K Miyazawa

Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid

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 μM) 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.

The JAK2 V617F tyrosine kinase mutation in myelodysplastic syndromes (MDS) developing myelofibrosis indicates the myeloproliferative nature in a subset of MDS patients

The JAK2 V617F tyrosine kinase mutation in myelodysplastic syndromes (MDS) developing myelofibrosis indicates the myeloproliferative nature in a subset of MDS patients

Apoptosis/differentiation-inducing effects of Vitamin K2 on HL-60 cells : dichotomous nature of Vitamin K2 in leukemia cells

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1–50 μM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 μM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

Vitamin K2 selectively induces apoptosis of blastic cells in myelodysplastic syndrome: flow cytometric detection of apoptotic cells using APO2.7 monoclonal antibody

We have previously reported that vitamin K2 (VK2) but not VK1 has a potent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometric analysis using monoclonal antibody (mAb), APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patients with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post-MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positive Jurkat cells were consistently detectable by flow cytometry when present at levels of at least 5% in the CTB-1 suspension. In patient samples the gating area for leukemic clone was determined using cell surface antigen-specific mAbs conjugated with either fluorescein isothionate (FITC) or phycoerythrin (PE) and subsequently the cells stained with phycoerythrin cyanine (PE-Cy5)-conjugated APO2.7 mAb were assessed within the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3–10 μM of VK2 (menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the leukemic blast cells as compared with the untreated control cells in all 15 MDS patients tested. This effect was more prominent on blastic cells than that on mature myeloid cells such as CD34−/CD33++ gated cells. In addition, VK2 performed much less effectively on CD3-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-inducing activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.

Successful treatment with cyclosporin A for myelodysplastic syndrome with erythroid hypoplasia associated with T‐cell receptor gene rearrangements

Myelodysplastic syndrome (MDS) with erythroid hypoplasia, a rare form of MDS, has not yet been clearly defined. We report four patients with MDS with erythroid hypoplasia who received immunosuppressive therapy. All were elderly, had severe transfusion‐dependent anaemia, morphological evidence of myelodysplasia and a low percentage (3·2–13·6%) of erythroid precursors. Administration of cyclosporin A (CsA) improved their anaemia; all transfusion‐dependent patients achieved transfusion‐independence. An inverted CD4/8 ratio was seen in three patients who also demonstrated T‐cell receptor (TCR)‐β and ‐γ gene rearrangements by Southern blotting and clonality by polymerase chain reaction. Treatment with CsA can be an attractive alternative treatment for patients with MDS with erythroid hypoplasia, which may be associated with a clonal abnormality in T cells.

Vitamin K2 therapy for myelodysplastic syndromes (MDS) and post-MDS acute myeloid leukemia: information through a questionnaire survey of multi-center pilot studies in Japan

Vitamin K 2 therapy for myelodysplastic syndromes (MDS) and post-MDS acute myeloid leukemia: information through a questionnaire survey of multi-center pilot studies in Japan

Vitamin K2 induces apoptosis of a novel cell line established from a patient with myelodysplastic syndrome in blastic transformation

We have previously reported that vitamin K2 (VK2) has a potent apoptosis inducing activity toward various types of primary cultured leukemia cells including acute myelogenous leukemia arising from myelodysplastic syndromes (MDS). We established a novel cell line, designated MDS-KZ, from a patient with MDS in blastic transformation, and further investigated the effects of VK2 using this novel cell line. MDS-KZ shows complex chromosomal anomaly including −4, 5q−, −7, 13q+, 20q−, consistent with that seen in the original patient. Culture of MDS-KZ cells in RPMI1640 medium containing 10% FBS lead to steady but very slow proliferation with a doubling time of 14 days. However, the cellular growth rate was significantly accelerated in the presence of various growth factors such as granulocyte colony-stimulating factor, stem cell factor, granulocyte–macrophage colony-stimulating factor, interleukin-3, and thrombopoietin. Most of the cultured cells show the morphological features of myeloblasts. They are positive for CD7, CD33, CD34, CD45, CD117, and HLA-DR. However, about 10% of the cells are more mature metamyelocytes and neutrophils with various dysplastic characteristics such as pseudo-Pelger nuclear anomaly and hypersegmentation, suggesting a potential for differentiation in this cell line. As previously reported for cultured primary leukemia cells, exposure to VK2, but not to VK1, resulted in induction of apoptosis of MDS-KZ cells in a dose-dependent manner (IC50: 5 μM). In addition, VK2 treatment induced down-regulation of BCL-2 and up-regulation of BAX protein expression with concomitant activation of caspase-3 (CPP32). A tetrapeptide functioning as antagonist of caspase-3, Ac-DEVD-H, suppressed the VK2-induced inhibition of cell growth, suggesting that caspase-3 is, at least in part, involved in VK2-induced apoptosis. These observations suggest that the MDS-KZ cell line can serve as a model for the study of the molecular mechanisms of VK2-induced apoptosis.

Combination of 22-oxa-1,25-dihydroxyvitamin D 3 , a vitamin D 3 derivative, with vitamin K 2 (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells

We originally reported that vitamin K2 (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D3derivative, 22-oxa-1,25-dihydroxyvitamin D3 (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1α,25(OH)2D3. In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.

Combination of granulocyte colony-stimulating factor and low-dose cytosine arabinoside further enhances myeloid differentiation in leukemia cells in vitro.

We examined the differentiation-inducing effect on freshly isolated myeloid leukemia cells in liquid suspension culture by combined treatment with granulocyte colony-stimulating factor (G-CSF) plus low-dose cytosine arabinoside (Ara-C; 5-10 ng/ml) in 25 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in leukemic transformation. Culture with G-CSF alone showed leukemic cell growth stimulation in 15 out of the 25 cases (60%) and induction of cell differentiation in 19 out of the 25 cases (76%), respectively. In 23 cases (92%), either growth stimulation and/or differentiation induction of leukemia cells was observed in response to G-CSF. This suggests that most myeloid leukemia cells are able to respond to G-CSF stimulation. In addition, treatment of cells with low-dose Ara-C alone resulted in the enhancement of myeloid specific antigens expression in 16 cases (64%). Treatment of leukemia cells with higher concentrations of Ara-C (over 50 ng/ml) alone resulted in cytocidal effects but not in the induction of differentiation. Furthermore, 15 cases (60%) showed pronounced myeloid differentiation of leukemia cells after combined exposure to G-CSF plus low-dose Ara-C as compared with cells treated with either G-CSF or Ara-C alone. The enhanced effect of differentiation induction by combining G-CSF plus low-dose Ara-C was also observed in a murine myeloid leukemia cell line WEHI-3B in vitro. These data suggest that treatment with G-CSF plus low-dose Ara-C is capable of inducing differentiation of leukemic cells in vitro, and also appears to be useful for the differentiation-based therapy of patients with AML and MDS.

Vitamin K2 therapy for a patient with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is an acquired stem cell disorder most likely arising from a genetic lesion in a single mulipotent hematopoietic stem cell that often progresses to overt acute myeloid leukemia (post-MDS AML). The syndrome predominantly affects the elderly, with a median age of 60 to 70 years. Intensive chemotherapy achieves an acceptable response rate, but the high incidence of treatment-related death and the short duration of complete remission mean that there is no survival benefit.1 High-dose chemotherapy supported by allogenic bone marrow transplantation is the only potentially curative treatment for MDS, however, such therapy can only be offered to a few younger patients. Since the prognosis of MDS in the elderly is poor, establishment of a novel strategy for treating these patients has been an urgent issue. We have previously reported that vitamin K2 (VK2) analogs such as menaquinone (MK)-3, -4 and -5, but not vitamin K1, have a potent apoptosis-inducing effect on various types of primary cultured leukemic cells, including post-MDS AML cells.3 In addition, we recently demonstrated that this apoptosis-inducing effect is rather selective for blast cells, as assessed by flow cytometry of whole marrow mononuclear cells from patients who had refractory anemia with excess of blast (RAEB) or RAEB in transformation. In contrast, VK2 was reported to show some differentiation-inducing activity on acute myeloid leukemic cell lines such as HL-60 and U937 in vitro. MK4 has been used clinically for the treatment of the patients with osteoporosis in Japan. The safety of daily doses of MK4 up to 135 mg orally over 24 weeks has already been confirmed in a late phase II trial for osteoporosis.6 These findings suggested that VK2 may be useful for the treatment of patients with MDS in blastic transformation. A 65-year-old man was referred to our department because of thrombocytopenia and anemia. Peripheral blood examination showed leukoerythroblastosis with morphological abnormalities such as hypogranulation and pseudo-Pelger nuclear abnormality of neutrophils, giant platelets, micromegakaryocytes, and binuclear erythroblasts with megaloblastic change. He was diagnosed as having MDS with myelofibrosis by bone marrow trephine. The percentage of blast cells in his peripheral blood soon reached over 30%, indicating progression to the overt acute leukemic phase (Table 1). As we also found previously in other MDS patients, culture of his peripheral mononuclear cells in the presence of 3 and 10 mm MK4 for 72 h resulted in selective elimination of CD34++/CD33dull+ blast cells in a dose-dependent manner (Figure 1). Because his fibrotic marrow made him unable to tolerate intensive chemotherapy, oral administration of 90 mg of MK4 daily was started after obtaining informed consent. Six weeks after initiation of MK4 therapy, the peripheral blast cell count has significantly decreased and the platelet count has increased from 31 × 109/l to 133 × 109/l (Table 1). Thereafter, the dose of MK4 was reduced to 45 mg/day, and treatment was continued. This has resulted in maintaining a good per-