Byung-Seon Chung

Increase of NKG2D ligands and sensitivity to NK cell-mediated cytotoxicity of tumor cells by heat shock and ionizing radiation

In this study, we have investigated if current cancer therapeutic modalities including hyperthermia and ionizing radiation can increase the expression of NKG2D ligands in human cancer cell lines. The expressions of NKG2D ligands were induced by both heat shock and ionizing radiation in various cell lines including KM12, NCI-H23, HeLa and A375 cells with peaks at 2 h and 9 h after treatment, respectively, although inducibility of each NKG2D ligand was various depending on cell lines. During the induction of NKG2D ligands, heat shock protein 70 was induced by heat shock but not by ionizing radiation. These results were followed by increased susceptibilities to NK cell-mediated cytolysis after treatment with heat shock and ionizing radiation. These results suggest that heat shock and ionizing radiation induce NKG2D ligands and consequently might lead to increased NK cell-mediated cytotoxicity in various cancer cells.

The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells

In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.

Direct injection of immature dendritic cells into irradiated tumor induces efficient antitumor immunity

Although there are several ways to load tumor antigens to DCs, in vitro preparation of tumor antigens and manipulation of DCs are usually required. Therefore, to develop a simple antitumor immunization method, we examined if direct injection of DCs into tumor apoptosed by ionizing IR could induce efficient antitumor immunity. Ionizing IR with 15 Gy induced apoptosis in tumor maximally after 6 hr. Injection of DCs i.t. into IR tumor induced strong cytotoxicity of splenocytes against tumor cells compared to i.t. injection of DCs or ionizing IR of tumor, both of which induced weak cytotoxicity. In an animal study, i.t. injection of DCs into IR tumor induced therapeutic antitumor immunity against a tumor established at a distant site. Moreover, when TNF‐α or LPS was added as a danger/maturation signal to DC suspension before i.t. injection, antitumor immunity was significantly potentiated compared to a group treated with i.t. injection of DCs into IR tumor. Our results suggest that injection of DCs into tumor apoptosed by ionizing IR might be a simple and efficient method of immunization against tumor.

Activation of c-jun N-terminal kinase/stress-activated protein kinase and the decreased ratio of Bcl-2 to Bax are associated with the auto-oxidized dopamine-induced apoptosis in PC12 cells.

Current concepts of the pathogenesis of Parkinsons disease center on the formation of reactive oxygen species (ROS). Dopamine is one of the major sources of ROS. In this study, the molecular events during the dopamine-induced apoptosis in PC-12 cells were studied using auto-oxidized dopamine. Auto-oxidized-dopamine induced DNA fragmentation and activation of c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) faster and stronger than dopamine. Furthermore, N-acetylcysteine, an antioxidant, prevented the auto-oxidized dopamine-induced JNK/SAPK activation and DNA fragmentation. Meanwhile, Bcl-2 started to decrease after onset of apoptosis, and Bax was increased up to beginning of apoptosis, and thereafter decreased. Therefore, these results suggested that activation of JNK/SAPK and the decreased ratio of antiapoptotic Bcl-2 to proapoptotic Bax appear to be associated with the dopamine-induced apoptosis.

Preventive effect of antioxidants in MPTP-induced mouse model of Parkinson's disease.

Oxidative stress to dopaminergic neurons is believed to be one of the causes of neurodegeneration in Parkinsons disease (PD). It was investigated whether N-acetylcysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) have a preventive effect in an oxidative stress-induced model of PD. We found that NAC and OTC prevent degradation of PARP during auto-oxidized dopamine- or auto-oxidized L-DOPA-induced apoptosis in PC12 cells. In an animal model study, NAC and OTC showed a preventive effect against MPTP-induced loss of tyrosine hydroxylase-positive neurons, and suppressed the nuclear translocation of c-jun N-terminal kinase (JNK), suggesting that NAC and OTC can prevent MPTP-induced apoptosis by suppressing JNK activation. Therefore, these results suggest that NAC and OTC can be used as potential agents to prevent the progression of PD.

Suppression of Multidrug resistance via Inhibition of Heat Shock Factor by Quercetin in MDR Cells

MDR1 promoter has been shown to contain heat shock elements (HSE), and it has been reported that FM3A/M and P388/M MDR cells show a constitutively activated heat shock factor (HSF), suggesting that HSF might be an important target for reversing the multidrug resistance. Therefore, it was examined whether quercetin, which has been shown to interfere with the formation of the complex between HSE and HSF, and to downregulate the level of HSF1, can sensitize MDR cells against anticancer drugs by inhibition of HSF DNA-binding activity. In this study, quercetin appeared to inhibit the constitutive HSF DNA-binding activity and the sodium arsenite-induced HSF DNA-binding activity in the MDR cells. The basal and sodium arsenite-induced MDRCAT activities were remarkably suppressed by the treatment of quercetin. These results were well consistent with the finding that the treatment of quercetin decreased the expression level of P-gp, MDR1 gene product, in dose-dependent manner, and markedly increased the sensitivity of MDR cells to vincristine or vinblastine. These results suggest that quercetin can decrease the expression of P-gp via inhibition of HSF DNA-binding activity, and might be useful as a chemosensitizer in MDR cells.

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.

Potentiation of chemosensitivity in multidrug-resistant human leukemia CEM cells by inhibition of DNA-dependent protein kinase using wortmannin.

DNA-dependent protein kinase (DNA-PK) is activated by DNA strand breaks and participates in DNA repair. Its regulatory subunit, Ku autoantigen, binds to DNA and recruits the catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the development of multidrug resistance (MDR). The Ku-DNA binding activity, the levels of Ku70/Ku80 and DNA-PKcs in MDR variants, CEM/VLB(10-2), CEM/VLB(55-8) and CEM/VLB100 were higher than those in their parental drug-sensitive CEM cells in a drug resistance-dependent fashion. Also, CEM/VLB100 cells showed about 3-fold increase of DNA-PK enzyme activity as compared with CEM cells. Similar results were observed in another MDR cell line, FM3A/M mouse mammary carcinoma cells. Moreover, we observed that CEM/VLB100 cells were about 11-fold sensitive to wortmannin, which inhibits DNA-PK, compared with the CEM cells, and sensitized the MDR cells when combined with either bleomycin or vincristine, but have a little effect on CEM cells. Wortmannin was shown to inhibit DNA-PK and Ku-DNA binding activity in CEM/VLB100 cells dose dependently but had a little or no effect on their parental cells. Our results suggested that enhanced expression of DNA-PK participates in the development of MDR, and the use of DNA-PK inhibitors such as wortmannin is likely to improve the effectiveness of anticancer drugs and thus could partially overcome drug resistance in MDR cells, through its ability to inhibit Ku/DNA-PK activity.

Antiapoptotic role of NF-κB in the auto-oxidized dopamine-induced apoptosis of PC12 cells

Current concepts of the pathogenesis of Parkinsons disease (PD) center on the formation of reactive oxygen species (ROS), and dopamine has been considered to be a major source of ROS. Recently, it has been shown in a postmortem study that nuclear translocation of nuclear factor‐kappa B (NF‐κB) was observed in dopaminergic neurons of patient with PD. However, its role is not known. The present study examined the possible role of NF‐κB in ODA (auto‐oxidized dopamine)‐induced apoptosis to understand the process of PD. Using the electrophoretic mobility shift assay, it was found that ODA activated the DNA binding activity of NF‐κB. Suppression of the transcriptional activity of NF‐κB in PC12 cells by overexpression of a wild‐type and a dominant negative mutant form (S32A/S36A) of inhibitor kappa B (IκB)‐α led to increase of apoptotic cell death induced by treatment of ODA. In addition, overexpression of NF‐κB in PC12 cells blocked ODA‐induced cell death. However, JNK/SAPK activities, which mediate various stress signals, were similar among the parental, NF‐κB‐ or dominant negative mutant IκBα‐transfected cells. Therefore, these results suggest that activation of NF‐κB during ODA‐induced apoptosis may have a counteracting activity against the signals mediating apoptotic cell death and thereby delay the process of Parkinsons disease.

Effect of the activated Raf protein kinase on the human multidrug resistance 1 (MDR1) gene promoter

Revealing the regulatory mechanism of the multidrug resistance 1 (MDR1) gene is important to gain understanding of MDR in tumor cells. Using MDR1 deletion constructs and the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we examined the effect of the activated Raf on human MDR1 promoter activity in transient expression assay and stable transfectants of GHE-L cells. A DNA sequence exhibiting strong activation of MDR1 promoter by 22W was located between -197 and -136 containing the upstream heat shock element (HSE) motifs without other regulatory elements, whereas the MDR1 deletion construct containing downstream HSE motif showed a relatively weaker activation by 22W. We observed that the activated Raf significantly potentiated the induction of MDRCAT activity in GHE-L cells by sodium arsenite or heat shock, which stimulates heat shock factor (HSF) binding to HSE. In addition, protein kinase A inhibitor (H-87) blocked the activation of the MDR1 promoter by 22W in GHE-L cells in a dose-dependent manner. From these results, we propose the possibility that Raf- and protein kinase A-dependent pathways control the transcription of MDR1 gene via a mechanism involving the modulation of HSF activity.