Induction of NKG2D ligands and subsequent enhancement of NK cell-mediated lysis of cancer cells by arsenic trioxide.

Natural killer (NK) cells are important effector cells in immune responses to tumor cells and the activation of NK cells is mediated through specific interactions between activating receptors and their cognate ligands. Recently, it has been demonstrated that induction of NKG2D ligands on tumor cells by various stresses render them more sensitive to NK cell-mediated killing. Therefore, in this study, it was investigated whether arsenic trioxide (ATO) could up-regulate NKG2D ligands on tumor cells and increase the susceptibility of cancer cells against NK cells. ATO increased transcription of NKG2D ligands, predominantly ULBP1, in various cancer cell lines, such as K562 chronic myelogenous leukemic cells, NB4 acute promyelocytic leukemic cells, and MCF7 breast cancer cells, and subsequently the surface expression of NKG2D ligands. These results were followed by increased susceptibility of cancer cells to NK cell-mediated cytotoxicity after treatment with ATO. This increase in cytotoxicity was abolished by addition of a blocking NKG2D monoclonal antibody, indicating that the increased susceptibility of ATO-treated cancer cells to cytotoxicity of NK cells was mediated through up-regulation of NKG2D ligands. In addition, abrogation of heat shock proteins induction with KNK437 would sensitize the ATO-treated MCF-7 cells to NK cell-mediated killing. This study suggests that the immunomodulatory property of ATO would be an attractive strategy to improve the effectiveness of NK cell-based cancer immunotherapy.

Comparison of level of NKG2D ligands between normal and tumor tissue using multiplex RT-PCR.

The abilities of NKG2D ligands to specifically mark stressed or transformed cells and activate NK cells suggest the possibility that the expression levels of NKG2D ligands in cancers may be helpful to predict the efficacy of NK cell-based cancer immunotherapy. Therefore, a multiplex RT-PCR was developed and used for rapid and simultaneous analysis of the expression level of NKG2D ligands in cancer cells and tissues. With total RNAs isolated from various cancer cell lines, the multiplex RT-PCR revealed various expression patterns of NKG2D ligands. With total tissue RNAs, the gastrointestinal tumors showed consistently increased NKG2D ligands, compared with adjacent normal tissues. However, NKG2D ligands were not always consistently increased in tumor tissues and expression patterns of NKG2D ligands were heterogeneous between patients, especially in breast and lung cancers. In addition, expression patterns of NKG2D ligands were very similar between various paired primary and their multidrug-resistant/metastatic cells, except MCF-7 sublines. These results demonstrated that the multiplex RT-PCR might be a useful diagnostics to detect the expression levels of NKG2D ligands in tissues as well as cells, and suggested that the gastrointestinal tumors might be good candidates for NK cell-based cancer immunotherapy, since it showed significantly higher levels of NKG2D ligands than adjacent normal tissues.

Bcr-Abl-independent imatinib-resistant K562 cells show aberrant protein acetylation and increased sensitivity to histone deacetylase inhibitors.

Bcr-Abl-independent signaling pathways are known to be involved in imatinib resistance in some patients with chronic myelogenous leukemia (CML). In this study, to find new targets for imatinib-resistant CML displaying loss of Bcr-Abl kinase target dependence, we isolated imatinib-resistant variants, K562/R1, K562/R2, and K562/R3, which showed profound declines of Bcr-Abl levels and its tyrosine kinase activity, from K562 cells. Importantly, the imatinib resistance mechanism in these variants also included aberrant acetylation of nonhistone proteins such as p53, Ku70, and Hsp90 that was due to upregulation of histone deacetylases (HDACs) and down-regulation of histone acetyltransferase (HAT). In comparison with K562 cells, the imatinib-resistant variants showed up-regulation of HDAC1, -2, and -3 (class I HDACs) and class III SIRT1 and down-regulation of CBP/p300 and PCAF with HAT activity, and thereby p53 and cytoplasmic Ku70 were aberrantly acetylated. In addition, these were associated with down-regulation of Bax and up-regulation of Bcl-2. In contrast, the class II HDAC6 level was significantly decreased, and this was accompanied by an increase of Hsp90 acetylation in the imatinib-resistant variants, which was closely associated with loss of Bcr-Abl. These results indicate that alteration of the normal balance of HATs and HDACs leads to deregulated acetylation of Hsp90, p53, and Ku70 and thereby leads to imatinib resistance, suggesting the importance of the acetylation status of apoptosis-related nonhistone proteins in Bcr-Abl-independent imatinib resistance. We also revealed that imatinib-resistant K562 cells were more sensitive to suberoylanilide hydroxamic acid, an HDAC inhibitor, than K562 cells. These findings may have implications for HDAC as a molecular target in imatinib-resistant leukemia cells.

Suppressive effect of a standardized mistletoe extract on the expression of activatory NK receptors and function of human NK cells.

Despite long-term use of mistletoe extracts for cancer treatment, their mode of action remains elusive. In this study, it was studied in vitro if mistletoe extract is able to modulate the expression of natural cytotoxic receptors (NCRs) and NKG2D receptor, which stimulate natural killer cell-mediated cytotoxicity. Unexpectedly, a mistletoe extract, ABNOBA viscum Fraxini, inhibited the expression level of NKp46 and NKG2D receptors in dose- and time-dependent manners. The levels of NKp30 and NKG2D receptors were remarkably induced and NKp44 was slightly induced after 48 h treatment with IL-2 and IL-15 in both mRNA and surface expression. The activatory NK receptors were not induced significantly after treatment with IL-12, IL-18, and IL-21 for 48 h. Induction of activatory NK receptors by IL-2 and IL-15 was suppressed almost to the untreated levels by treatment with mistletoe extract, which appeared to induce apoptosis of NK cells in a dose-dependent manner. However, the treatment with IL-2 and IL-15 did not prevent the mistletoe-induced NK-cell death. Mistletoe extract inhibited significantly the cytotoxic activity of resting and IL-2- or IL-15-stimulated NK cells. These results suggest that inhibition of survival and function of NK cells by mistletoe extract may curtail in part the therapeutic effects of mistletoe.

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells.

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.

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.

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells.

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.

Fractionated irradiation leads to restoration of drug sensitivity in MDR cells that correlates with down-regulation of P-gp and DNA-dependent protein kinase activity.

We showed that the drug sensitivity of multidrug-resistant (MDR) cells could be enhanced by fractionated irradiation. The molecular changes associated with fractionated radiation-induced chemosensitization were characterized. Irradiated cells of the multidrug-resistant CEM/MDR sublines (CEM/MDR/IR1, 2 and 3) showed a loss of P-glycoprotein (P-gp) and concurrent reduction of Ku DNA binding and DNA-PK activities with decreased level of Ku70/80 and increased level of DNA-PKcs, and these changes were followed by an increased susceptibility to anticancer drugs. These irradiated MDR cells also exhibited the reduction of other chemoresistance-related proteins, including BCL2, NF-kappaB, EGFR, MDM2 and Ku70/80, and the suppression of HIF-1alpha expression induced by hypoxia. In contrast, fractionated irradiation increased the levels of these proteins and induced drug resistance in the parental drug-sensitive CEM cells. These results suggest that the chemoresistance-related proteins are differentially modulated in drug-sensitive and MDR cells by fractionated irradiation, and the optimized treatment with fractionated radiation could lead to new chemoradiotherapeutic strategies to treat multidrug-resistant tumors.

Relationship between antiapoptotic molecules and metastatic potency and the involvement of DNA-dependent protein kinase in the chemosensitization of metastatic human cancer cells by epidermal growth factor receptor blockade.

The failure to treat metastatic cancer with multidrug resistance is a major problem for successful cancer therapy, and the molecular basis for the association of metastatic phenotype with resistance to therapy is still unclear. In this study, we revealed that various metastatic cancer cells showed consistently higher levels of antiapoptotic proteins, including Bcl-2, nuclear factor-kappaB, MDM2, DNA-dependent protein kinase (DNA-PK), and epidermal growth factor receptor (EGFR), and lower levels of proapoptotic proteins, including Bax and p53 than low metastatic parental cells. This was followed by chemo- and radioresistance in metastatic cancer cells compared with their parental cells. EGFR and DNA-PK activity, which are known to be associated with chemo- and radioresistance, were demonstrated to be mutually regulated by each other. Treatment with PKI166, an EGFR inhibitor, suppressed etoposide-induced activation of DNA-PK in A375SM metastatic melanoma cells. In addition, PKI166 enhanced markedly the chemosensitivities of metastatic cancer cell sublines to various anticancer drugs in comparison with those of low metastatic cancer cells. These results suggest that the activities of DNA-PK and EGFR, which is positively correlated with each other, may contribute to metastatic phenotype as well as therapy resistance, and the EGFR inhibitor enhances the effect of anticancer drugs against therapy-resistant metastatic cancer cells via suppression of stress responses, including activation of DNA-PK.

Association of DNA-dependent protein kinase with hypoxia inducible factor-1 and its implication in resistance to anticancer drugs in hypoxic tumor cells.

Tumor hypoxia contributes to the progression of a malignant phenotype and resistance to ionizing radiation and anticancer drug therapy. Many of these effects in hypoxic tumor cells are mediated by expression of specific set of genes whose relation to therapy resistance is poorly understood. In this study, we revealed that DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double strand break repair, would be involved in regulation of hypoxia inducible factor-1 (HIF-1). HIF-1beta-deficient cells showed constitutively reduced expression and DNA-binding activity of Ku, the regulatory subunit of DNA-PK. Under hypoxic condition, the expression and activity of DNA- PK were markedly induced with a concurrent increase in HIF-1alpha expression. Our result also demonstrated that DNA-PK could directly interact with HIF-1, and especially DNA-PKcs, the catalytic subunit of DNA-PK, could be involved in phosphorylation of HIF-1alpha, suggesting the possibility that the enhanced expression of DNA- PK under hypoxic condition might attribute to modulate HIF-1alpha stabilization. Thus, the correlated regulation of DNA-PK with HIF-1 could contribute to therapy resistance in hypoxic tumor cells, and it provides new evidence for developing therapeutic strategies enhancing the efficacy of cancer therapy in hypoxic tumor cells.

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 Parkinson's 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.

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-alpha 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.

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I-mediated antitumor immunity.

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I-restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I- and II-restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I-mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.

Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction.

To provide an improved understanding of the molecular basis of the aging process, it is necessary to measure biological age on a tissue-specific basis. The role of DNA damage has emerged as a significant mechanism for determination of life span, and DNA repair genes and stress-response genes are also implicated in the aging process. In the present study, we investigated the changes of DNA-PK activity, especially Ku activity, in the various tissues including kidney, lung, testis and liver during aging and its correlation with mtHSP70 expression. We showed that the modulation of Ku activity during the aging process was highly tissue-specific as shown with highly impaired Ku activity in testis and unaffected Ku activity in liver with age, and the level of Ku70 or Ku80 was differentially expressed in each aging tissue. We found also that age-associated alteration of Ku70/80 was prevented or not prevented by caloric restriction (CR) in a tissue-specific manner. Age-related decline in Ku70 during the aging process was associated with the increase of mtHSP70, which could play a role as a predictive marker for aging related to Ku regulation, and CR retarded aging-induced mtHSP70.

Involvement of DNA-dependent protein kinase in regulation of the mitochondrial heat shock proteins.

Since DNA-dependent protein kinase (DNA-PK) has been known to play a protective role against drug-induced apoptosis, the role of DNA-PK in the regulation of mitochondrial heat shock proteins by anticancer drugs was examined. The levels of basal and drug-induced mitochondrial heat shock proteins of drug-sensitive parental cells were higher than those of multidrug-resistant (MDR) cells. We also demonstrated that the development of MDR might be correlated with the increased expression of Ku-subunit of DNA-PK and concurrent down-regulation of mitochondrial heat shock proteins. The basal mtHsp70 and Hsp60 levels of Ku70(-/-) cells, which were known to be sensitive to anticancer drugs, were higher than those of parental MEF cells, but conversely these mitochondrial heat shock proteins of R7080-6 cells over-expressing both Ku70 and Ku80 were lower than those of parental Rat-1 cells. Also, the mtHsp70 and Hsp60 levels of DNA-PKcs-deficient SCID cells were higher than those of parental CB-17 cells. Our results suggest the possibility that mitochondrial heat shock protein may be one of determinants of drug sensitivity and could be regulated by DNA-PK activity.