Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells

Objective Hydroquinone(HQ),one of the phenolic metabolites of benzene,is widely recognized as an important participant in benzene-induced hematotoxicity.However,there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn’t been fully understood yet.Methods In this study,we treated K562 cells with 40μmol/L HQ for 72 h,examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring(PRM),and performed bioinformatics analysis to identify interaction networks.Results One hundred and eighty-seven upregulated differentially expressed proteins(DEPs)and 279 downregulated DEPs were identified in HQ-exposed K562 cells,which were involved in neutrophilmediated immunity,blood microparticle,and other GO terms,as well as the lysosome,metabolic,cell cycle,and cellular senescence-related pathways.Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways,we constructed the network of protein interactions and determined 6 DEPs(STAT1,STAT3,CASP3,KIT,STAT5B,and VEGFA)as main hub proteins with the most interactions,among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity.Conclusion Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

牛蒡子苷元对白血病K562/A02细胞耐药性的逆转作用及机制

目的:研究牛蒡子苷元对白血病耐药细胞株K562/A02阿霉素耐药性的影响及其作用机制。方法:体外培养人白血病细胞株K562及阿霉素耐药细胞株K562/A02,使用2.5-50μmol/L阿霉素处理,CCK-8检测细胞生长情况并计算药物半数抑制浓度(IC_(50))。采用不同浓度的牛蒡子苷元(1、2、4、8、16 mmol/L)处理K562/A02细胞,检测牛蒡子苷元对K562/A02细胞的影响,筛选适用浓度用于后续实验。在2 mmol/L牛蒡子苷元处理的K562/A02细胞中加入5μmol/L阿霉素,流式细胞术检测细胞凋亡,Western blot检测P-gp、MRP、cleaved caspase-3、Bax、Bcl-2以及TLR4/NF-κB信号通路蛋白的表达。在牛蒡子苷元和阿霉素共处理的K562/A02细胞中转染TLR4过表达质粒,检测细胞的药物敏感性和凋亡水平。结果:阿霉素对K562/A02细胞的IC_(50)为36.57μmol/L,高于K562细胞(1.30μmol/L)。当牛蒡子苷元浓度≤2 mmol/L时,K562/A02的细胞生长不会受到明显抑制。经2 mmol/L的牛蒡子苷元处理后,阿霉素对K562/A02细胞的IC_(50)明显降低。与对照组相比,牛蒡子苷元组K562/A02细胞凋亡率明显上升,cleaved caspase-3、Bax蛋白的表达明显上调,P-gp、MRP、Bcl-2、TLR4、My D88和p-NF-κB蛋白的表达明显下调,差异均具有统计学意义(P<0.05)。在转染TLR4过表达质粒后,牛蒡子苷元处理的K562/A02细胞对阿霉素的敏感性明显降低(P<0.05),细胞凋亡下降,并显著提升了P-gp、MRP、Bcl-2和TLR4/NF-κB信号通路蛋白的表达(P<0.05),同时降低了cleaved caspase-3和Bax蛋白的表达(P<0.05)。结论:牛蒡子苷元可能通过抑制TLR4/NF-κB信号通路从而逆转人白血病耐药细胞株K562/A02对阿霉素的耐药性。

Expression of Histone H2AX Phosphorylation and Its Potential to Modulate Adriamycin Resistance in K562/A02 Cell Line

DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy. Histone H2AX phosphorylation （ser139） （γH2AX） occurs rapidly at the onset of DNA double strand break （DSB） and is critical to the regulation of DSB repair. If DNA repair is successful, cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs. In this study, we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy. K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin （ADR） and γH2AX formed. Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR. In order to test the potential of γH2AX to reverse drug resistance, K562/A02 cells were treated with PI3K inhibitor LY294002. It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR. Additionally, the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.

Effect of Hydroxyapatitc Nanoparticles on K562 Cells in vitro

Stable and single-dispersed hydroxyapatite （HAP） nanoparticles were synthesized with ultrasonic-assisted method. HAP nanoparticles were characterized by dynamic light scattering, XRD （X-ray diffraction） and TEM （Transmission Electron Microscopy）. The effect of HAP nanoparticles on the K562 human myelogenous leukemia cell line was investigated by MTT assay and cell count test, and the mechanism was studied through the changes of cell cycle and ultrastructure. The results showed that HAP nanoparticles inhibited the proliferation of K562 cells dramatically in vitro. HAP nanoparticles entered the cytoplasm of K562 cells and the cells were arrested at G/M phase, thus, the cells died directly.

Effect on Proliferation and Erythroid Differentiation of K562 Cells by IER3IP1-Knockdown

Objective： To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene. Methods： The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid differentiation correlated genes Gfi-lB, GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days. Results： The shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76% （P〈0.01）. Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group （P〈0.01）. The proliferation ability was enhanced （P〈0.01） and the proportion of cells at G0/G1 phase decreased but S phase increased （P〈0.05） in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L STI571 for two days in K562/shRNA-IER3IP1 group （P〈0.01）. Conclusion： IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.

Lethal Effect of Benzene Nitrogen Mustard Glucoside Derivate on K562 Cells

A new synthesized benzene nitrogen mustard was converted into glycosyl donor-trichloroacetimidate that was glycosylated with p-nitrophenol（glycosyl donors） to form β-lactosyl p-nitrobenzene under the protection of acetyl in a stereoselective manner, was prepared and evaluated for its cytotoxicity towards cultured K562 cell line. Methylthiazoy tetrazolium（MTT） assay, transmission electron microscopy（TEM）, flow cytometry（FCM） and immunohistochemistry were utilized to explore the mechanisms of how the compound arrests the growth of HCT-T cells. This new synthesed benzene nitrogen mustard glucoside derivate（BNMGD） presented a lower toxicity to normal cells, but is significantly more toxic to K562 cells compared with nitrogen mustard, meanwhile it can induce the apoptosis of K562 cells. These results indicate that the new synthesized BNMGD can inhibit the growth of K562 cells and induce the apoptosis, and its cytotoxicity towards cultured K562 cell line is much more effective than that of nitrogen mustard.

Zeylenone promotes apoptosis of chronic myelogenous leukemia-derived K562 cells by a mechanism involving Jak2 and src kinase

OBJECTIVE The present study was designed to investigate anticancer effect of zeylenone(Zey)on K562 cells derived from chronic myelogenous leukemia(CML)both in vitro and in vivo,followed by exploring the underlying mechanisms.METHODS Initially,the effects of Zey on cel viability,proliferation,and apoptosis were measured in K562 cells by MTT,soft agar assay,AO/EB staining,hoechst 33258 staining and flow cytometric analysis after they were treated with Zey for indicated time,the involving signaling pathways were then investigated by JC-1,real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence analysis.Furthermore,the in vivo anti-tumoractivity of Zey was assessed with nude xenografts and the involving mechanism was confirmed by immunohistochemical(IHC)and histopathological analysis.RESULTS We identified that Zey dose-dependently decreased cell viability,colony formation and expression of Proliferating Cell Nuclear Antigen(PCNA),and significantly induced K562 cell apoptosis via regulating Bcl-2 family members,decreasing mitochondrial transmembrane potential,and activating caspase-3,caspase-9,and caspase-8(P<0.05 or P<0.01).Further study revealed that Zey significantly inhibited phosphorylation of Jak2 and Src and downregulated their downstream proteins,including stat3,PI3K/AKT/m TOR,and ERK1/2 signaling pathways(P<0.05 or P<0.01).Zey also suppressed tumor growth with low toxicity in mouse xenograft model of K562cells through decreasing expression of Jak2 and Src.CONCLUSION Our data demonstrated that Zey substantially suppressed K562 cells both in vitro and in vivo through Jak2 and Src pathways.These findings suggest the potential of Zey as an effective anticancer agent in CML treatment.

Suppression of Amino Acid Transporter LAT3 Expression on Proliferation of K562 Cells

The activity of the mTOR pathway is frequently increased in acute myeloid leukemia, and is tightly related with cellular proliferation. Leucine is tightly linked to the mTOR pathway and can acti- vate it, thereby stimulating cellular proliferation. LAT3 is a major transporter for leucine, and suppres- sion of its expression can reduce cell proliferation. Here, we show that suppression of LAT3 expression can reduce proliferation of the acute leukemia cell line, K562. We investigated the mRNA and protein expression of LAT3 in several leukemia cell lines and normal peripheral blood mononuclear cells （PBMNCs） using RT-PCR and Western blotting. We also evaluated cell viability using a methyl thia- zolyl tetrazolium （MTT） assay after blocking LAT3 expression with either shRNA targeted to LAT3 or a small molecular inhibitor BCH （2-aminobicyclo-（2,2,1）-heptane-2-carboxylic acid）. LAT3 mRNA and protein expression was detected in leukemia cell lines, but not in normal PBMNCs. Using K562 cells, it was found that cellular proliferation and mTOR pathway activity were significantly reduced when LAT3 was blocked with either shRNA or BCH. Our results suggest that leukemia cell proliferation can be sig- nificantly suppressed by blocking LAT3. This finding may lead to a new strategy to develop clinical therapy for the treatment of acute myeloid leukemia.

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-κB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI double-labeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blo ing.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.

In vitro antileukemia activity of ZSTK474 on K562 cells

Aim Chronic myelogenous leukemia （CML） is a hematopoietic stem cell cancer caused by the Bcr-Abl tyrosine kinase which arises from Philadelphia chromosome （Ph） translocation. Imatinib showed potent antitumor efficacy on CML but caused resistance, therefore, other chemotherapeutic drugs for CML are expected. Phosphati-dylinositol 3-kinases （PI3Ks） are lipid kinases that preferentially phosphorylate phosphatidylinositol 4,5-bisphos- phate （PIP2） to generate phosphatidylinositol 3,4,5-trisphosphate （PIP3） which activates the downstream Akt and mammalian target of rapamycin （mTOR） , and therefore play important roles in controlling signal pathways involved in cell proliferation, etc. ZSTK474, a specific PI3K inhibitor, was reported to show potent antitumor efficacy on various solid tumors, while the anti｜eukemia effect was not yet reported. Herein, the effects of ZSTK474 on K562 CML cells as well as the adriamycin-resistant human leukemia cells （K562/ADR） are reported. Methods Cell proliferation inhibition was detected by MTT assay. Cell cycle was analyzed by FACS. The expression of cell cycle related molecules like p27 and p21 was detected by western blot and qRT-PCR. Synergistic effect of ZSTK474 and Imatinib was evaluated by MTT assay and analyzed using Calcusyn. Results MTT assay showed that ZSTK474 could inhibit the proliferation of K562 and K562/ADR cells with ICs0 as 4 69 μmol · L^-1 and 7 57 μmol·L^-1 re- spectively. ZSTK474 induced cell cycle G1 arrest in the above two cell lines dose-dependently after 48 h treatment. Western blot analysis demonstrated ZSTK474 treatment decreased the level of cyclin D1 and increased the expres- sion of p27 and p21. Similar results in mRNA level were obtained by qRT-PCR assay. Combination of ZSTK474 and Imatinib indicated synergistic effect in both cell lines. Conclusion ZSTK474 exhibited anti-leukemia activity alone, and showed synergistic effect when combined with Imatinib, on CML K562 cells. These findings suggest possible application of ZSTK474 in CML treatment.

Anti-cancer Effects of Deguelin on Human Leukemia K562 and K562/ADM Cells In Vitro

In order to investigate the anti-cancer effects of deguelin and on K562 and K562/ADM cells in vitro and the underlying molecular mechanism and compare the cytotoxicity of deguelin on K562, K562/ADM cells and human peripheral blood mononuclear cells （PBMCs）. The effects of deguelin on cell proliferation were assessed by MTT assay. Apoptosis were detected by Annexin V/PI double-labeled cytometry. The effects of deguelin on the cell cycle were studied by a propidium iodide method. Our study showed that deguelin inhibited the proliferation of K562 cell and K562/ADM cell in a time- and dose-dependent manner and had minimal effects on normal human peripheral blood mononuclear cells. The ratio of IC50 value of deguelin of 24 h on K562/ADM cells to K562 cells was only 1.27, which was significantly lower than the ratio of IC50 value of ADM （higher than 20）. Deguelin could induce apoptosis of K562 cells and K562/ADM cells. K562 cells were arrested at G2/M phase while K562/ADM cells were arrested at G0/G~ phase. Our results suggested that deguelin was a novel anti-leukemia agents with high efficacy and low toxicity and it is also a promising agent for reversing drug resistance.

The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

The difference of sensitivity to indole- 3-acetic acid （ IAA ） combined with horseradish peroxidase （HRP） in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTF assay. The cell cycle and apoptosis of K562 and BXPC-3 cells were examined by a fluorescence flow cytometer （FCM） and terminal deoxynacleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC-3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

Antitumor Effect of Betulinic Acid on Human Acute Leukemia K562 Cells in vitro

The effects of betulinic acid (BA), a pentacyclic lupane-type triterpene, on the cell viability, cell cycle and apoptosis in human leukemia K562 cells were investigated. The effects of BA on the growth of K562 cells were studied by MTT assay. Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry. The effects of BA on the cell cycle of K562 cells were studied by a PI method. The expression of Bax and capase-3 was detected by using Western blot. The results showed that BA was cytotoxic to K562 cells with an IC50 of 21.26 μg/mL at 24 h. After treating K562 cells with 10 μg/mL BA for 72 h, the number of cells was reduced by 58%. BA induced apoptosis of K562 cells in a time-and dose-dependent manner. The proportion of cells in G0/G1 and G2/M phases was decreased and that in S phase was increased after K562 cells were treated with BA for 24 h. BA treatment also increased the expression of the pro-apoptotic proteins Bax and caspase-3. It suggested that BA could inhibit the proliferation of K562 cells through the induction of cell cycle arrest and apoptosis. The antitumor effects of BA were related with up-regulation of the expression of Bax and caspase-3 proteins. BA may qualify for the development of new therapies for leukemia.

EFFECT OF BcL-2 ANTISENSE DRUG WITH DIFFERENT STRUCTURE ON THE BIOLOGICAL FUNCTION OF K562 CELLS

Objective: To study the differences and similarities of the antisense drugs with different structures on the biological functions of K562 cells. Methods: Cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis and agarose gel electrophoresis of DNA fragmentation were also performed. The expression level of protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Results: PNA targeting the coding region of the Bcl-2 messenger RNA could effectively inhibit K562 cell viability, down-regulate the synthesis of the Bcl-2 protein and increase cell apoptosis. By 72 h after the Bcl-2 antisense PNA treatment, K562 cells showed more reduction in the level of Bcl-2 protein compared with cells treated with the antisense ODN. After treatment with 10 μmol/L of Bcl-2 antisense PNA or antisense ODN for 72 h, apoptotic rates of K562 cells were 13.15±1.13 and 11.72±1.12, respectively. Furthermore, there was significant difference in the percentage of apoptotic cells between antisense PNA group and antisense ODN group. Conclusion: The results suggest that antisense PNA targeting the coding region of Bcl-2 mRNA has better antisense effects than the antisense oligonucleotides on inducing apoptosis of K562 cells. Key words Bcl-2 - Antisense peptide nucleic acid - Antisense oligonucleotide - K562 cells - Apoptosis CLC number Q255 Foundation item: This work was supported by the Key Foundation of Science & Technology Program of Guangzhou (No.2001-Z-037-01), and the Nature Science Key Foundation of Guangdong Province (No. 021195).Biography: LEI Xiao-yong(1970–), male, associate professor, doctor of medicine, Institute of Pharmacy and Pharmacology, Nanhua University, majors in tumor pharmacology.

Wortmannin Inhibits K562 Lukemic Cells by Regulating PI3k/Akt Channel In Vitro

The inhibitory effect ofwortmannin on leukemic cells and the possible mechanisms were examined. K562 cells were treated with wortmannin of various concentrations （3.125-100 nmol/L） for （0-72 h. MTT assay was used to evaluate the inhibitory effect of wortmannin on the growth of K562 cells. Cell apoptosis was detected by both Annexin-V FITC/PI double-labeled cytometry and transmission electron microscopy （TEM）. The expression of p-Akt, T-p-Akt, NF-kBp65 and IKK-κB was determined by Western blotting and reverse transcription-polymerase chain reaction （RT-PCR）. Our results showed that wortmannin obviously inhibited growth and induced apoptosis of K562 cells in vitro in a time- and dose-dependent manner. The IC50 value of wortmannin for 24 h was 25±0.14 nmol/L. Moreover, wortmannin induced K562 cells apoptosis in a dose-dependent manner. TEM revealed typical morphological changes of apoptosis in wortmannin-treated K562 cells, such as chromatin condensation, karyopyknosis, karyorhexis and apoptotic bodies. Additionally, several important intracellular protein kinases such as p-Akt, NF-κBp65 and IKK-κB experienced degradation of vari- ous degrees in a dose-dependent manner both at protein level and transcription level when cultured with wortmannin, but the expression of total Akt showed no change. It is concluded that wortmannin can inhibit the proliferation and induce apoptosis of K562 leukemia cells possibly by down-regulating the survival signaling pathways （PI3K/Akt and NF-κB channels）.

Effects of Root Extracts from <i>Panax ginseng</i>C. A. Meyer (Araliaceae) of Different Ages on K562 Cells

It is well accepted in China that elder ginsengs have more bioactivity and value than younger ones. However, there is little research about the comparison of beneficial effects of ginsengs with different ages. In this study, ginseng root extracts (GRE) were extracted from ginsengs of 5, 8, 12, 14, and 16 years old, respectively, using 55% ethanol and their effects on human leukemic K562 cells within 48 hours were tested by using Cell Counting Kit-8. The results show that there are significant increases in the cell viability of all the GRE groups compared with Control group within 32 hours. Furthermore, the growth curves of GRE groups were obviously distinct from each other. The cell viability of 5-year-old and 8-year-old GRE groups kept a rapid increase while that of 16-year-old GRE group showed a strong fluctuation within 28 hours. Our results demonstrate that root extracts from ginsengs of different ages contain different bioactivity constituents and have different effects on cell.

Study of Celastrol on Akt Signaling Pathway and Its Roles in the Apoptosis of K562 Cells

The purpose Celastrol, the main active compound of the Celastrus genus plants, belonging to Celastraceae, has recently marked antitumour potency on solid tumours of various derivations, Methods: We demonstrate here that Celastrol also present powerful antileukaemic potency through both growth arrest and apoptosis induction in K562 cells, which was accompanied by typical apoptotic morphological and sharp decreased expression of phosphorylation level of Caspase family members and Akt signaling pathway related proteins were determined by western blot before and after celastrol treatment, and further the effect of AKT signaling pathway on celastrol-induced-apoptosis was analyzed. However, in vitro treatment with Celastrol resulted in significantly reduced expression of phophorylation of Akt, Survivin and Bcl-2 significantly in K562 cells. Results: 25 nmol/L WORT (PI3K-Akt inhibitor) can significantly augmented cell apoptosis induced by Celastrol in K562 cells in dose-dependent manner, Moreover, most Caspase3,8,6 were activated in K562 cells during Celastrol treatment, 50 μmol/Lz-VAD-fmk (Caspase inhibitor) can to enhance the apoptosis induced by Celastrol. Discussion: These results suggest that the fact that Akt signaling pathway might act as new targets of Celastrol, correlates well with the sensitivity to Celastrol, as well as the rate of apoptosis induced by Celastrol, Mechanisms that regulate Akt signaling pathway may be provide novel opportunities for drug development.

Antiproliferation Effects of Curcumin on the STAT5 Signaling Parthway in K562 Cells

OBJECTIVE Curcumin is the major component of the spice turmeric and the yellow pigment in curry powder. Many studies have shown that curcumin （diferuloylmethane） has significant antiproliferative and apoptotic effects in cancer cells by several mechanisms. Signal transducers and activators of transcription （STAT） proteins are critical in mediating a response in hematopoietic cells. This study was designed to investigate whether curcumin is associated with proteins involved in signal transduction and activation of transcription （STAT） and to investigate the expression of signal transducers and activators of transcription and the significance of the STAT5 signaling pathway of by treating k562 cells and cells from CML patients with curcumin. METHODS The study was divided into the following groups： normal control cells （human bone marrow cells）, untreated K562 cells, curcumin treated K562 cells, IFN-γ treated K562 cells, curcumin plus IFN-γ treated K562 cells, and CML patient cells with and without curcumin treatment. Cell proliferation was measured by the MTT assay. The expression of STAT5 mRNA was determined by RT-PCR. The expression of the STAT5 protein was assayed by Western-blotting and the expression of STAT5 in K562 cells was examined under confocal laser-scanning microscopy. The expression of STAT5 mRNA of K562 cells was determined with in situ hybridization. EMSA was used to assess the change in binding of STAT5 with DNA in CML patient cells. RESULTS The proliferation of the K562 cells and CML primary cells was decreased in the curcumin-treated group and/or IFN-γ group. The expression of STAT5 mRNA and protein were decreased the curcumin-treated group as compared with the K562 untreated group （P〈0.01）. STAT5 mRNA and protein expression was decreased in the IFN-γ group compared to the untreated K562 group （P〈0.01）. Combined use of curcumin with IFN-γ inhibited the proliferation of K562 cells and decreased the expression of STAT5 mRNA and protein of the K562 cells. For the CML patient cells, the OD value of STAT5-DNA binding in the curcumin treated cells was less than that compared to untreated cells. CONCLUSION The antiproliferation effects of curcumin may partly be mediated through signal transduction and activation of transcription and may involve the STAT5 signaling pathway.

藤黄酸对白血病K562/A02细胞的耐药逆转作用

本研究旨在探讨藤黄酸(gambogic acid,GA)对K562/A02细胞株的耐药逆转作用及其逆转机制。采用MTT法检测GA对K562和K562/A02细胞的增殖抑制作用及对K562/A02细胞阿霉素(ADM)耐药逆转效应;用流式细胞术检测GA联合ADM对K562及K562/A02细胞凋亡率的影响;DAPI荧光染色观察ADM联合GA作用后细胞的形态学改变;Western blot法检测K562及K562/A02细胞P-糖蛋白(P-gp)、存活蛋白(Survivin)基因的表达。结果表明:ADM作用48 h抑制K562和K562/A02细胞增殖的IC50值分别为(1.42±0.07)μg/ml和(28.42±1.40)μg/ml。GA≤0.0625μmol/L时,对K562及K562/A02细胞株无明显增殖抑制作用;0.0625μmol/L GA联合ADM作用于K562/A02细胞能增加其对ADM的敏感性,耐药逆转倍数为1.53。0.0625μmol/L GA联合ADM作用于K562/A02细胞48 h能提高细胞的凋亡率(P<0.05),下调Survivin及P-gp蛋白的表达(P<0.05)。结论:GA可以逆转K562/A02细胞的耐药性,增强耐药细胞对ADM的敏感性,其机制可能与提高K562/A02细胞凋亡、下调Survivin和P-gp蛋白的表达有关。