downregulated Bcl-2 family proteins in Schwann cells. In this signalling pathway, Bcl-2, Bcl-XL and Mcl-1 proteins were down-regulated while p-BAD and Puma proteins were up-regulated by high glucose treatment. Besides, we also proved that high glucose promoted apoptosis in Schwann cells through decreasing the p-NF-κB in the NF-κB signalling pathway. Key regulators of mTOR signalling pathway such as p-mTOR, Rictor and Raptor were also down-regulated after high glucose treatment. Additionally, high glucose treatment also decreased the Wnt signalling pathway downstream proteins (Wnt 5a/b, p-Lrp6 and Axin). Our results showed that melatonin treatment significantly inhibited high glucose-induced ROS generation, restored mitochondrial membrane potential and inhibited high glucose-induced apoptosis in Schwann cells. Furthermore, melatonin reversed the alterations of protein expression caused by high glucose treatment. Our results concluded that melatonin alleviates high glucose-induced apoptosis in Schwann cells through mitigating mitochondrial-related oxidative stress and the alterations of Bcl-2, NF-κB, mTOR and Wnt signalling pathways.

Role of TMPRSS4 Modulation in Breast Cancer Cell Proliferation.

Background:TMPRSS4 is a novel Type II transmembrane serine protease found at the surface of the cells and is involved in the development and cancer progression. However, TMPRSS4 functions in breast cancer remain poor understand. The present study investigated the function of TMPRSS4 in the breast cancer cells and the potential mechanistic action underling. Materials and Methods:The lentiviral vectors causing TMPRSS4 down-regulation and over-expression were established and transfected in MDA-MB-468 and MCF-7 cells, respectively. By using the CCK-8 assay, cell proliferation was analyzed. Moreover, western blot was used to detect the expression of certain proteins related to cell apoptosis (Bax and Bcl2) signaling pathway and telomere maintenance (POT1, TPP1, and UBE2D3). Cell cycle and cell apoptosis were also analyzed by using the Flow cytometry analysis. TMPRSS4 expression was detected at the mRNA level and protein level by performing qPCR and western blot technique, respectively. Results:TMPRSS4 expression is inhibited in stable transfected MDA-MB-468-shTMPRSS4 cells compared to the control MDA-MB-468-NC and its expression is up-regulated in stable transfected MCF-7-TMPTSS4 compared to its control MCF-7-NC. Moreover, TMPRSS4 silencing in breast cancer reduces cells proliferation by promoting cell cycle arrest in G2/M phase, cell apoptosis, and telomere maintenance impairment while the TMPRSS4 overexpression increases cells proliferation through cell apoptosis reduction and telomere maintenance reinforcement associated with insignificant change in cell cycle progression. Conclusion:TMPRSS4 plays important roles in cancer progression and may be considered as a good therapeutic target for cancer gene therapy especially breast cancer.

Influence of lncRNA HOTAIR on acute kidney injury in sepsis rats through regulating miR-34a/Bcl-2 pathway.

To study the regulatory effect of long non-coding ribonucleic acid (lncRNA) HOX transcript antisense RNA (HOTAIR) on acute kidney injury (AKI) in sepsis rats and its regulatory mechanism. The sepsis-induced AKI model was established in Sprague-Dawley (SD) male rats through cecal ligation puncture. A total of 30 SD rats were randomly divided into the control group, model group and lncRNA HOTAIR mimic group, with 10 rats in each group. Relative levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in kidney tissues were detected via enzyme-linked immunosorbent assay (ELISA). Apoptosis of kidney tissues was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Moreover, the target gene of miR-34a was searched using the miRNA online database. The messenger RNA (mRNA) expression levels of miR-34a and B-cell lymphoma-2 (Bcl-2) were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Compared with those in the control group, the rats in the model group showed injured pathological morphology of kidney, elevated contents of TNF-α and IL-1β, and apoptosis in kidney tissues. The target gene of miR-34a was Bcl-2, according to the miRNA online database. MiR-34a level in kidney tissues was upregulated, while the mRNA level of Bcl-2 significantly decreased in the model group. Compared with those in the model group, the pathological morphology of kidney tissues was improved, the content of TNF-α and IL-1β markedly declined, and the apoptotic rate of kidney tissues also reduced in lncRNA HOTAIR mimic group. The miR-34a level in kidney tissues decreased, while the Bcl-2 mRNA level remarkably increased in lncRNA HOTAIR mimic group. LncRNA HOTAIR overexpression can alleviate AKI in sepsis rats by inhibiting the apoptosis of kidney tissues by downregulating the miR-34a/Bcl-2 signaling pathway.

Combinational effect of curcumin and metformin against gentamicin-induced nephrotoxicity: Involvement of antioxidative, anti-inflammatory and antiapoptotic pathway.

Gentamicin (GM) is an antibiotic related to aminoglycoside group that is used in treating Gram-negative bacterial infections. However, treatment with gentamicin is considered to be limited as it induces an oxidative stress-mediated apoptosis in kidney which causes a nephrotoxicity. Metformin is a well-known biguanide that is used for treating diabetes mellitus, especially type 2. Supplement with plant metabolites or natural antioxidants produce a protective activity against many types of diseases in vivo. Curcumin is a main medicinal constituent of Curcuma longa, has reported for number of biological effects, such as antioxidant, anti-inflammatory, and antitumor. The study aims at evaluating the metformin and curcumin alone or in combination on nephrotoxicity induced by GM. The outcome of the study shows that both metformin and curcumin, when used unaided, were effectively decreasing GM-induced nephrotoxicity. The two drugs combination was showed synergistic effect in ameliorating a GM-induced kidney injury, as supported by expressively improved renal dysfunction. Metformin and curcumin showed strong protection against oxidative stress in GM treated animals through decreasing the activities and expression of various antioxidative enzymes. Moreover, combination of two drugs showed an anti-inflammatory response through reducing a level of pro-inflammatory cytokines including tumor necrosis factor-alpha, interleukin 1-beta, and interleukin 6 in GM intoxicated group of animals. Furthermore, GM agitated apoptosis was affectedly diminished by the combinational treatment of metformin and curcumin via down-regulating activity of cleaved Caspase-3 and pro-apoptotic factor Bax, whereas increasing anti-apoptotic factor Bcl-2 signaling pathways. The above results suggested that combinational treatment of metformin and curcumin might be have a synergizing effect and substantial potential against nephrotoxicity induced by GM. PRACTICAL APPLICATIONS: Curcumin and metformin combination exhibited substantial synergistic effect against GM-induced nephrotoxicity through reducing oxidative stress, inflammation, as well as apoptosis in kidney cells. Therefore, the method of combination of curcumin and metformin might be functional to treat or inhibit GM prompted nephrotoxicity in future.

Rosmarinic acid inhibits stem-like breast cancer through hedgehog and Bcl-2/Bax signaling pathways

Background: Rosmarinic acid (RA) is a natural phenolic acid present in various Lamiaceae herbs. RA shows anti-tumor effects on many tumors but has yet to be tested on triple negative breast cancer and its derived breast cancer stem-like cells (BCSCs). Objective: This study aimed to detect whether RA could inhibit the proliferation and migration of BCSCs through hedgehog (Hh) signaling while promoting apoptosis via Bcl-2/Bax. Materials and Methods: BCSCs from the human breast cancer cell line MD-MB-231 were isolated by fluorescence-activated cell sorting with the surface markers of CD44*/CD24-/low. The viability, migration, and apoptosis of BCSCs were assessed by the CCK-8 assay, cell wound healing test, and flow cytometry for positive staining for Annexin V-FITC and propidium iodine (PI), respectively. mRNA and protein levels of Hh and Bcl-2/Bax signaling pathways were obtained by real-time reverse transcriptase polymerase chain reaction and immunoblots. Results: RA inhibited the viability and migration of BCSCs and increased the numbers of late apoptotic cells. Consistent with the increased apoptosis, RA treatment downregulated Bcl-2 while upregulating Bax expression. In line with its effect to limit migration, RA treatment inhibited the expression of Hh-related genes smoothened and glioma-associated oncogene homolog 1. Conclusion: The present study suggests that RA exerts anti-cancer effects on BCSCs by inhibiting Bcl-2 and Hh signaling pathways.

Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.

The highest incidence of nasopharyngeal carcinoma (NPC) is in southeast China, including Taiwan. Many side effects have been observed following radiation therapy with chemotherapy; hence, exploring new treatment modalities for NPC is an important future direction. Angiotensin-(1–7) [Ang-(1–7)] is an endogenous heptapeptide hormone and important component of the renin–angiotensin system that acts through both the Mas receptor and AT2 receptor, exhibiting anti-proliferative and anti-angiogenic properties in cancer cells. However, the anti-cancer activity of Ang-(1–7) related to autophagy in NPC remains largely debated. The effects and signaling pathway(s) involved in the Ang-(1–7)/Mas receptor axis in NPC were investigated both in vitro and in vivo. Ang-(1–7) inhibited cell proliferation, migration, and invasion in NPC-TW01 cells. Ang-(1–7) induced autophagy by increasing the levels of the autophagy marker LC3-II and by enhancing p62 degradation via activation of the Beclin-1/Bcl-2 signaling pathway with involvement of the PI3K/Akt/mTOR and p38 pathways in vitro study. In addition, pre-treatment with Ang-(1–7) inhibited tumor growth in NPC xenografts by inducing autophagy, suggesting a correlation between PI3K/Akt/mTOR pathway inhibition and the abovementioned anti-cancer activities. However, no autophagy was observed following Ang-(1–7) post-treatment. Taken together, these data indicate that Ang-(1–7) plays a novel role in autophagy downstream signaling pathways in NPC, supporting its potential as a therapeutic agent for alleviation the incidence of NPC and preventive treatment of recurrent NPC.Key messages Ang-(1–7) inhibits cell proliferation, migration, and invasion by activating autophagyAng-(1–7)pre-treatment inhibits tumor growth via autophagy by suppressing PI3K/Akt/mTOR pathway.Ang-(1–7) may provide a novel preventative treatment for NPC and recurrent NPC Electronic supplementary materialThe online version of this article (10.1007/s00109-018-1704-z) contains supplementary material, which is available to authorized users.

Effect of HOXA6 on the proliferation, apoptosis, migration and invasion of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common types of tumor worldwide. The morbidity and mortality rates of CRC have increased significantly in adults <50years of age. In the present study, the effects of homeoboxA6(HOXA6) on the proliferation, apoptosis, migration and invasion of CRC cells were investigated. The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of HOXA6 in CRC tumor tissue was higher than that in adjacent normal tissue. Appropriate cell lines and plasmids were selected by RT-PCR and western blot analyses, and recombinant plasmids were transfected into Caco2 or HT-29 cells. The results of RT-qPCR and western blot analyses demonstrated that the expression of HOXA6 was effectively enhanced, or inhibited, following transfection. The rate of cell proliferation was measured with cell counting kit-8, colony formation assay and 5-ethynyl‑2'-deoxyuridine assay, apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry assays, and migration and invasion were evaluated using Transwell and wound-healing assays. The results demonstrated that the upregulation of HOXA6 promoted cell proliferation, migration and invasion, but inhibited apoptosis, whereas the downregulated expression of HOXA6 produced the opposite effects. In addition, the expression levels of apoptosis- and epithelial-mesenchymal transition(EMT)-related proteins were examined. The results of the western blot analysis revealed that the upregulated expression of HOXA6 suppressed the expression of B-cell lymphoma-2 (Bcl-2)-associated Xprotein, caspase-3, poly(ADP-ribose) polymerase and E-cadherin, but promoted the expression of Bcl-2, N-cadherin and Vimentin, whereas the opposite effect was observed in cells with downregulated HOXA6. These results indicated that HOXA6 regulated apoptosis through the Bcl-2 signaling pathway, and regulated migration and invasion through the EMT process. In conclusion, the present study confirmed that HOXA6 was involved in the regulation of CRC, which may inform the development of strategies for the diagnosis and treatment of CRC.

Overexpression of adrenomedullin protects mesenchymal stem cells against hypoxia and serum deprivation‑induced apoptosis via the Akt/GSK3β and Bcl‑2 signaling pathways.

The poor survival rate of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Adrenomedullin (ADM) has been identified as a potent apoptotic inhibitor. The present study aimed to investigate the protective effects of ADM on MSCs against hypoxia and serum deprivation (H/SD)-induced apoptosis, and to determine the potential underlying mechanisms. In the present study, a recombinant adenovirus expressing the ADM gene was established and was infected into MSCs. The infection rate was determined via microscopic detection of green fluorescence and flow cytometric analysis. The mRNA expression levels of ADM were detected by reverse transcription-polymerase chain reaction. In addition, a model of H/SD was generated. The MSCs were randomly separated into six groups: Control, enhanced green fluorescent protein (EGFP)-Adv, EGFP-ADM, H/SD, EGFP-Adv + H/SD and EGFP-ADM + H/SD. Cell viability and proliferation were determined using the Cell Counting kit-8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated-dUTP nick-end labeling assay and flow cytometric analysis using Annexin V-phycoerythrin/7-aminoactinomycin D staining. The protein expression levels of total protein kinase B (Akt), phosphorylated (p)-Akt, total glycogen synthase kinase (GSK)3β, p-GSK3β, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and cleaved caspase-3 were detected by western blot analysis. The results indicated that ADM overexpression could improve MSC proliferation and viability, and protect MSCs against H/SD-induced apoptosis. In addition, ADM overexpression increased Akt and GSK3β phosphorylation, and Bcl-2/Bax ratio, and decreased the activation of caspase-3. These results suggested that ADM protects MSCs against H/SD-induced apoptosis, which may be mediated via the Akt/GSK3β and Bcl-2 signaling pathways.

Simultaneously targeting Bcl-2 and Akt pathways reverses resistance of nasopharyngeal carcinoma to TRAIL synergistically.

Despite progress in treatment techniques, the five-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. Given the antiapoptotic functions of Bcl-2 and Akt, we examined the effects of targeting these pathways alone or simultaneously on TRAIL apoptosis in NPC cell lines. We first tested the cytotoxic effect of TRAIL and the expression of death receptors, Bcl-2, Akt, and p-Akt on four NPC cell lines by MTT and Western blotting, respectively. Small interfering RNAs (siRNAs) targeting Bcl-2 and PI3-K inhibitor (LY294002) were used alone or combined with TRAIL in the cell lines and cytotoxicity was examined by MTT. Apoptosis rates, mitochondrial transmembrane potential, and apoptotic pathway signals were detected by flow cytometric analysis, DiOC6(3) assays, and Western blotting after the various combination treatments on CNE-2, the cell line that was most resistant to TRAIL. Although no direct correlation between the sensitivity to TRAIL and the relative expression levels of Bcl-2 and activated Akt was found in the NPC cell lines examined, siRNA mediated the downregulation of Bcl-2 and LY294002-induced inactivation of Akt, increasing the sensitivity of all examined NPC cell lines to TRAIL. Synergistic enhancement of TRAIL-mediated cytotoxicity was observed in combination treatment of Bcl-2 siRNA and LY294002 compared to cells treated with each treatment alone. The synergistic effects were mediated through increased apoptotic signaling of the mitochondrial pathway, as was evident from the more increased mitochondrial depolarization, activation of caspase-9 and caspase-3, and suppression of XIAP. This study provides proof of principle that TRAIL combined with simultaneously targeting the Bcl-2 and Akt signaling pathways may have potential as a novel future treatment strategy for NPC.

Ginsenoside Rg1 protects rat bone marrow mesenchymal stem cells against ischemia induced apoptosis through miR-494-3p and ROCK-1

This study aimed to verify the cytoprotective effect of ginsenoside Rg1 in vivo, and to elucidate the mechanism of Rg1 in the ischemic microenvironment. Male rat bone marrow mesenchymal stem cells (rBMSCs) or rBMSCs treated with Rg1 were injected into ischemic region of the arterial embolism hind limb in female rats. Behavioral and histological data, obtained one-week post injection, showed that rBMSCs with Rg1 could improve the survival rate of BMSCs and enhance the therapeutic effects. rBMSCs treated with hypoxia and serum deprivation for 24h (H/SD-rBMSCs) showed the up-regulated expression of ras homolog family member A (RhoA), Rho associated coiled-coil containing protein kinase 1 (ROCK-1), myosin light chain 2 (MLC-2), Bcl2 associated agonist of cell death (Bad) and Bcl2 associated X, apoptosis regulator (Bax); while the expression of miR-148b-3p, miR-148b-5p and miR-494-3p was down-regulated. H/SD with Rg1 treatment (H/SD+Rg1-rBMSCs) inhibited the expression of ROCK-1, MLC-2, Bad and Bax, increased the expression of Bcl-2, miR-494-3p. After ROCK-1 knockout, the expression of Bad and Bax were downregulated and Bcl-2 upregulated, but Rg1 no longer altered their expression. Mir-494-3p functional study established that miR-494-3 mimic downregulated and miR-494-3 inhibitor upregulated ROCK-1 gene expression, Rg1 did not have the ability to change the ROCK gene expression after loss of function of miR-494-3p. Also, the function loss of mir-494-3p promoted apoptosis; otherwise reduced apoptosis. The anti-apoptotic effect of Rg1 disappeared after mir-494-3p loss or gain function. In conclusion, Ginsenoside Rg1 has shown to have protective effects on ischemic-induced rBMSCs apoptosis through mir-494-3p→ROCK-1→Bcl-2 signaling pathway.

Activated Yes-Associated Protein Accelerates Cell Cycle, Inhibits Apoptosis, and Delays Senescence in Human Periodontal Ligament Stem Cells.

Objectives: To provide insight into the biological effects of activated Yes-associated protein (YAP) on the proliferation, apoptosis, and senescence of human periodontal ligament stem cells (h-PDLSCs).Methods: h-PDLSCs were isolated by the limiting dilution method, and their surface markers were quantified by flow cytometry. Enhanced green fluorescence protein (EGFP)-labeled lentiviral vector was used to activate YAP in h-PDLSCs, then qRT-PCR and Western blotting were used to evaluate the expression level of YAP. Immunofluorescence was used to detect the location of YAP in h-PDLSCs. The proliferation activity was detected by cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU), and the cell cycle was determined by flow cytometry. Apoptosis was analyzed by Annexin V-APC staining. Cell senescence was detected by β-galactosidase staining. Proteins in ERK, Bcl-2, and p53 signaling pathways were detected by Western blotting.Results: h-PDLSCs were isolated successfully and were positive for human mesenchymal stem cell surface markers. After YAP was activated by lentiviral vector, the mRNA and protein of YAP were highly expressed, and more YAP translocated into the nucleus. When YAP was overexpressed in h-PDLSCs, proliferation activity was improved; early and late apoptosis rates decreased (P<0.05); the proportion of cells in G2/M phases increased (P<0.05), while that in G0/G1 phase decreased (P<0.05); cellular senescence was delayed (P<0.01); the expression of P-MEK, P-ERK, P-P90RSK and P-Msk increased, while the expression of Bcl-2 family members (Bak, Bid and Bik) decreased.Conclusions: Activated YAP promotes proliferation, inhibits apoptosis, and delays senescence of h-PDLSCs. The Hippo-YAP signaling pathway can influence ERK and Bcl-2 signaling pathways.