Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

MicroRNA-146a promotes apoptosis of gastric cancer SGC-7901 cells by targeting TAK1

AIM: To explore the effect of microRNA-146a (miR-146a) on apoptosis of human gastric cancer SGC-7901 cells and the underluing mechanism. METHODS: miR-146a mimic (up-regulated miR-146a expression) and miR-146a inhibitor (down-regulated miR-146a expression) were transfected into the SGC-7901 cells by liposome method. At the same time, miRNA nonsense sequence transfection group as the negative control group (NC group) was set up. RT-qPCR was used to evaluate the levels of miR-146a in the SGC-7901 cells after transfection. The effects of miR-146a on the cell apoptosis and growth were assessed by flow cytometry analysis and CCK-8 assay, respectively. The effect of over-expression or knockdown of miR-146a on transforming growth factor-β-activated kinase 1 (TAK1)/nuclear factor-kappa B (NF-κB) signaling was evaluated by RT-qPCR and Western blot. RESULTS: miR-146a modulated apoptosis of SGC-7901 cells. Over-expression of miR-146a significantly increased apoptosis, whereas knockdown of miR-146a inhibited the apoptosis of SGC-7901 cells. The expression of TAK1 at mRNA and protein levels was significantly decreased when miR-146a mimic was transfected into the SGC-7901 cells (P<0.05). On the contrast, the expression of TAK1 at mRNA and protein were significantly higher in miR-146a inhibitor transfection group than that in NC group (P<0.05), suggesting that miR-146a negatively regulated TAK1 expression. Moreover, knockdown of TAK1 enhanced the apoptosis of SGC-7901 cells (P<0.01), while over-expression of TAK1 inhibited the apoptosis of SGC-7901 cells(P<0.01). Additionally, both over-expression of miR-146a and knockdown of TAK1 led to a prominent increase in the expression of NF-κB inhibitor protein alpha (IκBα) and a significat decrease in B cell lymphoma-2 (Bcl-2) level in the SGC-7901 cells. CONCLUSION: miR-146a significantly promotes apoptosis of SGC-7901 cells by inhibition of NF-κB pathway via targeting TAK1.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effect of ruthenium-polypyridine complex on apoptosis of gastric cancer SGC-7901 cells

AIM:To study the effect of ruthenium-pyridine complex Ru1 on apoptosis of gastric cancer SGC-7901 cells. METHODS:MTT assay and crystal violet staining method were used to detect the viability and cell number of SGC-7901 cells treatment with Ru1. Annexin V-FITC and PI staining was performed to test the apoptosis rate of SGC-7901 cells. The protein expression of Bax and Bcl-2 was detected by Western blot. RESULTS:The results of MTT assay and crystal violet staining showed that the ruthenium-pyridine complexes significantly reduced the viability and cell number of SGC-7901 cells. Treatment with Ru1 for 24 h significantly increased the apoptotic rate of SGC-7901 cells (P<0.05). Ru1 up-regulated the expression of Bax protein and down-regulated the expression of Bcl-2 protein in the SGC-7901 cells (P<0.05). CONCLUSION:Ru1 induces apoptosis of SGC-7901 cells by affecting the expression of Bax and Bcl-2 proteins.     

Effect of ixazomib on apoptosis and NF-κB signaling pathway in pancreatic cancer cells

AIM:To investigate the effects of ixazomib on the apoptosis and NF-κB signaling pathway in pancreatic cancer cells. METHODS:Human pancreatic cancer cell lines CFPAC-1 and PANC-1 were cultured, and the cells were treated with ixazomib at 0, 10, 20, 30 and 40 nmol/L for 12, 18, 24 and 48 h. The expression of NF-κB p65, IκB kinase (IKK), Bax and caspase-3 in the cells at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by CCK-8 assay. The apoptosis was analyzed by flow cytometry. RESULTS:Treatment with ixazomib at 10~40 nmol/L inhibited the viability of PANC-1 cells and CFPAC-1 cells, and the inhibitory rate was increased significantly with the increases in the concentration and time (P<0.05). Compared with the control cells, treatment with ixazomib significantly increased the apoptotic rates of PANC-1 cells and CFPAC-1 cells in a dose- dependent manner (P<0.05), and significantly decreased the mRNA expression levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), while the mRNA expression levels of apoptotic factors Bax and caspase-3 in the PANC-1 cells and CFPAC-1 cells were significantly increased (P<0.05). The results of Western blot showed that treatment with ixazomib significantly decreased the protein levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), which was consistent with the results of mRNA expression. The protein levels of apoptosis factors Bax and caspase-3 in the CFPAC-1 cells were significantly increased (P<0.05), and the protein level of caspase-3 in the PANC-1 cells was increased significantly (P<0.05). However, Bax protein did not increase significantly in 10 nmol/L ixazomib group. CONCLUSION:Ixazomib, a proteasome inhibitor, inhibits the viability of pancreatic cancer cells and promotes apoptosis by inhibiting the activation of NF-κB signaling pathway in a time- and dose-dependent manner.     

Effects of all-trans retinoic acid on sensitivity of gastric cancer cells to radiotherapy

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on the viability of gastric cancer cell SGC-7901 and the sensitivity to radiotherapy. METHODS: MTT assay was used to examine the cell viability. Radio-sensitivity and cell cycle were determined by colony formation assay and flow cytometry, respectively. The mRNA levels of Bax, Bcl-2, survivin and NF-κB in the cells were measured by RT-qPCR. RESULTS: ATRA inhibited the viability of SGC-7901 cells in a concentration-dependent manner. The maximal inhibition was at concentration of 8 μmol/L. Colony formation assay revealed that the combination of ATRA with X-ray treatment significantly reduced the values of D0 and Dq, and shifted down the fitting survival curve, as compared with radiotherapy alone. Moreover, ATAR markedly decreased the percentage of G₂/M phase in the SGC-7901 cells (P<0.05). In addition, following ATRA treatment, the mRNA levels of Bcl-2 and survivin were decreased (P<0.05), whereas the mRNA levels of Bax and NF-κB were increased (P<0.05). CONCLUSION: ATRA enhances the sensitivity of SGC-7901 cells to radiotherapy, inhibits G₂/M arrest and regulates the mRNA expression of Bax, Bcl-2, survivin and NF-κB.     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

Expression of SCD-1 in cervical carcinoma and effect of its down-regulation on proliferation and apoptosis of cervical carcinoma cells

AIM:To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells. METHODS:The expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS:The protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells. CONCLUSION: SCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.     

Effects of TRAF3 on inhibiting cyst formation induced by NF-κB signaling in kidney

AIM: To investigate the effects of tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) on the signaling pathway of NF-κB and the expression of Bax and Bid in renal collecting duct epithelial cells of polycystic kidney disease (PKD), and to observe the tubulogenesis of TRAF3 transgenics for exploring the protective effect of TRAF3 on the cystogenesis and development of PKD. METHODS: The signaling changes of NF-κB and expression of Bax and Bid in PKD renal collecting duct epithelial cells and TRAF3 transgenic PKD renal collecting duct epithelial cells were determined by the Western blot. The percentage of apoptotic cells was measured by flow cytometry with Annexin V staining. The caspase-3 activity was also measured. The 3D Matrigel culture was performed to examine abnormal tubulomorphogenesis in vitro. RESULTS: The over-expression of TRAF3 significantly inhibited the signaling pathway of NF-κB in PKD renal collecting duct epithelial cells and significantly downregulated the expression of Bax and Bid, and also decreased the cell apoptosis. More branch structures were observed in the TRAF3 transgenic PKD renal collecting duct epithelial cells. CONCLUSION: TRAF3 is a negative regulatory inhibitor for Bax and Bid by regulating the NF-κB signaling and may be a new target for inhibiting the cyst formation in PKD.     

Perifosine inhibits cell proliferation and triggers apoptosis in human gastric cancer cell line SGC-7901 cells

AIM: To investigate the effect of perifosine, a novel inhibitor of Akt, on the cell proliferation and apoptosis in human gastric cancer cell line SGC-7901.METHODS: Cell growth inhibition was detected by MTT assay. Cell cycle was analyzed by flow cytometry. Annexin V-FITC apoptosis detection kit was used to determine apoptosis in the cells. Protein expression was examined by Western blotting.RESULTS: Akt phosphorylation was dose-dependently inhibited by perifosine in SGC-7901 cells. The results of MTT and cell cycle analysis indicated that perifosine inhibited the growth of human gastric cancer cells in a dose-dependent manner. Perifosine arrested the cell cycle progression at G₂ phase. Apoptosis induction became more effective with increasing the concentration of perifosine. The caspase cascade and its downstream effector poly(ADP-ribose) polymerase (PARP) were also activated upon perifosine treatment, and the level of Bcl-2 was down-regulated, whereas the protein level of Bax was up-regulated.CONCLUSION: The small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human gastric cancer cell line SGC-7901. Perifosine induces caspase-dependent apoptosis, and the key regulators include caspase-3, caspase-9 and Bcl-2.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

Role of Fas overexperssion in reversing cisplatin resistance in stomach cancer cells

AIM: To study the effect of Fas on cisplatin resistance in stomach cancer cells and its possible mechanisms.METHODS: The expression of Fas at mRMA and protein levels in SGC-7901 cells and SGC-7901/DDP cells was determined by RT-qPCR and Western blot. Fas-containing adenovirus vector was transfected into the SGC-7901/DDP cells to upregulate Fas expression. The cell viability was detected by CCK-8 assay. The cell cycle and cell apoptosis were analyzed by flow cytometry. The protein levels of Fas, P38/p-P38, JNK/p-JNK, cleaved caspase-8/caspase-8 and cleaved caspase-3/caspase-3 were detected by Western blot.RESULTS: The expression of Fas at both mRNA and protein levels was significantly downregulated in the SGC-7901/DDP cells. Fas expression was decreased by cisplatin in a dose-dependent manner in the SGC-7901 cells. Overexpression of Fas suppressed the viability and induced apoptosis in the SGC-7901/DDP cells, and upregulated the protein levels of p-P38, p-JNK, cleaved caspase-8 and cleaved caspase-3.CONCLUSION: Overexpression of Fas increases the sensitivity of the SGC-7901/DDP cells to cisplatin, and inhibits the cell growth and promotes cell apoptosis. The mechanism may be related to the activation of JNK and P38 pathway.     

Down-regulated BARF1 expression induces EBV-positive gastric carcinoma cell apoptosis via activating caspase-dependent mitochondrial pathway

AIM: To investigate the effects of BARF1 down-regulation on EBV-positive gastric carcinoma cell apoptosis, and the molecular mechanisms by BARF1 silencing-mediated apoptosis. METHODS: After NUGC3 and SNU719 cells were transfected with NCsiRNA and siRNA, respectively, the protein levels of BARF1, Bcl-2, Bax, cytochrome C, caspase 3 and capase 9 were detected by Western blot, and the mRNA expression of BARF1, Bcl-2 and Bax was determined by RT-PCR. The cell viability was measured by the method of Trypan blue exclusion and the cell apoptosis was analyzed by flow cytometry analysis with Annexin V-FITC/PI staining. The expression of the apoptosis-related proteins in the cells transfected with siRNA and NCsiRNA was examined by human apoptosis antibody arrays. Mitochondrial membrane potential was determined by flow cytometry. The interaction between Apaf-1 and caspase 9 was confirmed by immunoprecipitation. RESULTS: Compared with untreated and NCsiRNA groups, BARF1 gene silencing significantly inhibited the cell viability, induced apoptosis, and reduced the mitochondrial membrane potential in the NUGC3 and SNU719 cells transfected with siRNA. BARF1 gene silencing up-regulated the expression of pro-apoptotic proteins and down-regulated the expression of anti-apoptotic proteins, and the Bcl-2/Bax ratio was significantly decreased. In BARF1 gene silencing cells, the caspase inhibitor z-VAD-fmk inhibited BARF1 silencing-mediated apoptosis, and significantly increased the levels of cleaved caspase 3 and caspase 9. The concentration of cytochrome C significantly increased as compared with NCsiRNA group, and Apaf-1 interacted with caspase 9 in the cytoplasm. CONCLUSION: BARF1 silencing induces apoptosis via the mitochondrial pathway through regulating the expression of Bcl-2 and Bax proteins in a caspase-dependent manner in the NUGC3 and SNU719 cells.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.