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Aim To study the mechanism of action of the new derivative of podophyllotoxin(LN-13)in indu-cing the apoptosis of K562/A02 cells.Methods The MTT method was taken to detect the inhibition of LN-13 and VP-16 on K562/A02 proliferation and inhibi-tion rate and IC50 values were obtained 48 hours later. The K562/A02 cell morphological change induced by LN-13 were observed through Hochest33342 and PI staining after 48 hours later.Flow cytometry was taken to detect the apoptosis of K562/A02 cells induced by LN-13.The reverse transcription-polymerase chain re-action was taken to detect the Bcl-2,Bax,caspase-3 and mdr-1 mRNA expression.The expression of P-gp was detected by Western blot.Results The growth of K562 /A02 cells was obviously inhibited by LN-13 when IC50 value was 3.32 μmol · L-1 .LN-13 could obviously induced cell apoptosis observed by Ho-chest33342 and PI staining.Flow cytometry detection showed that LN-13(2,4,8 μmol·L-1 )could induce cell apoptosis and apoptosis ratio reached 15.0%, 48.0%,68.96%,respectively.The reverse transcrip-tion-polymerase chain reaction showed that LN-13 in-creased the Bax and Caspase-3 mRNA expression,and meanwhile the expression of mdr-1 mRNA decreased. Western blot showed that P-gp expression was de-creased as the LN-13 dose increased.The data were significantly different from those of control group.Con-clusion Podophyllotoxin derivative LN-13 can induce the apoptosis of K562 /A02 cells,which may be close-ly-related to regulating P-gp expression and apoptosis related gene mRNA expression.
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Aim Drug resistance is one of the major hinders on cancer treatments. α-enolase ( eno1 ) was closely related to the generation and development of drug resistance. This article aims to study the effect of eno1 on cell growth and drug resistance in human chro-nic myeloid leukemia cell line K562/A02 . Methods We screened three eno1 stable silencing cells K562/A02-sheno1 and its control cells K562/A02-shcon. Cell count assay was performed to test cell growth, MTT assay was used to test cell proliferation, flow cytometry was used to test the intra-cellular Rho123 content, the expression of genes were tested by real-time PCR assay and western blot assay on mRNA level and protein level, respectively. Results eno1 was o-ver-expressed in K562/A02 cells and its expression was increased by ( 2. 85 ± 0. 56 ) times and ( 1. 43 ± 0. 05 ) times on mRNA level and protein level com-pared to K562 cells. However, there was no difference in cell growth rate between K562/A02 cells and K562 cells. K562/A02-sheno1 cells showed lower cell growth rate and higher drug sensitivity to anti-cancer drugs taxol and doxorubicin. Moreover the Rho123 content was increased in K562/A02-sheno1 cells. The expression of MDR1 decreased in both mRNA level and protein level in K562/A02-sheno1 cells. Conclusion eno1 silencing could suppress cell growth, reverse drug resistance and increase its drug sensitivity in K562/A02 cells, and the mechanism was associated with the MDR1 gene.
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DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy.Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair.If DNA repair is successful,cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs.In this study,we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy.K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed.Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR.In order to test the potential of γH2AX to reverse drug resistance,K562/A02 cells were treated with PI3K inhibitor LY294002.It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR.Additionally,the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCAl.These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.
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Objective:To study the molecular mechanism for momordin in inducing apoptosis of multidrug-resistant human chronic leukemia K562/A02 cells. Methods:The growth inhibition value of K562/A02 cells was detected by CCK-8 method. Cell apoptosis was analyzed by Annexin Ⅴ flow cytometry (FCM) and cell morphological examination. FCM was also used in determining expression of P-glycoprotein, p53 protein, bcl-2 protein and caspase activity. Results:Momordin inhibited the proliferation of K562/A02 cells in a dose-dependent manner. It also induced cell apoptosis, reduced the expression of P-glycoprotein, p53 protein and bcl-2 protein, and increased caspase-3 and caspase-8 activity.Conclusion:Momordin reversed the inhibition of apoptosis in multidrug-resistant K562/A02 cells. The molecular mechanism may be related with down-regulation of expression of p53 protein, P-glycoprotein, and bcl-2 protein and up-regulation of caspase-3 and caspase-8 activities.
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Objective To explore the different expression of NF-κB in both K562 and its multidrug resistant cell line K562/A02 and discuss the mechanism of muhidrug resistance(MDR). Methods To detect the growing feature of the cells. Flow cytometry was used to analys the difference between the distribution profile of K562/S and K562/A02 cell. MTT colorimetry was used to determine the cytotoxic effect of adramycin, and expression of mdrl gene was detected by semi-quantitative reverse transcriptase poly-merase chain reaction (RT-PCR) in K562 and K562/A02 cells. FACS was used to determine the expression and function of glycoprotein (P-gp) on the cell membrane. Western blotting was used to determine the NF-κB p65protein in nueleus. Results There was a difference between K562 and K562/A02 cells growed in a halfadherent way rather than suspending ones, there were increases in the percentage number of cells at G0/G1 and S phases(P ＜0.05). This was mirrored by a decreasing number of cells within the G2/M phase(P＜0.05). Butthere was no difference in apoptosis rate(P ＞0.05). mdr1 mRNA was detected in K562/A02 cells, in which the expression P-gp was much higher [(94.17±0.89)%:(1.41 ±O.491)%]. NF-κB p65 protein in nucleus was overexpressed in K562/A02 cells. Conclusion The activation of NF-κB signaling pathway may attribute to the formation of MDR in K562/A02 cells.
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Objective To investigate the effects of Compound Zhe Bei granule (CZBG) combined with doxorubicin on the expression of GST and Topo - Ⅱ in K562/A02 cell line multidrug resistance tumor xenografts in mice. Methods Tumor xenografts model was established by injecting the multidrug resistance cell line K562/A02 in the axillary flank of BALB/c - nu - nu mice. Drug - comgbination of CZBG intragastric administration and doxorubicin intraperitoneal injection ( i. p. ) was given to the BALB/c - nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of GST and Topo - Ⅱ in K562/A02 tumor xenografts in mice was investigated by immunohistochemical technique. The integral optical density (IOD) of GST and Topo- lⅡ in K562/A02 tumor xenografts was measured by Image ProPlus 6.0. Results Compared with the single treatment of doxorubicin i. p group,the combination of the doxorubicin and CZBG with dosage classified by three types( high, middle, low) can decrease IOD of GSH and Topo - Ⅱ in K562/A02 tumor xenografts with statistical significance( P
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Objective To investigate the inhibitory effect of adriamycin in combination with hyperthermia on apoptosis and bcl-2 expression in the chronic leukemic cell line K562/AO2 in vitro.Methods The working con- centration of adriamycin against K562/AO2 determined by using methyl thiazolyl tetrazolium(MTT) assay was used to treat the chronic leukemic cell line K562/AO2 in vitro alone or in combination with hyperthermia induced using a hot water bath at 40,41 or 42℃.The inhibitory effect was evaluated by MTT assay.The apoptosis rates and bcl-2 ex- pression of K562/AO2 were determined by flow cytometry.Results The working concentration of adriamycin in the study was defined as its 50% inhibition concentration (IC50).A 60 min session of hyperthermia at 40℃,41℃or 42℃was associated with significant growth inhibition of the cell line K562/AO2.Adriamycin chemotherapy alone and with hyperthermia significantly inhibited the growth of K562/AO2.All treatments significantly increased apoptosis rates and down-regulated bcl-2 expression of the K562/AO2 cell line.Conclusion Adriamycin chemotherapy com- bined with 60 min sessions of hyperthermia showed significant suppression effect on K562/AO2 cell proliferation.The treatment can increase apoptosis rates and down-regulate bcl-2 expression.
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Background and Purpose:Recent studies have shown that survivin is an anti-apoptosis gene,which is involved in carcinogenesis and drug resistance of leukemia.Antisense oligodeoxynucleotide(ASODN) can be used to inhibit the expression of survivin,inducing apoptosis and enhancing the chemosensitivity of leukemic cells.This study was designed to explore the effect of survivin ASODN on the growth,apoptosis,and caspase-3 activity of leukemia cell line K562/A02 with the phenotype of drug resistance.Methods:Survivin ASOND was transfected to K562/A02 cells by liposomal reagent,The rate of inhibition,expression of survivin mRNA,apoptosis,and activity of Caspase-3 were detected by colormetric MTT,RT-PCR,flow cytometry and fluorometer,respectively.Results:Survivin ASODN could inhibite the cell proliferation in a dose and time dependent manner.Compared with controls,expression of survivin mRNA decreased by 36.2%(P
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Objective To explore the reversing mechanism of multidrug resistance of ciclosporin(CsA) on K562/A02 cell line,attenuating DNA damage repair through H2AX suppression.Methods K563 and K562/A02 respectively co-cultured with adriamycin(ADM) of different concentrations and CsA.MTT assay was employed to determine the inhibitory concentration of 50 percent(IC50),the resistance times and the reversal times.The K562/A02 cells treated with CsA,and reverse transcriptase(RT)-PCR technique was used to examine the mdr1 and H2AX mRNA level.Flow cytometry was used to measure P-glycoprotein(P-gp) and H2AX expression.Neutral comet assay was used to detect the level of DNA double strands break(DSBs) of the K562/A02 treated with ?H2AX antibody.Results 2?10-3g/L CsA could increase the sensitivity of K562/A02 to ADM,and the reversal times was 5.28.Mdr1 mRNA level and H2AX mRNA level were decreased when treated with CsA(P
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Objective To explore the relationship between nuclear factor-?B(NF-?B) and(P-glycoprotein)((P-gp)) on K562/A02 cells.Methods NF-?B and P-gp of K562/A02 cells cultured with PDTC in different concentrations and time were detected by immunohisochemisty,and the relationship between them was analyzed.Results In K562/A02 cells,the expression of NF-?B and P-gp were positively correlated,and were concentration-dependent and the time dependent.Conclusion The mechanism of NF-?B regulating anti-apoptosis of K562/A02 cells is related with MDR1.