Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

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.     

Effect of decitabine on resistance of K562/A02 cells to adriamycin

AIM: To investigate the effect of decitabine (DAC) on the resistance of human chronic myeloid leukemia cell line K562/A02 to adriamycin (ADR), and to explore the possible mechanism. METHODS: The K562/A02 cell line and its parental cell line K562 were treated with different concentrations of ADR or DAC alone, or in combination. The cytotoxic effects of these 2 agents were determined by CCK-8 assay. The degree of DNA methylation was evaluated by Sequenom MassARRAY system and colorimetric method. The cell cycle distribution and the apoptotic rate were determined by flow cytometry. RESULTS: K562/A02 cells were more significantly resistant to ADR than K562 cells.The half maximal inhibitory concentration of ADR for 24 h of the K562/A02 cells was about 50 times higher than that of the K562 cells. To DAC, in the concentration range of 0.5~8 μmol/L, K562/A02 cells were more sensitive than K562 cells. As compared with the same concentrations (4.31 μmol/L and 17.24 μmol/L) of ADR alone, the combination with 1 μmol/L DAC significantly improved the sensitivity of K562/A02 cells to ADR. Both DAC and ADR affected the cell cycle progression and apoptotic rate of K562/A02 cells. DAC (1 μmol/L) treatment mainly showed S phase arrest and increased early apoptotic rate for 24 h, and G₂/M phase arrest and increased late apoptosis and necrosis for 48 h in a time-related manner. ADR treatment showed G₂/M phase arrest and increased late apoptosis and necrosis in a concentration-dependent manner. In combination with 1 μmol/L DAC, the effect of ADR on the cell cycle distribution was further enhanced, showing more obvious G₂/M phase arrest, but no significant difference of the apoptotic rate was observed. The degree of methylation in the genome had no significant difference between the 2 cells, and it before and after DAC treatment had no significant change. CONCLUSION: DAC enhances the sensitivity of K562/A02 cells to ADR, showing drug resistance-reversing potential. The mechanism may be related to regulating cell cycle progression and promoting apoptosis and necrosis of K562/A02 cells.     

Inhibition of FoxM1 sensitizes leukemia K562 cells to homoharringtonine

AIM: To study whether inhibition of forkhead box protein M1(FoxM1) sensitizes leukemia K562 cells to homoharringtonine (HHT). METHODS: K562 cells were incubated with HHT at different concentrations (0μmol/L, 0.015μmol/L, 0.030μmol/L and 0.045μmol/L) for different time (0 h, 24 h, 48 h and 72 h). The mRNA and protein levels of FoxM1 were detected by real-time PCR and Western blot. FoxM1 siRNA was transfected into K562 cells with 0.015μmol/L HHT after 6 h. After 72 h incubation, the cell proliferation was detected by cell counting and soft agar assay, and the proportion of apoptotic K562 cells was determined by flow cytometry. The expression of c-Myc and Sp1 were detected by real-time PCR and Western blot. RESULTS: FoxM1 expression was reduced time-dependently and dose-dependently, suggesting that HHT mediated the downregulation of FoxM1 in K562 cells. In K562 cells, treatment with FoxM1 siRNA and HHT inhibited the cell proliferation and promoted the apoptosis significantly. Therefore, inhibition of FoxM1 sensitized leukemia K562 cells to HHT. The expression of c-Myc and Sp1 was positively regulated by FoxM1. CONCLUSION: HHT inhibits Forkhead box protein M1 expression in K562 cells. Inhibition of FoxM1 sensitizes K562 cells to HHT.     

Establishment of arsenic trioxide-resistant K562/AS2 cell line and the mechanism of multidrug resistance

AIM:To establish a arsenic trioxide (As₂O₃ )-resistant leukemic cell line to explore the mechanism of resistance to As₂O₃, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS:The arsenic trioxide (As₂O₃ )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As₂O₃. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS:The cell doublings time and the cell cycle of the arsenic trioxide (As₂O₃ )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As₂O₃, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As₂O₃, DNR, VP16 and Ara-C was 0.8、94、2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS:A cell line, K562/AS2, resistant to clinical achieving level (2 μmol/L) of As₂O₃ has been established. The relative resistant fold of K562/ AS2 to As₂O₃ is about 7.4 fold to the parent K562 line sensitive to As₂O₃. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).     

Antisense drug targeting with VEGF mRNA designed by computer aid and inhibitory effects on K562 cell line in vitro

AIM:The effective antisense sequences targeted VEGF mRNA with computer software would be screened and designed, and effect of them on growth K562 cells and protein expression of VEGF were studied with experiments.METHODS:Seven antisense sequences were selected and synthesized, which consisted of 18-20 deoxynucleotide acid and were modified with phosphorothioate, according to principle of low free energy of overall △G₃₇ Overall. Cell growth was assayed by trypan blue dye exclusion assay and level of VEGF protien in the media was determined by ELISA.RESULTS:Six of seven sequences were capable of inhibing growth of K562 cells and downregulating the VEGF protein expression significantly, compared with Scrambed control group. It was found that there was a close correlation between low level of overall △G₃₇ and antisense effectiveness (r=0.887,P<0.01).CONCLUSION:VEGF mRNA antisense oliogdeoxynucleotides, which were designed by computer software of RNAstructure, were able to inhibit growth of K562 and its protein expression. The VEGF mRNA may be new target attached by drugs. At same time, the computer aided design is useful methods to obtain the effective antisense.     

Effect of Liucha extract on tumor K562 cell growth

AIM:To investigate the effects of Liucha extract on the growth of tumor cells in vitro and its possible mechanism.METHODS:The capability of colony forming of human leukemia K562 cell in vitro and the cells metabolism were studied by semi-solid agar culture and MTT staining. Then , the changes in morphology in the tumor cells were examined using electronic microscope.RESULTS:Semi-solid agar culture and MTT colorimetric analysis showed that Liucha extrats could significantly inhibit the growth of the tumor cells and their capability of colony forming. Also,under the electronic microscope,it was found that the tumor K562 cell had a narrower perinuclear space,condensation of chromatin and an enlarged mitochondria , in which the cristase disappeared.CONCLUSION:The extract from Liucha possesses an inhibitory effect on K562 cell growth in vitro through affecting the metabolism of the tumor cells.     

Reversal effect of FG020318 on multidrug resistance in retinoblastoma cell line SO-Rb50

AIM: To investigate the multidrug resistance (MDR),reversal activity of 2-［4-(2-pyridin-2-yl-vinyl) henyl］-4,5-bis-(4-N,N-diethylaminophenyl)-1 (H)-imidazole (FG020318) in a retinoblastoma subline SO-Rb50/VCR,which is resistant to vincristine.METHODS: The procedure of stepwise increase in drug concentrations was used to obtain SO-Rb50/VCR,which was resistant to 200 μg/L vincristine.The chemosensitivity of this drug resistant cell line with and without FG020318 or cyclosporine A (CSA) were detected by MTT assay and the function of p-glycoprotein (P-gp) was examined by rhodamine 123 accumulation detected with flow cytometry (FCM).RESULTS: FG020318 (2.5 μmol/L) significantly reduced IC50 and increased the rhodamine accumulation in a concentration-dependent manner.It was much stronger than the positive control CSA in reversal of MDR.CONCLUSION: A new tumor MDR modulator FG020318 partly reverses MDR in SO-Rb50/VCR.It may be a promising new drug to tackling MDR.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

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.     

Effect of monocyte/macrophages activated by CpG-oligodeoxynucleotides of bacteria on K562 cells

AIM: To study the effect of monocyte/macrophages treated with CpG-oligodeoxynucleotides on leukemic K562 cells. METHODS: The monocytes/macrophages from peripheral blood cells were isolated and induced. The expressions of CD14 and CD16 on monocytes/macrophages were detected by means of flow cytometry. After treated with synthetic CpG-oligodeoxynucleotides, and nonCpG-oligodeoxynucleotides for 24 hours respectively, the inhibiting effect of monocyte/macrophages on K562 cells were detected using MTT method. The secretions of TNF-α and IL-12 from monocytes/macrophages were determined using ELISA method. RESULTS: The monocytes/macrophages treated with CpG-oligodeoxynucleotides enhanced their antitumor effect on K562 cells and increased the secretion levels of TNF-α and IL-12. Whereas, there was no significant difference between antitumor effect and cytokine secretion of the monocytes/macrophages treated with nonCpG-oligodeoxynucleotide. CONCLUSION: CpG-oligodeoxynucleotides increases the cytotoxicity of macrophages on K562 cells in vitro, as well as facilitates the IL-12 and TNF-α secretion. It provides a new approach for immunologic treatment of leukemia.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

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.     

Inhibitory effect of propolis on proliferation of K562 cells in vitro

AIM: To study the effect of propolis on the proliferation of K562 cells.METHODS: K562 cells were cultured in vitro. Cell proliferation was measured by MTT method. The apoptotic rate was determined by flow cytometry. RT-PCR was applied to detect mRNA expression of Nup98. The protein level of Nup98 was determined by Western blotting. RESULTS: The inhibitory rates of proliferation induced by propolis at the concentrations of 2 mg/L, 20 mg/L and 200 mg/L were obviously higher than that in control cells in a time-and dose-dependent manner. The apoptotic rate was increased in a dose-dependent manner. High concentration of propolis down-regulated the expression of Nup98 at mRNA and protein levels. CONCLUSION: Propolis inhibits the proliferation and induces apoptosis in K562 cells. The mechanism may be related with down-regulation of Nup98.     

Cellular biological effects of matrine on K562 and K562/Vin cells

AIM: To investigate the cellular biological effects of matrine on K562 and K562/Vin cells and discuss the anticancer mechanism of matrine. METHODS: MTT assay was used to detect the IC₅₀ of matrine on these two cell lines and the reversal effect of matrine on K562/Vin cell's resistance to vincristine. In addition, the growth curve of cells was drawed. The p-glycoprotein (P-gp) expression was determined by immunohistochemistry analysis. The morphological changes of cells under light microscopy and the structural changes under transmission electron microscope were observed. RT-PCR assay was used to detect the hTERT-mRNA expression. RESULTS: The IC₅₀ of matrine was 3.4, 4.6 mmol·L ^-1 for K562 and K562/Vin cells, respectively. Matrine (4.0 mmol·L ^-1) inhibited the growth of K562, K562/Vin cells, 2.0 mmol·L ^-1 matrine inhibited expression of P-gp and with 492.4 reversal index. Matrine killed K562 cells by inducing the apoptosis and the same effect on K562/Vin cells was also observed. The hTERT-mRNA expression of K562 cells were also inhibited by matrine. CONCLUSIONS: Matrine enhanced the cytotoxicity of vincristine in K562/Vin cells, induced the apoptosis of K562 and K562/Vin cells, also inhibited the hTERT-mRNA expression in K562 cells. It shows that matrine would be an effective anticancer medicine.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Reversal of apoptosis resistance of doxorubicin-resistant human myelogenous leukemia cell line K562/DOX by a cyclosporin D analogue PSC833

AIM:To study the reversal effect of a cyclosporin D analogue PSC833 on multidrug resistance of doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. METHODS:The reversal effects of PSC833 on resistance to doxorubicin (DOX)/vincristine (VCR) in K562/DOX cells were observed by MTT assay. The cell cycle analysis was performed by flow cytometry. Annexin V/PI staining was used to identify PSC833-induced apoptosis in K562/ DOX cells. These cells underwent incubation with DCFH-DA, JC-1 and Fluo-3/AM followed by flow cytometry for the measurement of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and intracellular calcium, respectively. The protein levels of cytochrome C (Cyt C), Bcl-2, Bax, and cleaved caspase-3 were detected by Western blotting. RESULTS:The DOX/VCR-induced cytotoxicity was significantly potentiated by PSC833. PSC833 arrested the cells in G2/M phase and increased the apoptosis induced by DOX in K562/DOX cells. During the apoptosis, the level of ROS and intracellular calcium increased, while the level of ΔΨm decreased. Furthermore, the release of Cyt C, activation of caspase-3, up-regulation of Bax and down-regulation of Bcl-2 were observed in K562/DOX cells treated with PSC833 and DOX. CONCLUSION: The reversal effect of PSC833 on multidrug resistance in K562/DOX cells is associated with the induction of apoptosis through a mitochondria-dependent pathway.     

Effects of Buzhong Yiqi decoction-medicated serum on drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin

AIM:To investigate the effects of Buzhong Yiqi decoction-medicated serum on the drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin. METHODS:Medicated serum containing Buzhong Yiqi decoction was prepared. The optimal dose of the medicated serum was selected by MTT assay. The A549 cells and A549/DDP cells were treated with the optimal medicated serum and cisplatin at different concentrations. The IC 50, resistance index and reversal index were determined. The cells were divided into control serum group, optimal medicated serum group, LY294002 group, LY294002 + optimal medicated serum group (combination group) and negative group. The expression of PI3K and Akt at mRNA and protein levels was detected by the methods of immunocytochemistry, Western blotting and real-time PCR. RESULTS:Treatment with 10% middle dose of medicated serum for 48 h was the optimal dose and time for medicated serum. The sensitivity of A549/DDP cells to cisplatin was obviously enhanced when the cells were exposed to the optimal medicated serum with the reversal index of 2.46. The expression of PI3K and Akt at mRNA and protein levels in optimal medicated serum group and combination group was significantly decreased. CONCLUSION:Buzhong Yiqi decoction decreases the resistance index by reducing the expression of PI3K in A549/DDP cells, thus increasing the sensitivity of A549/DDP cells to cisplatin.     

Role of Daxx in inhibiting megakaryocytic differentiation of K562 cells

AIM: To examine the effects of death domain-associated protein (Daxx) overexpression on the viability and megakaryocytic differentiation of K562 cells. METHODS: Daxx overexpression in the K562 cells was established. The expression of Daxx was detected by fluorescence microscopy, fluorescence quantitative real-time PCR and Western blot after transfection. CCK-8 assay was used to detect the cell viability after overexpression of Daxx. The expression of CD41 and CD61 in phorbol 12-myristate 13-acetate (PMA) induced K562 cells was detected by flow cytometry. The protein levels of Daxx and p-ERK were determined by Western blot. Nitroblue tetrazolium (NBT)-reducing test was used to assess leukemia cell differentiation in Daxx-overexpressing K562 cells and control cells. The expression of CD41 and CD61 induced by PMA in Daxx-overexpressing K562 cells was analyzed by flow cytometry. The protein levels of Daxx and p-ERK were also examined by Western blot. RESULTS: The stable overexpression of Daxx in the K562 cells was established. The viability was reduced in Daxx-overexpressing K562 cells. The expression of CD41 and CD61 was significantly increased after PMA induction in the K562 cells (P < 0.01). The protein expression of Daxx was reduced, but the protein level of p-ERK was increased. The expression of CD41 and CD61 was reduced after PMA induction in Daxx-overexpressing K562 cells (P < 0.01). The protein level of p-ERK was also reduced. CONCLUSION: Daxx overexpression inhibits the growth, megakaryocytic differentiation and production of p-ERK in the K562 cells.     

Inhibitory effect of cytotoxin 1 from Naja atra Cantor venom on K562 cells

AIM: To investigate the inhibitory effect of cytotoxin 1 (CTX1) from Naja atra Cantor venom on human chronic myeloid leukemia cell line K562.METHODS: The MTS and cell counting methods were used to detect cell relative viability and cell numbers of K562 cells treated with CTX1 at different concentrations. In the living culture system, the dead and dying cells stained by PI were observed by inverted fluorescence microscope. After treated with CTX1, the apoptotic cells were detected by flow cytometry with annexinV-FITC and PI double staining.RESULTS: The relative viabilities of K562 cells were (90.50±3.07)%, (58.33±3.08)% and (27.43±1.99)% when the cells were treated with CTX1 for 24 h at the concentrations of 2, 5 and 10 mg/L,respectively. The median inhibitory concentration of CTX1 on K562 cells was 5.77 mg/L after 24-hour treatment. Comparment with control group, the percentages of K562 cells by cell counting were (85.01±3.54)%, (56.65±3.59)% and (43.24±4.15)% after treatment with 8 mg/L of CTX1 for 6 h, 12 h, 24 h,respectively. As treatment concentration of CTX1 was elevated and treatment time was prolonged, the cells stained by PI were remarkably observed under inverted fluorescence microscope. After treatment with CTX1 at 8 mg/L for 6 h, 12 h and 24 h, the incidences of cell necrosis were (0.73±0.06)%, (13.90±0.46)% and (23.33±0.86)%, respectively, and the incidences of late apoptosis were (16.27±0.21)%, (26.90±1.23)% and (18.77±0.81)%, respectively.CONCLUSION: CTX1 possesses obvious inhibitory effect on K562 cells and it mainly causes the late phase of apoptosis and necrosis.     

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1 gene

AIM: To reverse multidrug resistance (MDR) of A549/DDP cells with short hairpin RNA (shRNA) expression vectors. METHODS: Two multidrug resistance-associated protein 1( MRP1 ) gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells. MRP1 mRNA was assayed by real-time fluorescent quantitative PCR. The MRP1 function was determined by rhodamine 123(Rho123) retention and the protein expression of MRP1 was detected by immunofluorescent staining. The viability of A549/DDP cells was evaluated by MTT method. RESULTS: MRP1 shRNA expression plasmids were successfully constructed. The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells. The intracellular accumulation of Rho123 significantly increased from(16.93±0.58)% to (89.02±0.59)% and (82.56±1.37)%. IC₅₀ of cisplatin were decreased from (101.45±0.64) μmol/L to (38.06±0.05) μmol/L and (53.72±0.36) μmol/L. IC₅₀ of 5-fluorouracil were decreased from (263.20±2.00) μmol/L to (98.82±1.16) μmol/L and (141.81±0.49) μmol/L. CONCLUSION: The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene. The sensitivity of A549/DDP cells to drug is reversed.