Effect of dimethyl sulfides on the induction of apoptosis in human leukemia Jurkat cells and HL-60 cells

Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me(2)S), dimethyl disulfide (Me(2)S(2)), dimethyl trisulfide (Me(2)S(3)) and dimethyl tetrasulfide (Me(2)S(4)) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 muM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me(2)S(4) asymptotically equal to Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for Jurkat cells and Me(2)S(4) > Me(2)S(3) > Me(2)S(2) asymptotically equal to Me(2)S for HL-60 cells. Me(2)S(3) and Me(2)S(4) significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Valproic acid sensitizes chronic lymphocytic leukemia cells to apoptosis and restores the balance between pro- and antiapoptotic proteins

Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in adults in the developed world. Despite significant advances in the treatment of cancer, CLL remains incurable. The main feature of the disease is the generation of circulating B-cells with prolonged survival caused by aberrant apoptosis. In this study, we observe that valproic acid (VPA), a well-established histone deacetylase (HDAC) inhibitor, mediates apoptosis in CLL cells ex vivo through caspase activation via both the extrinsic and the intrinsic apoptosis pathways, as indicated by the activation of the caspase proteins 8 and 9, and cleavage of the proapoptotic protein BID. The Bcl-2/Bax ratio was decreased as a consequence of decreased bcl-2 mRNA levels in response to treatment with VPA. With the results presented in this study, we have identified the HDAC inhibitor VPA as restoring the apoptotic pathways in CLL cells and thus their ability to undergo apoptosis.     

The synergistic effects of valproic acid and fluvastatin on apoptosis induction in glioblastoma multiforme cell lines

Glioblastoma multiforme (GBM) is the most common primary central nervous system malignant tumor. It responds poorly to standard therapies, such as surgical resection, radiation therapy and chemotherapy. Many chemotherapeutic drugs are focused on apoptosis induction and radiation sensitivity. Inhibition of histone acetylation via histone deacetylase inhibitor (HDACI) is one such strategy. Statins (or 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) are classical drugs used to lower cholesterol but also inhibitors of histone deacetylation activity. This study analyzes the combinatory effects of valproic acid (VPA) and fluvastatin on apoptosis induction in GBM8401 cells. The results show that they act synergistically in inducing γ-H2AX and apoptosis accompanied by higher acetylated histones H3 and H4. Downregulation of p53 occurred by VPA alone and fluvastatin alone, but not at their combined application; upregulation of p21 at the protein level was induced by each of the drugs alone and no further increase occurred at combined application. The drug BEZ235 inhibited phosphorylation of Akt and attenuated the level of γ-H2AX as well as cleaved PARP (cPARP) induced at combined application of VPA and fluvastatin. Induction of apoptosis within a 48 h incubation period was massive when measured as the subG1 peak (97%) and was detected after a 24 h incubation at low level when assayed with PE Annexin V. Synergistic apoptosis induction was demonstrated also after 24 h incubation by the appearance of cPARP. Partial silencing of p21 reduced cPARP as well as the percentage of apoptotic cells in the subG1 peak. However, partial silencing of p53 had no effect on apoptosis. Such findings offer a better understanding of the mechanism of action of HDACIs in combination with statins that may guide the development of a new combinatory reposition for the treatment of GBM.     

大田软海绵酸对人胚胎羊膜细胞FL凋亡的影响

大田软海绵酸（Okadaic acid,OA）是一种C38的长链脂肪酸,属聚醚类海洋毒素,是腹泻性贝毒（Diarrhetic shellfish poisoning,DSP）的主要成分,化学结构见下图。最初从大田软海绵（Halichondria Okadai）和隐瓜海绵（H．melanodocia）中分离得到,后发现实际上是由共生于上述两种海绵的利马原甲藻（Prorocentrum lima）所产生。     

Simultaneous treatment with camptothecin and valproic acid suppresses induction of Bcl-XL and promotes apoptosis of MCF-7 breast cancer cells

Camptothecin derivatives have been widely used for chemotherapy in patients with various cancers, but intrinsic and acquired drug resistance is major drawback to be overcome. In the present study, we demonstrated that simultaneous treatment with camptothecin and valproic acid induced apoptosis of MCF-7 cells, whereas neither agent alone could efficiently induce apoptosis. This induction of apoptosis was associated with loss of the mitochondrial membrane potential and was caspase dependent. Further investigation showed that concurrent treatment modulated the expression of pro-apoptotic and anti-apoptotic genes. Bcl-X_L expression was induced in MCF-7 cells treated with camptothecin alone, but not in cells treated simultaneously with camptothecin and valproic acid. Ectopic overexpression of Bcl-X_L in MCF-7 cells completely suppressed the induction of apoptosis, even with simultaneous treatment. On the other hand, efficient induction of apoptosis was achieved by treatment with camptothecin and Bcl-X_L inactivation (using siRNA or BH3 mimetic). The cytotoxic effect of camptothecin combined with valproic acid was more than additive for MCF-7 cells. Taken together, our results suggest that simultaneous administration of camptothecin and valproic acid might be useful for anticancer therapy.     

Humic acid induces apoptosis in human premyelocytic leukemia HL-60 cells

It has been shown that humic acid (HA), a phenolic polymer, exhibits pro-oxidant and cytotoxic effects. In this study, HA induction of apoptosis was studied using cultured human premyelocytic leukemia HL-60 cells. Treatment at a range of HA concentrations (50-400 microg/ml) resulted in dose-and time-dependent sequences of events marked by apoptosis, as demonstrated through by apoptotic features such as loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. This HA-induced apoptosis in the HL-60 cells was mainly associated with the release of cytochrome c from the mitochondria. Furthermore, apoptosis in the HL-60 cells was accompanied by the activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a major component in the apoptotic cell death mechanism. Although the HA-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data reported herein reveals that HA exerts antiproliferative action and growth inhibition on HL-60 cells through induction of apoptosis, which may have anticancer properties potentially useful for the development of new drug products.     

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As₂O ₃ (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As ₂O ₃ (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.     

Effect of 5-azacytidine on the differentiation of human leukemia K-562 cells

The treatment of K-562 cells with 10(-5) M to 10(-7) M 5-azacytidine induced a marked increase in benzidine-positive cells. Similarly, the exposure of K-562 cells to 2 X 10(-3) M butyric acid or 5 X 10(-7) M 1-beta-arabinofuranosylcytosine or 1 X 10(-3) M hydroxyurea induced an erythroid differentiation of K-562 cells. The activity of DNA-methyltransferase and the level of methylcytosine in newly synthesized DNA were significantly decreased when the cells were treated with 5-azacytidine or butyric acid, while 1-beta-arabinofuranosylcytosine or hydroxyurea had no inhibitory effect on DNA-methylation of K-562 cells. These results suggest that the inhibition of DNA-methylation is not necessarily a specific phenomenon for erythroid differentiation of K-562 cells.     

Fatty acid changes during the induction of differentiation of human promyelocytic leukemia (HL-60) cells by phorbolmyristate acetate.

We studied fatty acid changes that are likely to occur during phorbolmyristate acetate (PMA)-induced differentiation of HL-60 cells. It was observed that PMA-induced differentiation is associated with increased uptake, but not synthesis, of fatty acids. Fatty acid analysis revealed that arachidonic acid (AA), 20:5 n-3 and 22:6 n-3 levels are reduced with a concomitant increase in 22:5 n-6 in the phospholipid fraction. In the FFA fraction there are increases in free AA, free 20:5 n-3, 22:5 n-3 and 22:6 n-3, and a fall in free 22:5 n-6 in PMA-treated cells. PMA-induced differentiation and nitroblue tetrazolium reduction by PMA-treated cells was only partially inhibited (about 20-30%) by indomethacin and nordihydroguiaretic acid (cyclooxygenase and lipoxygenase inhibitors respectively), but not by superoxide dismutase, catalase or mannitol. These results indicate that PMA-induced differentiation of HL-60 cells is accompanied by specific changes in the fatty acid composition of the cells.     

Effect of calcium on differentiation of Friend leukemia cells

Induction of hemoglobin synthesis of Friend leukemia cells is inhibited by changing the ratio between internal and external Ca²⁺ concentrations. The concentration ratio can be successfully manipulated by the addition of the growth medium of (1) Ca²⁺ channel blocker D600 (90 nM-4 × 10² nM), (2) Ca²⁺ ionophore A23187 (1 × 10²–2 × 10² nM), and (3) EGTA at molar concentrations comparable to the Ca²⁺ concentration of the medium formulation (3 × 10² μM). The observations suggest that a specific ratio between intra- and extracellular Ca²⁺ is required for erythroid differentiation to proceed.     

Induction of differentiation of human leukemia cells]

Human myeloid leukemia cells do not differentiate into functional end-cells but remain in the proliferation pool. Human cell lines can serve as model for hematopoietic cells arrested at different stages of myeloid differentiation and helps to dissect the cellular and molecular events involved in leukemogenesis. Furthermore, several agents have been identified as inducers of differentiation of leukemia cells. Exciting new clinical observation have shown that patients with APL respond well to the treatment with all-trans retinoic acid. RAR-alpha gene was proved to translocated from chromosome 17 to a locus PML on chromosome 15. This new chimeric gene, PML-RAR alpha is extremely important to understand the leukemogenesis of APL.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 microg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC50 values: HL-60 (147.3 +/- 15.2 microg/ml), Raji (253.8 +/- 60.7 microg/ml) and NB-4 (269.3 +/- 12.4 microg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC50 > 800 microg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 μg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2α and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells. Jin and Song contributed equally to this article.     

The induction of apoptosis in human cervical carcinoma (HeLa) cells by gamma-linolenic acid

A high concentration (50 μg/ml) of gamma-linolenic acid (GLA) induced morphological lesions typical of apoptosis, as well as DNA fragmentation, in HeLa cells. A lower concentration of GLA (20 μg/ml), caused an increased proliferating cell nuclear antigen (PCNA) labelling, with 92.7% cells positive, compared to 27.7% at a concentration of 50 μg/ml GLA. In correlation with these results, the number of cells with degraded DNA below the G₀/G₁ peak increased significantly in the 50 μg/ml GLA-treated cells, but increased only slightly in cells exposed to the lower level of GLA. The high levels of PCNA induced by 20 μg/ml GLA, in both G₁ and S phases, may indicate a state of DNA repair synthesis, whilst at the higher concentration of GLA, most of the cells became apoptotic. Since apoptosis is associated with the deregulation of c-Myc expression, and as the Raf-1-MAP kinase cascade activates the expression of c-Myc and c-Jun, we investigated the effects of 20 and 50 μg/ml GLA on the Raf-1, c-Myc and c-Jun levels, and on the activity of MAP kinase. The results showed that 50 μg/ml GLA lowered the activity of MAP kinase. As expected with the decreased MAP kinase activity in the cells exposed to the higher level GLA, the c-Jun levels were also lowered. The levels of c-Myc, however, were increased. It is therefore possible that the deregulated expression of c-Myc in the HeLa cells exposed to the high level of GLA (50 μg/ml) may contribute to the induction of apoptosis in HeLa cells.     

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.