The synergistic reversal effect of multidrug resistance by quercetin and hyperthermia in doxorubicin-resistant human myelogenous leukemia cells

Purpose: This study aimed to evaluate the multidrug resistance (MDR) reversal activity of quercetin (Que) in combination with hyperthermia (HT) in human myelogenous leukemia cells K562/A.

Methods: The cytotoxicity of Que alone and the effect of Que and HT to doxorubicin (Dox) cytotoxicity were determined using MTT assay in K562 and K562/A cells. K562/A cells was heated with or without Que pretreatment, and the protein and mRNA levels of heat shock protein 70 (HSP70) and P-glycoprotein (P-gp) were determined by flow cytometry (FCM) and RT-PCR, respectively. Intracellular accumulation of Dox, cell cycle and apoptosis were monitored with FCM.

Results: Que alone inhibited cell growth in a dose-dependent manner in K562 and K562/A cells. Either Que or HT alone had a weak reversal effect on Dox resistance, however, combination HT and Que showed a much more significant reversal effect on Dox resistance (reverse fold 9.49). The elevated protein expression and mRNA level of HSP70 and P-gp in response to HT were inhibited by Que. Pretreatment with Que caused the cells to accumulate Dox 8.3-fold higher than in control cells. In addition, Que induced apoptosis and G2/M arrest in a dose-dependent manner, and the combination of Que and HT was found to have a synergistic efeect on apoptosis.

Conclusions: Que pretreatment could significantly inhance the MDR reversal activity of HT in resistant cell line, by sensitizing the cell to reversing MDR activity of HT.     

Targeting P-glycoprotein expression and cancer cell energy metabolism: combination of metformin and 2-deoxyglucose reverses the multidrug resistance of K562/Dox cells to doxorubicin

P-glycoprotein (P-gp) is one of the major obstacles to efficiency of cancer chemotherapy. Here, we investigated whether combination of metformin and 2-deoxyglucose reverses the multidrug resistance (MDR) of K562/Dox cells and tried to elucidate the possible mechanisms. The combination of metformin and 2-deoxyglucose selectively enhanced the cytotoxicity of doxorubicin against K562/Dox cells. Metformin was not a substrate of P-gp but suppressed the elevated level of P-gp in K562/Dox cells. The downregulation of P-gp may be partly attributed to the inhibition of extracellular signal-regulated kinase pathway. The addition of 2-deoxyglucose to metformin initiated a strong metabolic stress in both K562 and K562/Dox cells. Combination of metformin and 2-deoxyglucose inhibited glucose uptake and lactate production in K562 and K562/Dox cells leading to a severe depletion in ATP and a enhanced autophagy. Above all, P-gp substrate selectively aggravated this ATP depletion effect and increased cell apoptosis in K562/Dox cells. In conclusion, metformin decreases P-gp expression in K562/Dox cells via blocking phosphorylation of extracellular signal-regulated kinase. P-gp substrate increases K562/Dox cell apoptosis via aggravating ATP depletion induced by combination of metformin and 2-deoxyglucose. Our observations highlight the importance of combination of metformin and 2-deoxyglucose in reversing multidrug resistance.     

5-溴汉防己甲素与汉防己甲素对K562/A02细胞多药耐药性逆转作用的比较

背景与目的:5-溴汉防己甲素(5-bromotetrandrine,BrTet)是汉防己甲素(tetrandrine,Tet)的溴化产物,具有逆转P-糖蛋白(P-glyeoprotein,P-gp)介导的肿瘤多药耐药(muhidrug resistance,MDR)的作用。本研究旨在比较BrTet与Tet对人白血病细胞K562/A02多药耐药的逆转作用。方法:采用四甲基偶氮唑蓝法(MTT)法检测不同浓度BrTet对K562细胞和K562/A02细胞的增殖抑制效应;检测阿霉素(adfiamycin,ADM)对K562细胞和K562/A02细胞增殖的抑制作用,以及加用BrTet、Tet时上述抑制作用的变化,并计算半数抑制浓度(IC50)及逆转倍数。Westernblot法检测各组细胞P-gp的表达,流式细胞仪检测各组细胞内ADM的蓄积。结果:K562/A02细胞对ADM的耐药倍数为49.51倍。2.0μmol/L及更低浓度的BrTet和1.5μmol/L及更低浓度的Tet对K562细胞和K562/A02细胞抑制率均小于10%,无明显细胞毒性作用。加入1.0μmol/L的Tet后,K562/A02细胞对ADM的耐药倍数为12.17倍。加入0.25、0.5和1.0μmol/L的BrTet后,K562/A02细胞对ADM的耐药倍数分别为17.88、9.97和4.24倍。1.0"mol/L的BrTet和Tet分别使K562/A02细胞内ADM浓度提高了69.0%和51.6%,使P-gp表达分别下调了51.1%和43.73%,其差异具有统计学意义(P<0.05)。结论:BrTet及Tet均可逆转K562/A02细胞耐药,且前者较后者逆转作用更强,逆转机制与抑制P-gp的表达、增加细胞内抗肿瘤药物浓度有关。     

雷公藤红素逆转K562/A02细胞多药耐药的实验研究

目的探讨雷公藤红素逆转人慢性粒细胞白血病红白血病急变细胞株K562/A02多药耐药的效果。方法采用CCK-8法测定细胞的药敏性及耐药逆转性,应用流式细胞术检测细胞内ADM浓度、P-gp蛋白表达。结果雷公藤红素对K562/A02、K562的半数抑制率浓度（IC50）分别为（295.58±23.288）μmol/L、（411.59±26.551）μmol/L。K562/A02细胞对ADM的耐药性是K562细胞的79.78倍。细胞毒剂量的雷公藤红素作用后,ADM对K562/A02细胞的IC50显著下降（P〈0.05）,逆转倍数为117.860倍。细胞毒剂量（IC50）和非细胞毒剂量（IC10）的雷公藤红素处理后的K562/A02细胞内的ADM浓度显著增加（P〈0.05）,增加倍数分别为1.537倍和1.102倍。雷公藤红素能明显下调K562/A02细胞的P-gp表达。结论雷公藤红素对逆转K562/A02细胞的耐药性有一定的作用,其机制可能与下调P-gp表达有关。     

HSP27高表达与人白血病多药耐药细胞K562/VCR的关系

背景与目的:白血病细胞多药耐药(multidrug resistance,MDR)是白血病化疗失败的常见原因,虽然已有研究揭示了一些肿瘤MDR机制,但目前仍然不能完全解释MDR现象。本文旨在应用蛋白质组学方法筛选白血病耐药相关蛋白,并研究其与白血病MDR的关系,为进一步阐明白血病MDR发生的分子机制提供理论依据。方法:二维聚丙烯酰胺凝胶电泳(two-dimensional electrophoresis,2-DE)技术分离白血病细胞K562和人白血病耐药细胞K562/VCR的总蛋白,用基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption/ionization-time offlight-mass spectrometry,MALDI-TOF-MS)对差异表达的蛋白质点进行鉴定。应用反义核酸技术将分离鉴定差异表达蛋白的反义核酸转染至耐药细胞,Western blot检测转染后差异蛋白的表达情况,MTT法检测转染后细胞存活率,流式细胞仪检测转染后细胞凋亡率。结果:在K562/VCR细胞与K562细胞中鉴定出一差异表达蛋白点,并用质谱分析证实其为热休克蛋白27(heat shock protein27,HSP27)。用HSP27反义核酸转染K562/VCR细胞,在不同浓度长春新碱作用下,HSP27反义核酸转染的K562/VCR细胞的存活率较对照错义核酸转染组明显降低(P<0.05),流式细胞仪显示细胞凋亡率为16.37%,明显高于对照组(P<0.05)。结论:HSP27在K562/VCR细胞中高表达,其表达抑制后,K562/VCR细胞对长春新碱的敏感性增强。     

Experimental Study on the Mechanism of Reversal of Leukemia Multidrug Resistance by Proteasome Inhibitor Bortezomib

OBJECTIVE In this study, we applied multidrug resistant leukemia cell line expressing mdr1-mRNA to observe changes in mdr1-mRNA, the P-gp, cell cycle and apoptosis before and after bortezomib was used, in order to explore the mechanism of reversal of leukemia multidrug resistance by the proteasome inhibitor bortezomib.METHODS Flow cytometry (FCM) was used to detect the intracellular drug concentration, expression of P-gp, cell apoptosis and cell cycle status of K562/DNR cells before and a er treatment with different concentrations of bortezomib. Fluorescence quantitative PCR was applied to detect the mdr1-mRNA expression in K562/DNR and K562/S cells.RESULTS Bortezomib could increase the intracellular DNR content in K562/DNR cells, but showed no e. ect in K562/S cells.5-100 nmol/L bortezomib could significantly reduce the P-gp/mdr1-mRNA expression in K562/DNR cells in vitro, and showed a dose-dependent effect. There was a statistically significant di. erence (P < 0.05) between di. erent concentration groups and the control group. P-gp/mdr1-mRNA expression was negatively correlated with cell apoptosis (r = -0.912 and P < 0.01). After treatment with different concentrations of bortezomib for 24 h,K562/DNR cells in G2 + M phases were significantly increased,while cells in G0 + G1 phases and S phase were significantly decreased, accompanied by an increased apoptotic rate.CONCLUSION Bortezomib can induce G0 + G1 phase to G2 + M phase, and thereby enhance the chemosensitivity of leukemia, and may also reverse the multidrug resistance in leukemia mediated by P-gp overexpression encoded by mdr1 gene. This confi rms that bortezomib can reverse leukemia multidrug resistance at the levels of nucleic acid and protein molecules.     

汉防己甲素联合屈洛昔芬逆转K562/A02细胞耐药与诱导凋亡

目的探讨汉防己甲素(Tet)联合屈洛昔芬(Drol)对耐药细胞系K562/A02的逆转作用及其与诱导凋亡的关系.方法采用甲基四唑蓝法(MTT)测定柔红霉素(DNR)的细胞毒性;采用DNA凝胶电泳法观察Tet、Drol单独及联合应用对K562/A02细胞凋亡诱导作用的影响.结果 0.62 μg/ml Tet、1.94 μg/ml Drol均能增加DNR对K562/A02的细胞毒作用,其半数抑制量IC50分别为7.28±2.06 μg/ml和7.58±3.44 μg/ml,逆转倍数分别为2.94倍和2.82倍.两药联合作用明显增强,其IC50为1.66±0.41 μg/ml,逆转倍数达12.9倍.0.62 μg/ml Tet、1.94 μg/ml Drol单独及联合应用均不会诱导K562/A02细胞凋亡.结论单独应用Tet、Drol可部分逆转K562/A02细胞的耐药性,两药联用具有明显协同效应.Tet、Drol逆转耐药的机理与诱导K562/A02细胞凋亡无关.     

氟哌啶醇在K562/Dox细胞中对P-糖蛋白及氯离子通道的影响

目的　探讨氟哌啶醇 (Hal)对人红白血病耐药细胞K5 6 2 /Dox的逆转耐药作用 ,以及对P 糖蛋白 (P gp)和肿胀激活的氯离子通道的影响。方法　应用乳酸脱氢酶法 (LDH) ,测定Hal对瘤细胞增殖的抑制作用。以半定量逆转录聚合酶链反应 (RT PCR) ,分析经Hal处理后 3种耐药基因mRNA表达的变化。将瘤细胞荷载氯离子敏感染料MQAE后 ,以荧光分光光度计测定低渗环境中Hal对K5 6 2 /Dox细胞肿胀激活的氯离子通道的影响 ;应用ZM库尔特血球计数仪及 2 5 6频道测定仪 ,测定低渗环境中瘤细胞的体积变化 ,以判断Hal对细胞调节性体积减小 (RVD)的影响。结果　Hal对K5 6 2 /Dox细胞的耐药性具有明显的逆转作用 ,在 12 .5 0 ,6 .2 5和 3.12 μmol/L浓度时 ,其对K5 6 2 /Dox细胞耐药性的逆转倍数分别为 8.6 1,4 .35和 2 .2 5。RT PCR结果显示 ,用 12 .5 0 μmol/LHal处理K5 6 2 /Dox细胞后 ,P gp和多药耐药相关蛋白 (MRP)mRNA表达水平均降低 ,并呈现时间依赖性 ,分别较原水平下降 76 .3%和 6 4 .6 % (P <0 .0 5 ) ;谷胱甘肽硫转移酶π(GSTπ)mRNA的表达水平于用药后第 2天下降6 6 .1% ,第 3天回升 (P <0 .0 5 )。K5 6 2 /Dox细胞的氯离子浓度检测结果显示 ,单纯低渗刺激可使K5 6 2 /Dox细胞中MQAE荧光强度下降 (34.4 6± 5 .91     

异汉防己碱增强多药耐药肿瘤细胞对阿霉素的敏感性及其机制

目的:以K562/DOX和MCF-7/DOX细胞为对象,探讨异汉防己碱对化疗药物阿霉素（DOX）的增敏作用及其作用机制。方法:采用MTT法检测异汉防己碱的内在细胞毒性及其对阿霉素的增敏作用,并以RF值评价其增敏效果。应用流式细胞术（FCM）检测细胞膜上P-gp的表达以及细胞内DOX和罗丹明123（Rh123）的蓄积量。结果:异汉防己碱在10μg/ml的无毒剂量可明显增强DOX的细胞毒性。K562/DOX和MCF-7/DOX细胞膜上P-gp均呈强阳性表达,但异汉防己碱对该P-gp表达水平无明显影响。异汉防己碱可使K562/DOX和MCF-7/DOX细胞内DOX和123的荧光密度（FI）均明显增加,由此证明异汉防己碱可有效抑制P-gp的功能。结论:异汉防己碱可通过抑制P-gp的功能而增强阿霉素的敏感性,从而有效逆转肿瘤细胞的多药耐药性（MDR）,它可能成为有效多药耐药逆转剂的候选药物。     

Screening novel, potent multidrug-resistant modulators from imidazole derivatives

The overexpression of P-glycoprotein (P-gp) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was designed to screen potent MDR modulators from imidazole derivatives. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The intracellular accumulation of doxorubicin (Dox) was detected by fluorescence spectrophotometry. The function of P-gp was examined by Rhodamine 123 accumulation detected with flow cytometry (FCM). Among imidazole derivatives, FG020326, FG020327, and FG020318 were found to possess three- to fourfold stronger reversal MDR activity than verapamil, a well-known positive MDR modulator. Imidazole derivatives significantly increased the Dox accumulation and inhibited P-gp function exhibited by the increase of Rhodamine accumulation in MDR cells. The fold reversal of MDR was relative with the increase of Rhodamine accumulation. FG020326, FG020327, and FG020318 showed potent MDR reversal activity in vitro. Their mechanism of MDR reversal is associated with the inhibition of P-gp function and the increase of anticancer accumulation. These results suggest FG020326, FG020327, and FG020318 are promising to further study and develop.     

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

Synergistic effect of cyclosporin A and verapamil in overcoming vincristine resistance of multidrug-resistant cultured human leukemia cells

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC50). The degree of reversal of resistance was expressed as x-fold decrease by dividing the IC50 value without modifier(s) by that with modifier(s). CyA overcame P-gp-associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose-dependent. In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 microgram/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease). At a high concentration (5 micrograms/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.     

Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine

Purpose The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo.Methods Drug sensitivity was determined using the MTT assay. The in vivo effect of Tet was investigated using nude mice grafted with sensitive and resistant KB human epidermoid cancer cells. Doxorubicin (Dox) accumulation was analyzed by fluorospectrophotometry and the protein and mRNA levels of P-glycoprotein (P-gp) were determined by immunocytochemistry and RT-PCR, respectively.Results BrTet at 0.25, 0.5 and 1 M reversed Dox resistance in MDR human breast cancer MCF-7/Dox cells dose-dependently and its potency was greater than that of Tet at the same concentrations. BrTet reversed vincristine (VCR), Dox and paclitaxel resistance in MDR human oral epidermoid carcinoma KBv200 cells as well as innate VCR and Dox resistance in human hepatocellular carcinoma Bel₇₄₀₂ cells. However, BrTet showed no effect on the IC₅₀ values of the above-mentioned anticancer drugs in sensitive MCF-7 and KB cells. No reversal effect of BrTet on the cytotoxicity of 5-fluorouracil and cisplatin, non-P-gp substrates, was observed. In nude mice bearing KBv200 xenografts on the left flank and KB xenografts on the right flank, i.p. injection of 5 mg/kg and 10 mg/kg BrTet significantly enhanced the antitumor activity of Dox against KBv200 xenografts with inhibitory rates of 33.0% and 39.2%, while Dox alone inhibited the growth of KBv200 xenografts by only 11.6%. No enhancement by BrTet was seen in KB xenografts. Moreover, BrTet at 5 mg/kg reversed paclitaxel resistance in KBv200 xenografts. Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of Dox in MCF-7/Dox cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in MCF-7/Dox cells, but had no effect on mdr1 expression.Conclusions BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs. BrTet may be a promising MDR modulator for eventual assessment in the clinic.     

马钱子碱对白血病K562/A02细胞多药耐药性的逆转作用

目的：研究马钱子碱（vauqueline）对人白血病K562/A02细胞多药耐药性的逆转作用。方法：采用噻唑蓝（MTT）法检测马钱子碱的细胞毒作用;采用半定量逆转录聚合酶链反应（RT-PCR）和免疫印迹（Western blot）分别检测非细胞毒浓度（IC10）的马钱子碱对K562/A02细胞MDR1 （multidrug resistance gene 1）、多药耐药相关蛋白（multidrug resistance-associated protein,MRP）、拓扑异构酶Ⅱ（topoisomeraseⅡ,TopoⅡ）、谷胱苷肽-S-转移酶（glutathione s-transferase,GST-π）mRNA及其蛋白表达的影响。结果：非细胞毒浓度（IC10）的马钱子碱作用后,K562/A02细胞中MDR1mRNA及P-gp表达降低（P<0.01）。而MRP、TopoⅡ、GST-π mRNA及其蛋白的表达无明显变化（P>0.05）,同时马钱子碱能增加化疗药物在白血病细胞内的积累。结论：马钱子碱能部分逆转K562/A02细胞的耐药性,其作用机制可能与下调K562/A02细胞MDR1 mRNA的表达,导致细胞膜上P-gp的表达量减少,化疗药物从细胞内溢出减少有关。     

Synergistic Effect of Cyclosporin A and Verapamil in Overcoming Vincristine Resistance of Multidrug-resistant Cultured Human Leukemia Cells

Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK). Drug sensitivity was expressed as 50% inhibitory concentration (IC₅₀). The degree of reversal of resistance was expressed as x -fold decrease by dividing the IC₅₀ value without modifier(s) by that with modifier(s). CyA overcame P-gp-associated MDR significantly in all four MDR cell lines. Reversal of VCR resistance by CyA appeared to be dose-dependent. In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 μg/ml was still effective. The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease). At a high concentration (5 μg/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease). This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance. CyA overcame VCR resistance more efficiently than VER. The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety.     

P13-K抑制剂LY294002逆转P-gP介导的白血病和胃癌细胞耐药

背景与目的：磷脂酰肌醇3-激酶／蛋白激酶B[phosphatidylinositol-3-kinase（P13-K）／proteinkinaseB（Akt）,P13-K／Aktl通路是调控细胞生存的重要信号转导通路之一。本研究旨在探讨P13-K抑制剂LY294002对P-gP过表达的人类白血病K562／DNR和胃癌SGC7901／ADR细胞多药耐药性的逆转作用。方法：将细胞分为单纯药物组和LY294002预处理组,单纯药物组分别以柔红霉素（daunorubicin,DNR）、阿霉素（adriamycin,ADR）、长春新碱（vincristine,VCR）和依托泊甙（etoposide,VP-16）处理,LY294002预处理组在加药前以LY294002进行预处理。用台盼蓝拒染法及MTT法检测药物敏感性及LY294002对细胞耐药性的影响。Westernblot检测K562／DNR和SGC7901／ADR细胞中P．gP及p-Akt的表达。流式细胞术检测细胞内药物浓度。结果：2．5μmol／LLY294002预处理显著降低DNR、ADR、VCR和VP-16对K562／DNR细胞的IC50,相对逆转效率分别为72．4％、64．9％、60．4％和52．8％。此外,LY294002部分逆转SGC7901／ADR对ADR的耐药性,相对逆转效率为31．0％。LY294002预处理可部分抑制p-Akt和P-gP的表达。随着处理时间的延长．K562／DNR、SGC7901／ADR细胞内DNR、ADR的蓄积效应有增强的趋势。结论：LY294002通过抑制P13-K／Akt信号转导通路,部分逆转P-gp介导的白血病和胃癌细胞的多药耐药。     

屈洛昔芬与他莫昔芬对K562/A02耐药性影响的比较

目的:比较屈洛昔芬 (droloxifene,DRO)和他莫昔芬 (tamoxifen,TAM)对K562/A02耐 药性的逆转作用。方法:应用 MTT法、RT PCR和免疫细胞化学 染色观察细胞毒活性和MDR1、 GSTpi耐药基因表达变化,采用流 式细胞仪测定细胞内多柔比星 (ADR)浓度。结果:在20、10和5 mmol/L浓度时,DRO和TAM均 显著逆转K562/A02的耐药性, DRO和TAM的逆转倍数相比较 差异无统计学意义。MDR1和 GSTpi的mRNA和蛋白表达在 DRO和TAM处理后第2天开始 降低,第5天出现明显降低。20 mmol/L浓度的DRO和TAM分别 孵育K562/A02,可使其细胞内 ADR积累分别增加2.9和3.0 倍。结论:DRO与TAM类似,有 较强的逆转活性,可明显抑制 MDR1和GSTpi基因的表达及增 加细胞内ADR的积累。     

川芎嗪联合三氧化二砷逆转K562/ADM细胞多药耐药的实验研究

目的探讨川芎嗪与三氧化二砷联合逆转耐药人红白血病细胞株K562/ADM多药耐药的效果。方法采用WST-8法测定细胞的药敏性及抗药性逆转,应用流式细胞术检测细胞凋亡、细胞内ADM浓度、P-gp蛋白的表达,采用免疫细胞化学二步法检测细胞GST-π表达。结果非细胞毒性浓度的TMP（20μg/ml）及As2O3（0.5μmol/L）可降低ADM对K562/ADM细胞的IC50（P〈0.05）,2种药物联合应用对ADM的逆转倍数明显高于两者单独应用（P〈0.05）,而且也高于两者单独应用之和;两者以非细胞毒性浓度联合应用提高K562/ADM细胞内ADM浓度和细胞凋亡百分率,作用大于两药单独应用,并且明显下调细胞P-gp和GST-π表达（P〈0.05,P〈0.01）。结论非细胞毒性剂量的TMP和As2O3,均可部分逆转有多药耐药表型的细胞株K562/ADM对阿霉素的耐药性,两者联合应用效果优于单独应用,具有协同作用,其机制可能与下调P-gp和GST-π表达有关。     

Imatinib resistance in multidrug-resistant K562 human leukemic cells

The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-kappaB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-kappaB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC(50)(K562-Vinc)/IC(50)(K562-WT)) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0+/-0.3, 2.8+/-0.4 and 44.8+/-8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-kappaB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-kappaB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-kappaB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-kappaB pathway.     

NF-κB信号通路的激活对白血病细胞K562／A02多药耐药的影响

 目的　研究K562细胞株及其耐药细胞株K562／A02 NF-κB活性的差异性表达,探讨白血病多药耐药（MDR）发病机制。方法　用MTT法检测K562／A02的耐药倍数,观察细胞生长的形态学变化,PI单染法检测K562／S、K562／A02细胞周期分布,并用RT-PCR 方法检测mRNA的表达、流式细胞仪检测P糖蛋白（P-gp）表达及其功能,Western blotting方法检测细胞核NF-κB p65表达,比较K562与K562／A02白血病耐药细胞株之间的差异性。结果　与K562细胞不同,耐药细胞株K562／A02细胞生长特性发生改变,呈半贴壁生长,与K562／S细胞相比,K562／A02细胞的 G0／G1期、S期比例增高,G2／M期细胞比例减低,差异有统计学意义（P＜0.05）,凋亡细胞比例差异无统计学意义（P＞0.05）,且检测到mdr1 mRNA及细胞膜P-gp表达增高以及细胞核内NF-κB p65表达明显增加。结论　NF-κB信号通路的激活即NF-κB p65细胞核内转位可能参与了白血病MDR的形成。