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Objective: To explore the effects of tetramethylpyrazine injection on apoptosis of human leukemia cells and the expressions of apoptotic-relevant proteins. Methods: Leukemia cell line U937 cells were treated with different concentrations (0.5, 1.0, 1.5, 2.0 and 2.5 mg/mL) of tetramethylpyrazine for 24, 48 and 72 h. Then the proliferation inhibitory rate of U937 cells was detected by CCK-8 (cell counting kit-8) method. The cell cycle distribution and the apoptosis of U937 cells were detected by flow cytometry. The expressions of apoptotic-relevant proteins survivin, Bcl-2, Bax and caspase-3 in U937 cells were measured by Western blotting. Results: Tetramethylpyrazine significantly inhibited the cell growth of U937 cells in a time- and dose-dependent manner (P 2/M phase decreased (P < 0.05), as well as the apoptosis rate increased significantly in a dose-dependent fashion (P < 0.05). The expression levels of survivin and Bcl-2 proteins were reduced (P < 0.05), and the expression levels of Bax and caspase-3 proteins were elevated (P < 0.05) in a dose-dependent fashion in U937 cells after tetramethylpyrazine treatment. Conclusion: Tetramethylpyrazine injection can significantly induce apoptosis of U937 cells resulting in antineoplastic effect. Its mechamism may be related to down-regulation of survivin and Bcl-2 protein expressions and up-regulation of Bax and caspase-3 protein expressions.
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Objective To delineate the potency of YC-1 on the proliferation,apoptosis,cell cycle and the protein expression of Caspase-3,-8,-9 in U937 and THP-1 leukemia cell lines.Methods MTT assay was performed to detect proliferation.Flow cytometry was used to measure the apoptosis and cell cycle.The expression of Caspase-3,-8 and-9 were detected by Western blot.Results The MTT assay showed that cell proliferation was inhibited in a concentration-dependent manner in 1.0,3.0,10.0 μmol/L YC-l-treated U937 and THP-1 cells.The survival rates for YC-1 after 24 h in U937 cells were (76.5±4.4) ％,(68.7±6.8) ％,(60.9±13.2) ％ respectively and (94.1±1.4) ％,(81.4±2.0) ％,(72.7±3.0) ％ respectively in THP-1 cell,compared with the control group (100 ％),there were significant differences (F =15.870,126.629,P ＜ 0.01).The apoptosis rates for 1.0,3.0,10.0 μmol/L YC-1 after 24 h were (40.7±1.0) ％,(55.6±2.3) ％,(71.8±1.5) ％respectively in U937 cells and (34.6±2.0) ％,(50.3±3.5) ％,(59.6±4.6) ％ respectively in THP-1 cells.With the control group (4.7±1.4) ％,(1.8±1.0) ％,there were significant difference (F =937.229,200.447,P ＜ 0.01).However,there was no significant difference for cell cycle.In addition,Cleaved Caspase-8 and Cleaved Caspase-3 expression after 1.0,3.0,10.0 μmol/L YC-1 treated for 24 h were significantly higher than control,but the expression of Caspase-9 did not appear significant change in U937 cells.As the same concentration and time point,Cleaved Caspase-3 expression increased with no change of Caspase-9 or Caspase-8 in THP-1 cells.Conclusion YC-1 effectively suppress the proliferation with little effect on cell cycle,but induce the apoptosis,have no effect on cell cycle,and the mechanism of apoptosis may be related to the Caspase activation in U937 and THP-1 cell lines.
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Objective To investigate the effect of membrane-bound prostaglandin E2 synthase 1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60/A cells.Methods Flow cytometry,Western blot and ELISA were used to measure the difference of cell cycle,expression of cyclin D1, mPGES-1 among HL-60/A cells,MNC and HL-60 cells.The effect of MK886 on cell cycle,cyclin D1,mPGES-1,PGE2,P-Akt and c-myc of HL-60/A cells were observed.Results Compared with MNC and HL-60 cells,the expression of cyclin D1 and mPGES-1 were higher in HL-60/A cells,the percentage of G0-G1 phase was decreased [MNC (62.63±6.58) ％,HL-60 (38.86±2.25) ％,HL-60/A (30.53±2.15) ％]and S phase increased[MNC (12.18±4.43) ％,HL-60 (47.70±1.88）％,HL-60/A (57.56±1.54) ％](all P＜ 0.05).After treated with MK886,cell cycle was arrested in G0-G1 phase.The expression of mPGES-1,cyclin D1,P-Akt and c-myc and synthesis of PGE2 were decreased.Conclusion MK886 can arrest HL-60/A cell cycles in G0-G1 phase,which possibly through down-regulation of mPGES-1/PGE2,reduction cyclin D1,P-Akt and c-myc expression.
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Objective: To investigate the effects of vector-mediated targeting human telomerase reverse transcriptase ( hTERT) gene RNA interference (RNAi) on the proliferation and apoptosis of leukemia HL-60 cells. Methods: The targeting hTERT gene RNAi recombination vector (pSilencer1.0-U6/hTERT) was transfected into HL-60 cells. The HL-60 cells transfected with empty vector (pSilencer1.0-U6), RNAi-mate or HL-60 cells were used as the controls. The expression of hTERT protein was detected by Western blotting. The proliferation and apoptosis of HL-60 cells were measured by MTT assay and flow cytometry, respectively. Results: The expression levels of hTERT protein in HL-60 cells were lower and the proliferation inhibitory rate and apoptosis rate of HL-60 cells were higher than those of each control group after transfection with pSilencer1.0-U6/hTERT for 24, 72, and 120 h ( P0.05). Conclusion: Vector-mediated targeting hTERT gene RNAi can down-regulate the expression of hTERT protein and inhibit the proliferation and induce apoptosis of HL-60 cells in vitro.
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Objective: To observe emodin-induced molecular changes in PI3K/AKT signaling pathway in human leukemia K562 cells transplanted into BALB/c nude mice, and to explore whether emodin induces the apoptosis of K562 cells through PI3K/AKT signaling pathway. Methods: The subcutaneously transplanted tumor model of human K562 cells in nude mice was established. After continuously intraperitoneal injection with different doses of emodin for 12 d, the mice were sacrificed. Then the tumor weight and volume were measured, and the tumor inhibition rate was calculated. Emodininduced apoptotic morphological changes of K562 cells were detected by HE stain and scanning electron microscopy. RT-PCR and Western blotting were used to detect the expressions of PI3K, AKT and FoxO 3a mRNAs and proteins, respectively. Results: The relative tumor volumes (V/V0) were significantly smaller in the low-, moderate- and high-dose emodin-treated groups (8.90±0.24, 5.62±0.17 and 2.06± 0.31, respectively) than that in the untreated group (11.83±0.47; P <0.01). Significant apoptosis of K562 cells was found in emodin-treated groups under a light microscope and an electron microscope. RT-PCR revealed down-regulation of PI3K and AKT mRNAs expression and up-regulation of FoxO 3a mRNA expression induced by different concentrations of emodin in a dose-dependent manner. Western blotting analysis showed that the expression levels of PI3K and AKT proteins were markedly decreased and the expression level of FoxO 3a protein was significantly elevated in xenografted tumors treated with emodin. Conclusion: Emodin can significantly inhibit the growth of K562 cell xenografts in nude mice. The underlying mechanism may be associated with inhibition of PI3K/AKT signaling pathway. Copyright© 2011 by TUMOR.
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Objective: To investigate a new strategy to reverse multidrug resistance of chronic myeloid leukemia cell line K562 by RNA interference technique through constructing an eukaryotic vector of short hairpin RNA (shRNA) targeting homeobox A10 (HOXA10) gene. Methods: The eukaryotic vector pGPHI/GFP/Neo-HOXA10 with shRNA targeting HOXA10 gene was constructed and then transfected into K562 cells by positive ion liposome. The stable transfectants were vertificated by reverse transcriptase PCR (RT-PCR) 4 weeks after G418 pressure selection. The changes of sensitivity of K562 cells to leurocristine (VCR) and etoposide (VP-16) after transfection with shRNA-HOXA10 were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results: After selection with G418, the cell clones stably transfected with pGPHI/GFP/Neo-HOXA10 were successfully constructed and verificated by RTPCR. The half inhibitory concentration (IC50) values of VCR and VP-16 for K562 cells transfected with shRNA-HOXA10 were significantly reduced and the apoptosis rate of K562 cells after HOXA10 gene interference combining with chemotherapy was increased significantly as compared with those of shRNA-negative control group and the normal control group (P0.05). Conclusion: The eukaryotic vector of short hairpin RNA (shRNA) targeting HOXA10 gene can enhance the abilities of VCR and VP-16 to inhibit the cell proliferation and induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells. It is hinted that RNA interference targeting HOXA10 gene may reverse the multidrug resistance of leukemia cells in some degree. Copyright© 2011 by TUMOR.
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Objective: To investigate the apoptosis of human leukemia Jurkat cells induced by Youchasaponin in vitro and to explore its possible mechanism. Methods: The effects of Youchasaponin with different concentrations on the proliferation of Jurkat cells were detected by cell count assay. The cell cycle distribution and the apoptosis rate of Jurkat cells were determined by flow cytometry (FCM). The expression levels of caspase-3, poly (ADP-ribose) polymerase (PARP), p-Bcl-2, Bcl-2, Bax and caspase-9 proteins were analyzed by Western blotting. Results: The proliferation of Jurkat cells was significantly inhibited after treatment with Youchasaponin (1-16 μg/mL) in a dose-dependent manner. The percentage of Jurkat cells at G0/G1 phase and G2/M phase was decreased after treatment with Youchasaponin (1-4 μg/mL) for 24 h, while the percentage of Jurkat cells at S phase was increased. The apoptosis rate of Jurkat cells induced by Youchasaponin was increased in a dose-dependent manner. The expression levels of p-Bcl-2 and Bcl-2 proteins were down-regulated, and the expression levels of caspase-3, PARP and caspase-9 proteins were up-regulated, whereas the expression level of Bax protein had no change. Conclusion: Youchasaponin can inhibit the cell proliferation and induce the apoptosis of human leukemia cells. This effect may be related to the mitochondrial pathway of apoptosis. Copyright© 2011 by TUMOR.
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Objective To investigate the effects of simvastatin (SV) on the proliferation,differentiation and apoptosis of human promyelocytic leukemia cell line NB4.Methods NB4 cells were incubated with SV at different concentration with or without all-trans retinoic acid (ATRA),and NB4 cells without any treatment were taken as normal control.Cells of different groups were collected at 24 h,48 h and 72 h after incubation for further detection.Morphological changes by Wright stain were performed.MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the surface CD11b expression levels,the early stage apoptosis ratio and cell necrosis ratio.Results Treated with 15 μ mol/L SV,10 μ mol/L SV and 5 μ mol/L SV respectively,with the NB4 cells growth,the cell inhibition rates gradually increased (F =7.15,P =0.000),as well as CD11b expression levels (F =3.41,P =0.014) and AnnexinVexpression levels (F =43.38,P =0.000).Furthermore the NB4 cells treated with 15 μ mol/L SV exhibited the most significant changes with cell inhibition rate of 0.96±0.02,CD11b expression level increased to (62.41±6.37) ％ and AnnexinV expression level increased to (87.38±2.94) ％ after 72 h incubation.Combination of 15 μmol/L SV with 0.5 μmol/L ATRA displayed obvious interaction for increasing CD11b expression levels (F =4.093,P =0.025),while no significant interaction for cell inhibition rates and Annexin V expression levels were observed.After 72 h incubation,the CD11b expression levels (89.46±9.13) ％ in NB4 cells treated with 15 μ mol/L SV in combination with 0.5 μ mol/L ATRA were significantly higher than those treated with ATRA (71.27±7.27) ％ and SV (62.41±6.37) ％ (t =2.71,P =0.054; t =4.37,P =0.017)' solely.Conclusion Simvastatin in vitro inhibits NB4 cell proliferation,promotes cell apoptosis,and synergistically induces cell differentiation with ATRA dose-dependently in vitro,which indicates that SV may have the effect of synergistic anti-promyelocytic potency with ATRA.
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Objective To investigate the suppression effect, the apoptosis and TGF-β_1 mRNA expression of rhDCN and dororubicin(ADM) on leukemic K562 cell line. Methods K562 cells in Logarithmic growth phase were divided into Saline group, pcDNA3.1 (+)-DCN group, ADM group, and pcDNA3.1 (+)-DCN-ADM group. Morphology change of cell was detected by Wright stain, cell proliferation activity was assessed by MTT. The apoptosis index of K562 cells was assessed by FCM, and TGF-β_1 mRNA of cell was assessed by RT-PCR. Results Wright stain showed that more pronounced morphological apoptosis changes of K562 cells in combined group. MTT method results showed that the proliferation inhibition rate of the combined group was (61±1.32) % higher than that of individual intervention group [DCN group, (20±1.90) %; ADM group, (47±1.04) %](P <0.05). FCM results showed that the apoptosis index of the combined group was (61.30± 0.9) %, higher than that of Individual intervention group [DCN group, (28.25±1.3) %; ADM group, (31.85± 1.5) %](P <0.05). TGF-β_1 mRNA synthesis of combined group was significantly decreased. Conclusion rhDCN can markedly enhance cytotoxicity of ADM on K562 cells, and the mechanisms of apoptosis may be due to down-regulation of TGF-β_1 mRNA. Specific mechanisms will be further studied.
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Objective: To investigate the effect of cinobufacini on apoptosis of U937 cells and its possible mechanism. Methods: Cell viability was measured by MTT assay. The morphological changes were observed by Wrightś staining and immunofluorescence staining. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptotic rate was evaluated by teminal deoxynucleotidyl transferase (TdT) labeling in situ. Expression of bel-2 protein was analyzed by flow cytometry. The levels of Fas and Fas-1 mRNA were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results: Compared with the control group, treatment with cinobufacini at 0.225 to 1.8 μg/ mL for 1-3 d significantly inhibited growth of U937 cells in a time and dose dependent manner. The IC50 value was 1.36 μg/ mL at 24 h. The typical morphological changes of apoptosis and typical apoptotic DNA ladder were observed after incubation with cinobufacini at 0.9 μg/mL for 1 d. The apoptotic rate evaluated by TdT labeling in situ were 4.8%, 13.57%, and 24.33% after exposure to cinobufacini at 0.225, 0.45, and 0.9 μg/ mL for 1 d, respectively. The expression levels of bcl-2 protein and Fas-1 mRNA significantly decreased and the expression of Fas mRNA significantly increased in apoptotic cells. Conclusions: Cinobufacini inhibits growth and inducs apoptosis of U937 cells via inhibition of bcl-2 and Fas-1 genes and activation of Fas gene.
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Objective: To investigate whether tachyplesin, a kind of peptide isolated from the hemocytes of horseshoe crabs, can inhibit the growth of multidrug-resistant leukemia cell line HL-60/VCR. Methods: Through continuous exposures to low-concentration of vincristine (VCR) and intermittant exposures to gradually increased-concentrations of VCR, HL-60 cells were induced to form multi-drug-resistant cell line HL-60/VCR. The resistance of HL-60/VCR cells to chemotherapeutic agents and the effect of tachyplesin on the growth of HL-60/VCR cells were evaluated by MTT assay. The expressions of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) in HL-60/VCR cells were detected by flow cytometry. Results: HL-60/VCR cells had multidrug resistance to chemotherapeutic agents. The expressions of P-gp and MRP were up-regulated by 78.3% and 58.3%, respectively (P <0.01). Tachyplesin inhibited the growth of HL-60/VCR and HL-60 cells in a dose-dependent manner. HL-60/VCR cells had similar sensitivity to tachyplesin with HL-60 cells. Tachyplesin enhanced the sensitivity of HL-60/VCR cells to VCR but had no effects on the expressions of P-gp and MRP. Conclusion: Tachyplesin reverses multidrug resistance of HL-60/VCR cells and enhances its sensitivity to VCR.