Effects of epigallocatechin-3-gallate on 1-methyl-4-phenylpyridinium ion-induced apoptosis in rat PC12 cells

AIM: To investigate the effects of epigallocatechin-3-gallate (EGCG) on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in rat pheochromocytoma (PC12) cells and to explore the relationships between its roles of anti-oxidation, intracellular calcium homeostasis and anti-apoptosis. METHODS: Rat PC12 cells were pretreated with vehicle control or EGCG (10, 50, and 100 μmol/L) for 30 min, then cultured with MPP+ (900 μmol/L) for 24 h. The cell viability and apoptosis were monitored by MTT assay and flow cytometry using Annexin V and PI. The activity of intracellular reactive oxygen species (ROS), contents of superoxide dismutase (SOD) and malondialdehyde (MDA), cytoplasmic Ca2+ density and apoptotic morphology of mitochondria were examined by fluorescent plate-based assays, confocal microscope, and transmission electron microscope, respectively. RESULTS: MPP+ impaired the PC12 cells in a concentration-dependent pattern and induced apoptosis of the cells (31% versus control). Compared with the control, the cells pretreated with EGCG showed markedly higher rate of viability and lower apoptosis. Meanwhile, EGCG pretreatment significantly increased the SOD activity and decreased the levels of MDA and ROS. Interestingly, EGCG also decreased the concentration of cytoplasmic Ca2+ and improved the morphology of mitochondria. CONCLUSION: EGCG exhibits inhibitory effects on MPP+-induced apoptosis in rat PC12 cells, which is possibly associated with increasing the cell ability of anti-oxidation and decreasing the concentration of cytoplasmic Ca2+.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.     

Effects of human recombinant acidic fibroblast growth factor on cell proliferation and apoptosis in NIH3T3 cells

AIM:Through studying the differences betwe en wild-type acidic fibroblast growth factor (waFGF) with recombinant aFGF (raFG F ),the biological effect of raFGF concerned with mitogenic activity was evaluate d.METHODS:NIH3T3 cell line was used.Cell proliferation with MTT method was used to study the mitogenic activity.Flow cytometry was also used fo r detection of apoptosis,cell membrane permeability and mitochondria potential.The role of heparin sulfate (HS) on aFGF biological effect was studied at the s ame time in this research.RESULTS:The enhancement of raFGF on cell proliferation was sign ificantly lower than that of waFGF.The restriction of raFGF on apoptosis and th e enhancement of it on cell membrane permeability were all lower than those of w aFGF significantly.The enhancement of raFGF on mitochondria potential was lower than that of waFGF significantly.The HS improved the biological effect of aFGF .CONCLUSION:The mitogenic activity of raFGF is lower than that of waFGF and raFGF has little effect on apoptosis.     

Effects of phycocyanin on apoptosis of human laryngeal cancer HEP-2 cells

AIM: To investigate the effect of phycocyanin on the apoptosis of human laryngeal cancer HEP-2 cells and to explore the inhibitory mechanism of phycocyanin to tumor. METHODS: Highly purified phycocyanin was extracted from spirulina. The effects of phycocyanin at different concentrations on the growth of human laryngeal cancer HEP-2 cells were detected by MTT assay. In addition, the cell structures were observed under electron microscope. The cell apoptosis was analyzed by flow cytometry. The production of reactive oxygen species (ROS) was measured by flow cytometry. Enzymatic activities of caspase-3, -8 and -9 were measured by chemical colorimatry. The expression of Bax, Bcl-2, Fas, P53, caspase-3 and caspase-9 at mRNA and protein levels was determined by RT-PCR and Western blot. RESULTS: MTT test confirmed that phycocyanin inhibited the cell activity of HEP-2 cells with time and dose dependent manners. The result of electron microscope observation and flow cytometry indicated that phycocyanin induced the apoptosis of HEP-2 cells. The intracellular content of ROS was increased. The activities of caspase-3, -8 and -9 were increased. RT-PCR showed that the mRNA expression of Bax, Fas, P53, caspase-3, caspase-9 was increased and Bcl-2 was decreased. The results of Western blot were consistent with the results of RT-PCR. CONCLUSION: Phycocyanin might induce apoptosis of HEP-2 cells by down-regulating Bcl-2, up-regulating Bax, Fas and P53, and the transduction of apoptotic signals in the human laryngeal cancer cells.     

Effects of low-dose mifepristone on apoptosis in human ovarian luteinized granulosa cells

AIM: To investigate the effect of different low-dose mifepristone on apoptosis in granulosa cells and to test low-dose mifepristone as an orally contraceptive drug. METHODS: By using immunofluorescence, terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) and flow cytometry technique, the nuclear morphologic features and ratio of apoptosis and fluorescent intensity of caspase-3 in granulosa cells cultured in vitro treated with different low-doses of mifepristone were observed, respectively. RESULTS: By the display of immunofluorescence, the granulosa cells in treatment group were classified as apoptotic cells on the basis of their morphologic features contained a single condensed chromatin, multiple nuclear fragments. The results of TUNEL showed significant difference between control group and groups treated with different concentration of mifepristone (P<0.01). A significant difference (P<0.01) was also observed between the treatment groups with 1.25 μmol/L and 2.50 μmol/L mifepristone. The fluorescent intensity of caspase-3, observed by flow cytometry showed significant difference (P<0.01) between control group and treatment groups. CONCLUSION: Granulosa cells are induced to apoptosis by low-dose mifepristone, which may be regulated by the activation of caspase-3.     

Effects of silymarin on homocysteine-induced apoptosis in human umbilical vein endothelial cells

AIM:To investigate the effect of silymarin on homocysteine-induced cell viability and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS:Cell viability was analyzed by using MTT and LDH assay. Apoptotic cells were detected by using DNA fragmentation and flow cytometric analysis. The level of intracellular reactive oxygen species (ROS) and the potential of mitochondrial membrane were determined by flow cytometric assay. The activity of caspase-3, -6 and -9 were measured with microplate spectrofluorometer. Protein levels were examined by Western blotting. RESULTS:Treatment of cultured HUVECs with HCY for 48 h induced a significant decrease in cell viability, and the percentage of apoptosis increased to 76.8%. The level of intracellular ROS and activity of caspase-3, -6 and -9 enhanced, and the red/green ratios of mitochondrial membrane decreased. However, simultaneous treatment with silymarin exhibited cytoprotective effects, reduced formation of the DNA ladder, prevented the levels of Bax and Bcl-2 proteins and the accumulation of ROS as well as caspase-3, -6 and -9 activation, reconverted the potential of mitochondrial membrane, and the percentage of apoptosis/necrosis was significantly decreased to 12.7% in a dose-dependent manner. CONCLUSION:These results demonstrate that silymarin has the protective capacity to antagonize HCY-induced apoptosis in HUVECs. The antiapoptotic action of silymarin may be partially dependent on an anti-oxidative stress effects, inhibition of caspases activity, and maintenance of mitochondria function.     

Effects of andrographolide on invasion and apoptosis of ovarian cancer SKOV-3 cells

AIM:To investigate the effects of andrographolide on the invasion and apoptosis of ovarian cancer cell line SKOV-3,and to explore the possible mechanisms.METHODS:SKOV-3 cells were treated with different concentrations (0,5,10,20 or 40 μmol/L) of andrographolide for different time (12,24,36 or 48 h),and then the cell viability was determined by CCK-8 assay.The cell invasion ability was analyzed by Transwell assay and cell apoptosis was detected by TUNEL staining.The protein levels of p-PI3K,p-Akt and p-mTOR were examined by Western blot.RESULTS:The results of CCK-8 assay revealed that andrographolide inhibited the growth of SKOV-3 cells in a dose-and time-dependent manner.Treatment with andrographolide at 20 μmol/L for 36 h significantly decreased the invasion ability of SKOV-3 cells,while increased cell apoptosis.In addition,the protein levels of p-PI3K,p-Akt and p-mTOR were reduced after andrographolide treatment.CONCLUSION:Andrographolide inhibits the growth and invasion of ovarian cancer SKOV-3 cells by suppression of PI3K/Akt/mTOR signaling pathway.     

Inhibitory effect of quercetin on apoptosis of PC12 cells induced by rotenone

AIM: To observe the effects and mechanisms of quercetin on the apoptosis of PC12 cells induced by rotenone. METHODS: PC12 cells were used in the study. Quercetin at the concentration of 300 μmol/L was added into the PC12 cells cultured in DMEM-F12 medium with 10% fetal calf serum. The morphological changes of the cells were observed under fluorescence microscope. The apoptotic rate was determined by flow cytometry assay. The protein levels of Bax and Bcl-2 were determined by Western blotting, and the mitochondrial membrane potential was measured by ratiometric probe JC-1.RESULTS: In the cells treated with rotenone+quercetin, the morphology of the cells was significantly improved, and the apoptotic rate was decreased to 6.7%, significantly lower than that in the cells treated with rotenone alone (P<0.01). The expression of Bcl-2 was up-regulated and Bax was down-regulated in rotenone+quercetin group (P<0.01), while the mitochondrial membrane potential was also increased (P<0.01) as compared to those in rotenone group.CONCLUSION: Pretreatment of quercetin inhibits the development of apoptosis in PC12 cells induced by rotenone. One of the mechanisms may be correlated with up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, thus maintaining mitochondrial membrane potential.     

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

AIM：To investigate the role of human wings apart-like (hWAPL) protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA (shRNA) duplexes. METHODS：The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell proliferation was detected by MTT assay, and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining. Western blotting was used to analyze the expression of cleaved caspase-3, p21 and p27. The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model. RESULTS：Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA. The shRNA-mediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells. Additionally, the expression levels of cleaved caspase-3, p21 and p27 were up-regulated in hWAPL knockdown cells. Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells. CONCLUSION：hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo, and might serve as a therapeutic target in cancer treatment.     

Effects of microRNA-483-3p on human glioma cells

AIM:To investigate the effects of microRNA (miRNA)-483-3p on the growth and migration of human glioma cell line A172 and its potential mechanisms.METHODS:The abundance of miRNA-483-3p in human embryonic kidney 293 cells and different human glioma cell lines (A172,U251 and SHG44) was measured by RT-qPCR.After down-regulation of miRNA-483-3p by transfection of inhibitor in the A172 cells,the cell viability,cell cycle distribution and cell migration were detected by CCK-8 assay,flow cytometry and Transwell assay,respectively.Furthermore,the protein levels of cell cycle-related molecules and epithelial-mesenchymal transition markers were measured by Western blot.Luciferase reporter assay was used to predict and verify the target gene of miRNA-483-3p.RESULTS:miRNA-483-3p was highly expressed in human glioma cells.Knockdown of miRNA-483-3p inhibited A172 cell viability,arrested cell cycle and decreased cell migration rate.Furthermore,the protein levels of cyclin D1,cyclin-dependent kinase 4,phoshorylated retinoblastoma protein,N-cadherin and vimentin were significantly decreased after knockdown of miRNA-483-3p,accompanied with the up-regulation of E-cadherin and β-catenin protein expression.Luciferase reporter assay demonstrated that Smad4 was a potential target gene of miRNA-483-3p.Down-regulation of Smad4 in the A172 cells transfected with miRNA-483-3p inhibitor partially reversed the effect of miRNA-483-3p on cell viability and migration.CONCLUSION:Knockdown of miRNA-483-3p restrains the growth and migration of A172 cells by targeting Smad4.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Effects of matrine on apoptosis and related protein expression in human medulloblastoma D341 cells

AIM: To investigate matrine-induced apoptosis of human medulloblastoma D341 cells and the expression of Bax, Bcl-2, serine/threonine kinase Akt and phosphorylated Akt (p-Akt) in vitro. METHODS: D341 cells were divided into experimental groups (added with matrine at different concentrations) and control group (under the same conditions without matrine). The proliferation of D341 cells was analyzed by CCK-8 assay. Apoptosis was detected by Annexin V-FITC/PI double staining and the expression of Bax, Bcl-2, Akt and p-Akt was detected by Western blotting. RESULTS: Matrine significantly inhibited the proliferation of D341 cells and increased the apoptosis in a dose- and time-dependent manner. The cell apoptosis was characterized by chromatin condensation with margination of chromatin to the nuclear membrane, increased when and larger cytoplasmic vacuoles, and formation of apoptotic body after treatment with matrine. The expression of Bax increased, while the expression of Bcl-2 and p-Akt decreased when the drug concentration gradually increased. CONCLUSION: Matrine induces the apoptosis of human medulloblastoma D341 cells in vitro by activation of Bax, down-regulation of Bcl-2 and reduction of p-Akt expression level in the PI3K/Akt signaling pathway.     

Exogenous ATP induces formation of membrane pore in PC12 cells

AIM：To investigate the formation of membrane pore in PC12 cells induced by exogenous adenosine triphosphate (ATP) and to identify the key molecular targets. METHODS：PC12 cells were treated with different concentrations of ATP to establish the injury model. The morphological change was observed under an inverted phase-contrast microscope. The viability of the PC12 cells was measured by CCK-8 assay. Fluorescent dye YO-PRO-1 was used to detect the membrane permeability. The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was assessed by real-time PCR and Western blotting. RESULTS：After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous, and the number of adherent cells decreased gradually in a dose-dependent manner. The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with control group (P<0.05). YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner. The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G (a P2X7 receptor inhibitor) pretreatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0.05). The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expression of Panx1 was not changed (P>0.05) when PC12 cells were exposed to ATP for 3 h. CONCLUSION：Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P2X7 receptor in PC12 cells.     

Effects of GSK-3β inhibitor on proliferation and apoptosis of SW480 cells

AIM: To investigate the mechanism and the effect of glycogen synthase kinase 3β (GSK-3β) inhibitor (2’Z,3'E)-6-bromoindirubin-3'-oxime (BIO) on the protein expression of β-catenin and Bcl-2, and proliferation and apoptosis in colon carcinoma SW480 cells.METHODS: The immunohistochemical staining and Western blotting were performed to detect the protein expression of β-catenin, cyclin D1 and Bcl-2. The cell cycle distribution and apoptotic rate were detected by flow cytometry. The morphologic features of SW480 cells before and 24 h after BIO exposure at different concentrations were observed under microscope with HE staining.RESULTS: Compared with the untreated SW480 cells, the protein expression of β-catenin significantly increased and some β-catenin positive nuclear staining positive cells appeared in BIO treated cells. and The cells exposed to BIO showed that the cyclin D1 protein and the cells in S stage and G₂/M stage moderately increased, the protein level of Bcl-2 moderately decreased, and the cell apoptosis rate was significantly lower than those in control cells. Furthermore, the morphological changes of the SW480 cells were observed 24 h after BIO treatment. CONCLUSION: Our results indicate that GSK-3β inhibitor BIO participates in the cellular processes of promoting proliferation and inhibiting apoptosis in colon carcinoma cells. The mechanisms are mainly associated with activating the β-catenin pathway and regulating the balance of Bcl-2 pathway, and the up-regulation of β-catenin is most likely the possible factor for SW480 cell regression.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.