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      Anti-tumor effects of CIK combined with oxaliplatin in human oxaliplatin-resistant gastric cancer cells in vivo and in vitro

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          Abstract

          Background

          Drug resistance remains a great challenge in the treatment of gastric cancer. The goal of this study was to explore the anti-tumor effects and mechanism of cytokine-induced killer (CIK) cell combined with oxaliplatin (L-OHP) in human oxaliplatin-resistant gastric cancer cells.

          Methods

          After producing oxaliplatin-resistant gastric cancer cells, cell morphology, growth and doubling time were observed, followed by detection of cell cycle distribution and apoptosis, drug sensitivity (e.g., L-OHP) and expression of P-gp and livin. MTT assay, in vivo pharmacodynamics and pathomorphology experiments were used to detect killing activities of CIK combined with L-OHP.

          Results

          Compared with parental gastric cancer cells, oxaliplatin-resistant gastric cancer cells in S phase were reduced and cell apoptosis rate was increased (P < 0.05), the inhibition rate of 10 chemotherapeutics on oxaliplatin-resistant gastric cancer cells was significantly lower and the expression of P-gp was significantly higher (P < 0.05). However, there was no significant difference in livin expression between parental gastric cancer cells and oxaliplatin-resistant gastric cancer cells (P > 0.05). The in vitro killing activity of CIK combined with L-OHP on parental cells and oxaliplatin-resistant cells were significantly enhanced compared with L-OHP or CIK alone. And it showed greater synergetic effects against oxaliplatin-resistant cells compared with parental cells (P < 0.05). In addition, survival rate, abdominal circumference and pathomorphology results revealed stronger in vivo anti-tumor effects when the two therapies were combined.

          Conclusions

          The mechanism of oxaliplatin-resistant cell secondary multidrug resistance was correlated with the variation of cell cycle distribution, extension of doubling time and upregulation of P-gp expression. The synergistic effect of CIK in combination with L-OHP on killing activity against oxaliplatin-resistant cells was shown in vivo and in vitro.

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          Most cited references34

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          Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity

          C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti- CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine- induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.
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            XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs.

            The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.
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              A novel population of expanded human CD3+CD56+ cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency.

              P Lu, R Negrin (1994)
              Recently, we have reported a novel protocol for the generation of highly efficient cytotoxic effector cells by culturing PBLs in the presence of IFN-gamma, IL-2, mAb against CD3, and IL-1 alpha. We have termed these cultures cytokine-induced killer (CIK) cells because the phenotype of the cells with the greatest cytotoxicity expresses both the T cell marker CD3 and the NK cell marker CD56. Cells with this phenotype are rare (approximately 1 to approximately 5%) in uncultured PBLs. CD3+CD56+ cells expand nearly 1000-fold under these culture conditions. The majority of the CD3+CD56+ cytotoxic cells in CIK cultures were derived from CD3+CD56- T cells, and not CD3-CD56+ NK cells. Expression of CD56, but not CD8, on CD3+ cells correlated with the greatest cytotoxicity against various cellular targets. We have used mice with severe combined immunodeficiency (SCID) injected with human lymphoma cells to evaluate the in vivo antitumor effects of CIK vs lymphokine-activated killer (LAK) cells. Groups of animals inoculated with 1 x 10(6) SU-DHL4 cells (a human B lymphoma cell line with a t(14;18) chromosomal translocation), injected 1 day later with CIK cells either i.v. or i.p., had significantly prolonged survival compared with control animals injected with tumor cells alone (median survival 90 days vs 58 days, p < 0.001) or animals treated with LAK cells (median survival 90 days vs 68 days, p < 0.002). Approximately 30% of the SCID mice challenged with SU-DHL4 cells and treated with CIK cells became long-term survivors compared with none of the animals treated with LAK cells. No molecular evidence of occult lymphoma was found in the CIK cell-treated long-term survivors when their bone marrow, spleen, liver, and lung were analyzed by t(14;18) PCR at the end of 6 mo. By using these culture conditions, a novel population of cytotoxic cells can be generated readily from T cells that have superior in vivo antitumor activity in SCID mice, as compared with LAK cells.
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                Author and article information

                Journal
                J Exp Clin Cancer Res
                Journal of Experimental & Clinical Cancer Research : CR
                BioMed Central
                0392-9078
                1756-9966
                2010
                30 August 2010
                : 29
                : 1
                : 118
                Affiliations
                [1 ]Department of Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China
                [2 ]Department of Gynecology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China
                Article
                1756-9966-29-118
                10.1186/1756-9966-29-118
                2939545
                20799994
                c8a9fbb2-cbc5-48c4-8d56-5b6c68f64271
                Copyright ©2010 Zhao et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 May 2010
                : 30 August 2010
                Categories
                Research

                Oncology & Radiotherapy
                Oncology & Radiotherapy

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