Overexpression of sorcin in multidrug resistant human leukemia cells and its role in regulating cell apoptosis

In an attempt to identify novel proteins involved in the emergence of multidrug resistance (MDR) in leukemia cells, we adopted a proteomics approach to analyze protein expression patterns in leukemia cell lines, K562, and its MDR counterpart, K562/A02. Combining high-resolution two-dimensional gel electrophoresis and mass spectrometry, we compared the protein expression profiles between K562 and K562/A02. A total number of 22 protein spots with altered abundances of more than 2-fold were detected and 14 proteins were successfully identified. Consistent with our previous observations by cDNA microarray, sorcin, a 22-kDa calcium-binding protein, was also identified by this proteomic approach with a 10.4-fold up-regulation in K562/A02 cells. Overexpression of sorcin protein in K562 cells by gene transfection led to significantly reduced cytosolic calcium level and increased resistance to cell apoptosis. Further, leukemia cell lines over-expressing sorcin also showed up-regulation of Bcl-2, along with decreased level of Bax. Taken together, our results suggest that sorcin plays an important role in the emergence of MDR in leukemia cells via regulating cell apoptosis pathways, thus may represent both a new MDR marker for prognosis and a good target for anti-MDR drug development.     

神经酰胺诱导药物敏感型和耐药型细胞凋亡机制的探讨

以药物敏感型细胞株Ｋ５６２／Ｓ和耐药型细胞株Ｋ５６２／Ａ０２为对象．观察原癌基因Ｂｃｌ－２的表达量在两种细胞中的差异,以及神经酰胺作为一个新的脂质第二信使诱导细胞凋亡的能力,并利用酪氨酸激酶抑制剂ｇｅｎｉｓｔｅｉｎ,酪氨酸磷酸酯酶抑制剂ｖａｎａｄａｔｅ,观察酪氨酸可逆磷酸化与细胞凋亡间的关系．结果显示：在Ｋ５６２／Ａ０２中Ｂｃｌ－２的表达量明显高于Ｋ５６２／Ｓ;外源性神经酰胺能成功地诱导Ｋ５６２／Ｓ,Ｋ５６２／Ａ０２细胞凋亡,凋亡细胞具有典型的形态学改变和ＤＮＡ“Ｌａｄｄｅｒ”形成,ＦＣＭ检测出现凋亡细胞峰,但在同样的诱导条件下,Ｋ５６２／Ｓ细胞凋亡明显高于Ｋ５６２／Ａ０２细胞．ＦＣＭ检测ｇｅｎｉｓｔｅｉｎ能显著改变这两种细胞生长周期,但细胞阻滞于Ｇ２／Ｍ期,便对神经酰胺诱导的细胞凋亡无明显作用,ｖａｎａｄａｔｅ单独对细胞地明显作用,但与神经酰胺共同作用能明显提高细胞凋亡率．以上结果表明在药物诱导的细胞调亡中Ｂｃｌ－２基因起重要作用,神经酰胺能诱导Ｋ５６２／Ｓ和Ｋ５６２／Ａ０２细胞调亡．     

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.     

Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells

Curcumin (CUR), a polyphenol derived from the plant Curcuma longa, displays potential anti-cancer activity. One of the mechanisms stems from its ability to elicit cell cycle arrest followed by suppression of cell proliferation. Herein, we reported that CUR significantly induced DNA damage and mediated S and G2/M phase arrest in colorectal carcinoma HCT116 cells. Unlike etoposide, a classical topoisomerase II inhibitor, CUR-triggered G2/M phase arrest was hardly reversed by caffeine (CAFF) which is an inhibitor of activated ataxia-telangiectasia-mutated (ATM)/ATM- and Rad3-related (ATR), indicating that ATM and ATR signaling pathways may be not involved in CUR-mediated S and G2/M phase arrest in HCT116 cells. Furthermore, we demonstrated that CUR caused mitosis arrest in HCT116 cells by using mitotic protein monoclonal antibody-2 as a mitosis marker and the surface plasmon resonance assay. The findings provide new mechanisms of cell proliferation inhibition triggered by CUR in HCT116 cells.     

Rapid diagnosis of multidrug resistance in cancer by electrochemical sensor based on carbon nanotubes-drug supramolecular nanocomposites

The multidrug resistance (MDR) in cancer is a major chemotherapy obstacle, rendering many currently available chemotherapeutic drugs ineffective. The aim of this study was to explore the new strategy to early diagnose the MDR by electrochemical sensor based on carbon nanotubes-drug supramolecular interaction. The carbon nanotubes modified glassy carbon electrodes (CNTs/GCE) were directly immersed into the cells suspension of the sensitive leukemia cells K562 and/or its MDR cells K562/A02 to detect the response of the electrochemical probe of daunorubicin (DNR) residues after incubated with cells for 1h. The fresh evidence from the electrochemical studies based on CNTs/GCE demonstrated that the homogeneous, label-free strategy could directly measure the function of cell membrane transporters in MDR cancer cells, identify the cell phenotype (sensitive or MDR). When the different ratios of the sensitive leukemia cells K562 and its MDR ones K562/A02 were applied as a model of MDR levels to simulate the MDR occurrence in cancer, the cathodic peak current showed good linear response to the fraction of MDR with a correlation coefficient of 0.995. Therefore, the MDR fraction can be easily predicted based on the calibration curve of the cathodic peak current versus the fraction of MDR. These results indicated that the sensing strategy could provide a powerful tool for assessment of MDR in cancer. The new electrochemical sensor based on carbon nanotubes-drug supramolecular nanocomposites could represent promising approach in the rapid diagnosis of MDR in cancer.     

Downregulation of microRNA let-7f mediated the Adriamycin resistance in leukemia cell line

Multidrug resistance (MDR) has become the major cause of failure chemotherapy for leukemia and high mortality of leukemia. The study aimed to investigate whether the let-7f mediate the Adriamycin (ADR) resistance of leukemia, and to explore the potential molecular mechanism. Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the soft agar clone formation assay. Flow cytometry was performed to detected cell cycle and apoptosis. The targeted regulationship was analyzed by dual-luciferase assay. Real-time polymerase chain reaction and Western blot were used to measure the expressions of let-7f, ABCC5, ABCC10, cell cycle-related proteins, and apoptosis-related proteins. The xenograft mouse model was used to conduct the tumor formation assay in vivo. The results demonstrated that the expression of let-7f was lower in multidrug-resistant K562/A02 cell lines compared to that in K562, while ABCC5 and ABCC10 were upregulated. Overexpression of let-7f in K562/A02 cell lines downregulated the ABCC5 and ABCC10 expression, enhanced cell sensitivity to ADR, promoted cell apoptosis, and inhibited cell proliferation. let-7f was proved to negatively regulate ABCC5 and ABCC10. Tumor formation assay further determined that let-7f overexpression increased sensitivity to ADR. Taken together, the let-7f downregulation induced the ADR resistance of leukemia by upregulating ABCC5 and ABCC10 expression. Our study provided a novel perspective to study the mechanism of MDR and a new target for the reversal of MDR.     

Effect of the o-methyl catechols apocynin,curcumin and vanillin on the cytotoxicity activity of tamoxifen

Apocynin (APO), curcumin (CUR) and vanillin (VAN) are o-methyl catechols widely studied due their antioxidant and antitumour properties. The effect of treatment with these o-methyl catechols on tamoxifen (TAM)-induced cytotoxicity in normal and tumour cells was studied. The cytotoxicity of TAM on red blood cells (RBC) was performed by haemoglobin or K⁺release and on polymorphonuclear leukocytes (PMNs) by trypan blue dye exclusion method. Cytotoxic activity was assessed in human chronic myeloid leukemia (K562) cell line by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). According the release of haemoglobin and K⁺, the CUR showed a decrease in TAM cytotoxicity on RBC; however, in PMN, APO, CUR and VAN showed increased of these cells viability. VAN presented the highest cytotoxicity on K562 cells, followed by APO and CUR. These results point the potential therapeutic value of these o-methyl catechols with TAM, particularly of CUR, which potentiates the cytotoxic effects of TAM on K562 cells and also decreases TAM-associated cytotoxicity on RBC and PMN.     

Synthesis and evaluation of substituted dibenzo[c,e]azepine-5-ones as P-glycoprotein-mediated multidrug resistance reversal agents

A series of substituted dibenzo[c,e]azepine-5-ones (7a-h) were synthesized and evaluated as P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal agents. The most potent compound 7h could significantly and selectively enhance the chemo-sensitivity of drug-resistant K562/A02 cells to the cytotoxic effect of adriamycin (ADR) in a dose-dependent manner. Further studies indicated that 7h could markedly increase intracellular accumulation of both rhodamine 123 and ADR in K562/A02 cells and inhibit their efflux from the cells. And 7h had little effect on the levels of P-gp mRNA and protein in K562/A02 cells. These results suggest that the anti-MDR effect of 7h might be attributed to the inhibition of drug efflux function of P-gp, leading to the increased drug accumulation in K562/A02 cells, and thus the compound could be served as a lead for developing P-gp-mediated MDR reversal agents.     

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。     

Toxicity mechanisms of onion (Allium cepa) extracts and compounds in multidrug resistant erythroleukemic cell line

Onion (Allium cepa) is being studied as a potential anticancer agent, but little is known regarding its effect in multidrug resistance (MDR) cells. In this work, the cytotoxicity of crude onion extract (OE) and fractioned extract (aqueous, methanolic and ethyl acetate), as well as some onion compounds (quercetin and propyl disulfide) were evaluated in Lucena MDR human erythroleukemic and its K562 parental cell line. The capacity of OE to induce apoptosis and/or necrosis in these cells, the possible participation of oxidative stress and DNA damage were also assessed. Similar sensitivities were obtained for both tumoral cells, however only OE caused significant effects in the cells. In K562 cells, a significant increase of apoptosis was verified while the Lucena cells experienced a significant increase of necrosis. An antioxidant capacity was verified for OE discarding oxidative damage. However, OE provoked similar significant DNA damage in both cell lines. Thus, the OE capacity to overcome the MDR phenotype suggests anti-MDR action of OE.     

Synthesis and evaluation of furoxan-based nitric oxide-releasing derivatives of tetrahydroisoquinoline as anticancer and multidrug resistance reversal agents

Multidrug resistance in tumor cells poses a major obstacle to efficient chemotherapy. Several types of agents have been recognized as multidrug resistance inhibitors, among which the tetrahydroisoquinolines is the most studied. In current study 16 furoxan-based nitric oxide-releasing derivatives of tetrahydroisoquinoline were synthesized. Their cytotoxic activities and effects in reversing multidrug resistance have been evaluated. The results revealed that these compounds had moderate cytotoxic effects. Compounds 7a-f, 7h, and 7l showed higher cytotoxicities than the rest, but lower than adriamycin on K562 cell line. Compounds 7d, 7f, and 7l exhibited potent MDR reversal activities on K562/A02 cell line. The accumulation assay indicated that compounds 7d, 7f, and 7l significantly increased the intracellular accumulation of rhodamine123 in K562/A02 cells. Furthermore, these three compounds produced high concentrations of NO in K562/A02 cells. Potentially, the high concentrations of NO produced by NO donor moieties will lead to an increased cytotoxicity to K562/A02 cells. Our results suggested that compounds 7d, 7f, and 7l had anticancer effects, as well as multidrug resistance reversal effects.     

三氧化二砷对K562/A02 细胞的凋亡及细胞周期的影响

目的:研究三氧化二砷对多药耐药急性白血病细胞株K562/A02凋亡与细胞周期的影响及可能机制。方法:取阿霉素(Adr)的耐药白血病细胞株分为未加药的对照组及加入不同浓度的三氧化二砷(其终浓度为4.0μmol/L、5.0μmol/L)组,流式细胞仪检测细胞凋亡及细胞周期分布,Western blot方法检测不同浓度三氧化二砷对K562/A02细胞核NF-κBp65蛋白水平。结果:与对照组比较,三氧化二砷可显著增加Adr对K562/A02细胞凋亡率,阻滞细胞于G0/G1期,降低K562/A02细胞胞核中NF-kB p65的表达(P均<0.05)。结论:三氧化二砷可能是通过抑制NF-kB的胞内活化转位,从而促进K562/A02细胞凋亡及抑制细胞增殖。     

Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3

Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10-60 microg/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 microg/ml at 24 h and 31.5 microg/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 microg/ml GLA for 48 h and with 40 microg/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pan-caspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation.     

Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential antileukemia agents against drug resistant K562/A02 cell overexpressing P-gp. Among them, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could significantly inhibit K562/A02 cells proliferation with an IC₅₀ value of 0.96 μM. Interestingly, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity (CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression, reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide might be a promising lead to discover novel antileukemia agents against P-gp overexpressing leukemic cells.     

Cleistanthin A causes DNA strand breaks and induces apoptosis in cultured cells

Cleistanthin A is a novel anticancer agent isolated from Cleistanthus collinus (Rox B). It caused chromatid aberrations in a dose dependent manner. However, the concentrations that induced the aberrations, neither affected viability nor induced DNA strand breaks. Only at higher concentrations and after long exposure, DNA strand breaks were observed. Cleistanthin A induced apoptosis in Chinese hamster ovary (CHO) cells, in cervical carcinoma (Si Ha) cells and in a p53 deficient cell line K562. Cleistanthin A-induced cell death was low in bcl-2 transfected cells. Cleistanthin A inhibited the incorporation of [3H]thymidine into DNA; however, it did not affect the transport of [3H]thymidine into these cells. These studies indicate that the cytotoxic effects of cleistanthin A are mediated by the inhibition of DNA synthesis, induction of DNA damage and apoptosis.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.     

Discovery of aromatic amides with triazole-core as potent reversal agents against P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major impediment for clinical cancer therapy. 19 novel aromatic amides with triazole-core as MDR reversal agents were designed and synthesized via click chemistry to reverse MDR. Among them, compound 42 was identified as the most promising candidate with high potency (EC₅₀ = 78.1 ± 5.4 nM), low cytotoxity (SI > 1282) and persistent duration in reversing doxorubicin (DOX) resistance in K562/A02 cells. 42 also enhanced the potency of other P-gp associated cytotoxic agents with different structures. In further study, remarkably increased intracellular accumulation of Rh123 and DOX in K562/A02 cells was achieved by compound 42, while CYP3A4 activity had no change by compound 42. These results indicate that compound 42 as a relatively safe modulator of P-gp-mediated MDR has good potential for further development.     

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aims

Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM.