The antisense phosphorothioate oligodeoxynucleotides of bcl- 2 oncogene enhances the apoptotic effect of As2O3 on NB4 leukemic cells

AIM: To study and explore whether the antisense phosphorothioate oligodeoxynucleotides (ASODN) of bcl-2 oncogene would increase the apoptotic effect of As₂O₃ on NB4 leukemic cells. METHODS: The biological and morphological changes in NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were observed. The changes in DNA content and Bcl-2 protein of NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were determined by tissue chemistry and flowcytometry. RESULTS: There was much more apoptotic effect of As₂O₃ on NB4 cells while it combined with bcl- 2 ASODN (ASODN 10.0 μmol/L+ As₂O₃ 0.25 μmol/L) than alone(ASODN 10.0 μmol/L or As₂O₃ 0.25μmol/L),and so did inhibitory effect of Bcl-2 protein expression by flow cytometry. CONCLUSION:The bcl-2 ASODN can enhance the apoptotic effect of As₂O₃ on NB4 leukemic cells.     

Different inhibition of hepatocarcinoma cell growth by As2O3 in SMMC-7721 and BEL-7402 cell lines

AIM: To explore the different inhibitory effect of arsenic trioxide (As₂O₃) on hepatocarcinoma cell growth in SMMC-7721 and BEL-7402 cell lines and its mechanism. METHODS: The cell culture and trypan blue staining were used to study the inhibitory effect of arsenic trioxide on cell growth, and the glutathione (GSH) contents in hepatocarcinoma cells treated with arsenic trioxide were detected. RESULTS: Arsenic trioxide inhibited the growth of BEL-7402 cells in a time and dose-dependent manner. The inhibitory effect was significant at a lower dose of 0.50 μmol/L for 24 h, however, to SMMC-7721 cells, a higher dose of 2.00 μmol/L for 96 h was needed. The inhibitory rate of arsenic trioxide (0.25-2.00 μmol/L) on BEL-7402 cell growth was higher than that on SMMC-7721 cells. The content of GSH in SMMC-7721 cells was much higher than that in BEL-7402 cells . CONCLUSION: There was a significant difference in inhibition of hepatocarcinoma cell growth by arsenic trioxide between BEL-7402 and SMMC-7721 cell lines, the cause of which may be due to the difference in GSH content in BEL-7402 and SMMC-7721 cells.     

Study of establishment of K562/HHT cell line and reversal of multidrug resistance by antiprogestin drug mifepristone

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.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

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.     

中晚熟抗黑星病梨新品种--秋水晶

‘秋水晶’梨是陕西省果树研究所用‘砀山酥’和‘栖霞大香水’杂交培育的中晚熟抗黑星病的梨新品种。果实发育期 14 5d ,平均单果质量 2 0 8g ,大果可达 359g ,椭圆形 ,淡黄色。肉质细 ,酥脆 ,风味香甜 ,汁液多 ,口感佳。丰产、稳产。成熟期正值瓜果淡季 ,可在中秋节消费高峰期上市     

Effect of decitabine on resistance of K562/A02 cells to adriamycin

AIM: To investigate the effect of decitabine (DAC) on the resistance of human chronic myeloid leukemia cell line K562/A02 to adriamycin (ADR), and to explore the possible mechanism. METHODS: The K562/A02 cell line and its parental cell line K562 were treated with different concentrations of ADR or DAC alone, or in combination. The cytotoxic effects of these 2 agents were determined by CCK-8 assay. The degree of DNA methylation was evaluated by Sequenom MassARRAY system and colorimetric method. The cell cycle distribution and the apoptotic rate were determined by flow cytometry. RESULTS: K562/A02 cells were more significantly resistant to ADR than K562 cells.The half maximal inhibitory concentration of ADR for 24 h of the K562/A02 cells was about 50 times higher than that of the K562 cells. To DAC, in the concentration range of 0.5~8 μmol/L, K562/A02 cells were more sensitive than K562 cells. As compared with the same concentrations (4.31 μmol/L and 17.24 μmol/L) of ADR alone, the combination with 1 μmol/L DAC significantly improved the sensitivity of K562/A02 cells to ADR. Both DAC and ADR affected the cell cycle progression and apoptotic rate of K562/A02 cells. DAC (1 μmol/L) treatment mainly showed S phase arrest and increased early apoptotic rate for 24 h, and G₂/M phase arrest and increased late apoptosis and necrosis for 48 h in a time-related manner. ADR treatment showed G₂/M phase arrest and increased late apoptosis and necrosis in a concentration-dependent manner. In combination with 1 μmol/L DAC, the effect of ADR on the cell cycle distribution was further enhanced, showing more obvious G₂/M phase arrest, but no significant difference of the apoptotic rate was observed. The degree of methylation in the genome had no significant difference between the 2 cells, and it before and after DAC treatment had no significant change. CONCLUSION: DAC enhances the sensitivity of K562/A02 cells to ADR, showing drug resistance-reversing potential. The mechanism may be related to regulating cell cycle progression and promoting apoptosis and necrosis of K562/A02 cells.     

Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

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 (ΔΨ_m), 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 ΔΨ_m, 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.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

AIM: To investigate the synergetic inhibitory effect of sorafenib and daunorubicin (DNR) on K562 and U937 cells. METHODS: The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry (Annexin Ⅴ/PI staining) and Hoechst 33258 staining assay. p-ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jins Method. RESULTS: Combination of sorafenib and DNR showed synergistic growth inhibition (q>1.15, P<0.01) and synergistic promotion of apoptosis (q>1.15, P<0.05) in K562 and U937 cells. The level of p-ERK1/2 in K562 cells was obviously higher than that in U937 cells (P<0.01). p-ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 cells for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION: Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti-leukemic activity of DNR in K562 and U937 cells via down-regulation of p-ERK1/2 expression.     

Total flavonoids of onion attenuate hydrogen peroxide-induced oxidative damage in retinal pigment epithelial cells

AIM: To investigate the effects of total flavonoids of onion (FO) on hydrogen peroxide (H₂O₂)-induced oxidative damage in retinal pigment epithelial cells. METHODS: The retinal pigment epithelium ARPE-19 cells were divided into 5 groups:control group, H₂O₂ group (treated with H₂O₂), FO-L+H₂O₂ group (treated with H₂O₂ and low concentration of FO), FO-M+H₂O₂ group (treated with H₂O₂ and medium concentration of FO) and FO-H+H₂O₂ group (treated with H₂O₂ and high concentration of FO). The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. DCFH-DA staining was used to detect reactive oxygen species (ROS) level in the cells. WST assay was used to detect superoxide dismutase (SOD) activity. The content of malonaldehyde (MDA) was measured by TBA method. Mitochondrial membrane potential was analyzed by JC-1 staining. The protein levels of cytochrome C (Cyt C) in the cytoplasm, and cleaved caspase-3 and cleaved caspase-9 in the cells were determined by Western blot. RESULTS: Treatment with H₂O₂ decreased ARPE-19 cell viability, increased the apoptotic rate and the level of ROS in the cells, decreased SOD activity, increased the content of MDA, decreased mitochondrial membrane potential, and increased the protein levels of Cyt C in the cytoplasm and cleaved caspase-3 and cleaved caspase-9 in the cells (P<0.05). Compared with H₂O₂ group, the cell viability in FO-L+H₂O₂ group, FO-M+H₂O₂ group and FO-H+H₂O₂ group was increased, the apoptotic rates were decreased, the levels of ROS were decreased, SOD activity was increased, the content of MDA was decreased, mitochondrial membrane potential was increased, the protein level of Cyt C was decreased in the cytoplasm, and the protein levels of cleaved caspase-3 and cleaved caspase-9 protein in the cells were decreased gradually (P<0.05). CONCLUSION: Total flavonoids of onion reduce H₂O₂-induced oxidative damage in retinal pigment epithelial cells.     

Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells

AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H₂O₂) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H₂O₂ group, 6-gingerol group (6-gingerol + H₂O₂) and LY294002 group (6-gingerol + H₂O₂ + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H₂O₂ at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H₂O₂ group were significantly increased compared with control group. The mitochondrial edema was obvious in H₂O₂ group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H₂O₂-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H₂O₂ induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H₂O₂-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

Alteration of circulating endothelial cells from acute promyelocytic leukemia patients before and after treatment and its influential factors

AIM: To determine the biological feature of circulating endothelial cells (CECs) in acute promyelocytic leukemia (APL) patients before and after treatment, and to analyze the relationship between CECs and the clinical characteristics. METHODS: The CECs were sorted from peripheral blood by magnetic-activated cell sorting and then counted by 3-color flow cytometry. The cells were identified by immunofluorescence staining for the expression of CD146, CD31, CD144, VEGFR-2, CD45 and CD133. The CECs were cultured in vitro, and the tube formation and colony-forming rate were determined. RESULTS: Increased quantity of CECs was observed in CD34 positive group and group with WBC>10×10⁹/L (P<0.05). The quantity of CECs had a significant difference among low risk, medium risk and high risk groups (P<0.05). The positive rate of CD133 and quantity of CECs significantly reduced in 32 APL patients when they gain complete remission after treatment (P<0.05). The amount of tube formation and colony-forming rate were significantly reduced after treatment (P<0.05). The ratio of CECs quantity from APL patients after treatment to that before treatment had a negative correlation with arsenic concentration in urine on day 7 during As₂O₃ treatment (P<0.05). CONCLUSION: Accurately counting CECs may be helpful for evaluating prognosis and designing treatment strategy.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM:To investigate the effects of rapamycin (Rapa) on hydrogen peroxide (H₂O₂)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS:The human umbilical vascular endothelial cells (HUVECs) were divided into 4 groups:control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine (3-MA)+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase (SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope (TEM). The protein levels of phosphorylated Rb (p-Rb), Rb, p21, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS:Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells (P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group (P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone (P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-Ⅱ were increased (P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected (P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-Ⅱ was detected. CONCLUSION:Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.     

Establishment of gastric cancerous multi-drug resistance cell stain BGC823/5-FU and its resistance mechanism

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.     

An oxidative injury model of human kidney epithelial cells

AIM: To establish an injured cell model using human kidney proximal tubule epithelial cell line (HKC) to mimic the oxidative injury by hydrogen peroxide (H₂O₂). METHODS: The cell viability, the content of malondialdehyde (MDA) in the culture supernatant and the activity of intracellular superoxide dismutase (SOD) were detected to investigate the degree of cell injury. Osteopontin (OPN) expressed on the cell membrane surface were observed by laser confocal microscopy before and after cell injury. The changes of cellular morphology and the ultrastructure of membrane surface were observed under scanning electronic microscope. RESULTS: After HKC cells were treated with H₂O₂ at the concentration of 1 mmol/L for different time, the cell viability and the activity of SOD decreased and the content of MDA increased. The expression level of OPN significantly increased and reached to maximae at 1 h. The injured cells appeared shriveled and rough surface, and the shedding of most flagellae was also observed. CONCLUSION: H₂O₂ induces severer injury in HKC cells, including not only the cell viability and membrane surface ultrastructure, but also the OPN expression on the membrane, which could bind calcium oxalate crystal. Therefore, treatment with H₂O₂ at the concentration of 1 mmol/L for 1 h can be used to establish an oxidative injury model in HKC cells.     

Effects of PDGFRα on melanocyte apoptosis induced by hydrogen peroxide

AIM: To investigate the effects of platelet-derived growth factor receptor α (PDGFRα) on melanocyte apoptosis induced by hydrogen peroxide (H₂O₂). METHODS: Melanocyte PIGI was used as the research object. After exposed to H₂O₂ at different concentrations, the cell viability was detected by MTT assay. The PIGI cells were transfec-ted with empty vector pCMV6 or PDGFRα over-expression vector pCMV6-PDGFRα. The transfection efficiency was determined by RT-qPCR and Western blot. The effect of H₂O₂ on the viability of the PIGI cells after over-expression of PDGFRα was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. The protein levels of p38, p-p38 and cleaved caspase-3 in the cells were detected by Western blot. DCDHF-DA was used to estemate the generation of reactive oxygen species (ROS) in the cells. RESULTS: The viability of PIGI cells decreased after exposed to H₂O₂ (P<0.05), and the half maximal inhibitory concentration of H₂O₂ was 0.7 mmol/L. Transfection with PDGFRα over-expression vector successfully induced high expression of PDGFRα at mRNA and protein levels in the PIGI cells, and increased the viability of the cells with H₂O₂ treatment (P<0.05). Over-expression of PDGFRα decreased the apoptotic rate of PIGI cells treated with H₂O₂ (P<0.05), and the level of ROS in the cells (P<0.05). The protein levels of cleaved caspase-3 and p-p38 were also decreased (P<0.05). CONCLUSION: PDGFRα inhibits the apoptosis of melanocytes induced by H₂O₂, partially reverses the growth inhibition of melanocytes by H₂O₂, and decreases the ROS level. The mechanism may be related to regulating the protein levels of p-p38 and cleaved caspase-3 in the cells.