Resveratrol, an ingredient of wine, acts synergistically with Ara-C and tiazofurin in HL-60 human promyelocytic leukemia cells

Resveratrol (RV), a naturally occurring stilbene derivative, is a potent free radical scavenger causing a number of biochemical and antineoplastic effects. It was shown to induce differentiation and apoptosis in leukemia cells and was also identified as an inhibitor of ribonucleotide reductase (RR), a key enzyme of DNA synthesis.In this study, we report about the biochemical effects of RV in HL-60 human promyelocytic leukemia cells. RV effectively inhibited in situ RR activity. Furthermore, incubation of HL-60 cells with RV significantly decreased intracellular dCTP, dTTP, dATP and dGTP concentrations. In growth inhibition and clonogenic assays, RV acted synergistically with both Ara-C and tiazofurin in HL-60 cells. We conclude that RV could become a viable candidate as one compound in the combination chemotherapy of leukemia and therefore deserves further in vitro and in vivo testing.     

Biochemical modulation of Ara-C effects by amidox, an inhibitor of ribonucleotide reductase in HL-60 promyelocytic human leukemia cells

Amidox, a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for anti cancer chemotherapy. We investigated the biochemical and antineoplastic effects of amidox as a single agent and in combination with Ara-C in human HL-60 promyelocytic leukemia cells. Amidox inhibited the growth of HL-60 cells in a growth inhibition assay with an IC50 of 25 microM. In a soft agar colony forming assay, amidox yielded a 50% inhibition of colony formation at 13 microM. We also investigated the effects of amidox treatment on the formation of deoxynucleosidetriphosphates. Amidox (50 and 75 microM for 24 hours) could significantly decrease intracellular concentrations of dCTP, dATP and dGTP pools, whereas dTTP levels increased. We then tested the combination effects of amidox with Ara-C; this combination yielded additive cytotoxic effects both in growth inhibition and in soft agar colony formation assays. This effect was due to the increased formation of Ara-CTP, the active metabolite of Ara-C, after preincubation with amidox. Preincubation of HL-60 cells with 75 and 100 microM amidox for 24 hours caused an increase in the intracellular Ara-CTP concentrations by 576% and 1143%, respectively. Therefore amidox might offer an additional option for the treatment of leukemia and thus be further investigated in in vivo studies as a single agent and in combination with Ara-C.     

Epigallocatechin gallate,ellagic acid,and rosmarinic acid perturb dNTP pools and inhibit de novo DNA synthesis and proliferation of human HL-60 promyelocytic leukemia cells: Synergism with arabinofuranosylcytosine

Epigallocatechin gallate (EGCG), ellagic acid (EA) and rosmarinic acid (RA) are natural polyphenols exerting cancer chemopreventive effects. Ribonucleotide reductase (RR; EC 1.17.4.1) converts ribonucleoside diphosphates into deoxyribonucleoside diphosphates being essential for DNA replication, which is why the enzyme is considered an excellent target for anticancer therapy.EGCG, EA, and RA dose-dependently inhibited the growth of human HL-60 promyelocytic leukemia cells, exerted strong free radical scavenging potential, and significantly imbalanced nuclear deoxyribonucleoside triphosphate (dNTP) concentrations without distinctly affecting the protein levels of RR subunits (R1, R2, p53R2). Incorporation of ¹⁴C-cytidine into nascent DNA of tumor cells was also significantly lowered, being equivalent to an inhibition of DNA synthesis. Consequently, treatment with EGCG and RA attenuated cells in the G0/G1 phase of the cell cycle, finally resulting in a pronounced induction of apoptosis. Sequential combination of EA and RA with the first-line antileukemic agent arabinofuranosylcytosine (AraC) synergistically potentiated the antiproliferative effect of AraC, whereas EGCG plus AraC yielded additive effects.Taken together, we show for the first time that EGCG, EA, and RA perturbed dNTP levels and inhibited cell proliferation in human HL-60 promyelocytic leukemia cells, with EGCG and RA causing a pronounced induction of apoptosis. Due to these effects and synergism with AraC, these food ingredients deserve further preclinical and in vivo testing as inhibitors of leukemic cell proliferation.     

Combination of diethyldithiocarbamate with 12-O-tetradecanoyl phorbol-13-acetate inhibits the growth of human myeloid leukemia HL-60 cells in vitro and in xenograft model

ABSTRACT

12-O-tetradecanoylphorbol-13-acetate (TPA), is a major active constituent of the seed oil of Croton tiglium L., has pharmacological activity for the treatment of acute myeloid leukemia patients. Diethyldithiocarbamate (DTC) is a potent inhibitor of NF-κB show activity of anticancer. In this study, we determined the effect of DTC and TPA in combination on HL-60 cells cultured in vitro and in vivo. In this study, we have shown that DTC and TPA synergistically inhibited the growth of HL-60 cells and strongly induced apoptosis in the cells. Mechanistic studies showed that the combined effects of DTC and TPA were associated with a decrease in Bcl-2. The animal experiment showed that the combination of DTC and TPA more potently inhibited the growth of HL-60 tumors than either agent alone. Our results indicate that the administration of TPA and DTC in combination may be an effective strategy for inhibiting the growth of acute myeloid leukemia cells.     

Ganoderma lucidum polysaccharide exerts anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells

In this study, we investigated the anti-tumor activity both in vitro and in vivo of a polysaccharide obtained from Ganoderma lucidum on HL-60 acute myeloid leukemia cells, and focused on its targeting effect on mitogen-activated protein kinase (MAPK) pathways. It was found by the methods such as western blot and flow cytometry (FCM), that G. lucidum polysaccharide (GLP) blocked the extracellular signal-regulated kinase/MAPK signaling pathway, simultaneously activated p38 and JNK MAPK pathways, and therefore regulated their downstream genes and proteins, including p53, c-myc, c-fos, c-jun, Bcl-2, Bax, cleaved caspase-3 and cyclin D1. As a result, cycle arrest and apoptosis of HL-60 cells were induced. Therefore, GLP exerted anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.     

Anti-c-myc DNA increases differentiation and decreases colony formation by HL-60 cells

Summary The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me₂SO), which is also know to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examine and compared with Me₂SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me₂SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation. This work was supported by grants to E. W. from the National Institutes of Health, Bethesda, MD (CA 42960), and the Leukemia Society of America.     

Capillin,a major constituent of Artemisia capillaris Thunb. flower essential oil,induces apoptosis through the mitochondrial pathway in human leukemia HL-60 cells

BackgroundNatural products are one of the most important sources of drugs used in pharmaceutical therapeutics. Screening of several natural products in the search for novel anticancer agents against human leukemia HL-60 cells led us to identify potent apoptosis-inducing activity in the essential oil fraction from Artemisia capillaris Thunb. flower.MethodsThe cytotoxic effects of extracts were assessed on human leukemia HL-60 cells by XTT assay. Induction of apoptosis was assessed by analysis of DNA fragmentation and nuclear morphological change. The plant name was checked with the plant list website (http://www.theplantlist.org).ResultsA purified compound from the essential oil fraction from Artemisia capillaris Thunb. flower that potently inhibited cell growth in human leukemia HL-60 cells was identified as capillin. The cytotoxic effect of capillin in cells was associated with apoptosis. When HL-60 cells were treated with 10⁻⁶ M capillin for 6 h, characteristic features of apoptosis such as DNA fragmentation and nuclear fragmentation were observed. Moreover, activation of c-Jun N-terminal kinase (JNK) was detected after treatment with capillin preceding the appearance of characteristic properties of apoptosis. Release of cytochrome c from mitochondria was also observed in HL-60 cells that had been treated with capillin.ConclusionCapillin induces apoptosis in HL-60 cells via the mitochondrial apoptotic pathway, which might be controlled through JNK signaling. Our results indicate that capillin may be a potentially useful anticancer drug that could enhance therapeutic efficacy.     

氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。     

Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.     

Antitumor Effects of 3,3′,4,4′,5,5′-Hexahydroxystilbene in hl-60 Human Promyelocytic Leukemia Cells

Resveratrol (3,4′,5-trihydroxystilbene, RV) exerts remarkable cytostatic and cytotoxic effects against a multitude of human cancer cell lines. Since the introduction of additional hydroxyl groups was supposed to increase the biological activity of RV, we have synthesized a number of polyhydroxylated stilbene analogues as potential antitumor agents. In this study, the activity of 3,3′,4,4′,5,5′-hexahydroxystilbene (M8) was investigated in HL-60 human promyelocytic leukemia cells. Employing a growth inhibition assay, incubation with M8 and RV resulted in IC₅₀ values of 6.25 and 12 μM, respectively. Using a specific Hoechst/propidium iodide double staining method, we found that M8 was able to induce apoptosis in concentrations significantly lower than those of RV. In addition, M8 arrested cells in the S phase and totally depleted cells in the G2-M phase of the cell cycle (143% and 0% of control after treatment with 12.5 μM M8, respectively). We therefore believe that this promising agent deserves further preclinical and in vivo testing.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

Anaplasma phagocytophilum infection induces apoptosis in HL-60 cells

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that survives in neutrophils by delaying apoptosis. The human promyelocytic leukemia cell line HL-60 has been the ultimate choice for culturing Anaplasma in vitro. In this study, we assessed the various events of drug-induced apoptosis in A. phagocytophilum-infected HL-60 cells. Anaplasma infection reduced the cell viability and increased the apoptosis in HL-60 cells and staurosporine or etoposide-induced apoptosis was further exacerbated with Anaplasma infection. Altogether our results suggest that A. phagocytophilum infection is proapoptotic in HL-60 cells unlike in neutrophils where it is antiapoptotic.     

DNA甲基化通过锌指蛋白185调控慢性粒细胞白血病细胞的增殖

锌指蛋白185(ZNF185)属于LIM结构域蛋白,参与细胞的增殖和分化,在多种肿瘤细胞中具有抑癌基因的功能.ZNF185在正常人血液系统细胞中高表达,但目前对白血病细胞的作用未见研究.采用Western blot检测人外周血中性粒细胞、急性粒细胞白血病细胞系HL-60和慢性粒细胞白血病细胞系K562细胞中ZNF185的表达,发现ZNF185在HL-60和K562细胞中的表达水平显著低于外周血中性粒细胞.为了阐明ZNF185对慢性粒细胞白血病细胞增殖的影响,从人外周血中性粒细胞克隆ZNF185编码序列,转染K562细胞,MTT检测细胞增殖,发现过表达ZNF185显著抑制K562细胞的增殖.甲基化特异PCR分析表明:ZNF185启动子在HL-60和K562细胞中高甲基化,用5-氮杂-2′-脱氧胞苷处理K562细胞,促进ZNF185的表达,显著抑制细胞增殖.研究结果表明,ZNF185启动子高甲基化导致其在K562细胞中的表达降低和细胞增殖抑制作用减弱.可能是慢性粒细胞白血病发生或发展的原因之一.     

Antitumor effects of 3,3',4,4',5,5'-hexahydroxystilbene in HL-60 human promyelocytic leukemia cells

Resveratrol (3,4',5-trihydroxystilbene, RV) exerts remarkable cytostatic and cytotoxic effects against a multitude of human cancer cell lines. Since the introduction of additional hydroxyl groups was supposed to increase the biological activity of RV, we have synthesized a number of polyhydroxylated stilbene analogues as potential antitumor agents. In this study, the activity of 3,3',4,4',5,5'-hexahydroxystilbene (M8) was investigated in HL-60 human promyelocytic leukemia cells. Employing a growth inhibition assay, incubation with M8 and RV resulted in IC50 values of 6.25 and 12 microM, respectively. Using a specific Hoechst/propidium iodide double staining method, we found that M8 was able to induce apoptosis in concentrations significantly lower than those of RV. In addition, M8 arrested cells in the S phase and totally depleted cells in the G2-M phase of the cell cycle (143% and 0% of control after treatment with 12.5 microM M8, respectively). We therefore believe that this promising agent deserves further preclinical and in vivo testing.     

<Emphasis Type="Italic">N,N</Emphasis>-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells,to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome <Emphasis Type="Italic">c</Emphasis> and AIF

Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.     

Differential expression patterns of SUMO proteins in HL-60 cancer cell lines support a role for sumoylation in the development of drug resistance

Small ubiquitin-like modifier (SUMO) proteins are involved in a variety of cellular processes. Alterations in SUMO conjugation have been implicated in several human diseases, including cancer. Although the main cause of failure in cancer treatment is the development of drug resistance by cancer cells, the mechanisms of drug resistance are not fully understood. SUMO proteins are thought to play roles in various cellular pathways, but no studies have as yet compared the expression of the different SUMO proteins in chemosensitive and drug-resistant cancer cells. To determine the relationship between protein sumoylation and drug resistance, the expression of various SUMO isoforms has been studied and compared in the HL-60 cell line (a model for leukemic cells) and in HL-60RV cells (resistant to vincristine). Co-immunostaining of cells by anti-SUMO antibodies and antibodies against various nuclear subdomains has been examined by an advanced type of bioimaging analysis. Whereas SUMO-2/3 co-localizes exclusively with nuclear bodies containing promyelocytic leukemia protein in both cell types, SUMO-1 has also been seen in nucleolar regions of HL-60, but not in HL-60RV, cells. In HL-60 cells, SUMO-1 occurs adjacent to, but not co-localized with, the nucleolar marker fibrillarin. Western blot analysis has revealed higher levels of free SUMO and sumoylated products in drug-resistant cells and the presence of specific SUMO-1 conjugates in drug-sensitive HL-60 cells, possibly consistent with a specific nucleolar signal. Shortly after the induction of ethanol and oxidative stress, HL-60RV, but not HL-60, cells show increased accumulation of high-molecular-weight SUMO-2/3 conjugates. Thus, SUMO-1 probably has a specific role in the nucleoli of HL-60 cells, and the alteration of sumoylation might be a contributing factor in the development of drug resistance in leukemia cells. The author thanks Stern College for Women, Yeshiva University and the Joseph Alexander Foundation for supporting this research project.     

Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in HL-60 human promyelocytic leukemia cells

Amidox (3,4-dihydroxybenzamidoxime), a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for cancer chemotherapy. In the present study we investigated the antineoplastic effects of Amidox alone and in combination with Arabinofuranosylcytosine (Ara-C) in HL-60 human promyelocytic leukemia cells. In growth inhibition experiments Amidox yielded an IC50 of 30 microM, colony formation was inhibited at an IC50 of 20 microM as determined by a soft agar assay. Exposure of the cells to 75 and 100 microM Amidox for 24 hours was shown to significantly decrease intracellular dCTP, dGTP and dATP pools, whereas dTTP concentration increased, as determined by HPLC. The combination of Amidox with Ara-C yielded more than additive cytotoxic effects both in growth inhibition assays and in soft agar assays. We could show that--after preincubating the cells with 75 and 100 microM Amidox and subsequent exposure to Ara-C--intracellular Ara-CTP levels increased by 576% and 1143%, respectively. In conclusion, Amidox might offer an additional option for the treatment of leukemia and thus be further investigated in vitro and in vivo.     

12-o-Tetradecanoylphorbol 13-acetate prevents baicalein-induced apoptosis via activation of protein kinase C and JNKs in human leukemia cells

In the present study, we found that baicalein (BE), but not its glycoside baicalin (BI), induced apoptosis in human leukemia HL-60 and Jurkat cells, but not in primary murine peritoneal macrophages (PMs) or human polymorphonuclear (PMN) cells, by the MTT assay, LDH release assay, and flow cytometric analysis. Activation of the caspase 3, but not caspase 1, enzyme via inducing protein processing was detected in BE-induced apoptosis. The ROS-scavenging activity of BE was identified by the anti-DPPH radical, DCHF-DA, and in vitro plasmid digestion assay, and none of chemical antioxidants including allpurinol (ALL), N-acetyl-cystein (NAC), and diphenylene iodonium (DPI) affected BE-induced apoptosis in HL-60 cells. This suggests that apoptosis induced by BE is independent of the production of ROS in HL-60 cells. Interestingly, the apoptotic events such as DNA ladders formation and activation of the caspase 3 cascade were significantly blocked by TPA addition in the presence of membrane translocation of PKCα, and TPA-induced protection was reduced by adding the PKC inhibitors, GF-109203X and staurosporin. TPA addition induces the phosphorylation of JNKs and ERKs, but not p38, protein in HL-60 cells, and incubation of HL-60 cells with JNKs inhibitor SP600125, but not ERKs inhibitor, PD98059 or the p38 inhibitor SB203580, suppressed the protective effect of TPA against BE-induced apoptotic events including DNA ladders, apoptotic bodies, caspase 3 and D4-GDI protein cleavage in according with blocking JNKs protein phosphorylation. In addition, PKC inhibitor GF-109203X treatment blocks TPA-induced ERKs and JNKs protein phosphorylation, which indicates that activation of PKC locates at upstream of MAPKs activation in TPA-treated HL-60 cells. Additionally, a loss in mitochondrial membrane potential with a reduction in Bcl-2 protein expression, the induction of Bad protein phosphorylation, and translocation of cytochrome c from mitochondria to the cytosol were observed in BE-treated HL-60 cells, and these events were prevented by the addition of TPA. GF-109203X and SP600125 suppression of TPA against cytochrome c release induced by BE was identified. This suggests that activation of PKC and JNKs participate in TPA's prevention of BE-induced apoptosis via suppressing mitochondrial dysfunction in HL-60 cells.     

Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.