Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells

Lycopene, the major tomato carotenoid, has been found to inhibit proliferation of several types of cancer cells, including those of breast, lung, and endometrium. By extending the work to the HL-60 promyelocytic leukemia cell line, we aimed to evaluate some mechanistic aspects of this effect. Particularly, the possibility was examined that the antiproliferative action of the carotenoid is associated with induction of cell differentiation. Lycopene treatment resulted in a concentration-dependent reduction in HL-60 cell growth as measured by [3H]thymidine incorporation and cell counting. This effect was accompanied by inhibition of cell cycle progression in the G0/G1 phase as measured by flow cytometry. Lycopene alone induced cell differentiation as measured by phorbol ester-dependent reduction of nitro blue tetrazolium and expression of the cell surface antigen CD14. Results of several recent intervention studies with beta-carotene, which have revealed no beneficial effects of this carotenoid, suggest that a single dietary component cannot explain the anticancer effect of diets rich in vegetables and fruits. Thus another goal of our study was to examine whether lycopene has the ability to synergize with other natural anticancer compounds, such as 1,25-dihydroxyvitamin D3, which when used alone are therapeutically active only at high and toxic concentrations. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibited a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression. Such synergistic antiproliferative and differentiating effects of lycopene and other compounds found in the diet and in plasma may suggest the inclusion of the carotenoid in the diet as a cancer-preventive measure.     

植物黄酮对白血病细胞HL-60氧化-还原电位影响

目的 观察植物黄酮3',4',5',7'-四羟黄酮(1uteolin)和3'-甲氧基,3,7,4'-三羟黄酮(geraldol)对白血病细胞HL-60氧化-还原电位的影响.方法 四甲基偶氮噻唑蓝(MTT)法检测细胞活力变化;邻苯二醛(POT)比色法测定胞内还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平,计算GSH/GSSG及氧化-还原电位.结果 10μmol/L的luteolin或Geraldol作用0 h时细胞氧化-还原电位为-(295.5 ±31.2)mV,随后逐渐升高,分别作用4和2 h后达到-(278.6 ±28.7)和-(279.6±26.3)mV,与对照组比较,差异有统计学意义(P<0.05);10μmol/LNAC干预能够降低luteolin和geraldol对HL-60细胞的增殖抑制效应,干预后其24 h增殖抑制率分别由(25.4±1.8)%,(21.8±1.5)%降低至(16.8±1.1)%,(12.1±1.0)%,差异有统计学意义(P<0.05).结论 luteolin和ger-aldol能够明显升高HL-60细胞的氧化-还原电位,降低其GSH水平,可能在其抗肿瘤机制中发挥作用.     

Antioxidant and cytotoxic properties of lyophilized beer extracts on HL-60 cell line

An impressive number of studies have suggested that red wine can be considered the protective beverage of choice against chronic and degenerative pathologies. Only few and controversial data are available on a potential, similar role for beer, which represents a more cost-effective, safe, and widely available beverage. Starting from the evidence that many antioxidant compounds present in red wine are also present at similar or even higher concentrations in beers, we first screened 48 commercially available beers and selected one (Mrt-HP) with very high polyphenol concentration and antioxidant activity estimated by ferric reducing antioxidant power. We demonstrated that a lyophilized preparation of Mrt-HP beer was cytotoxic with respect to a beer with low polyphenolic content (Trt-LP) when assayed on HL-60 human leukemia cell line. We measured a 60% decrease in cell viability at a polyphenol concentration of 250 microM quercetin equivalents. We also demonstrated that Mrt-HP cytotoxicity was not an artifact due to cell growth conditions because addition of Mrt-HP extracts to cell medium generated peroxide levels indistinguishable from controls. By means of cytofluorimetric analysis of pre-G1 population and caspase 3 activation, we demonstrated that Mrt-HP extracts activated apoptosis in HL-60 cell line. Finally, we found that the concentration of quercetin, resveratrol, and gallic acid in Mrt-HP was 10, 4.6, and 4.6-fold higher, respectively, than in Trt-LP, suggesting that the presence of these molecules might be responsible for the observed cytotoxicity. These data, together with the low in vivo beer toxicity reported in the literature, suggest a possible chemopreventive role for this beverage that requires further studies in animal models.     

表没食子儿茶素没食子酸酯体外诱导HL-60细胞凋亡

目的 为研发天然抗白血病新药，研究茶多酚主要活性成分表没食子儿茶素没食子酸酯(EGCG)体外诱导细胞凋亡的作用。方法 采用体视显微镜、DNA凝胶电泳及透射电镜技术，观察EGCG对体外诱导人急性早幼粒白血病细胞(HL-60)凋亡的影响及其诱导凋亡的最佳作用时间和最佳作用浓度。结果 发现EGCG实验组中，HL—60细胞生长被显著抑制，DNA凝胶电泳中可见DNA条带，其细胞超微结构明显改变。当250ug／m1 EGCG作用细胞6h时，其诱导细胞凋亡的作用最明显。结论 EGCG可体外诱导HL-60细胞凋亡．可能是抗白血病的侯选药。     

Inhibition of 5-lipoxygenase induces cell death in anti-inflammatory fatty acid-treated HL-60 cells

BACKGROUND: Enteral nutrition containing eicosapentaenoic (20:5 omega‐3) and gamma‐linolenic acid (18:3 omega‐6) decreases leukotriene B4 levels and neutrophils in bronchoalveolar lavage fluid of patients and animals with acute respiratory distress syndrome. Reduction in pulmonary inflammation may be caused by decreased neutrophil migration or survival. We showed that apoptosis increases in eicosapentaenoic/gamma‐linolenic acid‐treated HL‐60 cells. We hypothesize that eicosapentaenoic/gamma‐linolenic acid‐induced apoptosis involves downstream metabolic products of lipoxygenase and cyclooxygenase enzymes. This study determined the effects of inhibitors of lipoxygenase and cyclooxygenase enzymes on eicosapentaenoic/gamma‐linolenic acid‐treated HL‐60 cells. METHODS: Cells were incubated with 50 microM eicosapentaenoic/20 microM gamma‐linolenic acid in the presence of an enzyme inhibitor (1–10 microM) for 12 hours. Compounds were used to inhibit cyclooxygenase (ibuprofen), 12‐lipoxygenase (baicalein), or 5‐lipoxygenase (AA‐861). Flow cytometry assessed viability, apoptosis, and necrosis. RESULTS: 5‐Lipoxygenase inhibition decreased cell viability and increased cell death (apoptosis + necrosis) in eicosapentaenoic/gamma‐linolenic acid‐treated HL‐60 cells. Inhibition of cyclooxygenase 1 and 2 and 12‐lipoxygenase had no significant effect on cellular viability and death in eicosapentaenoic/gamma‐linolenic acid‐treated HL‐60 cells. Adding leukotriene B4 counteracted the effect of 5‐lipoxygenase inhibition on apoptosis in eicosapentaenoic/gamma‐linolenic acid‐treated HL‐60 cells. CONCLUSIONS: These data suggest that the processing of eicosapentaenoic and gamma‐linolenic acid by 5‐lipoxygenase is critical to HL‐60 cell survival.     

乙醇诱导HL－60细胞凋亡的研究

为了解乙醇导致血液粒细胞、单核细胞、淋巴细胞数量减少的原因,以ＨＬ－６０细胞为 凋亡研究模型,采用流式细胞术和电子显微术等方法探讨了乙醇对细胞凋亡的影响。结果表明,ＨＬ－６０细胞在５０、１００、１５０、２００、２５０ｍｍｏｌ／Ｌdisplay structure     

RRR-alpha-tocopheryl succinate modulation of human promyelocytic leukemia (HL-60) cell proliferation and differentiation.

HL-60 human promyelocytic leukemia cells can be induced to differentiate to granulocytes by retinoic acid and dimethyl sulfoxide or monocyte-macrophages by phorbol esters and 1,25-dihydroxyvitamin D3. These studies show that RRR-alpha-tocopheryl succinate (TS) inhibits HL-60 cell proliferation and induces the HL-60 cells to differentiate toward a functionally deficient macrophage-like cell. TS at (15 micrograms/ml) was found to suppress HL-60 cell proliferation by 63% and 89% at 24 and 48 hours, respectively. This suppression of proliferation, however, is not permanent and requires the presence of TS. HL-60 cells treated for 48 hours with TS (15 micrograms/ml) were found to be blocked in the G2/M phase of the cell cycle. HL-60 cells blocked in the G2/M cell cycle phase by TS expressed normal levels of the transferrin receptor. TS-treated HL-60 cells exhibited binucleated morphological appearance; however, the cells did not exhibit chemotaxis, phagocytosis, or changes in the expression of the cell surface markers, CD11a and CD18. However, HL-60 cells treated for 48 hours with TS (15 micrograms/ml) could be stimulated to produce superoxide radicals and exhibited nonspecific esterase activity, two characteristics of macrophages. These results suggest a role for TS as an antitumor proliferative agent and as a modifier of human leukemia cell differentiation.     

Effects of adipocyte-secreted factors on cell cycle progression in HT29 cells

Background

Obesity is a chronic sub-inflammatory condition which is a risk factor for several cancer diseases, e.g. colon cancer. Adipose tissue secretes biologically active factors like leptin with a known pro-inflammatory or mitogenic activity. Both, chronic inflammation and an increased cell proliferation are considered to play an important role in colon carcinogenesis. Diverse phytochemicals were shown to have cell growth inhibiting effects.

Aim of the study

The aim was to investigate whether adipocytes could mediate a proliferative capacity to HT29, a human colon adenocarcinoma cell line, and whether phytochemicals could modulate this effect.

Methods

Infranatants of adipocyte cultures from different donors were prepared and the effects of those conditioned adipocyte media (CAM) on HT29 cell growth were measured. Additionally, cell cycle progression was analyzed by flow cytometry after CAM treatment and ERK 1/2 phosphorylation was analyzed.

Results

CAM from a subgroup of adipose tissue donors stimulated HT29 cell growth, whereas others did not. This effect seems to be mediated via the ERK 1/2 pathway. Furthermore, CAM caused changes in cell cycle distribution with a shift of HT29 cells from G1- into the S-phase. This effect could be mimicked by leptin (1 nM). Co-incubation of CAM-treated HT29 cultures with β-carotene or EGCG did not have a significant impact on cell cycle progression, whereas genistein (30 µM) tended to inhibit the CAM-stimulated transition of cells into the S-phase.

Conclusion

This study confirmed the mitogenic activity of leptin in HT29 cells, although leptin secretion from adipocytes is not likely to be responsible for CAM-stimulated cell growth in our test system. The investigated phytochemicals seem to have only a minor influence on CAM-mediated cell cycle progression.

     

Lycopene and apo-12'-lycopenal reduce cell proliferation and alter cell cycle progression in human prostate cancer cells

Lycopene is associated with a reduced risk of prostate cancer. However, lycopene may not be wholly responsible for the effects seen in vivo or in cell culture systems. Apo-lycopenals or other lycopene metabolites, whether produced by cleavage enzymes within the body or consumed with tomato products, can be found in tissues at concentrations equivalent to physiological retinoid concentrations. Therefore, it is plausible that lycopenoids, like retinoids, are bioactive within tissues. Androgen-independent DU145 prostate cancer cells were treated with lycopene, apo-8'-lycopenal, or apo-12'-lycopenal. DU145 cell proliferation was significantly reduced by supra-physiological levels of lycopene and apo-12'-lycopenal, in part, through alteration of the normal cell cycle. Levels of the gap junction protein, connexin 43, were unaltered by lycopene or apo-lycopenal treatment while cell apoptosis rates significantly decreased. We further confirmed that connexin 43 protein levels were unaltered by lycopene treatment in mouse embryonic fibroblasts, or in Dunning R3327-H rat prostate tumor. The present data indicate that lycopene and apo-12'-lycopenal reduce the proliferation of prostate cancer cells, in part, by inhibiting normal cell cycle progression.     

Neoplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibited by mango (Mangifera indica L.) juice and mango juice extracts

The mango, Mangifera indica L., is a fruit with high levels of phytochemicals, suggesting that it might have chemopreventative properties. In this study, whole mango juice and juice extracts were screened for antioxidant and anticancer activity. Antioxidant activity of the mango juice and juice extracts was measured by 3 standard in vitro methods. The results of the 3 methods were in general agreement, although different radicals were measured in each. Anticancer activity was measured by examining the effect on cell cycle kinetics and the ability to inhibit chemically induced neoplastic transformation of mammalian cell lines. Incubation of HL-60 cells with whole mango juice and mango juice fractions resulted in an inhibition of the cell cycle in the G(0)/G(1) phase. A fraction of the eluted mango juice with low peroxyl radical scavenging ability was most effective in arresting cells in the G(0)/G(1) phase. Whole mango juice was effective in reducing the number of transformed foci in the neoplastic transformation assay in a dose-dependent manner. These techniques provide valuable screening tools for health benefits derived from mango phytochemicals.     

三羟基苯甲酸对淋巴瘤细胞凋亡及周期影响

目的 观察不同剂量3,4,5三羟基苯甲酸化合物(TAD)对小鼠淋巴瘤细胞株EL-4细胞的存活率、细胞凋亡及细胞周期进程的影响,探讨其对肿瘤生长抑制的机制.方法 应用四甲基偶氮噻唑蓝(MTT)比色法和流式细胞仪检测经不同浓度(3.125~25μg/ml)TAD处理的EL-4细胞存活率、细胞凋亡率及细胞周期的变化.结果 EL-4细胞经过3.125,6.25,12.5和25μg/ml TAD处理后,各观察组细胞存活率依次为(80.60±6.01)%,(75.39±4.97)%,(71.42±3.73)%和(54.96±4.57)%;与对照组100%存活率相比明显降低(P<0.01,P<0.001).细胞凋亡率依次为(4.95±1.76)%,(19.58±7.91)%,(35.85±7.01)%,(11.67±2.96)%;明显高于对照组的(0.36±0.33)%(P<0.01,P<0.001).TAD引起细胞周期进程的改变,G1期细胞百分数明显高于对照组(P<0.05,P<0.01,P<0.001),S期细胞百分数明显低于对照组(P<0.05,P<0.01,P<0.001);G2期细胞无明显变化.结论 TAD能够降低EL-4细胞存活率,诱导EL-4细胞凋亡,阻止G1期细胞向S期进程,其抑制作用可能通过G0/G1期阻滞引起.     

雄黄对白血病HL-60细胞的生长抑制及其机制

以不同浓度（7.5～60 mmol/L）的雄黄（As4S4）作用于体外培养HL-60细胞12～48 h,MTT法检测细胞生长抑制率,流式细胞仪法观察细胞凋亡率,免疫组化法检测bc1-2,突变型p53蛋白表达,应用RT-PCR法检测HL-60细胞中突变p53 mRNA表达。结果不同浓度的雄黄作用不同时间后,可显著抑制HL-60细胞的生长,呈时间-剂量依赖性,并诱导细胞发生凋亡,凋亡率为30.18%～70.98%（P〈0.01）。雄黄作用24 h后,下调突变型p53 mRNA的表达,且bc1-2、突变型p53蛋白表达逐渐降低,并呈浓度依赖性。这可能是其重要机制之一。     

Benzene metabolite 1,2,4-benzenetriol induces halogenated DNA and tyrosines representing halogenative stress in the HL-60 human myeloid cell line

Background: Although benzene is known to be myelotoxic and to cause myeloid leukemia in humans, the mechanism has not been elucidated.Objectives: We focused on 1,2,4-benzenetriol (BT), a benzene metabolite that generates reactive oxygen species (ROS) by autoxidation, to investigate the toxicity of benzene leading to leukemogenesis.Methods: After exposing HL-60 human myeloid cells to BT, we investigated the cellular effects, including apoptosis, ROS generation, DNA damage, and protein damage. We also investigated how the cellular effects of BT were modified by hydrogen peroxide (H₂O₂) scavenger catalase, hypochlorous acid (HOCl) scavenger methionine, and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase (MPO)-specific inhibitor.Results: BT increased the levels of apoptosis and ROS, including superoxide (O₂^•−), H₂O₂, HOCl, and the hydroxyl radical (^•OH). Catalase, ABAH, and methionine each inhibited the increased apoptosis caused by BT, and catalase and ABAH inhibited increases in HOCl and ^•OH. Although BT exposure increased halogenated DNA, this increase was inhibited by catalase, methionine, and ABAH. BT exposure also increased the amount of halogenated tyrosines; however, it did not increase 8-oxo-deoxyguanosine.Conclusions: We suggest that BT increases H₂O₂ intracellularly; this H₂O₂ is metabolized to HOCl by MPO, and this HOCl results in possibly cytotoxic binding of chlorine to DNA. Because myeloid cells copiously express MPO and because halogenated DNA may induce both genetic and epigenetic changes that contribute to carcinogenesis, halogenative stress may account for benzene-induced bone marrow disorders and myeloid leukemia.     

Iron deficiency alters the progression of mitogen-treated murine splenic lymphocytes through the cell cycle.

The influence of iron deficiency on the progression of mitogen-treated splenic lymphocytes through the cell cycle was studied in 16 control, 16 pair-fed, 15 iron-deficient (ID) and 16 ID mice that were repleted for up to 3 d (R3). The test and control diets differed only in iron concentrations (0.09 vs. 0.9 mmol/kg). When mice were killed (68 d of feeding), the hemoglobin concentration and liver iron stores of ID and R3 mice were <50% those of control mice (P < 0.05). Iron deficiency did not reduce the percentage of CD3(+) cells, but decreased CD3(+) cells/mg spleen (P < 0.05). In concanavalin A-treated and nonactivated cultures, there were no significant differences among groups in the percentages of cells in resting phase of the cell cycle (G0) to cell cycle initiation phase (G1), DNA synthesis phase (S) and exit from the S phase (G2) to mitosis phase (M) phases. In anti-CD3 and anti-CD3/anti-CD28-treated cultures, higher percentages of lymphocytes from ID and R3 mice than those from control and pair-fed mice were in the G0--G1 phase (P < 0.05). Conversely, lower percentages of activated cells from ID and R mice than those from control and pair-fed mice were in S and G2--M phases (P < 0.05). Incubation of lymphocytes with mitogens decreased the percentages of cells in G0--G1 phase from 90% to 80% in control and pair-fed but not in ID and R3 mice (P < 0.05). In activated cells, indices of iron status negatively correlated with the percentages of cells in G0--G1 (r = -0.306 to -0.597) but positively with those in S (r = 0.166--0.511) and G2--M phases (r = 0.265-0.59; P < 0.05). Data suggest that altered cell cycle progression likely contributes to impaired lymphocyte proliferation usually associated with iron deficiency.     

凋亡细胞细胞膜和线粒体的动态变化

应用拓扑异构酶Ⅱ抑制剂ＶＰ－１６及化学毒物叠氮钠分别诱导ＫＬ－６０细胞凋亡及坏死,在０,２,４,８,１６及２４ｈ各时间点,应用Ｈｏｅｃｈｓｔ３３２５８染色,荧光显微镜及透射电镜对核形态进行观察,通过流式细胞术检测二乙酸荧光素（ＦＤＡ）,罗丹明１２３（Ｒｈ１２３）和碘化丙啶（ＰＩ）荧光强度的变化,观察细胞凋亡及坏死过程中细胞膜通透性和线粒体膜电位的动态变化。     

Malnutrition suppresses cell cycle progression of hematopoietic progenitor cells in mice via cyclin D1 down-regulation

ObjectiveProtein malnutrition (PM) often is associated with changes in bone marrow (BM) microenvironment leading to an impaired hematopoiesis; however, the mechanism involved is poorly understood. The aim of this study was to compare the cell cycle progression of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) and evaluate the cell cycle signaling in malnourished mice to assess the mechanism of cell cycle arrest.MethodsC57Bl/6J mice were randomly assigned in control and malnourished groups receiving normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5-wk period. Nutritional and hematologic parameters were assessed and BM immunophenotypic analysis was performed. Cell cycle of HPC (Lin^–) and HSC (Lin^–Sca-1⁺c-Kit⁺) were evaluated after 6 h of in vivo 5-bromo-2'-deoxyuridine (BrDU) incorporation. Cell cycle regulatory protein expression of HPC was assessed by Western blot.ResultsMalnourished mice showed lower levels of serum protein, albumin, glucose, insulin-like growth factor-1, insulin, and higher levels of serum corticosterone. PM also caused a reduction of BM myeloid compartment resulting in anemia and leukopenia. After 6 h of BrDU incorporation, malnourished mice showed G0-G1 arrest of HPC without changes of HSC proliferation kinetics. HPC of malnourished mice showed reduced expression of proteins that induce cell cycle (cyclin D1, cyclin E, pRb, PCNA, Cdc25a, Cdk2, and Cdk4) and increased expression of inhibitory proteins (p21 and p27) with no significant difference in p53 expression.ConclusionPM suppressed cell cycle progression mainly of HPC. This occurred via cyclin D1 down-regulation and p21/p27 overexpression attesting that BM microenvironment commitment observed in PM is affecting cell interactions compromising cell proliferation.     

漆姑草醇提物对HL-60细胞诱导分化作用

目的 探讨漆姑草醇提物（HSJ）对人早幼粒白血病细胞（HL-60）的诱导分化作用。方法 实验设漆姑草250.0、125.0、62.5 μg/mL组、阳性对照组（吡柔比星100 μg/mL）、对照组;噻唑蓝（MTT）法检测HL-60细胞增殖抑制率;瑞氏-吉姆萨染色法观察细胞形态;硝基四氮唑蓝（NBT）还原实验法测定细胞分化能力;流式细胞术检测分化相关抗原CD11b、CD14阳性细胞的表达;Western blot法检测c-myc蛋白表达。结果 与对照组比较,漆姑草组HL-60细胞核浆比例减小,核仁变为肾形或蚕豆形,出现明显的细胞分化形态,细胞增殖受到不同程度抑制;对照组HL-60细胞NBT还原率为（0.83±0.29）%,CD11b和CD14阳性细胞表达率分别为（41.13±0.42）%、（53.30±7.23）%,c-myc蛋白表达量为（0.71±0.02）;漆姑草250.0、125.0、62.5 μg/mL组HL-60细胞NBT还原率[分别为（16.67±1.76）%、（10.83±1.26）%、（6.00±0.87）%]升高,CD11b和CD14阳性细胞表达率[分别为（66.77±4.27）%、（52.27±0.95）%、（46.27±0.38）%和（96.63±0.23）%、（85.25±3.80）%、（63.84±0.78）%]增加,c-myc蛋白表达量[分别为（0.38±0.01）、（0.45±0.01）、（0.58±0.02）]降低,差异均有统计学意义（均P<0.05）。结论 漆姑草醇提物可诱导HL-60细胞向成熟粒细胞、单核细胞方向分化。