menaquinone-6 and Leukemia

Vitamin K2 induces differentiation and apoptosis in a wide array of human cancer cell lines. Vitamin K2-mediated apoptosis proceeds much more slowly than the apoptosis induced by conventional anticancer agents. Thus, it is possible to analyze the underlying mechanism in detail. In this chapter, we focus on the pro-apoptotic effects of vitamin K2 on mitochondrial physiology with particular emphasis on changes in mitochondrial membrane potential (DeltaPsim). Upon treatment of ovarian cancer TYK-nu cells with vitamin K2, superoxide is produced after two to three days, followed shortly thereafter by release of mitochondrial cytochrome c. This is accompanied by other apoptotic features such as characteristic morphological changes and DNA fragmentation by day four. Data suggest that superoxide production might cause damage to mitochondrial membranes, open permeability transition pores, and result in disruption of DeltaPsim with subsequent release of cytochrome c. Both vitamin K2-induced production of superoxide and reduction of DeltaPsim are completely inhibited by alpha-tocopherol such that cell viability is retained. Thus, we propose that the loss of DeltaPsim caused by superoxide might be the major cause of apoptosis following exposure to vitamin K2. However, other pathways may be involved since cyclosporin A failed to completely inhibit vitamin K2-induced apoptosis.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Line, Tumor; Humans; Leukemia; Membrane Potential, Mitochondrial; Neoplasms; Vitamin K 2

Although all-trans retinoic acid (ATRA) is a standard and effective drug used for differentiation therapy in acute promyelocytic leukemia, ATRA-resistant leukemia cells ultimately emerge during this treatment. Therefore, the development of new drugs or effective combination therapy is urgently needed. We demonstrate that the combined treatment of vitamin K2 and cotylenin A synergistically induced monocytic differentiation in HL-60 cells. This combined treatment also synergistically induced NBT-reducing activity and non-specific esterase-positive cells as well as morphological changes to monocyte/macrophage-like cells. Vitamin K2 and cotylenin A cooperatively inhibited the proliferation of HL-60 cells in short-term and long-term cultures. This treatment also induced growth arrest at the G1 phase. Although 5 µg/ml cotylenin A or 5 µM vitamin K2 alone reduced c-MYC gene expression in HL-60 cells to approximately 45% or 80% that of control cells, respectively, the combined treatment almost completely suppressed c-MYC gene expression. We also demonstrated that the combined treatment of vitamin K2 and cotylenin A synergistically induced the expression of cyclin G2, which had a positive effect on the promotion and maintenance of cell cycle arrest. These results suggest that the combination of vitamin K2 and cotylenin A has therapeutic value in the treatment of acute myeloid leukemia.

Topics: Cell Differentiation; Cell Proliferation; Cyclin G2; Diterpenes; Drug Synergism; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia; Monocytes; Proto-Oncogene Proteins c-myc; Vitamin K 2

Altered metabolism is increasingly acknowledged as an important aspect of cancer, and thus serves as a potentially fertile area for the identification of therapeutic targets or leads. Our recent work using transcriptional data to predict metabolite levels in cancer cells led to preliminary evidence of the antiproliferative role of menaquinone (vitamin K2) in the Jurkat cell line model of acute lymphoblastic leukemia. However, nothing is known about the direct metabolic impacts of menaquinone in cancer, which could provide insights into its mechanism of action. Here, we used metabolomics to investigate the process by which menaquinone exerts antiproliferative activity on Jurkat cells. We first validated the dose-dependent, semi-selective, pro-apoptotic activity of menaquinone treatment on Jurkat cells relative to non-cancerous lymphoblasts. We then used mass spectrometry-based metabolomics to identify systems-scale changes in metabolic dynamics that are distinct from changes induced in non-cancerous cells or by other chemotherapeutics. One of the most significantly affected metabolites was phosphoethanolamine, which exhibited a two-fold increase in menaquinone-treated Jurkat cells compared to vehicle-treated cells at 24 h, growing to a five-fold increase at 72 h. Phosphoethanolamine elevation was observed prior to the induction of apoptosis, and was not observed in menaquinone-treated lymphoblasts or chemotherapeutic-treated Jurkat cells. We also validated the link between menaquinone and phosphoethanolamine in an ovarian cancer cell line, suggesting potentially broad applicability of their relationship. This metabolomics-based work is the first detailed characterization of the metabolic impacts of menaquinone treatment and the first identified link between phosphoethanolamine and menaquinone-induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Ethanolamines; Humans; Jurkat Cells; Leukemia; Metabolome; Metabolomics; Vitamin K 2

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.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; HL-60 Cells; Humans; Leukemia; Vitamin K 2

Vitamin K2 (VK2) effectively induces apoptosis in leukemia cell lines, including HL-60 and U937. However, combined treatment of cells with VK2 plus 1alpha,25-dihydroxy vitamin D3 (VD3) resulted in suppression of VK2-inducing apoptosis and pronounced induction of monocytic differentiation as compared with that by VD3 alone. After achieving monocytic differentiation by pre-exposure to VK2 and VD3, the cells became resistant to various apoptotic stimuli including VK2- and H2O2-treatment and serum deprivation. Accumulation of cytoplasm p21CIP1 along with disappearance of nuclear p21CIP1 was detected in cells in response to 96-h treatment with VK2 plus VD3. A stable transfectant, U937-deltaNLS-p21CIP1, which lacked the nuclear localization signal of p21CIP1 and showed overexpression of cytoplasm p21CIP1 without monocytic differentiation, was resistant to apoptosis. These data suggest that a change of intracellular distribution of p21CIP1 from nucleus to cytoplasm along with differentiation appears to be anti-apoptotic. Clinical benefits of using VK2 for treatment of patients with leukemia and myelodysplastic syndrome (MDS) have been reported. Our data suggest that VK2 plus VD3 may be an effective combination for differentiation-based therapy for leukemia and also MDS whose cytopenias are mediated though apoptosis.

Topics: Apoptosis; Calcitriol; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor p21; Cytoplasm; Flow Cytometry; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Hydrogen Peroxide; Immunoblotting; Immunoprecipitation; Leukemia; MAP Kinase Kinase 4; Models, Biological; Monocytes; Myelodysplastic Syndromes; Time Factors; Transfection; U937 Cells; Vitamin K 2

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.

Topics: Apoptosis; Bone Marrow; Diterpenes; Drug Synergism; Farnesol; Flow Cytometry; Gefarnate; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Molecular Structure; Myelodysplastic Syndromes; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Vitamin K; Vitamin K 1; Vitamin K 2