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Aim To investigate the effect of 2-deoxy-D-glucose(2-DG)on the sensitivity of leukemia multi-drug resistant K562/ADMcells to adriamycin by inhib-iting glycolytic pathway as well as its molecular mecha-nisms.Methods The leukemia drug-resistant K562/ADM cells and parental K562 cells were used as the target cell models.The cell proliferating activity was assessed with an MTT colorimetric assay,and the gly-colysis including glucose consumption,lactate export, and hexokinase activity was determined by glucose, lactic acid and hexokinase (HK)testing kits.The ex-pression and phosphorylation of mammalian target of rapamycin(mTOR)and glucose transporter-4 (GLUT-4)expression were analyzed by western blot.Results K562/ADM drug-resistant cells possessed higher HK activity,GLUT-4 expression level and aerobic glycolic ability than K562 sensitive cells. 2-DG treatment markedly inhibited HK activity,glucose consumption, and lactate export both in K562 cells and K562/ADM cells,and suppressed the proliferation of the two cells in a time-and concentration-dependent manner.Low concentration of 2-DG or adriamycin could increase the expression and phosphorylation of mTOR.However, the co-administration of 2-DG and adriamycin markedly counteracted adriamycin-mediated enhancement of mTOR expression and phosphorylation and down-regu-lated GLUT-4 expression in K562/ADM cells,and 2-DG dramatically improved the sensitivity of K562/ADM cells to cytotoxicity.Conclusion 2-DG inhibits the proliferation of drug-resistant K562/ADM cells and en-hances the sensitivity to adriamycin by blocking aerobic glycolysis pathway through inhibiting hexokinase activi-ty,counteracting adriamycin-stimulated increased ex-pression and phosphorylation of mTOR and downregu-lating GLUT-4 expression.
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Objective: To study the expression levels of forkhead transcription factor(FoxO1) mRNA and protein in the insulin resistance (IR) HepG2 cells model (HepG2/IR) and IR reversal HepG2 cells model (HepG2/IR-PH), and to explore its mechanism in IR.Methods:The HepG2/IR was induced with different doses of insulin (1×10-10, 1×10-9, 1×10-8, 1×10-7, 1×10-6 and 1×10-5 mol·L-1) for different time(24, 36 and 48 h)in the HepG2 cells.The cells in control group were not treated with insulin.The glucose levels in supernant were determined by glucose oxidase method, and the glucose consumption in HepG2 in various groups were calculated to confirm the optimum induction conditions of HepG2/IR.The HepG2/IR-PH was induced with different doses of pioglitazone hydrochloride (PH) (0.156, 0.313, 0.625, 1.250, 2.500, 5.000, 10.000 and 20.000 mmol·L-1) in the HepG2 cells, and control group was set up at the same time. The proliferation activities of cells were observed by MTT assay to confirm the optimum reversal concentration of PH.The FoxO1 mRNA and protein expression levels were detected by Real-time PCR and Western blotting methods.Results: The glucose consumption decreased by 45.84% in HepG2/IR after treated with 1×10-7 mol·L-1 insulin for 36 h, and there was significant difference compared with control group(P0.05).Compared with control group, the expression levels of FoxO1 mRNA and protein in HepG2/IR were significantly increased(P0.05).Conclusion:The IR of HepG2/IR is associated with the FoxO1 mRNA expression.The detection of FoxO1 mRNA seems to be an indicator to evaluate the efficacy of insulin sensitizer, and inhibiting the expression of FoxO1 mRNA may be developed as a potential therapy for type 2 diabetes.
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Objective To establish the insulin resistant HepG2(HepG2/IR)cells model,and investigate the relationship of insu-lin resistance and reduced susceptibility to chemotherapy in hepatoma cells and its mechanism.Methods HepG2 cells were cultured in medium containing 0.5μmol/L insulin for different hours to induce insulin resistance.Glucose consumption of HepG2/IR cells were measured by Hitachi 7600 automatic biochemical analyzer.The cis-dichlorodiamineplatinum(DDP)sensitivity of the HepG2 and HepG2/IR cells were determined by MTT assay,the Annexin Ⅴ/PI assay was adopted to measure the apoptosis rate.In addi-tion,real-time PCR,flow cytometry (FCM)and Western-Blot were employed to detect the mRNA and protein levels of insulin re-ceptor(InsR)and endoplasmic reticulum chaperonin 78(GRP78).Results The glucose consumption decreased and expression of In-sR was down-regulated in HepG2/IR cells.The HepG2/IR cells had reduced sensitivity to DDP (P<0.05 ).The IC50 s of the HepG2/IR cells treated by DDP for 48 h and 72 h were 158.8% and 165.9% of HepG2 cells respectively,while the apoptosis rate was 50.29% lower.The mRNA and protein level of GRP78 in HepG2/IR cells were 2.12 and 2.27 times of that in HepG2 cells. Conclusion The stable HepG2/IR cells showed stronger resistance to DDP were established from HepG2 cell induced with insulin, and its mechanism may be related to the increased expression of GRP78.
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Aim To investigate the role of autophagy and its mechanism in Raji cell death induced by arse-nic trioxide. Methods Transmission electron micros-copy ( SEM) and MDC fluorescence staining were used to observe autophagy. MTT colorimetry was employed to assay the cellular proliferating activity. Cell apopto-sis and cell cycle analysis were performed using FITC-Annexin-V/PI double staining and flow cytometry ( FCM) . The expressions of LC3 and the conversion of LC3-I to LC3-II were measured by western bloting. The expression of bcl-2 mRNA and p53 mRNA were detected by reverse transcription-polymerase chain re-action ( RT-PCR ) . Results Arsenic trioxide could obviously inhibit the proliferation of Raji cells, arrest the cells at G2/M phase and induce apoptosis. Mean-while, arsenic trioxide markedly inhibited the expres-sion of bcl-2 mRNA and enhanced the expression of p53 mRNA in Raji cells. Arsenic trioxide also induced autophagy synchronously which paralleled with the in-duction of apoptosis in Raji cells, and 3-MA, an auto-phagy inhibitor, was able to reverse the arsenic triox-ide-activated autophagic activity, up-regulate bcl-2, down-regulated p53 expression and suppress the lethal effect of arsenic trioxide on Raji cells to reduce their sensitivity to arsenic trioxide. In contrast, the Rapamy-cin, an autophagy inducer, possessed the completely opposite effects on Raji cells compared with 3-MA. Conclusions The apoptosis and autophagic cell death are coexistent in arsenic trioxide-triggered death of Raji lymphoma cells, and Bcl-2 and p53 may play a key regulating role in this process.
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<p><b>OBJECTIVE</b>To investigate the inhibitory and apoptosis-inducing effects of parthenolide (PTL) on human leukemia K562 cells and its leukemia stem cells (LSC).</p><p><b>METHOD</b>MTT assay was used to detect the proliferating activity of K562 cells, and the cellular apoptosis was assayed with Annexin V/PI double staining. Flow cytometry (FCM) was employed to determine the relative proportion of LSC in K562 cells. The self-renewal and proliferating potential were examined with methylcellulose colony-forming units (CFU) assay.</p><p><b>RESULT</b>By use of MTT assay, we found PTL had significant inhibitory effect on the proliferation of K562 cells, the 50% inhibitory concentration (IC50) values were 17.1, 8.67, 9.42 micromol x L(-1) for 24, 48 and 72 h, respectively. After administration with 5 micromol x L(-1) and 10 micromol x L(-1) PTL, the apoptotic rate of K562 cells was (49.56 +/- 5.11)% and (71.88 +/- 2.12)%, and (52.63 +/- 4.14)% and (57.50 +/- 4.47)% in LCS-like (CD34 + CD38-) cells in K562 cell population, respectively. A slightly increase of relative content of LSC in K562 cells was observed. There was an 15-fold increase in the higher concentration of the PTL-treated cells. The methylcellulose colony-forming units assay showed a 24.1% to 89.2% decrease in the CFU of K562 cells administrated with 0.5 micromol x L(-1) to 4.0 micromol x L(-1) PTL, and the CFU of the surviving cells increased by 5.0% to 50.0% on condition that K562 cells were pre-treated with 5 micromol x L(-1) to 15 micromol x L(-1) PTL for 48 h.</p><p><b>CONCLUSION</b>PTL eminently inhibits proliferation of K562 cells and LSC in K562 cells, and induces the cell apoptosis.</p>
Subject(s)
Humans , Apoptosis , Cell Proliferation , K562 Cells , Leukemia , Drug Therapy , Neoplastic Stem Cells , Cell Biology , Plant Extracts , Pharmacology , Sesquiterpenes , PharmacologyABSTRACT
Objective To explore whether arsenic trioxide(As2O3)-induced apopotosis in drug-resistant leukemia K562/ADM cells may induce in through endoplasmic reticulum stress leukemia cell apopotosis.Methods The apoptosis of K562/ADM cells was identified by double staining of FITC-Annexin V and propidium iodide(PI),the ultrastructure of the cells,endoplasmic reticulum and mitochondria were observed by transmission electron microscopy.Flow cytometry(FCM) was employed to assess mitochondrial inner membrane potential(??m),intracellular calcium concentration,cytochrome c(Cyt c) release and caspase-3 activity.The expression of GRP78 protein was analyzed by Western blot.Results During the apoptotic process of K562/ADM cells induced with 2 ?mol/L and 5 ?mol/L As2O3,the endoplasmic reticulum exhibited obvious expansion and degranulation,and the mitochondria illustrated inner and outer membranes fusion,reduced and confused cristae,swelling and vacuolization.The mitochondrial ??m decreased,the intracellular calcium concentration and releasing of cytochrome c from mitochondria increased,and caspase-3 was activated.Western blot result indicated upregulation of GRP78 protein at endoplas-mic reticulum in apopototic K562/ADM cells.Conclusion As2O3 can initiate the endoplasmic reticulum stress in K562/ADM cells,and induces to apoptosis of the drug-resistant cell via endoplasmic reticulum-mitochondrial pathway.
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Aim To explore the apoptotic effect of mimics of manganese superoxide dismutase(MnSODm)on human leukemia cell line K562 in vitro and the possible molecular mechanisms.Methods Human leukemia K562 cells were used as the target cells.The cell proliferating activity was examined by a MTT colorimetric assay,and the apoptosis of K562 cells was assessed with FITC-Annexin V and propidium iodide(PI)double staining and morphological changes.The expressions of bcl-2 and bax mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR),and flow cytometry(FCM)was employed to measure the expressions of Bcl-2 and Bax protein,mitochondrial inner membrane potential(??m),Cytochrome C(Cyt C)release and Caspase-3 activity.Results The proliferation of K562 cells was obviously inhibited by 0.5~10 mg?L-1 MnSODm(P
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<p><b>OBJECTIVE</b>To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and p-glycoprotein (P-gp) expression of multidrug-resistant human leukemia cells.</p><p><b>METHODS</b>Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry.</p><p><b>RESULTS</b>Zero point five to 20 micromol/L As(2)O(3) inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than the parental K562 cells. As(2)O(3)-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As(2)O(3) significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin.</p><p><b>CONCLUSIONS</b>As(2)O(3) induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Apoptosis , Arsenicals , Pharmacology , Drug Resistance, Multiple , Gene Expression , Genes, MDR , Leukemia , Genetics , Metabolism , Oxides , PharmacologyABSTRACT
Background and purpose:Drug-resistance is the main obstacle in terms of efficacy of chemotherapy for leukemia, RNA interference(RNAi) strategy possesses the characteristics of specilization, high-efficiency and low-toxicity, and can effectively and specifically inhibit the overexpression of given gene. This study was designed to investigate the effect of small interfering RNA (siRNA) on expression of mdr1 gene and drug-resistance in multidrug-resistant human leukemia K562/ADM cell.Methods:Human multidrug-resistant leukemia cell line K562/ADM over-expressing mdr1 gene was used as the target cells, Two siRNAs (si-mdr1-1 and si-mdr1-2) targeted mdr1 gene were chemically synthesized and transfected into K562/ADM cells. Expression of mdr1 mRNA was determined by RT-PCR, P-glycoprotein (P-gp) expression was measured using flow cytometry (FCM), and the sensitivity of K562/ADM cells to adriamycin was assessed with a MTT colorimetric assay.Results:Two siRNAs (si-mdr1-1 and si-mdr1-2) specially designed in this study could markedly down-regulate the expression of mdr1 mRNA and its product P-gp in K562/ADM cells. After cells transfected with si-mdr1-1 or si-mdr1-2 for 24h and 48h, the inhibition of mdr1 mRNA expression in the cells for si-mdr1-1 was 55.5% and 22.5%; and for si-mdr1-2, 16.0% and 57.6%, respectively. Treated with siRNA for 72h, the expression intensity of P-gp in the two transfected cell lines decreased 74% and 85%, respectively. Both si-mdr1-1 and si-mdr1-2 significantly enhanced the sensitivity of K562/ADM cells to adriamycin and reversed their drug-resistance, the reversal efficiency was 2.52-folds and 1.96-folds, respectively.Conclusions:The siRNA could effectively and specifically silence the expression of mdr1 gene and overcome the drug-resistance mediated by P-gp in K562/ADM cells.
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In order to study the enhancement of immune functions and autologous tumor-killing (ATX) activity by kappa-selenocarrageenan (KSC) in mice bearing sarcoma 180, the effects of KSC and/or Cyclophosphamide (Cy) on natural killer(NK) activity, lymphokine activated killer(LAK) activity, the production of interleukin-2 (IL-2), ATK activity and the growth of sarcoma 180 (S180) were observed. The results showed that KSC promoted NK activity, LAK activity and ATK activity in vivo, increased IL-2 production at 40mg/kg ?d x 9d. It also enhanced the antitumor action of Cy (20mg/kg?d x 9d) and offsetted the inhibition of Cy on immunocompetent cells. The ATK activity in splenocytes of SI80-bearing mice could be induced and augmented by recombinant interleukin-2 (rIL-2) in vitro. In conclution, KSC had a up-regulating effect on immune functions and ATK activity in tumor-bearing mice, therefore, can be used as a biological response modifier (BRM) in cancer biotherapy.
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Object To study the augmentation of the T activated killer (T AK) cell proliferating and tumor killing activities of the polysaccharide fraction from Rhodiola kirilowii (Regel) Regel, Lycium barbarum L., Hedysarum polybotrys Hand. Mazz., Glehnia littoralis F. Schmidt ex Miq., Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. and Aloe barbadensis Mill. in vitro. Methods The T AK cells were induced by anti CD 3 antibody (CD 3McAb) and rIL 2 from human peripheral blood mononuclear cells (PBMC). The effects of the above six plant polysaccharides (1～100 ?g/mL) on the proliferation of T AK cell, the cytotoxicity to Raji cells and L 1210 cells, and the IL 2 receptor (IL 2R) expression in T AK cells were determined. Results The six polysaccharides alone had no obvious effect on the proliferation of T AK cells. In the presence of rIL 2 and CD 3McAb, they could reinforce the proliferation of T AK cells and its tumor killing activities against Raji cells and L 1210 cells at a different extent, and a 21%～68% increase of IL 2R expression in T AK cells was observed. Conclusion The plant polysaccharides significantly enhance the proliferation and the tumoricidal activities of T AK cells and the enhancing actions related to the increase of IL 2R expression.