Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells

Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood.     

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.     

Preferential localisation of elastase in cytosol of human myeloid leukemia HL-60 cells.

The subcellular distribution of the elastase in human myeloid leukemia HL-60 cells was studied in comparison with that in normal leukocytes. On differential centrifugation, most of the elastase activity of HL-60 cell lysates was recovered in the 105,000 x g supernatant, while that of human peripheral blood leukocyte lysates was recovered in the 500 x g precipitate (azurophil granule-rich fraction). Moreover, on Percoll density gradient centrifugation, the elastase activity in HL-60 cell extracts was recovered in the lightest fraction with none in the azurophil granule-rich fractions, whereas most of the activity in leukocyte extracts was recovered in the azurophil granule-rich fractions. This subcellular localization of elastase did not change when HL-60 cells differentiated into monocytes and granulocytes by induction with 12-O-tetradecanoyl phorbol-13-acetate and retinoic acid, respectively. Furthermore, on Sephadex G-75 gel filtration, the elastase activity in HL-60 cell extracts was eluted earlier than that in leukocyte extracts. The size estimation indicated that the elastase of HL-60 cells was 36-30 kDa, corresponding to the size of an elastase precursor reported. The relevance of a large form of the elastase in HL-60 cells to its subcellular localization is discussed.     

1,1-Diphenyl-2-picrylhydrazyl radical-scavenging compounds from soybean miso and antiproliferative activity of isoflavones from soybean miso toward the cancer cell lines

Guided by their DPPH radical-scavenging activity, nine compounds were isolated from soybean miso. Of these, 8-hydroxydaidzein, 8-hydroxygenistein and syringic acid had as high DPPH radical-scavenging activity as that of alpha-tocopherol. The antiproliferative activity of four of the isolated isoflavones toward three cancer cell lines was examined. 8-Hydroxygenistein showed the highest activity (IC50=5.2 microM) toward human promyelocytic leukemia cells (HL-60).     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

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。     

Cytotoxicity and mode of action of vanada- and niobatricarbadecaboranyl monohalide complexes in human HL-60 promyelocytic leukemia cells

Vanada- and niobatricarbadecaboranyl monohalide complexes proved to be potent cytotoxic agents against murine and human leukemia and lymphoma growth as well as HeLa suspended uterine carcinoma. The vanada complex reduced the growth of KB nasopharynx, Hepe liver, HCT-8 ileum and 1-A9 ovary solid carcinomas. A mode of action study in human HL-60 promyelocytic leukemia cells showed that DNA and purine de novo syntheses were significantly inhibited with suppression of the regulatory enzymes activities of DNA polymerase alpha and PRPP-amido transferase. There was moderate inhibition of RNA synthesis and m-RNA polymerase activity. These complexes did not inhibit human topoisomerase I or II activity, although the niobium complex nicked the DNA. The complexes did activate caspases 3, 6 and 9 which are linked to apoptosis programmed cell death. These vanada- and niobatricarbadecaboranyl monohalide complexes appear to be more specific in their effects on leukemia cell metabolism than other sandwich complexes which have broad effects on multiple enzymes.     

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 immunoblotting 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.     

Dioscin (saponin)-induced generation of reactive oxygen species through mitochondria dysfunction: a proteomic-based study

It is generally believed that traditional Chinese medicine such as saponins has great value as potent cancer prevention and chemotherapeutic agents; however, the molecular basis for their activities is for the most part lacking. In the present study, we used proteomics to examine the cytotoxic effect of dioscin, a glucoside saponin, on human myeloblast leukemia HL-60 cells. Dioscin induced apoptosis in HL-60 cells in a time-dependent manner. Protein profiling of the microsomal fraction with enriched plasma membrane proteins isolated from HL-60 cells revealed that proteins act as chaperones and/or mediators of protein folding and were substantially altered in expression cells upon dioscin stimuli. Further biochemical study indicated that mitochondria dysfunction caused generation of reactive oxygen species (ROS), leading to the changes in protein expression. The mitochondrial transmembrane potential (DeltaPsi m) inhibitor aristolochic acid (ArA) partially abrogated the dioscin-initiated death receptor apoptosis pathway and cell death. The current study provided detailed evidence to support that dioscin is capable of inducing apoptosis in mammalian cells, in which the mitochondria-initiated apoptosis pathway plays an important role.     

Control of differentiation and apoptosis of human myeloid leukemia cells by cytokinins and cytokinin nucleosides, plant redifferentiation-inducing hormones.

We examined the effects of various adenine analogues on the growth and differentiation of human myeloid leukemia HL-60 cells. Some of these analogues inhibit growth and induce nitroblue tetrazolium reducing activity in HL-60 cells. Cytokinins such as kinetin, isopentenyladenine, and benzyladenine were very effective in inducing nitroblue tetrazolium reduction and morphological changes in the cells into mature granulocytes. On the other hand, cytokinin ribosides such as kinetin riboside, isopentenyladenosine, and benzylaminopurine riboside were the most potent for growth inhibition and apoptosis. Cytokinin ribosides greatly reduced the intracellular ATP content and disturbed the mitochondrial membrane potential and the accumulation of reactive oxygen species, whereas cytokinins did not. When the cells were incubated with cytokinin ribosides in the presence of O(2)(-) scavenger, antioxidant or caspase inhibitor, apoptosis was significantly reduced and differentiation was greatly enhanced. These results suggest that both cytokinins and cytokinin ribosides can induce granulocytic differentiation of HL-60 cells, but cytokinin ribosides also induce apoptosis prior to the differentiation process.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.     

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.     

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.     

Effects of recombinant human PLD2 on proliferation and apoptosis of HL-60 cells

In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells

We have previously reported that Brazilian propolis extracts inhibited growth of HL-60 human myeloid leukemia cells, which is partly attributed to the induction of apoptosis associated with granulocytic differentiation. In this study, we isolated three compounds which induce granulocytic differentiation evaluated by nitroblue tetrazolium (NBT)-reducing assays from the water extract of propolis and identified as 4,5-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 3,4-di-O-caffeoylquinic acids by NMR analysis. Cell growth inhibitory activity of these caffeoylquinic acids was found in HL-60 cell, which was mainly attributed to the induction of apoptosis. Furthermore, the potency of caffeoylquinic acid derivatives to induce granulocytic differentiation was examined in HL-60 cells. Caffeic, quinic, and chlorogenic acids had no effects on the NBT-reducing activity, while 3,4,5-tri-O-caffeoylquinic acid induced more than 30% of NBT-positive cells. These results suggest that the number of the caffeoyl groups bound to quinic acid plays an important role in the potency of the caffeoylquinic acid derivatives to induce granulocytic differentiation. This is the first report demonstrating that the caffeoylquinic acid derivatives induce granulocytic differentiation of HL-60 cells.     

In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Halocynthiaxanthin and fucoxanthinol isolated from Halocynthia roretzi induce apoptosis in human leukemia, breast and colon cancer cells

The sea squirt Halocynthia roretzi metabolizes fucoxanthin, and subsequently accumulates its derived carotenoids with characteristic structures. In the present study, we isolated halocynthiaxanthin and fucoxanthinol as carotenoids having antiproliferative activity from H. roretzi. Halocynthiaxanthin and fucoxanthinol inhibited the growth of HL-60 human leukemia cells in a dose- and time-dependent manner. Viability of HL-60 treated with 12.5 microM halocynthiaxanthin and fucoxanthinol was decreased by 12.1% and 5.7% of control after 48 h incubation, respectively. Furthermore, halocynthiaxanthin and fucoxanthinol induced apoptosis in HL-60 cells, MCF-7 human breast cancer cells, and Caco-2 human colon cancer cells. When HL-60 cells were incubated with 12.5 microM halocynthiaxanthin and fucoxanthinol for 48 h, relative DNA fragmentations were enhanced to 5- and 7-fold compared to that in control cells, respectively. The activities of apoptosis induction by halocynthiaxanthin and fucoxanthinol were higher than that of fucoxanthin which we have previously reported as a carotenoid possessing the ability to induce apoptosis. Fucoxanthinol exhibited the highest apoptosis-inducing activity among the three carotenoids. Furthermore, the expression levels of apoptosis-suppressing protein Bcl-2 were decreased in HL-60 cells treated with halocynthiaxanthin and fucoxanthinol. These results suggest that halocynthiaxanthin and fucoxanthinol exhibited potential antiproliferative effects via apoptosis induction in several cancer cell lines.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Structure–activity relationship (SAR) of parthenin analogues with pro-apoptotic activity: Development of novel anti-cancer leads

Analogues of parthenin were synthesized by substitutions at different reaction centres to establish a structure–activity relationship (SAR). Some of the molecules have displayed significant cytotoxicity in human cervical carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compounds also induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction. Also one of the lead molecules has been shown to be the inhibitor of both telomerase and topoisomerase-II.