Down-regulated miR-331-5p and miR-27a are associated with chemotherapy resistance and relapse in leukaemia

Multidrug resistance (MDR) and disease relapse are challenging clinical problems in the treatment of leukaemia. Relapsed disease is frequently refractory to chemotherapy and exhibits multiple drug resistance. Therefore, it is important to identify the mechanism by which cancer cells develop resistance. In this study, we used microRNA (miRNA) microarray and qRT-PCR approaches to investigate the expression of miRNAs in three leukaemia cell lines with different degrees of resistance to doxorubicin (DOX) compared with their parent cell line, K562. The expression of miR-331-5p and miR-27a was inversely correlated with the expression of a drug-resistant factor, P-glycoprotein (P-gp), in leukaemia cell lines with gradually increasing resistance. The development of drug resistance is regulated by the expression of the P-gp. Transfection of the K562 and, a human promyelocytic cell line (HL) HL60 DOX-resistant cells with miR-331-5p and miR-27a, separately or in combination, resulted in the increased sensitivity of cells to DOX, suggesting that correction of altered expression of miRNAs may be used for therapeutic strategies to overcome leukaemia cell resistance. Importantly, miR-331-5p and miR-27a were also expressed at lower levels in a panel of relapse patients compared with primary patients at diagnosis, further illustrating that leukaemia relapse might be a consequence of deregulation of miR-331-5p and miR-27a.     

Epigenetic regulation of microRNA-128a expression contributes to the apoptosis-resistance of human T-cell leukaemia Jurkat cells by modulating expression of Fas-associated protein with death domain (FADD)

Increased expression of miR-128a is often observed in acute lymphoblastic leukaemia (ALL) compared with its expression in acute myeloid leukaemia (AML). The objective of this study was to investigate the role of miR-128a, especially that in the Fas-signalling pathway, in T-cell leukaemia cells. The role of miR-128a in Fas-mediated apoptosis was examined by using Fas-activating antibody (CH-11)-susceptible Jurkat cells and -resistant Jurkat/R cells. Whereas ectopic expression of miR-128a conferred Fas-resistance on Jurkat cells by directly targeting Fas-associated protein with death domain (FADD), antagonizing miR-128a expression sensitized Jurkat/R cells to the Fas-mediated apoptosis through derepression of FADD expression. Myeloid leukaemia HL60 and K562 cells were also CH-11-resistant, sharing a similar resistant mechanism with Jurkat/R cells. Furthermore, CH-11 induced demethylation of the promoter region of miR-128a with resultant up-regulation of miR-128a expression in Jurkat/R cells, which was shown to be a mechanism for the resistance of Jurkat/R cells to Fas-mediated apoptosis. Our results indicate that the induction of miR-128a expression by DNA demethylation is a novel mechanism of resistance to Fas-mediated apoptosis.     

Synergistic effect of the combination of nanoparticulate Fe3O4 and Au with daunomycin on K562/A02 cells

In this study, we have explored the possibility of the combination of the high reactivity of nano Fe3O4 or Au nanoparticles and daunomycin, one of the most important antitumor drugs in the treatment of acute leukemia clinically, to inhibit MDR of K562/A02 cells. Initially, to determine whether the magnetic nanoparticle Fe3O4 and Au can facilitate the anticancer drug to reverse the resistance of cancer cells, we have explored the cytotoxic effect of daunomycin (DNR) with and without the magnetic nano-Fe3O4 or nano-Au on K562 and K562/A02 cells by MTT assay. Besides, the intracellular DNR concentration and apoptosis of the K562/A02 cells was further investigated by flow cytometry and confocal fluorescence microscopic studies. The MDR1 gene expression of the K562/A02 cells was also studied by RT-PCR method. Our results indicate that 5.0 x 10(-7) M nano-Fe3O4 or 2.0 x 10(-8) M nano-Au is biocompatible and can apparently raise the intracellular DNR accumulation of the K562/A02 cells and increase the apoptosis of tumor cells. Moreover, our observations illustrate that although these two kinds of nanoparticles themselves could not lower the MDRI gene expression of the K562/A02 cells, yet they could degrade the MDR1 gene level when combining with anticancer drug DNR. This raises the possibility to combine the nano-Fe3O4 or nano-Au with DNR to reverse the drug resistance of K562/A02 cells, which could offer a new strategy for the promising efficient chemotherapy of the leukemia patients.     

Role of ZNRD1 (zinc ribbon domain-containing 1) in angiogenesis of leukaemia cells

Here, we have first investigated the roles of ZNRD1 in angiogenesis of leukaemia. The leukaemia cell line K562 was transfected with the vector that included the full-length cDNA of ZNRD1, then the growth and angiogenesis of cells were detected. Up-regulation of ZNRD1 could significantly inhibit the growth of cells, reduce tumour microvessel densities and inhibit the VEGF (vascular endothelial growth factor) production. The results of human miRNA array and real-time PCR showed that ZNRD1 could significantly up-regulate the expression of miR-214 and down-regulate the expression of miR-296. Taken together, ZNRD1 might inhibit tumour angiogenesis and could be considered as a target for leukaemia therapy.     

Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.     

Nrf2/HO-1/HMGB1信号通路在白血病细胞K562/A02化疗耐药中的机制研究

目的:通过观察高迁移率族蛋白1(HMGB1)、转录因子NF-E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)基因沉默对白血病化疗耐药细胞(K562/A02细胞株)的影响,探讨该信号通路在白血病化疗耐药中的作用及其可能机制。方法:将HMGB1基因、Nrf2基因及HO-1基因的特异性干扰RNA分别转染阿霉素耐药细胞株K562/A02,荧光实时定量(RT-PCR)方法检测HMGB1、Nrf2及HO-1的mRNA表达水平,Western blot方法检测HMGB1、Nrf2及HO-1的蛋白表达水平,免疫荧光方法检测Nrf2的蛋白表达,并使用CCK-8方法检测转染前后K562/A02细胞株的细胞活性。结果:HMGB1基因、Nrf2基因或HO-1基因沉默的K562/A02细胞活性皆显著低于对照组及空白组(P0.05),化疗敏感性恢复。结论:HMGB1高表达导致了白血病细胞株K562/A02对阿霉素的化疗耐药,Nrf2/HO-1信号通路参与了HMGB1诱导的K562/A02细胞的化疗耐药,其表达上调可恢复K562/A02细胞对阿霉素的敏感性。     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

DC-CIK对K562/A多药耐药基因mdr1表达的影响

目的：体外观察树突状细胞（dendritic cell,DC）联合细胞因子诱导的杀伤细胞（cytokine inducedkiller,CIK）对K562/A细胞株多药耐药基因mdr1表达的影响。方法：采集健康人的外周血,分离出单个核细胞（peripheral blood mononuclear cell,PBMC）,在体外加入多种细胞因子经诱导生成DC及CIK细胞,以流式细胞仪检测其表面标志,将DC细胞内加入K562/A细胞裂解物致敏后,再与CIK细胞混合培养48小时。将致敏后的DC-CIK细胞与K562/A及K562分组培养后以荧光定量PCR检测其mdr1基因表达的情况,PBMC作为对照组。结果：RT-PCR中可见K562/A＋DC-CIK组中mdr1 mRNA表达较K562/A明显降低,经荧光定量PCR观察到K562/A内mdr1 mRNA表达为K562的10.27倍、K562/A/PBMC略低于未处理的K562/A（P〉0.05）,K562/A/DC-CIK细胞中mdr1 mRNA含量较K562/A、K562/A/PBMC少（P〈0.05）。DC-CIK细胞与细胞株混合培养后,mdr1基因表达较混合培养前明显降低。结论：实验数据显示DC-CIK可使耐药细胞株内mdr1基因表达下调。但K562与DC-CIK混合培养后该基因降低不明显,提示该基因在细胞中存在着基础表达,意义在于维持细胞内稳态。目前针对逆转白血病耐药的研究较少,需要多进行相关研究以拓宽细胞免疫治疗在逆转耐药领域的应用。DC-CIK是具有发展潜力的抗肿瘤方法。本实验将为下一阶段研究逆转耐药的机制提供依据,DC-CIK细胞免疫疗法有望成为逆转肿瘤耐药的新方法。     

LncRNA UCA1 elevates the resistance of human leukemia cells to daunorubicin by the PI3K/AKT pathway via sponging miR-613

Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.     

X连锁凋亡抑制蛋白反义寡核苷酸逆转K562细胞及难治性癫痫大鼠对卡马西平和苯妥英钠耐药性

凋亡在癫痫发生机制中起重要作用,但其在难治性癫痫耐药机制中的作用尚不清楚．为研究X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)反义寡核苷酸对K562/Dox(阿霉素诱导)耐药细胞及难治性癫痫大鼠耐药性的影响,首先建立耐药的K562/Dox细胞株,比较XIAP在耐药细胞株和正常K562细胞株的表达情况,观察转染XIAP反义寡核苷酸后,线粒体膜电位变化以及对卡马西平和苯妥英钠耐药性的影响．另外,建立慢性杏仁核点燃癫痫模型,筛选出耐药组和药物敏感组,通过侧脑室注射XIAP反义寡核苷酸,对照组注射生理盐水．观察其对各组大鼠后放电阈值(after discharge threshold,ADT)、后放电时程(after discharge duration,ADD)等电生理指标的影响．结果发现,XIAP在K562/Dox耐药细胞上的表达明显高于正常K562细胞,XIAP反义寡核苷酸转染K562/Dox耐药细胞后,XIAP的表达明显下降．导致了K562/Dox细胞线粒体跨膜电位的下降,而且对苯妥英钠和卡马西平的耐药性明显下降,IC₅₀分别由(1 978.2 ± 90.3) mg/L和(1 875.6 ± 83.2) mg/L,降低到(1 123.5 ± 54.2) mg/L和(1 084.5 ± 60.6) mg/L,逆转倍数分别为1.76和1.73．同时动物实验发现,耐药组大鼠在给予XIAP反义寡核苷酸后,ADT明显高于对照组(P < 0.05), ADD时程也明显缩短．上述结果证明,XIAP在耐药的K562/Dox细胞株存在高表达,下调XIAP在K562/Dox细胞株表达可以改善K562/Dox对卡马西平和苯妥英钠的耐药性．而且下调XIAP表达可以协助AEDs改善耐药大鼠的电生理活动,提示XIAP参与了难治性癫痫的耐药．     

AKR1C3 decreased CML sensitivity to Imatinib in bone marrow microenvironment via dysregulation of miR-379-5p

BackgroundDrug resistance is an important cause of death for most patients with chronic myeloid leukemia (CML). The bone marrow microenvironment is believed to be mainly responsible for resistance to BCR-ABL tyrosine kinase inhibitors. The mechanism involved, however, is still unclear.MethodsBioinformatic analysis from GEO database of AKR1C3 was utilized to identify the AKR1C3 expression in CML cells under bone marrow microenvironment. Western blot and qPCR were performed to detect the AKR1C3 expression in two CML cell lines K562 and KU812 cultured +/‐ bone microenvironment derived stromal cells. CCK-8, soft agar colony assay, and Annexin V/PI assay were performed to detect the sensitivity of CML cells (K562 and KU812) to Imatinib under a gain of or loss of function of AKR1C3 treatment. The CML murine model intravenous inoculated with K562-OE-vector and K562-OE-AKR1C3 cells were established to estimate the effect of AKR1C3 inhibitor Indomethacin on Imatinib resistance. The bioinformatic analysis of miRNA databases was used to predict the potential miRNAs targeting AKR1C3. And the luciferase assay was utilized to validate the target relationship between miR-379-5p and AKR1C3. And, the soft agar colony assay and Annexin V/PI were used to validate the effect of miR-379-5p in AKR1C3 induced Imatinib resistance.ResultsIn present study, we investigated AKR1C3 was highly expressed in CML under bone marrow microenvironment. AKR1C3 decreased Imatinib activity in K562 and KU812 cells, while inhibition of AKR1C3 could enhance Imatinib sensitivity in vitro study. Furthermore, murine model results showed combination use of AKR1C3 inhibitor Indomethacin effectively prolong mice survival, indicating that AKR1C3 is a promising target to enhance Imatinib treatment. Mechanically, AKR1C3 was found to be suppressed by miR-379-5p, which was down-expression in bone marrow microenvironment. Besides, we found miR-379-5p could bind AKR1C3 3’UTR but not degrade its mRNA level. Further, gain of miR-379-5p rescued the imatinib resistance induced by AKR1C3 overexpression in CML cells.ConclusionsAltogether, our study identifies a novel signaling regulation of miR-379-5p/AKR1C3/EKR axis in regulating IM resistance in CML cell, and provides a scientific base for exploring AKR1C3 as a biomarker in impeding IM resistance in CML.     

Establishment and molecular characterization of decitabine‐resistant K562 cells

The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.     

TCP1 increases drug resistance in acute myeloid leukemia by suppressing autophagy via activating AKT/mTOR signaling

T-complex protein 1 (TCP1) is one of the subunits of chaperonin-containing T complex (CCT), which is involved in protein folding, cell proliferation, apoptosis, cell cycle regulation, and drug resistance. Investigations have demonstrated that TCP1 is a factor being responsible for drug resistance in breast and ovarian cancer. However, the TCP1 role in acute myeloid leukemia (AML) remains elusive. In the present study, we discovered that the TCP1 expression was elevated in AML patients and high TCP1 expression was associated with low complete response rate along with poor overall survival. TCP1 showed higher expression in the adriamycin-resistant leukemia cell line HL60/A and K562/A, comparing to their respective parent cells HL60 and K562 cells. TCP1 inhibition suppressed drug resistance in HL60/A and K562/A cells, whereas TCP1 overexpression in HL60 cells incremented drug resistance, both in vitro and in vivo. Mechanistic investigations revealed that TCP1 inhibited autophagy and adriamycin-induced cell apoptosis, and TCP1-mediated autophagy inhibition conferred resistance to adriamycin-induced cell apoptosis. Furthermore, TCP1 interacted with AKT and mTOR to activate AKT/mTOR signaling, which negatively regulates apoptosis and autophagy. Pharmacological inhibition of AKT/mTOR signal particularly activated autophagy and resensitized TCP1-overexpressing HL60 cells to adriamycin. These findings identify a novel role of TCP1 regarding drug resistance in AML, which advise a new strategy for overcoming drug resistance in AML through targeting TCP1/AKT/mTOR signaling pathway.Subject terms: Prognostic markers, Acute myeloid leukaemia     

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Comparison of the binding of anthracycline derivatives to purified DNA and to cell nuclei

Fluorescence method was used to study the interactions of anthracyclines with purified DNA and with cell nuclei at 37 degrees C, at pH ranging from 6.8 to 8. Four anthracyclines were used; adriamycin (ADR), 4'-o-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR) and aclacinomycin (ACM). The values of pKa of deprotonation of these four drugs in the pH range 6.5-8.5 are 8.4, 7.7, 8.4 and 7.0 for ADR, THP-ADR, DNR and ACM, respectively. The overall binding constants K* of these four drugs to purified DNA was determined at various pH values. The binding constants K0 and K+ of the respectively neutral form and once protonated form of the drugs to DNA were calculated. Using cell nuclei from K562 cells, the amount of drug intercalated (CN) within the nuclei of K562 cells and the amount of free drug (CE) in the solution were determined at various pH values: measuring at the same pH values, a linear correlation occurred between K* and CN/CE.