XIAP and P-glycoprotein co-expression is related to imatinib resistance in chronic myeloid leukemia cells

P-glycoprotein (Pgp) and XIAP co-expression has been discussed in the process of the acquisition of multidrug resistance (MDR) in cancer. Here, we evaluated XIAP and Pgp expression in chronic myeloid leukemia (CML) samples, showing a positive correlation between them. Furthermore, we evaluated the effects of imatinib in XIAP and Pgp expression using CML cell lines K562 (Pgp⁻) and K562-Lucena (Pgp⁺). Imatinib increased XIAP and Pgp expression in K562-Lucena cells, while in K562 cells a downregulation of these proteins was observed, suggesting that imatinib induces an increment of MDR phenotype of CML cells that previously exhibit high levels of Pgp/XIAP co-expression.     

Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [(14)C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [(3)H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.     

Effect of imatinib mesylate on chronic myelogenous leukemia hematopoietic progenitor cells

Treatment of chronic myelogenous leukemia (CML) has been greatly enhanced by the development of Imatinib mesylate, a specific inhibitor of the BCR/ABL tyrosine kinase. While it is clear that imatinib effectively targets BCR/ABL positive hematopoietic cells, studies examining its effect on primitive hematopoietic progenitors are much more limited. As CML arises in a primitive hematopoietic progenitor cell, it is especially important to understand the effect of imatinib on these cells. Here we review studies investigating the effect of imatinib on the proliferation and viability of primitive and committed hematopoietic progenitors in CML. We describe evidence that BCR/ABL positive progenitors may persist in patients responding to imatinib and discuss problems of resistance to imatinib. Finally we discuss studies evaluating new approaches to overcome resistance of CML progenitor cells to imatinib.     

P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

Imatinib (Glivec), STI571) is an intracellular acting drug that demonstrates high activity against BCR-ABL-positive chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). However, many patients, especially with advanced disease, develop drug resistance. Here, we show by a novel high-performance liquid chromatography-based method that intracellular levels of imatinib decrease in P-glycoprotein (Pgp)-positive leukemic cells. In a model of K562 cells with gradually increasing Pgp expression, a Pgp-dependent decline of intracellular imatinib levels was observed. Decreased imatinib levels were associated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of imatinib on cellular proliferation and apoptosis. The modulation of Pgp by cyclosporin A (CSA) readily restored imatinib cytotoxicity in these cells. Finally, we provide first data showing a biological effect of Pgp modulation in the imatinib treatment of a patient with BCR-ABL-positive ALL. MDR1 overexpression must therefore be considered as an important clinical mechanism in the diversity of resistance development to imatinib treatment.     

Loss of P-glycoprotein expression in hematopoietic stem cells does not improve responses to imatinib in a murine model of chronic myelogenous leukemia.

Selective inhibition of the BCR/ABL tyrosine kinase by imatinib has become a first-line therapy for chronic myelogenous leukemia (CML). However, BCR/ABL-positive progenitors often persist despite treatment, and relapse associated with resistance to imatinib has been described in many patients with advanced disease. Drug efflux by P-glycoprotein (P-gp), as well as point mutations in BCR/ABL oncoprotein, has been implicated in the mechanism of resistance to imatinib. In this study, we established a murine transplantation model of CML-like myeloproliferative disease using Mdr1a/1b-null mice and analyzed the effects of loss of P-gp on resistance to imatinib. We found that mice transplanted with Mdr1a/1b-null bone marrow (BM) that had been transduced with a BCR/ABL retroviral vector displayed similar responses to imatinib, compared with those transplanted with BCR/ABL-transduced wild-type BM. In the absence of P-gp, the incidence and latency of disease in secondary recipients was not changed in imatinib-treated mice, relative to wild-type controls. Furthermore, K562 cells engineered to overexpress P-gp remained sensitive to imatinib-induced growth inhibition and cell death. Together, our findings suggest that P-gp expression in hematopoietic stem cells does not significantly contribute to imatinib resistance in CML.     

p21 Confers resistance to imatinib in human chronic myeloid leukemia cells

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21^Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21^Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21^Cip1 in the response to imatinib in CML.     

甲磺酸伊马替尼治疗慢性粒细胞白血病的临床观察

目的:总结甲磺酸伊马替尼(IM)治疗Ph阳性慢性粒细胞白血病(CML)患者的临床观察体会。方法:收集110例Ph阳性CML慢性期(CP)患者、6例加速期(AP)患者和4例急变期(BC)患者分别口服IM 400、600或800 mg/d。通过血液学、细胞遗传学和分子遗传学指标来判断疗效。结果:CP患者完全血液学反应(CHR)率、主要细胞遗传学反应(MCyR)率和完全细胞遗传学反应(CCyR)率分别为98.2%、90.9%和80.9%;AP分别为66.7%、33.3%和16.7%,P值分别为0.002、0.000和0.000。其中CP患者中肝肾功能不全的患者需减少IM剂量。27例经干扰素治疗失败的CP患者IM治疗仍有效。第1年高随访(1次/月)CP患者CCyR达81.9%,非高随访则为63.6%,P=0.005 2。服药前肾功能不全和肝功能不全较脏器功能正常的CP患者服药6个月内发生3~4级血液学毒副反应概率增高,服药6个月后有所减少。结论:IM对CP患者包括干扰素治疗失败的有较高疗效,对AP和BC患者有一定近期疗效。肝肾功能不全的患者易出现血液学毒副反应,需要药物剂量调整。     

Resistance to imatinib in chronic myelogenous leukemia: Mechanisms and clinical implications

The introduction of imatinib represented a breakthrough in the treatment of chronic myelogenous leukemia (CML). However, about 20% of patients treated in early chronic-phase CML are off therapy after 6 years because of resistance or intolerance, and most patients taking imatinib remain BCR-ABL-positive at the molecular level, indicating primary refractoriness of a leukemic subpopulation. Patients with advanced disease often do not respond, or they eventually relapse. Resistance frequently is associated with mutations in the kinase domain of BCR-ABL. Other mechanisms leading to reactivation of BCR-ABL or preventing sufficient BCR-ABL inhibition also exist. Resistance of patients with continued BCR-ABL inhibition despite leukemic progression indicates clonal evolution triggered by BCR-ABL-independent mechanisms. Current efforts to optimize BCR-ABL-targeted treatment focus on the difficulty in reaching CML stem cells. Success will most likely depend on integration of combined treatment algorithms—whether they be a combination of molecules interfering with signaling pathways or additional immune-based treatment adjuncts.     

慢性粒细胞白血病伊马替尼与尼洛替尼代谢组学差异性研究

目的 应用代谢组学技术研究服用伊马替尼与尼洛替尼的慢性粒细胞白血病(CML)患者血浆代谢差异,探讨其对CML患者血浆内源性代谢物干预作用的差异性.方法 选取2015年3月至2015年6月在苏州大学附属第一医院就诊的获得最佳疗效的CML慢性期患者34例,按服用药物分为伊马替尼组(19例)和尼洛替尼组(15例).采用回顾性病例对照研究方法,应用气相色谱-质谱(GC-MS)技术,对两组患者血浆样本中内源性小分子代谢物进行全面检测,采集代谢物指纹图谱,利用多变量数据分析及t检验筛选差异代谢物,并通过MetPA分析平台探讨差异代谢物相关的代谢通路.结果 应用GC-MS代谢组学方法可以区分伊马替尼与尼洛替尼.结合变量权重重要性(VIP)值及t检验筛选到差异代谢物,伊马替尼组中苯丙氨酸、吲哚丙酸、肉豆蔻酸、半乳糖、葡萄糖、软脂酸、色氨酸、硬脂酸、邻苯二甲酸及邻苯二甲酸酯含量较低,其VIP值分别为1.633(t=6.099,P<0.001)、1.338(t=4.367,P<0.001)、1.557(t=6.716,P<0.001)、1.154(t=3.056,P=0.005)、1.941(t=2.196,P=0.035)、1.207(t=3.785,P=0.001)、1.625(t=6.398,P<0.001)、1.555(t=6.553,P<0.001)、1.633(t=7.679,P<0.001)、1.633(t=8.374,P<0.001).主要涉及色氨酸代谢、苯丙氨酸代谢及半乳糖代谢通路.结论 应用代谢组学方法找到了CML患者伊马替尼与尼洛替尼的血浆内源性差异代谢物,这些差异可能与药物成分配比及药物体内代谢差异有关.     

Triptolide down-regulates bcr-abl expression and induces apoptosis in chronic myelogenous leukemia cells

Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide, a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis in a variety of malignant cell lines. K562 cells are usually resistant to apoptosis induction, probably because of the expression of bcr-abl, the hybrid gene characteristic of the Philadelphia chromosome t (9;22). Present studies demonstrate that triptolide inhibited K562 cells proliferation and induced apoptosis in a dose and time-dependent manner. The growth-inhibitory IC50 value for triptolide treatment was 40 ng/ml. Characteristic apoptotic features were confirmed by morphology, internucleosomal DNA fragmentation, and Annexin V Staining. Significantly, triptolide-induced apoptosis of K562 cells was associated with a decline in bcr-abl expression levels, at the concentrations of 20 ng/ml, 40 ng/ml and 80 ng/ml, triptolide was able to decrease the expression of bcr-abl down to 50%, 30% and 20% respectively of the basal value after 72 h. Our findings strongly suggest that triptolide might be an effective therapeutic agent against CML cells.     

Contribution of BCR–ABL-independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells

BCR–ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR–ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR–ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR–ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR–ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR–ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR–ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism. ( Cancer Sci 2009)     

伊马替尼联合干扰素-α治疗慢性髓细胞性白血病的疗效

目的探讨伊马替尼联合干扰素-α治疗慢性髓细胞性白血病的疗效。方法回顾性分析50例慢性髓细胞性白血病患者的临床资料,分为接受伊马替尼联合干扰素-α治疗的观察组(28例)和接受干扰素-α治疗的对照组(22例),观察治疗后缓解情况、不良反应例数以及生活质量。结果两组患者血液学缓解情况无明显差异,观察组细胞遗传学反应情况明显好于对照组,各类不良反应例数明显少于对照组,KPS评分、躯体功能、心理功能、社会功能、认知功能和总体生活质量评分明显高于对照组。结论伊马替尼联合干扰素-α能够改善治疗的细胞遗传学反应、减少不良反应例数、提高患者生活质量,是行之有效的治疗方式。     

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.