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1.
AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.  相似文献   

2.
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   

3.
AIM:To observe the effect of protein kinase C-α(PKCα)antisense oligonucleotide on cell growth, cell cycle and the expression of cyclin E in human poor-differentiated nasopharyngeal carcinoma(NPC) cell line CNE-2Z. METHODS:Antisense PKCα was transfected by cationic liposome(LP) in CNE-2Z cells to analyze the cell growth and cell cycle by MTT colorimetric assay and flow cytometry, respectively. Moreover, the expression of cyclin E was determined by immunocellularchemistry and scanning the result of dot-blotting. RESULTS:①With the concentration of antisense PKCα increasing, the relative cell growth index was decreased gradually(P＜0.01). ②After treated with antisense PKCα, the percentage of cells in G1 phase enhanced(P＜0.05). ③Compared with the control group, the expressing intensity of cyclin E reduced in antisense PKCα group, and the expression of cyclin E decreased to 66.5%±18.4％(P＜0.05) of the control by scanning quantitative analysis. CONCLUSION:These results indicated that antisense PKCα may inhibit cell growth in CNE-2Z via suppressing the expression of cyclin E and hindering cell process in G1 phase.  相似文献   

4.
AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.  相似文献   

5.
AIM: To study the effect of hexamethylene bisacetamide(HMBA) on the proliferation and expression of KLF6 and related proteins in human tongue carcinoma Tca8113 cells. METHODS: After cultured with HMBA, the growth of the Tca8113 cells was assayed by MTT method, and the morphology of the cells was observed under microscope. The cell cycle was determined by flow cytometry. The mRNA expression of KLF6 was detected by RT-PCR. The protein levels of KLF6, p53, cyclin D1 and c-Jun were measured by the method of immunohistochemistry. RESULTS: The number of adherent cells obviously decreased along with the concentration of HMBA, and the growth inhibition of Tca8113 cells was in a concentration/time-effect relationship after treated with HMBA. Some reversal features of the Tca8113 cells developed to normal cells in morphology after induced by HMBA. The proportion of the cells in G1 phase was (52.00?0.02)% before treating with HMBA. The proportion of the cells in S phase was (34.00?0.08)%, and (14.00?0.10)% of G2 phase cells. After treated with HMBA, the cell number in G1 phase significantly increased with the exposure time going on, while the cell number in S phase significantly reduced, so did the cell number in G2 phase. The cell cycle was significantly arrested in G1 phase (P<0.05). The apoptosis peak also appeared. The mRNA expression of KLF6 significantly increased after induced by HMBA (P<0.05), so did the protein levels of KLF6 and p53 (P<0.05), while the expression of cyclin D1 and c-Jun was significantly decreased (P<0.05). CONCLUSION: HMBA inhibits the proliferation of Tca8113 cells by arresting the cell cycle in G0/G1 phase and resuming Tca8113 cells to normal and apoptosis at last.  相似文献   

6.
AIM: To study the effect and mechanism of chlorophyllin (CHL) inhibiting HT29 cells. METHODS: IC50 value and growth curve of HT29 cells were detected with MTT method. Apoptosis was detected with Wright-Giemsa staining, FCM and DNA electrophoresis. Telomerase was detected by PCR-ELISA, and protein and mRNA expression of COX-2 gene were detected through RT-PCR and Western blot. RESULTS: CHL inhibited the growth of HT29 in a dose-dependent manner. CHL blocked HT29 cells in G1 phase but did not induce apoptosis. Different concentration of CHL inhibits the expression of telomerase and COX-2 in HT29 cells. CONCLUSION: CHL inhibited the growth of HT29 cells by inhibiting the expression of telomerase and COX-2 and blocking cells in G1 phase.  相似文献   

7.
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8.
AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G0/G1 phase were significantly decreased with increased percentage of the cells in S and G2/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G0/G1 phase to S phase and G2/M phases.  相似文献   

9.
AIM:To investigate the effect of antisense oligonucleotides(ASON) of c-sis on cellular cycle and proliferation of pulmonary artery vascular smooth muscle cells(VSMC).METHODS:Tissue mass culture was done to get VSMC of pulmonary artery. Different concentrations of antisense oligonucleotides of c-sis were added into the cultures to observe the VSMC proliferation curve using MTT test. The changes of VSMC cellular cycle were also observed by flow cytometry.RESULTS:ASON with mid-to high concentrations restrained the proliferation of VSMC apparently with the peak of cell growth being attenuated or eliminated. Affected by mid-concentration ASON, PDGF-BB showed significant accelerating effect on the proliferation of VSMC. The ratio of G0／G1 in cellular cycle was increased significantly in VSMC culture with ASON in comparison with control. The G0/G1 ratio also showed significant differences among different concentration of ASON groups(P＜0.05).CONCLUSION:Mid-to high concentration of ASON was a powerful inhibitor of cellular proliferation for pulmonary artery VSMC. ASON increased the ratio of G0／G1 significantly and the increase seems to be ASON dosage dependent.  相似文献   

10.
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11.
AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.  相似文献   

12.
AIM:To observe the effect of simvastatin on the proliferation of vascular smooth muscle cells(VSMCs) induced by serum and growth factor PDGF-BB and the effect of simvastatin on the expression of PTEN,a important regulator of G1/S cell cycle transition. METHODS:The DNA synthesis was determined by [3H]-TdR incorporation, cell cycle was examined with flow cytometry, the protein level of PTEN was measured by Western blot method. RESULTS: (1)Simvastatin inhibited [3H]-TdR incorporation in a dose dependent manner. (2) Flow cytometric DNA analysis revealed that simvastatin induced significantly enhancement of G0/G1 phase and decrease in S phase VSMCs.(3)Simvastatin increased protein level of PTEN and mevalonate, a metabolite of HMG-COA, reversed the effect of simvastatin on PTEN protein expression. CONCLUSION:Simvastatin may inhibit proliferation of VSMCs and retarded cell cycle in G0/G1 phase by increasing PTEN expression through inhibiting synthesis of mevalonate.  相似文献   

13.
AIM: To investigate the effect of wild-type PTEN transfection on the sensitivity of human leukemia K562 cells to artesunate (ART) and its molecular mechanism. METHODS: The adenovirus containing wild-type PTEN (Ad-WT-PTEN) or empty vectors (Ad) were transfected into K562 cells[with multiplicity of infection (MOI)=200]. The untransfected cells served as normal control. The effect of wild-type PTEN on the inhibition of K562 cell growth by ART was observed. The sensitizing ratio of PTEN combined with ART based on IC50 was calculated. The viability of K562 cells was detected by MTT assay. The apoptosis was analyzed by flow cytometry. The mRNA level of PTEN was assessed by real-time PCR. The protein expression of PTEN, p-Akt and Akt was detected by Western blot. The activity of caspase-3/7 was measured by caspase activity kits. RESULTS: The sensitivity of K562 cells to ART was significantly increased by 2.25 folds after transfected with PTEN based on the IC50. The cell viability in Ad-WT-PTEN+ART group was significantly lower than that in Ad+ART group after transfection for 3 d (P<0.01). The apoptotic rate in Ad-WT-PTEN+ART group was significantly higher than that in Ad+ART group (P<0.01). The expression of PTEN at mRNA and protein levels in the K562 cells after transfection with PTEN was significantly increased, and the protein level of p-Akt and caspase-3/7 activity were down-regulated, particularly in PTEN combined with ART group. CONCLUSION: The wild-type PTEN gene enhances the sensitivity of the K562 cells to ART by down-regulating the level of p-Akt and up-regulating the caspase-3/7 activity.  相似文献   

14.
AIM: To observe the effects of exogenous zinc on the biological behavior of hepatocellular carcinoma (HCC) cell line BEL-7404. METHODS: BEL-7404 cells were cultured with zinc sulfate at various concentrations. The intracellular concentration of zinc, cell viability, cell cycle, cell apoptosis and migration and invasion abilities were measured by TSQ fluorescent probe, MTT assay, DNA ploid analysis, acridine orange/ethidium bromide fluorescence staining and Transwell assay, respectively. The mRNA and protein expression levels of albumin in the BEL-7404 cells were determined by real-time PCR and Western blot, respectively. RESULTS: With the elevated concentration of zinc in culture condition, the concentration of zinc in the BEL-7404 cells was increased (P<0.05). The cell viability and migration and invasion abilities were decreased, while the apoptotic rate was increased (P<0.05). The cells in G0/G1 phase were decreased, while the cells in G2/M phase were increased. Additionally, the mRNA and protein expression of albumin also increased (P<0.05). CONCLUSION: The zinc ion inhibits the cell viability as well as migration and invasion abilities, blocks the cells in G2/M phase, and may reduce cell malignant phenotype.  相似文献   

15.
AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G0/G1 phase, arrested the cells in G2/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.  相似文献   

16.
SONG Ling  ZHOU Qiang  LI Na  YU Jie  LI Yang  ZHANG Chi 《园艺学报》2017,33(11):2015-2019
AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G0/G1 phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.  相似文献   

17.
AIM: To investigate whether the PI3K/Akt signaling pathway regulates the expression of ABC transporter through the downstream glycogen synthase kinase-3β (GSK-3β) pathway and participates in the multidurg resistance of colorectal cancer (CRC) HCT-15 cells. METHODS: Colorectal cancer HCT-15 cells were cultured and then treated with GSK-3β inhibitor (HY-19807) and PI3K/Akt pathway inhibitor (HY-13898), respectively. The median inhibitory concentration (IC50) of oxaliplatin for HCT-15 cells in each group was detected by CCK-8 assay, the inhibition rate and resistance index were also calculated. The protein levels of Akt, p-Akt, GSK-3β, p-GSK3β-Ser9 and ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP-2) in the HCT-15 cells were determined by Western blot. The mRNA expression of ABC transporter in the HCT-15 cells was detected by RT-qPCR. The cell cycle distributions were analyzed by flow cytometry assasy. RESULTS: After GSK-3β inhibitor HY-19807 was used in the HCT-15 cells, the median inhibitory concentration of oxaliplatin was significantly increased, the protein levels of p-GSK3β-Ser9, P-gp and MRP-2 were up-regulated compared with control group (P<0.05), the changes of Akt and p-Akt were not obvious compared with control group (P>0.05). The results of RT-qPCR also showed that the mRNA levels of ABCB1 and ABCC2 were increased (P<0.01). Meanwhile, analysis of the cell cycle distribution showed that GSK-3β inhibitor HY-19807 promoted HCT-15 cell transition from G1 phase to S phase, and cell proliferation was vigorous. After the PI3K/Akt pathway inhibitor HY-13898 was applied to HCT-15 cells, the IC50 of oxaliplatin was decreased compared with control group (P<0.05). Moreover, the protein levels of p-Akt, p-GSK3β-Ser9, P-gp and MRP-2 were down-regulated (P<0.01). RT-qPCR results also showed that the mRNA expression of ABCB1 and ABCC2 was decreased (P<0.01). At the same time, G1 phase was prolonged, which inhibited cell transition from G1 phase to S phase, and inhibited cell proliferation. The protein expression of total GSK-3β was consistent in each group. CONCLUSION: The PI3K/Akt signaling pathway is involved in the proliferation and multidrug resistance of colorectal cancer HCT-15 cells by regulating the phosphorylation of GSK-3β and changing the expression of ABC transporter.  相似文献   

18.
AIM: To investigate the effect of decitabine (Dacogen, DAC) on the proliferation and differentiation of K562 cells. METHODS: The K562 cells were treated with different concentrations of DAC. The colony formation ability of the cells was detected by the colony formation assay with semi-solid culture. The cell viability was detected with MTT assay. The morphologic features were observed under inverted microscope with Wright's staining. The changes of the cell cycle distribution and the expression of CD11b and CD42b were analyzed with flow cytometry. The protein expression of CDK2, cyclin E1, P27, GATA-1 and PU.1 in the K562 cells was determined by Western blot. RESULTS: DAC significantly decreased the colony number of the cells and cell viability in a dose-dependent manner. The morphological changes of the cells displayed partial differentiation. After treated the K562 cells with DAC for 72 h, the cell proportion in S phase was obviously decreased, while the cell proportion in G2/M phase was obviously increased in a dose-dependent manner. After treated the K562 cells with DAC for 7 d, the percentage of CD11b and CD14 positive cells was further elevated, and the protein expression of P27, GATA-1 and PU.1 was increased. However, the protein expression of CDK2 and cyclin E1 was decreased. CONCLUSION: DAC inhibits the proliferation and induces differentiation of the K562 cells via regulation of cell cycle.  相似文献   

19.
AIM: To study the role of c-myc oncogene in L6565 leukemia oncogenesis and the effects of therapy by inhibition of its expression with antisense c-myc. METHODS: A recombinant retroviral vector containing antisense c-myc of the murine (pGNCas) was constructed and then transfected into PA317 cells by the method of calcium phosphate precipitation. L6565 clone cells were infected with retrovirus particles. Stable integretion of antisense c-myc was shown by PCR. The change of the malignance and phenotype of L6565as were detected by the examination of the growth, morphology, cells cycle, agar assay and expression of c-myc. RESULTS: The shape of most L6565as cells became spherical. The growth of L6565as was inhibited compared to control cells. The analysis of cells cycle: L6565as cells were arrest in G0/G1 phase, decreased in S phase. The ability of L6565as cells to form colony in soft agarose was significantly suppressed. c-myc in L6565as cells was lowly expressed. CONCLUSION: (1)c-myc plays a critical role in L6565 leukemia oncogenesis; (2)Inhibition of expression of c-myc makes partly reversion of malignant phenotype of L6565 murine leukemia clone cells.  相似文献   

20.
AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G0/G1 phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G0/G1 phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.  相似文献   

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