You-Soo Park

Quercetin enhances susceptibility to NK cell-mediated lysis of tumor cells through induction of NKG2D ligands and suppression of HSP70.

It is known that treatments with heat shock, some anticancer drugs, and ionizing radiation increase the expression of heat-shock proteins (HSPs) and natural killer group 2D (NKG2D) ligands in tumor cells. The increased HSPs may make the tumor cells resistant to apoptosis and reduction of HSPs may make the tumor cells more susceptible to natural killer (NK)-cell mediated lysis of tumor cells. In this study, we investigated whether quercetin which has inhibitory activities against heat-shock factor, protein kinase C, nuclear factor-κB, and phosphatidyl inositol 3-kinase, can modulate the expression of NKG2D ligands and suppress the HSPs in tumor cells. The results of this study showed that quercetin significantly induced the expression of several NKG2D ligands including major histocompatibility complex class I-related chain B, UL16-binding protein 1, and UL16-binding protein 2 in K562, SNU1, and SNU-C4 cells. The quercetin-treated K562, SNU1, and SNU-C4 cells showed an enhanced susceptibility to NK-92 cells through induction of NKG2D ligands. This increased expression of NKG2D ligands seemed to be due to the inhibition of the nuclear factor-κB and phosphatidyl inositol 3-kinase pathways. The findings of this study suggest that the induced NKG2D ligands with the decrease of HSP70 protein by quercetin may provide an attractive strategy to improve the effectiveness of NK cell-based cancer immunotherapy.

Phase I study of autologous dendritic cell tumor vaccine in patients with non-small cell lung cancer

BACKGROUND A dendritic cell vaccine has been developed as a novel strategy for generating antitumor immunity in the treatment of cancer. The purpose of this study was to assess the maximal tolerated dose, safety, and immunologic response of a new dendritic cell vaccine (DC-Vac) into which tumor lysate was loaded by electroporation and pulse in patients with advanced non-small cell lung cancer (NSCLC). PATIENTS AND METHODS Fifteen patients with inoperable stage III or IV NSCLC were assigned to cohorts that received 3, 6, or 12 × 10(6) DC-Vac intradermally 3 times at 2 week intervals. We also evaluated immunologic and tumor responses. RESULTS The maximum dose of DC-Vac (12 × 10(6)) was shown to be safe. In 5 of 9 patients, the vaccine resulted in increased interferon (IFN)-γ production by CD8+ cells after exposure to tumor lysate. Additionally, there were mixed responses which do fulfill progressive disease definition but demonstrate some clinical benefit in two patients. CONCLUSION The administration of tumor lysate-loaded autologous dendritic cells by electroporation and pulse was non-toxic and induced immunologic responses to tumor antigens. The two mixed tumor responses which were achieved may represent a potential benefit of this new DC-Vac.

Synergistic enhancement of NK cell-mediated cytotoxicity by combination of histone deacetylase inhibitor and ionizing radiation

BackgroundThe overexpression of histone deacetylase (HDAC) and a subsequent decrease in the acetylation levels of nuclear histones are frequently observed in cancer cells. Generally it was accepted that the deacetylation of histones suppressed expression of the attached genes. Therefore, it has been suggested that HDAC might contribute to the survival of cancer cells by altering the NKG2D ligands transcripts. By the way, the translational regulation of NKG2D ligands remaines unclear in cancer cells. It appears the modulation of this unclear mechanism could enhance NKG2D ligand expressions and the susceptibility of cancer cells to NK cells. Previously, it was reported that irradiation can increase the surface expressions of NKG2D ligands on several cancer cell types without increasing the levels of NKG2D ligand transcripts via ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related (ATM-ATR) pathway, and suggested that radiation therapy might be used to increase the translation of NKG2D ligands.MethodsTwo NSCLC cell lines, that is, A549 and NCI-H23 cells, were used to investigate the combined effects of ionizing radiation and HDAC inhibitors on the expressions of five NKG2D ligands. The mRNA expressions of the NKG2D ligands were quantitated by multiplex reverse transcription-PCR. Surface protein expressions were measured by flow cytometry, and the susceptibilities of cancer cells to NK cells were assayed by time-resolved fluorometry using the DELFIA® EuTDA cytotoxicity kit and by flow cytometry.ResultsThe expressions of NKG2D ligands were found to be regulated at the transcription and translation levels. Ionizing radiation and HDAC inhibitors in combination synergistically increased the expressions of NKG2D ligands. Furthermore, treatment with ATM-ATR inhibitors efficiently blocked the increased translations of NKG2D ligands induced by ionizing radiation but did not block the increased ligand translations induced by HDAC inhibitors. The study confirms that increased NKG2D ligand levels by ionizing radiation and HDAC inhibitors could synergistically enhance the susceptibilities of cancer cells to NK-92 cells.ConclusionsThis study suggests that the expressions of NKG2D ligands are regulated in a complex manner at the multilevel of gene expression, and that their expressions can be induced by combinatorial treatments in lung cancer cells.

Combination Effect of Regulatory T-Cell Depletion and Ionizing Radiation in Mouse Models of Lung and Colon Cancer

PURPOSE To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. METHODS AND MATERIALS We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. RESULTS Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. CONCLUSIONS Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy.

Improvement of antitumor effect of intratumoral injection of immature dendritic cells into irradiated tumor by cyclophosphamide in mouse colon cancer model.

Recently, chemotherapy and radiotherapy are known to directly affect some immunosuppressive barriers within a tumor microenviroment. We used cyclophosphamide (CTX), which is known to enhance the immune response by suppressing CD4+CD25+ regulatory T cells (Treg cells) when used at a low dose, as a chemotherapeutic agent to provide a synergic effect in the irradiation and dendritic cells (DC) combination therapy. Some previous studies observed that a single-dose CTX treatment significantly reduced the number of Treg cells in 3–5 days, however, the reduced Treg cells increased rapidly after 5 days. To overcome the disadvantages of a single-dose CTX, we used 30 mg/kg dose of CTX, which was treated intraperitoneally to mice 3 days before every immature DC (iDC) injection (known as “metronomic schedule CTX”). Irradiation was applied at a dose of 10 Gy to the tumor on the right thigh by a linear accelerator. Then, iDC was intratumorally injected into the irradiated tumor site. Growth of a distant tumor on the right and left flank was suppressed by an injection of iDC into the irradiated tumor, and this effect was increased by the metronomic schedule CTX. Also, combinations treated with the metronomic schedule CTX and ionizing radiation (IR)/iDC, showed the longest survival time compared with other groups. This antitumor immune response of IR/iDC was improved by metronomic schedule CTX and this result was associated with decreasing the proportion of CD4+CD25+ Treg cells and increasing the number of tumor-specific interferon-&ggr;-secreting T cells. Our results demonstrated that metronomic schedule CTX improves the antitumor effect of immunization with an injection of DC s into the irradiated tumor.

The cryopreservation of high concentrated PBMC for dendritic cell (DC)-based cancer immunotherapy☆

Peripheral blood mononuclear cells (PBMC) have been accepted as a unique material for cancer immunotherapy using dendritic cells (DC) or activated lymphocytes that are being developed as an alternative or adjuvant to conventional therapies such as surgery, chemotherapy and radiation treatment. Although successful cryopreservation of large numbers of PBMC is critical for the immunotherapy, subsequent functional study of the effects of PBMC cryopreservation on differentiation into immune cells has not been well defined. In this study, over 1.0 x 10(8)cells/ml PBMC were cryopreserved as long as 52 weeks using a controlled-rate freezer (CRF) and stored in a vapor phase of liquid nitrogen tank. The effect of PBMC cryopreservation on differentiation into DC was studied by comparing the phenotypic and functional properties of immature DC (iDC) and mature DC (mDC) derived from cryopreserved PBMC to those from fresh PBMC. The results show that cryopreservation of PBMC at a fairly high cell concentration does not significantly affect cell recovery, viability, or phenotypes of PBMC. After differentiation into DC, iDC and mDC derived from cryopreserved PBMC had their typical phenotypes and function equivalent to those derived from fresh PBMC. Therefore, the improved cryopreservation process of PBMC described in this study is available for DC-based cancer immunotherapy.

Cyclophosphamide Potentiates the Antitumor Effect of Immunization with Injection of Immature Dendritic Cells into Irradiated Tumor

Growth of a tumor on the left flank was suppressed by direct injection of immature DCs (iDCs) into the irradiated tumor on the right thigh (IR/DC). This antitumor immune effect of IR/DC was enhanced by pretreatment with CTX (CTX+IR/DC) and this effect was related with increased number of tumor-specific IFN-γ secreting T cells and decreased ratio of CD4+CD25+/CD4+ T cells. The treatment with CTX+IR/DC increased or decreased the levels of IL-2 or IL-10, respectively. These results demonstrated that antitumor effect of IR/DC could be augmented by pretreatment with low-dose CTX, suggesting a new antitumor therapeutic modality of chemoradioimmunotherapy.

Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals.

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206+ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206+ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.

Enhanced dendritic cell-based immunotherapy using low-dose cyclophosphamide and CD25-targeted antibody for transplanted Lewis lung carcinoma cells.

Regulatory T cells (Tregs) is one of the main obstacles to the success of cancer immunotherapy. The effect of dendritic cell (DC)-based immunotherapy can be attenuated by immune suppressive functions of Tregs. We used a CD25-targeted antibody and low-dose cyclophosphamide (CTX) as immunomodulators to increase the antitumor effect of intratumoral injection of immature DCs into the irradiated tumor cells (IR/iDC). CTX or CD25-targeted antibody alone showed a significant reduction in the number of Tregs within the tumor microenvironment. When they are combined with IR/iDC, the number of Tregs was further reduced. Although IR/IDC showed strong antitumor effects such as reduction in tumor growth, increase in Th1 immune response, and improvement of survival, the therapeutic effect was further improved by combining treatments with immunomodulators. CTX and CD25-targeted antibody showed no significant difference in tumor growth when combined with IR/iDC, but CTX further increased the number of interferon (IFN)-&ggr;-secreting T cells, cytotoxicity, and survival rate. Although irradiation induced depletion of T lymphocytes, administration of DCs recovered this depletion. Particularly, the lymphocytes were more significantly increased when CTX and IR/iDC were combined. Low-dose CTX has already been used as an immunomodulator in clinical trials, and it offers several advantages, including convenience, low-cost, and familiarity to clinicians. However, CD25-targeted antibody cannot only deplete Tregs, but also may affect IL-2–dependent effector T lymphocytes. Therefore, CTX is an effective means to inhibit Tregs, and an effective immunomodulatory agent for multimodality therapy such as combination treatment of conventional cancer therapy and immunotherapy.

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.