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Aim To explore whether propofol attenuates neuroinflammation and brain damage via modulating PI3K/Akt signaling pathway following focal cerebral ischemia in rats, and further investigate the possible mechanisms. Methods Sprague-Dawley rats which underwent the cerebral ischemic injury by the suture occlusion model were randomly divided into sham operation, MCAO, propofol-treated and LY294002 groups. Neurological deficit scores, cerebral infarct size, and cerebral water content were measured , then the myeloperoxidase (MPO) activities in rat brain were measured as an index of neutrophil infiltration. The content of TNF-α and IL-1β in blood was determined using ELISA; the expressions of p-Akt and Akt in rat brain were detected by Western blot. Results Propofol reduced neurological deficit scores, cerebral infarct size, cerebral water content, MPO activity , TNF-α and IL-1β content, which were all abolished by LY294002. Propofol up-regulated the expression of p-Akt, which was inhibited by LY294002. Conclusion Propofol attenuates neuroinflammation and ischemic brain damage via modulating the PI3K/Akt signaling pathway.
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Objective To explore the effects of phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (AKT) signal pathway inhibitor LY294002 on retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) mice.Methods Totally 60 C57BL/6J mice were collected and randomly divided into the experimental group and control group,with 30 mice in each group.Then OIR model was induced by Smith methods.Rats in the experimental group were intravitreally injected with 0.5 μL LY294002,while the control group was given the same amount of phosphate buffer saline (PBS) one day before out of the incubator.Retinal sections with HE staining were applied to count the number of neovascular cell nuclei breaking through the inner limiting membrane,as well as the protein and mRNA expression of pAKT and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry and RT-PCR.Results The number of retinal neovascular cell nucleus in the experimental group was obviously smaller than that in the control group (P ＜ 0.05).The protein expression of pAKT and VEGF was weakly expressed,and the absorbance (A) value of the positive cells was decreased in the experimental group compared with the control group (all P ＜ 0.05).The mRNA expression of AKT and VEGF was obviously decreased in the experimental group compared with the control group (all P ＜ 0.05).Conclusion The development of RNV in OIR mice can be markedly inhibited by LY294002 inhibiting PI3K/AKT pathway,and therefore LY294002 is expected to be an effective method for preventing vascular proliferative retinopathy.
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Objective: To investigate the effects of hydroxysafflor yellow A (HSYA) on the proliferation, apoptosis and migration of hepatocellular carcinoma cells, and to explore whether HSYA plays a role through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt) signaling pathway. Methods: After the treatment with HSYA and PI3K inhibitor LY294002, CCK-8 assay, clone formation assay and scratch healing assay were used to detect the proliferation, clone formation and migration abilities of human hepatoma HepG2, Hep3B and SMMC7721 cells, respectively. FCM was used to detect the effect of HSYA on the apoptosis of HepG2 cells. Western blotting was used to detect the expressions of matrix metalloprotein 2 (MMP2), caspase 3, cleaved-caspase 3 and phospho-Akt (p-Akt). Results: Both 50 μmol/L HSYA and 10 μmol/L LY294002 inhibited the proliferation of human hepatoma HepG2, Hep3B and SMMC7721 cells (all P < 0.05). The clone formation rate and migration rate of HepG2, Hep3B and SMMC7721 cells treated with 50 μmol/L HSYA, 10 μmol/ L LY294002 and 50 μmol/L HSYA in combination with 10 μmol/L LY294002 were lower than those in the untreated control group (all P < 0.05). The clone formation rate and migration rate of HepG2, Hep3B and SMMC7721 cells in HSYA combined with LY294002 treatment group were lower than those in HSYA or LY294002 alone treatment group (all P < 0.01). The apoptosis rates of HepG2 cells in HSYA, LY294002 and HSYA combined with LY294002 treatment groups were higher than those in the untreated control group (all P < 0.05). The apoptosis rate of HepG2 cells in HSYA combined with LY294002 treatment group was higher than that in HSYA or LY294002 alone treatment group (P < 0.01). After the treatment with HSYA, LY294002 and HSYA combined with LY294002, the expression levels of p-Akt, MMP2 and caspase 3 in HepG2 cells were lower than those in the untreated control group (all P < 0.01), and the expression level of cleaved-caspase 3 was higher than that in the untreated control group (P < 0.01). The effects of HSYA combined with LY294002 treatment on the expressions of p-Akt, MMP2, caspase 3 and cleaved-caspase 3 were better than those of HSYA or LY294002 alone treatment group (all P < 0.01). Conclusion: HSYA can inhibit the proliferation and migration abilities of hepatocellular carcinoma HepG2, Hep3B and SMMC7721 cells, and induce the apoptosis of HepG2 cells by blocking PI3K/Akt signaling pathway.
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Objective:To examine the role of LY294002 in cardiac function and myocardial structure in dilated cardiomyopathy (DCM) rats.Methods:Fifty-two male SD rats were randomly assigned to a control group (n=16) and a DCM group (n=36).The DCM rats were induced by intraperitoneal injection of adriamycin,and the control rats were given normal saline.After observation for 2 weeks,6 rats from each group were killed randomly.In the end of the 8th week,the 24 DCM rats were randomly assigned to a DCM group (n=12) and a LY294002 group (n=12),which were given normal saline and LY294002,respectively.In the end of the 8th week and 16th week,the cardiac function was analyzed by ultrasonic cardiogram (UCG) and the plasma was collected to test the level of N-terminal pro-brain natriuretic peptide (NT-pro BNP).HE and Van Gieson (VG) staining were performed to calculate the collagen volume fraction (CVF).Results:Compared with the control group,the left ventricular end-diastolic dimension (LVEDD),left ventricular end-systolic dimension (LVESD) and NT-proBNP level of in the DCM rats were increased obviously,while the left ventricular ejection fraction (LVEF),left ventricular fractional shortening (LVFS) in the DCM rats were decreased obviously (P＜0.01).These changes were consistent with DCM characteristics.Compared with the DCM group,the LVEDD,LVESD and NT-proBNP levels in the LY294002 group were decreased,while the LVEF and LVFS were increased (P＜0.05).Histopathology showed that the myocardium in the DCM rats was fibrotic and the CVF was increased compared with the control rats (P＜0.01).The myocardial structure was improved in the LY294002 group compared to the DCM group.Conclusion:LY294002 can reduce the myocardial fibrosis in the DCM rats and improve the cardiac function.
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Aim To investigate the effect of CD44 anti-body-A3 D8 on the expression of IL-3 Rα and down-stream PI3K/Akt in NB4 cells. Methods The ex-pression of IL-3 Rα mRNA was detected by real-time quantitative RT-PCR, the IL-3Rα protein expression and changes of PI3 K/Akt signal pathway in NB4 cells treated with A3D8 were analyzed by Western blot. An-nexin-V-FITC/PI double staining flow cytometry was u-tilized to detect the apoptotic cells. The inhibitor of PI3 K/Akt signaling LY294002 combined with A3 D8 was used to inhibit the PI3K/Akt in NB4 cells. Re-sults After treated with A3 D8 , both the transcription-al level and translational level of IL-3 Rα were remark-ably reduced, and the PI3K/Akt pathway was inhibi-ted. LY294002 improved the inhibitory and apoptotic effects of A3D8 on NB4 cells. Conclusion CD44 antibody A3 D8 can downregulate the expression of IL-3Rα and inhibit the downstream PI3K/Akt pathway.
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Objective To explore the potential mechanisms of non-small cell lung carcinoma cells to rsTRAIL protein-induced apoptosis by phosphatidylinositol 3′-kinase (PI3K/Akt)inhibitor LY294002,and to provide new ways to increase killing activities of rsTRAIL protein for non-small cell lung cancer.Methods The A549 cells at logarithmic growth phase were selected and randomly divided into rsTRAIL group and LY294002+rsTRAIL group. The inhibitory rate of growth of the A549 cells was tested by MTT assay.The cell cycle and apoptotic rate were detected by flow cytometry analysis. The expression levels of Ser473 phosphorylated form of Akt (p-Akt),c-FLIPL protein and Bcl-2 protein in the A549 cells in two groups were analyzed by Western blotting method. Results The inhibitory rate of growth of the A549 cells in LY294002+rsTRAIL group (74.6 %± 2.63%)was higher than that in rsTRAIL group (5.61% ± 0.32%) (P< 0.05 ). Compared with rsTRAIL group, the percentage of the cells at G0/G1 phase in LY294002+rsTRAIL group was increased(P<0.05)and the percentage of the cells at S phase was decreased(P<0.05).The apoptotic rate of the A549 cells in LY294002+rsTRAIL group (61.5%±3.02%)was higher than that in rsTRAIL group (3.21%±0.96%)(P<0.05). The Western blotting results showed that the expression levels of p-Akt, c-FLIPL and Bcl-2 proteins in the A549 cells in LY294002+rsTRAIL group were decreased (P<0.05 )and the ratio of Bax/Bcl-2 was increased (P<0.05 ) compared with rsTRAIL group.Conclusion LY294002 can increase the killing activity of rsTRAIL protein in A549 cells by inhibiting the activity of PI3K.
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Aim To investigate the effects of 17β-es-tradiol on the apoptosis induced by ketamine in primary cultured cortical neurons. Methods Primary cultured cortical neurons were treated with different concentra-tions of ketamine or 17β-estradiol respectively. 24 hours after various treatments, neuron viability was measured by MTT assay. The structure of neurons was analyzed using microscope. Apoptotic neurons were de-termined by the TUNEL assay. The level of pAkt ex-pression was analyzed by Western blot. ResultsCompared with the control group, ketamine decreased neuron viability in a dose-dependent manner. Com-pared with ketamine group, 17β-estradiol increased neuron viability in a dose-dependent manner. Lack of three-dimensional sense,faded color,uncleared outline were observed, and fractured neuron axons or neurons death were also observed in neurons treated by 100μmol · L-1 ketamine. 100 μmol · L-1 ketamine in-creased the number of apoptotic neurons and decreased the expression of pAkt. 0.1 μmol · L-1 17β-estradiol decreased the number of apoptotic neurons and in-creased the expression of pAkt. LY294002 inhibited the protective effects of 17β-estradiol, the number of apoptotic neurons increased, and the level of pAkt de-creased significantly. Conclusion 17β-estradiol ex-erts the neuroprotective effects against ketamine-in-duced neuroapoptosis by activating the PI3 K/Akt sig-naling pathway.
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Objective To investigate the effect of LY294002 on proliferation of cultured human glomerular mesan-gial cells, and to study on the mechanism underlying this proliferation. Methods Different concentrations of LY294002 (0.02,0.2,2,20,100 mg/L) were administrated to the cultured human glomerular mesangial cells. The effects of mesangial cell proliferation were measured using CCK-8 colorimetric assay. Under appropriate concentrations, proliferation of cultured mesangial cells were measured using CCK-8 at 0.5, 1, 24, 48 hours of drug administration time. Results LY294002(0.02 mg/L)didn’t obviously inhibited the growth of mesangial cells(P>0.05), the inhibition rate was 5.05%. The effect of LY294002 on the cells decreased with rising concentration (0.2 to 20 mg/L) in a dose-dependent manner (P0.05), but inhibited proliferation gradually from 1 to 48 h (P<0.05). Conclu-sion Within the certain range of concentration and time, the LY294002 inhibits the proliferation of human glomerular me-sangial cells, and PI3K/Akt/mTOR signaling pathway may regulate the proliferation of human mesangial cell.
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Background Protein kinase B (Akt) is the center of multiple cellular signaling pathways,and it participates in the regulation of cell function,such as proliferation,migration,and metabolism of cells.Phosphoinositide 3-kinase (PI3K) promotes activation of Akt and therefore triggers many signal pathways.PI3K inhibitor can silent Akt,but whether it can affect the biological behavior of lens epithelial cells (LECs) during the posterior capsular opacity (PCO) is worthy of investigation.Objective This study was to explore the effect of LY294002,a PI3K inhibitor,on Akt activation in human LECs.Methods Human LECs strain,HLEC-B3 cells,were cultured and passaged.The cells were incubated to 96-well plate for 24 hours,then LY294002 was added with the final concentration at 0,10,20,30,40,50,60,70,80 μmol/L,respectively.After incubation for 24 hours,cell counting kit-8 (CCK-8) was used to detect the inhibitory rate of cell proliferation.Transforming growth factor-β2 (TGF-β2) of 10 μg/L was added in the medium of cells as TGF-β2 group,TGF-β2 + LY294002 (20 μmol/L) was used to coculture the other group of cells,and the cells without TGF-β2 and LY294002 served as the control group.Phosphorylation of Akt (p-Akt) in the cells was detected by laser scan confocal immunofluorescence microscope.The expression level of p-Akt was evaluated using Western blot.Results CCK-8 assay showed that the A value of the cells was gradually reduced with the increase of LY294002 concentration (Fgroup =9.72,P =0.00),but the A value was significant raised along with the lapse of time (Ftime =1737.54,P=0.00).Confocal immunofluorescence revealed that a little of p-Akt was expressed in the control cells.After induced by TGF-β2,lots of red fluorescence of p-Akt was seen in cells around the cell membranes.But in the TGF-β2+LY294002 co-culture cells,the fluorescence of Akt was much weaker.Western blot showed that the expression level of p-Akt was 0.91±0.08,1.48±0.13 and 0.95±0.19 in the control group,TGF-β2 group and TGF-β2 +LY294002 group,respectively,with a significant difference among the three groups (F =15.04,P =0.00).Conclusions LY294002 can inhibit the activation of Akt induced by TGF-β2.LY294002 may have utility in the prevention and treatment of PCO.
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Objective To study the effects of tyrosine kinase receptor B-brain-derived neurotrophic factor (TrkB-BDNF) signal pathway on the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9(MMP-9) of neuroblastoma.Methods We used all-trans retinoic acid (ATRA) to induce the high expression of TrkB in the SH-SY5Y cell line,and then added the ectogenid BDNF to activate the TrkB-BDNF and its three downstream signal pathways.TrkB-BDNF signal pathway was inhibited by specific tyrosine kinase inhibitor K252a.The three downstream signal pathway was respectively inhibited by LY294002 (the phosphatidylinositol 3-hydroxy kinase (PI3 K) pathway inhibitor)、U73122 (the phospholipase C pathway inhibitor) 、U0126(the mitogen activated protein kinase pathway inhibitor).Enzyme linked immunosorbent assay was used to detect the concentration of VEGF and MMP-9 protein in the SY5Y cell culture supernatants.Results VEGF [(485.89 ± 109.99) pg/ml] and MMP-9 [(15.73 ± 1.72) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF were significantly higher than that of the control group and ATRA alone group(P ＜0.05).VEGF [(272.42 ±86.33) pg/ml]and MMP-9 [(5.25 ± 1.44) pg/ml] protein levels in the group of ATRA + BDNF + K252a were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(314.12 ±24.68) pg/ml] and MMP-9 [(4.91 ± 1.08) pg/ml] protein levels in the group of ATRA + BDNF + LY294002 were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(444.08 ±64.49) pg/ml] and MMP-9 [(13.28 ±3.38) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA +BDNF + U73122 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).VEGF [(429.97 ± 19.95) pg/ml] and MMP-9 [(13.96 ± 4.45) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF + U0126 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).Conclusion Activation of TrkB-BDNF signal pathway can increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.TrkB-BDNF signal pathway may be through activating its downstream PI3K pathway to increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.The synthesis and secretion of VEGF and MMP-9 can be inhibited by blocking the TrkB-BDNF signal pathway with K252a or blocking its downstream signal pathway PI3 K with LY294002.
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Objective To investigate the effects of gemcetabine and LY294002 monotherapy or combination on the proliferation and poptosis of pancreatic cancer cell lines BxPc-3 and MiaPaCa-2.Methods Cell proliferation and poptosis were detected by MTT and Annexin V-FTTC,respectively.Results Both gemcetabine and LY294002 could inhabit the proliferation of the two cell lines.Their inhibitory effects were increased accompanied with increased drug concentrations and the cell survival rates was negatively correlated with logarithmic of the drug concentrations (r＜-0.95,P＜0.01).The inhibitory effects of gemcetabine and LY294002 to the BxPc-3 proliferation were significantly stronger than to the MiaPaCa-2(P＜0.05).For BxPc-3 and MiaPaCa-2,the IC50 of gemcetabine were(10.07±1.83),(36.45±2.71)μmol/L(P＜0.05),and the IC50 of LY294002 were(7.84±1.48),(17.89±1.98)μmol/L(P＜0.05),respectively.Gemcetabine and LY294002 could induce cell apoptosis(P＜0.01).Though both the concurrent or consecutive use of these two drugs could promote cell apoptosis,the effect of the concurrent group was significantly stronger(P＜0.05).The order of these two drugs in the concurrent group had no significant influence on their effects(P＞0.05).Conclusion Both gemcetabine and LY294002 could inhibit the proliferation of pancreatic cancer cell lines.Their concurrent application shows a significant inhibitory effect on the cell apoptosis.
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Objective To investigate the role of Akt signal pathway on the expression of monocyte chemoattractant protein-1 ( MCP-1 ) and nuclear transcription factor-κB (NF-κB) in renal tubular epithelial cells (HK-2) stimulated by albumin and to explore the mechanisms of action. Method The HK-2 cells were incubated in the presence of albumin (5,15,30 mg/mL) with or without Ly294002 (an inhibitor of Akt). Expression of mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).Expression of Akt and protein MCP-1 were assessed by Western blot. Electrophoretic mobility shift assay (EMSA) was used to detect the activation of NF-κB. q-test was used to evaluate the differences in means between groups. Results Compared with control group, the expression of MCP-1 mRNA remarkly increased. [Control group: 0.233 ±0.01; BSA(5 mg/mL) group: 0.285 ±0.04; BSA( 15 mg/mL) group:0.387 ± 0.02; BSA ( 30 mg/mL) group: 0.473 ± 0.05; BSA ( 30 mg/mL) + Ly294002 group: 0. 325 ±0.05, P ＜ 0.05 ]. The expression of MCP-1 protein in renal interstitum of operation group were remarkly increased too. [ Control group: 100 ± 15.1; BSA ( 5 mg/mL) group: 148 ± 19.3; BSA ( 15 mg/mL) group: 176±20.7; BSA(30 mg/mL) group: 263 ± 18.1; BSA(30 mg/mL) + Ly294002 group: 175 ± 18.0, P ＜0.05 ]. Albumin stimulated the expression of MCP-1mRNA and protein in a dose-dependent manner. Albumin remarkably increased the activity of NF-κB. Albumin enhanced the expression of Akt. Ly294002 inhibited albumin-induced the expression of NF-κB and partially decreased the level of MCP-1. Apositive correlation was noted between NF-κB activation and MCP-1 expression( r = 0.68 ,P ＜ 0.01 ). Conclusions Albumin-induces MCP-1 and NF-κB production via Akt signal pathway in renal tubular epithelial cells.
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This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells.The 50% inhibition concentration(IC50)of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay(MTT)in vitro.HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC20 alone or combined with LY294002 for 24 h,and then radiated by different doses of X-ray.The cell survival ratio was obtained by means of clone formation.One-hit multi-target model was fitted to the cell survival curve to calculate dose quasithreshold (Dq),mean lethal dose(D0),2Gy survival fraction(SF2)and sensitization enhancement ratio(SER).The pAkt and total Akt expression was detected by Western blotting and DNA damage by neutro-comet electrophoresis.The HeLa cells were randomly divided into 7 groups in terms of different treatments: Control; radiation treatment(RT)group; LY294002+RT group; cisplatin+RT group; docetaxel+RT group; LY294002+cisplatin+RT group; LY294002+docetaxel+RT group.The apoptosis ratio of each group was measured by flow cytometry.The results showed that docetaxel and cisplatin significantly enhanced the phosphorylation of Akt in radiation-treated HeLa cells.The Dq,Do and SF2 in LY294002-contained groups were lower than those in docetaxel or cisplatin+RT group.The SER in the LY294002+docetaxel+RT group was 1.35 times that of the docetaxel+RT group,and that in the LY294002+cisplatin+RT group 1.26 times that of the cisplatin+RT group.The Comet electrophoresis showed that tail distance in the LY294002+cisplatin+RT group or LY294002+do-cetaxel+RT group was longer than in the cisplatin+RT group or docetaxel+RT group.The apoptosis ratio in the LY294002+cisplatin+RT group or LY294002+docetaxel+RT group was higher than in the cisplatin+RT group or docetaxel+RT group.It was concluded that inhibiting PI3K/Akt pathway can increase the effect of docetaxel and cisplatin on the radiosensitivity of HeLa cells and DNA damage resulted from radiation.
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Objective To elucidates the regulation of Notch1 activity by Rab5 which controls endosome fusion.Methods Rab5 expression of BxPC3 cells were inhibited by siRNA interference,the changes of Notch ICD protein were measured by Western blot.We use Wortmannin and LY294002 which inhibit the acticity of Phosphatidylinositol(PI) 3-kinases to investigate Notch ICD protein level.Results After inhibiting Rab5 expression or the activity of Phosphatidylinositol(PI) 3-kinases,Notch ICD protein level and the growth of BxPC3 cells decreased.Conclusion In BxPC3 cells,Rab5 and Phosphatidylinositol(PI) 3-kinases are important to regulate Notch1 activity and the activation of Notch is dependent on endocytosis.
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OBJECTIVE:To explore the effect of the specific inhibitor-LY294002 of the signaling pathway PI3K/AKT on cell cycle and apoptosis of HL60/VCR cells and its mechanism.METHODS:Vincristine(VCR) resistant HL60/VCR were treated with LY294002(final concentration at 1.0,2.5,5.0,or 10 ?mol?L-1) or without LY294002(control).IC50 was detected by MTT;flow cytometry was used to evaluate apoptosis and cell cycle distribution,and changes of Bcl-2,CyclinD1 gene mRNA levels were detected by reverse transcription polymerase chain reaction.RESULTS:As compared with control,LY294002 significantly reduced the IC50 value in HL60/VCR to vincristine in a concentration dependent manner(P
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Objective To evaluate whether Ly294002,suppressing phosphatidylinositol 3 kinase (PI3K)/AKT survival signaling pathway,can change the sensitivity of breast cancer cells to radiotherapy. Methods Breast cancer cultured MCF7 cells treated with:radiation alone;Ly294002;or the combination of radiation and Ly294002.The inhibition of PI3K/AKT by Ly294002 was confirmed by Western blot.Clo- nogenic assay was used quantitatively to measure the mitotic cell death,and caspase-3 assay was used to e- valuate apoptosis.Results 1.Ly29400 could partially inhibit phosphorylated AKT but not radiation,the combination of both could enhance the inhibition of phosphorylated AKT,2.Timing of exposing cells to Ly294002 had some impact on clonogenic survival by radiation,one hour pre-radiation and 10 days post-ra- diation exposing to Ly294002 could maximally sensitize the cells to irradiation,3.Ly29400 combined with radiation could synergistically enhance mitotic death and apoptosis of MCF7 cells,with SER of SF_4 and D_0, being equal to 1.25 and 1.42.Conclusions PI3K/AKT pathway may be a potential target for enhancing the response of breast cancer cells to radiotherapy.
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Aim To observe whether the inhibition of PI3K/PKB signal pathway by LY294002[PI3K pathway inhibitor] could improve the sensitivities of human gastric carcinoma cell line SGC7901 and SGC7901/VCR to anti-cancer drugs.Methods The sensitivities of SGC7901 and SGC7901/VCR to chemotherapeutic drug VCR were detected by MTT.The MDR1 and XIAP mRNA expression levels were evaluated by semi-quantitative RT-PCR,and their protein levels were detected by immunocytochemistry.The PKB and phospho-PKB protein levels were detected by Western blot and the apoptosis ratio was detected by flow cytometry.Results 2?10-5mol?L-1LY294002 enhanced the sensitivities of SGC7901 and SGC7901/VCR cells to VCR(P
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Objective To investigate the reversing effects on drug resistance of gastric carcinoma by suppressing PI3K/PKB signal pathway,and explore its implicated mechanism.Methods MTT assay was used to test the inhibitory effects of VCR alone or VCR in combination with PI3K/PKB inhibitor,LY294002 on SGC7901/VCR cells.The SGC7901 treated with or without LY294002 served as control.The protein levels of PKB and phospho-PKB in the above VCR cells were determined by Western blot analysis.The mRNA expressions of MDR1,Bax and Bcl-2 were evaluated by semi-quantitative PCR with ?-actin as the inner control.The apoptosis was detected by flow cytometry.Tumor volume on the tumor-bearing mice transplanted by SGC7901/VCR cells or cells treated by VCR,LY294002 and their combination respectively was measure for the in vivo effect of LY294002.Results LY294002 enhanced the sensitivities of SGC7901/VCR cells to VCR significantly,and promoted the apoptosis rate induced by VCR prominently,with their corresponding drug resistant index decreasing from 40.45 to 8.50.The protein level of phospho-PKB was reduced,and the mRNA expression levels of MDR1 and Bcl-2 were inhibited(P