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1.
AIM: To study the mechanism of multidrug resistance (MDR) of leukemia cells induced by homoharringtonine (HHT) and the reversal effect of mifepristone on MDR.METHODS: Human leukemia cell line K562 was induced into MDR cell line by intermittent administration of high dose of HHT.MTT assay was used to detect the sensitivity of these MDR cells to all sorts of chemotherapeutic agents with or without mifepristone.The cytotoxicity of mifepristone was also observed.RT-PCR was used to detect the expression of MDR1 gene and glucosylceramide synthase (GCS) gene.Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells with or without mifepristone.Immunohistochemistry was used to detect the expression of Bcl-2,Bax and caspase-3 in these MDR cells with or without mifepristone.RESULTS: MDR cell line K562/HHT was acquired after induced by HHT for 2 months.This MDR cell line possessed the ability of 462.6 fold resistance to HHT and cross-resistance to adriamycin,vincristine and etoposide.The expression of MDR1 gene,GCS gene,P-glucoprotein and Bcl-2/Bax ratio in K562/HHT cells were significantly higher than those in K562 cells (P<0.05).The caspase-3 expression and the accumulative value of intracellular DNR in K562/HHT cells were significantly lower than those in K562 cells (P<0.05).10 μmol/L mifepristone reversed the resistance of K562/HHT cells to HHT,adriamycin,vincristine and etoposide at different levels.The Bcl-2/Bax ratio,caspase-3 expression and accumulative value of intracellular DNR in K562/HHT cells treated with RU486 were significantly different compared with K562/HHT cells without RU486 treatment (P<0.05).CONCLUSIONS: Leukemia cell line K562 can be induced into MDR cell line K562/HHT by HHT.P-glucoprotein,GCS,Bcl-2/Bax ratio and caspase-3 may play an important role in K562/HHT cells.Mifepristone can reverse MDR in K562/HHT cells by decreasing the accumulative value of intracellular drug and regulating the expression of Bcl-2,Bax and caspase-3.  相似文献   

2.
AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47phox and p22phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47phox and p22phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.  相似文献   

3.
AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨm), reactive oxygen species (ROS) and the concentration of intracellular Ca2+ ([Ca2+]i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured by the method of colorimetry. The levels of cytochrome C, Bcl-2, Bax, β-catenin and phosphorylated low-density lipoprotein receptor-related protein 6 (p-LRP6) were determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨm, and increased the levels of ROS, cytochrome C and[Ca2+]i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.  相似文献   

4.
AIM: To investigate the role of reactive oxygen species(ROS)-mediated mitochondrial oxidative injury in isonicotinyl hydrazide(INH)-induced DNA damage and the protective effect of quercetin on L-02 cells. METHODS: The injury model of hepatocyte L-02cells in vitro induced by INH was established. The cells were divided into control group, INH group, low-dose quercetin group and high-dose quercetin group. The DNA damage of L-02 cells was evaluated by the comet test. The mitochondrion was prepared, and the level of mitochondrial ROS and the value of mitochondrial membrane potential(ΔΨm) were detected by fluorescent probes DCFH-DA and rhodamine 123. The content of MDA was measured by TBA method. The activity of SOD was assessed with the xanthine oxidase method. The protein expression of Bcl-2 and Bax was determined by Western blotting, and the value of Bax/Bcl-2 was calculated. RESULTS: INH induced obvious DNA damage, increased the level of mitochondrial ROS, the content of MDA and the value of Bax/Bcl-2, and markedly reduced the value of ΔΨm and the activity of SOD in the L-02 cells. Quercetin attenuated DNA damage, reduced the level of mitochondrial ROS, elevated the value of ΔΨm, declined the content of MDA, increased the activity of SOD and decreased the value of Bax/Bcl-2 in the L-02 cells. CONCLUSION: INH induces DNA damage in L-02 cells by generation of mitochondrial oxidative stress. Quercetin has a protective effect on L-02 cells to attenuate the INH-induced DNA damage by inhibiting ROS-mediated mitochondrial oxidative damage.  相似文献   

5.
WANG Li-min 《园艺学报》2015,31(9):1715-1719
AIM: To investigate the effect of Ginsenoside Rh2(Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS: The cell viability was determined by MTT assay. MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining. The expression of Bcl-2, Bax, cytochrome C(Cyt C) and cleaved caspase-3 were measured by Western blot.RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner. Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased, while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group(P<0.05). The protein level of cleaved caspase-3 was also increased(P<0.05).CONCLUSION: Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway, suggesting that Rh2 is a novel approach for the treatment of osteosarcoma.  相似文献   

6.
ZHENG Ying  JIANG Kai 《园艺学报》2015,31(9):1578-1583
AIM: To determine whether imperatorin would enhance the effect of doxorubicin therapy on cervical cancer in vitro.METHODS: The viability of HeLa cells treated with imperatorin and doxorubicin was determined by MTT assay in vitro. The expression of Bcl-2 protein family(Mcl-1, Bcl-2, Bcl-xL, Bad and Bax) in HeLa cells treated with imperatorin and doxorubicin was evaluated by Western blot analysis. The apoptosis and mitochondrial membrane potential(ΔΨm) in the HeLa cells treated with imperatorin and doxorubicin were analyzed by flow cytometry. A Mcl-1 expression vector was constructed, and its role in the cytotoxicity of imperatorin plus doxorubicin to HeLa cells was detected by MTT assay.RESULTS: Addition of imperatorin significantly enhanced the cytotoxicity of doxorubicin to HeLa cells in vitro. Mcl-1 expression was down-regulated by imperatorin but was not influenced by doxorubicin in the HeLa cells. A combination of imperatorin and doxorubicin induced apoptosis and ΔΨm collapse more significantly compared with the treatment with imperatorin or doxorubicin alone. Furthermore, the imperatorin-induced sensitization for doxorubicin cytotoxicity to HeLa cells was abolished by the transfection with Mcl-1 expression plasmid.CONCLUSION: The combination of doxorubicin with imperatorin enhances the antitumor effect of doxorubicin on cervical cancer cells via targeting Mcl-1.  相似文献   

7.
LI Xue-yan  XU Xia 《园艺学报》2015,31(11):1933-1942
AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.  相似文献   

8.
AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.  相似文献   

9.
AIM: To establish the gastric cancerous multidrug resistance cell stain BGC823/5-FU and investigate the relationship between the resistance and the expression of apoptosis related protein Survivin, Bcl-2, Bax and caspase-3. METHODS: Human gastric cancer cell line BGC823 was induced into MDR cell line by intermittent administration of high dose of 5-FU. MTT assay was used to detect the sensitivity of these MDR cells to some chemotherapeutic agents. Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells. Western blotting was used to detect the expression of Survivin, Bcl-2, Bax and caspase-3. RESULTS: The resistance cell stain BGC823/5-FU was established, which possessed the ability of 10.82 fold resistance to 5-FU and cross-resistance to adriamycin, mitomycin C and cisplatin. The expression of P-glucoprotein was higher in BGC823/5-FU cells than that in BGC823 cells, while the accumulative value of intracellular DNR was decreased in BGC823/5-FU cells. Compared with its parent cells, expressions of Bax and caspase-3 in BGC823/5-FU cells were significantly down-regulated, surviving and Bcl-2 were upregulated in BGC823/5-FU cells. CONCLUSION: Gastric cancer cell line BGC823 has been induced into MDR cell line BGC823/5-FU. P-glucoprotein, Survivin, Bcl-2/Bax ratio and caspase-3 may play an important role in MDR of BGC823/5-FU cells.  相似文献   

10.
AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH,the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.  相似文献   

11.
AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC50 of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G2 phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.  相似文献   

12.
AIM To investigate the effect of nisin on apoptosis of human osteosarcoma MG63 cells and its related oxidative stress mechanism. METHODS The MG63 cells were cultured in the medium containing different concentrations of nisin with or without antioxidant N-acetyl-L-cysteine （NAC）. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry with annexin-V/PI staining. The production of intracellular reactive oxygen species （ROS） was detected by redox-sensitive dye 2',7'-dichlorofluorescein diacetate （DCFH-DA）. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide （JC-1） was used to detect the changes of mitochondrial membrane potential （MMP）. The protein levels of apoptosis-associated molecules Bax, Bcl-2 and cleaved caspase-3 were determined by Western blot. RESULTS Nisin decreased the viability of MG63 cells and promoted the apoptosis in a dose-dependent manner. It also up-regulated the protein level of cleaved caspase-3, increased the protein expression ratio of Bax/Bcl-2, triggered a large amount of intracellular ROS generation and reduced the MMP （P<0.05）. Moreover, antioxidant NAC significantly inhibited nisin-induced apoptosis of MG63 cells, down-regulated the protein level of cleaved caspase-3, decreased Bax/Bcl-2 ratio, reduced intracellular ROS level, and restored the MMP （P<0.05）. CONCLUSION Nisin may promote oxidative stress in human osteosarcoma cells, activate mitochondrial apoptosis pathway, and eventually induce apoptosis.  相似文献   

13.
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.  相似文献   

14.
AIM：To investigate the effect of ethyl docosahexaenoate (Et-DHA) on the apoptosis of human hepatocarcinoma HepG2 cells. METHODS：HepG2 cells were used to test the anticarcinogenicity of Et-DHA. The direct inhibition of HepG2 cells by Et-DHA was detected by MTT. Nuclear morphological features of the HepG2 cells were observed under fluorescence microscope after staining with Hochest 33258. The levels of Bax, Bak, Bid, Bcl-2, Smac and cytochrome C (Cyt C) in mitochondria and cytosol, the cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in cytosol, as well as the release of reactive oxygen species (ROS), total superoxide dismutase (SOD) and caspase-9 activity in the Et-DHA-treated HepG2 cells were determined by Western blotting and ELISA. Furthermore, by co-culturing the HepG2 cells with T cells, the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were observed, and the level of granzyme B was detected. RESULTS：Et-DHA significantly inhibited the growth of HepG2 cells in a concentration- and time-dependent manner. The ROS release and caspase-9 activity increased markedly in Et-DHA-treated HepG2 cells, and no significant change of the total SOD activity was observed. The levels of the pro-apoptotic proteins Bax, Bak and Bid in mitochondria increased, the anti-apoptotic protein Bcl-2 as well as mitochondrial Cyt C and Smac levels decreased, and the cytoplasmic Cyt C, Smac, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved Bid levels showed dose-dependent increases. Additionally, the degree of Et-DHA-induced apoptosis in HepG2 cells in the co-culture group (T cells+HepG2 cells) showed a further increase as compared with the HepG2 cells treated with Et-DHA alone. Due to Et-DHA inducing elevation of granzyme B level in the T cells, the granzyme B released into HepG2 cells was significantly increased. CONCLUSION:Et-DHA might induce the apoptosis of HepG2 cells through activation of caspase-3 mainly via a mitochondrial intrinsic pathway and a caspase-8 pathway, and promote the increase in granzyme B indirectly by activating T cells, thus enhancing the cytotoxic effect on HepG2 cells.  相似文献   

15.
AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC50) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G0/G1. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G0/G1 phase.  相似文献   

16.
AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.  相似文献   

17.
SHAO Qi  CAO Fei  LI Mei  ZHANG Yan 《园艺学报》2016,32(12):2233-2238
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-AKTSer473 and p-AKTThr308, 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.  相似文献   

18.
AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.  相似文献   

19.
AIM：To investigate the effect of ferulic acid (FA) on the apoptosis of PC12 cells induced by kainic acid (KA) in vitro. METHODS：In order to establish an Alzheimer disease neuronal cell model, the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol/L. These model neurons were divided into KA model group and 3 groups treated with FA at doses of 25, 50 and 100 μmol/L, respectively. At the same time, normal group was established without KA pretreatment. The viability of the PC12 cells was detected by MTT assay. The expression of Bcl-2, Bax and cytochrome C (Cyt C) was determined by immunocytochemical method. Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V/PI double staining. The protein levels of Bcl-2, Bax and Cyt C were analyzed by Western blotting. RESULTS：The cell survival rate, the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased (P<0.01),while the expression of Bax and Cyt C was obviously increased compared with normal control group (P<0.01). The apoptotic rate in KA model group was obviously increased compared with normal control group (P<0.01) After the intervention of FA, the cell survival rates were increased and the apoptotic rates were decreased. Furthermore, the positive rate and expression of Bcl-2, and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased, while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group (P<0.05 or P<0.01). CONCLUSION：KA obviously induces apoptosis of PC12 cells. FA had obvious protective effect on PC12 cells against the toxicity of KA. FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax, thus improving the survival rate of PC12 cells.  相似文献   

20.
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.  相似文献   

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