Eun Shil Kim

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase.

EB1089, a novel 1,25‐dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI‐H929 human myeloma cells. EB1089 inhibited cell growth of NCI‐H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose‐dependent manner. We could also detect apoptosis in NCI‐H929 cells exposed to EB1089 (1 × 10−7 m for 72 h) using the sub‐G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down‐regulation of the Bcl‐2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089‐treated NCI‐H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose‐ and time‐dependent manner. In addition, EB1089 caused the down‐regulation of p44 extracellular signal‐related kinase (ERK) activity and up‐regulation of the p38 kinase activity during apoptosis of NCI‐H929 cells. These results suggest that EB1089 inhibits growth of NCI‐H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.

Monensin-mediated growth inhibition in human lymphoma cells through cell cycle arrest and apoptosis

Summary. Monensin, a Na+ ionophore, regulates many cellular functions, including apoptosis. We investigated the in vitro antiproliferative effect of monensin on nine human lymphoma cell lines. Monensin significantly inhibited the proliferation of all the lymphoma cell lines examined with a 50% inhibition concentration of about 0·5 μmol/l, and induced a G1 and/or a G2‐M phase arrest in these cell lines. To address the antiproliferative mechanism of monensin, we examined the effect of this drug on cell‐cycle‐related proteins in CA46 cells (both G1 and G2 arrest) and Molt‐4 cells (G2 arrest). Treatment with monensin for 72 h decreased CDK4 and cyclin A levels in CA46 cells, and cdc2 levels in Molt‐4 cells. In monensin‐treated CA46 cells, increased p21‐CDK2, p27‐CDK2 and p27‐CDK4 complex forms were observed. And, in monensin‐treated Molt‐4 cells, increased p21–cdc2 complex form was detected. Furthermore, the activities of CDK2‐ and CDK4‐associated kinases were reduced in association with Rb hypophosphorylation in monensin‐treated CA46 cells. The activity of cdc2‐associated kinase was decreased in both cell lines, which was accompanied by induction of Wee1. Also, monensin induced apoptosis in these cell lines, as evidenced by annexin V binding assay and flow cytometric detection of sub‐G1 DNA content. This apoptotic process was associated with loss of mitochondria transmembrane potential (Δψm). Taken together, these results demonstrated for the first time that monensin potently inhibits the proliferation of human lymphoma cell lines via cell cycle arrest and apoptosis.

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3‐sensitive HL‐60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down‐regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34− cells > leukaemic CD34+ cells > normal CD34− cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S‐phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.

Ontogeny of natural killer cells and T cells by analysis of BCR-ABL rearrangement from patients with chronic myelogenous leukaemia.

Chronic myelogenous leukaemia (CML) is a haematological malignant disorder characterized by the Philadelphia chromosome (Ph) and BCR–ABL gene rearrangement. This abnormal fusion gene can be considered to serve as a marker for the transformed cell clone in CML and is found in all cells arising from the same malignant precursor cell. It has been detected in CML cells of the myeloid, monocytic, erythroid and B‐lymphocytic lineages. However, it is still arguable as to whether T lymphocytes or natural killer (NK) cells carry this marker. Answering this question would clarify the ontogenic relationship between NK cells and T cells. We examined 12 CML patients and studied the expression of BCR–ABL rearrangement by fluorescence in situ hybridization (FISH) in both NK cells and T cells sorted by flow cytometry. The purity of T cells was 95·6–99·8% and that of NK cells was 95·3–99·3% after sorting. Neither NK cells nor T cells showed any positive BCR–ABL signal with the exception of one patient who recovered from a lymphoid blastic crisis. We speculate that T cells and NK cells originate from BCR–ABL‐negative stem cells.

Inhibitory effects of eupatilin on tumor invasion of human gastric cancer MKN-1 cells

Extracts of the whole herb of Artemisia asiatica Nakai (Asteraceae) are used in traditional oriental medicine to treat inflammation. Eupatilin (5,7-dihydroxy-3′,4′,6-trimethoxyflavone) is one of the pharmacologically active components found in A. asiatica, and has been shown to possess anti-tumoral effects in some malignancies, including gastric cancer. However, its anti-metastatic effect in gastric cancer is hardly known. In this study, anti-metastatic effect of eupatilin was investigated in the human gastric cancer cell line, MKN-1. Eupatilin inhibited MKN-1 growth in a dose- and a time-dependent manner, and induced apoptosis with a concomitant increase of caspase-3 activity. ELISA demonstrated that release of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8) was significantly reduced by eupatilin. And p-AKT and p-ERK (p44/42) was reduced. Expression level of β-catenin and integrin was reduced and p-GSKβ was increased. In transcription reporter system, the activity of the transcriptional factor, NF-κB, was reduced by eupatilin and the expression of p65 was down-regulated when MKN-1 cells were treated with eupatilin. Moreover, a zymography study revealed that this reduction in invasive potential resulted from a reduction in type IV collagenolytic (gelatinolytic) activity. The expressions of metalloproteinases (MMP-2 and MMP-9) were also reduced in MKN-1 cells treated with eupatilin. In vitro invasion assay, eupatilin inhibited MKN-1 penetrating reconstituted basement membrane barriers. These results suggest that eupatilin inhibits the MKN-1 gastric cancer cell proliferation via activation of caspase-3 and the metastatic potential of gastric cancer cells via down-regulation of NF-κB activity followed by reduction of pro-inflammatory cytokine-mediated MMPs expressions.

Anti-leukemic effect of a synthetic compound, (±) trans-dihydronarciclasine (HYU-01) via cell-cycle arrest and apoptosis in acute myeloid leukemia

(±) trans‐Dihydronarciclasine, isolated from Chinese medicinal plant Zephyranthes candida, has been shown to possess quite potent anti‐tumoral effect against selected human cancer cell lines. However, little is known about the anti‐tumoral effect of (±) trans‐dihydronarciclasine in acute myeloid leukemia (AML). This study was performed to investigate the effect of a novel synthetic (±) trans‐dihydronarciclasine (code name; HYU‐01) in AML. The HYU‐01 inhibited the proliferation of various AML cell lines including HL‐60 as well as primary leukemic blasts in a dose‐dependent manner. To investigate the mechanism of the anti‐proliferative effect of HYU‐01, cell‐cycle analysis was attempted in HL‐60 cells, resulting in G1 arrest. The expression levels of CDK2, CDK4, CDK6, cyclin E, and cyclin A were decreased in a time‐dependent manner. In addition, HYU‐01 up‐regulated the expression of the p27, and markedly enhanced the binding of p27 with CDK2, 4, and 6, ultimately resulting in the decrease of their kinase activities. Furthermore, HYU‐01 induced the apoptosis through the induction of proapoptotic molecules and reduction of antiapoptotic molecules in association with the activation of caspase‐3, ‐8, and ‐9. These results suggest that HYU‐01 may inhibit the proliferation of HL‐60 cells, via apoptosis, as well as G1 block in association with the induction of p27.