Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells.</p><p><b>METHODS</b>Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression.</p><p><b>RESULTS</b>Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.</p><p><b>CONCLUSIONS</b>The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.</p>

Reversal of multidrug resistance in leukemic cell line K562/AO2 by chlordelazine in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>Some recent studies revealed that phenthiazine might be able to reverse tumor cell drug-resistance. Chlorderazin belongs to the phenthiazine compounds. The study aimed to investigate the reversing effect and mechanism of chlorderazin on multidrug resistance of leukemic cell line K562/AO2.</p><p><b>METHODS</b>(1) The cytotoxicities of chlorderazin were assayed with the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. (2) The reverse effect of chlorderazin on K562/AO2 cells was analyzed with MTT method. The multidrug resistance reversal index (RI) was equal to the ratio of control group IC(50)/test group half inhibition concentration IC(50). (3) The intracellular daunorubicin (DNR) concentrations were measured by the flow cytometry. (4) Mdr1 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of mdr-1/beta-actin density was calculated.</p><p><b>RESULTS</b>(1) Chlorderazin 3 micro g/ml showed little toxicity to K562/AO2 cells and the suppression rate was less than 5%, so the concentration of 3 micro g/ml chlorderazin was selected as the experiment concentration. (2) The cytotoxicities of DNR to K562/AO2 were enhanced by 3 micro g/ml of chlorderazin (P < 0.05) and RI was 1.901. (3) Chlorderazin of 3 micro g/ml could increase the intracellular DNR accumulation significantly (P < 0.05), and the fluorescence staining by the flow cytometry was higher (250.95 +/- 18.96) than the control group (112.75 +/- 15.78) and shift right in K562/AO2 cells treated with chlorderazin, and the difference was significant (P < 0.05). (4) Chlorderazin has no significant influence to the expression level of mdr-1 mRNA. Both test group and control group showed a clear mdr-1 mRNA band located at the position of 157 kb. The ratios of mdr-1/beta-actin density were 0.414 +/- 0.012 in the test group and 0.447 +/- 0.027 in the control group, respectively, and the difference was not significant (P > 0.05).</p><p><b>CONCLUSION</b>Chlorderazin could reverse the multidrug resistance by increasing the intracellular DNR accumulation in K562/AO2 cells. The effects had no correlation to the mdr-1 gene. Further study is needed.</p>