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Combination treatment with decitabine and ionizing radiation enhances tumor cells susceptibility of T cells

View Article: PubMed Central - PubMed

ABSTRACT

Decitabine has been found to have anti-metabolic and anti-tumor activities in various tumor cells. Recently, the use of decitabine in combination with other conventional therapies reportedly resulted in improved anti-tumor activity against various tumors. Ionizing radiation (IR) is widely used as a cancer treatment. Decitabine and IR improve immunogenicity and susceptibility of tumor cells to immune cells by up-regulating the expression of various molecules such as major histocompatibility complex (MHC) class I; natural-killer group 2, member D (NKG2D) ligands; and co-stimulatory molecules. However, the effects of combining decitabine and IR therapies are largely unknown. Our results indicate that decitabine or IR treatment upregulates MHC class I, along with various co-stimulatory molecules in target tumor cells. Furthermore, decitabine and IR combination treatment further upregulates MHC class I, along with the co-stimulatory molecules, when compared to the effect of each treatment alone. Importantly, decitabine treatment further enhanced T cell-mediated cytotoxicity and release of IFN- γ against target tumor cells which is induced by IR. Interestingly, decitabine did not affect NKG2D ligand expression or NK cell-mediated cytotoxicity in target tumor cells. These observations suggest that decitabine may be used as a useful immunomodulator to sensitize tumor cells in combination with other tumor therapies.

No MeSH data available.


Related in: MedlinePlus

Effects of treatment with decitabine, ionizing radiation (IR), or their combination on activity of T cells against target tumor cells.T cells were co-cultured with decitabine, IR or both treated tumor cells (A549, HCT-116, and HepG2 cells) for 5days for MLR assay. Also, T cells were co-cultured with decitabine, IR or both treated tumor cells at various ratios for 4 h for cytotoxicity assay. For blockade experiments, T cells were co-cultured with combination-treated target tumor cells in the presence of 10 ug/ml anti-CD40, CD80, and/or HLA-A,B,C antibody. T cell proliferation (a) or inhibition (b) was assessed using Cell Counting Kit-8 (CCK8), and T cell cytotoxicity (c) or inhibition of cytotoxicity (d) was performed by flow cytometry. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. Results express the average T cell proliferation or T cell-mediated cytotoxicity ± SD in A549, HCT-116, and HepG2 cells. Experiments were independently performed from five healthy donors. The assay was performed in triplicated each donor. The statistical significance was determined using a one-way ANOVA. #P < 0.05, ##P < 0.005, ###P < 0.0005 (#DAC 0 versus other groups, mIgG versus other groups). *P < 0.05, **P < 0.005, ***P < 0.005 (*DAC 5 versus other groups, αCD40 versus other groups). **P < 0.05, P < 0.005, P < 0.0005 (**IR 8 Gy versus DAC 5 + IR 8 Gy, αCD80 versus αCD40 + αCD80). @P < 0.05, @@P < 0.005 (@αCD40 + αCD80 versus αCD40 + αCD80 + αHLA-ABC).
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f3: Effects of treatment with decitabine, ionizing radiation (IR), or their combination on activity of T cells against target tumor cells.T cells were co-cultured with decitabine, IR or both treated tumor cells (A549, HCT-116, and HepG2 cells) for 5days for MLR assay. Also, T cells were co-cultured with decitabine, IR or both treated tumor cells at various ratios for 4 h for cytotoxicity assay. For blockade experiments, T cells were co-cultured with combination-treated target tumor cells in the presence of 10 ug/ml anti-CD40, CD80, and/or HLA-A,B,C antibody. T cell proliferation (a) or inhibition (b) was assessed using Cell Counting Kit-8 (CCK8), and T cell cytotoxicity (c) or inhibition of cytotoxicity (d) was performed by flow cytometry. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. Results express the average T cell proliferation or T cell-mediated cytotoxicity ± SD in A549, HCT-116, and HepG2 cells. Experiments were independently performed from five healthy donors. The assay was performed in triplicated each donor. The statistical significance was determined using a one-way ANOVA. #P < 0.05, ##P < 0.005, ###P < 0.0005 (#DAC 0 versus other groups, mIgG versus other groups). *P < 0.05, **P < 0.005, ***P < 0.005 (*DAC 5 versus other groups, αCD40 versus other groups). **P < 0.05, P < 0.005, P < 0.0005 (**IR 8 Gy versus DAC 5 + IR 8 Gy, αCD80 versus αCD40 + αCD80). @P < 0.05, @@P < 0.005 (@αCD40 + αCD80 versus αCD40 + αCD80 + αHLA-ABC).

Mentions: Decitabine and IR combination treatment significantly increased MHC class I and co-stimulatory molecules in tumor cells (Fig. 2). To investigate role of treatment with decitabine and/or IR-induced co-stimulatory molecules expression in stimulation of T cells, we performed mixed lymphocyte culture using the decitabine, IR or both treated tumor cells and co-stimulatory molecules (CD40 and CD80) blockade experiments using the combination-treated tumor cells. Decitabine or IR-treated tumor cells induced stronger T cell proliferation compared with untreated tumor cells (Fig. 3a). However, decitabine-treated HepG2 cells induced very weak T cell proliferation. In particular, the combination treatment further significantly increased T cell proliferation compared with either treatment alone. In contrast, blockade of co-stimulatory molecules (CD40, CD80 or both) significantly decreased decitabine and IR-induced T cell proliferation (Fig. 3b). Thus, these results suggest that decitabine and IR-induced co-stimulatory molecules expression on tumor cells can directly enhance T cell stimulation. Also, we examined whether it can increase T cell-mediated cytotoxicity in the decitabine, IR or both treated tumor cells. A549, HCT-116, and HepG2 cells were treated with 5 μM decitabine for 24 h. Then, tumor cells were exposed to an 8 Gy radiation dose. T cells were co-cultured with the target tumor cells treated by decitabine, IR, or their combination for 4 h; the cytotoxicity assay was performed using flow cytometry. T cell-mediated cytotoxicity against target tumor cells significantly increased by treatment with decitabine and IR alone compared to that in untreated target tumor cells. Furthermore, the combination treatment significantly increased T cell-mediated cytotoxicity compared with either treatment alone (Fig. 3c). Although IR alone increased the cytotoxicity of T cells against target tumor cells than decitabine alone, there was no significant difference. To evaluate the effect of treatment with decitabine and IR-induced MHC class I and co-stimulatory molecules expression in the anti-tumor cytotoxicity of T cells, T cells were co-cultured with target tumor cells in the presence of blocking antibody against MHC class I, CD40 and CD80. As shown in Fig. 3d, blockade of MHC class I, CD40 or CD80 resulted in a substantial reduction of anti-tumor cytotoxicity against target tumor cells, although it was dependent on cell type. In particular, CD80 or MHC class I blockade strongly inhibited the anti-tumor cytotoxicity induced by combination treatment of decitabine with IR against all target tumor cells. Furthermore, the combination blockade of MHC class I and co-stimulatory molecules more significantly reduced T cell-mediated cytotoxicity compared with either blockade alone. These results suggest that decitabine and IR combination treatment further enhances the T cells-mediated cytotoxicity by increasing the expression of MHC class I and co-stimulatory molecules in tumor cells.


Combination treatment with decitabine and ionizing radiation enhances tumor cells susceptibility of T cells
Effects of treatment with decitabine, ionizing radiation (IR), or their combination on activity of T cells against target tumor cells.T cells were co-cultured with decitabine, IR or both treated tumor cells (A549, HCT-116, and HepG2 cells) for 5days for MLR assay. Also, T cells were co-cultured with decitabine, IR or both treated tumor cells at various ratios for 4 h for cytotoxicity assay. For blockade experiments, T cells were co-cultured with combination-treated target tumor cells in the presence of 10 ug/ml anti-CD40, CD80, and/or HLA-A,B,C antibody. T cell proliferation (a) or inhibition (b) was assessed using Cell Counting Kit-8 (CCK8), and T cell cytotoxicity (c) or inhibition of cytotoxicity (d) was performed by flow cytometry. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. Results express the average T cell proliferation or T cell-mediated cytotoxicity ± SD in A549, HCT-116, and HepG2 cells. Experiments were independently performed from five healthy donors. The assay was performed in triplicated each donor. The statistical significance was determined using a one-way ANOVA. #P < 0.05, ##P < 0.005, ###P < 0.0005 (#DAC 0 versus other groups, mIgG versus other groups). *P < 0.05, **P < 0.005, ***P < 0.005 (*DAC 5 versus other groups, αCD40 versus other groups). **P < 0.05, P < 0.005, P < 0.0005 (**IR 8 Gy versus DAC 5 + IR 8 Gy, αCD80 versus αCD40 + αCD80). @P < 0.05, @@P < 0.005 (@αCD40 + αCD80 versus αCD40 + αCD80 + αHLA-ABC).
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f3: Effects of treatment with decitabine, ionizing radiation (IR), or their combination on activity of T cells against target tumor cells.T cells were co-cultured with decitabine, IR or both treated tumor cells (A549, HCT-116, and HepG2 cells) for 5days for MLR assay. Also, T cells were co-cultured with decitabine, IR or both treated tumor cells at various ratios for 4 h for cytotoxicity assay. For blockade experiments, T cells were co-cultured with combination-treated target tumor cells in the presence of 10 ug/ml anti-CD40, CD80, and/or HLA-A,B,C antibody. T cell proliferation (a) or inhibition (b) was assessed using Cell Counting Kit-8 (CCK8), and T cell cytotoxicity (c) or inhibition of cytotoxicity (d) was performed by flow cytometry. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. Results express the average T cell proliferation or T cell-mediated cytotoxicity ± SD in A549, HCT-116, and HepG2 cells. Experiments were independently performed from five healthy donors. The assay was performed in triplicated each donor. The statistical significance was determined using a one-way ANOVA. #P < 0.05, ##P < 0.005, ###P < 0.0005 (#DAC 0 versus other groups, mIgG versus other groups). *P < 0.05, **P < 0.005, ***P < 0.005 (*DAC 5 versus other groups, αCD40 versus other groups). **P < 0.05, P < 0.005, P < 0.0005 (**IR 8 Gy versus DAC 5 + IR 8 Gy, αCD80 versus αCD40 + αCD80). @P < 0.05, @@P < 0.005 (@αCD40 + αCD80 versus αCD40 + αCD80 + αHLA-ABC).
Mentions: Decitabine and IR combination treatment significantly increased MHC class I and co-stimulatory molecules in tumor cells (Fig. 2). To investigate role of treatment with decitabine and/or IR-induced co-stimulatory molecules expression in stimulation of T cells, we performed mixed lymphocyte culture using the decitabine, IR or both treated tumor cells and co-stimulatory molecules (CD40 and CD80) blockade experiments using the combination-treated tumor cells. Decitabine or IR-treated tumor cells induced stronger T cell proliferation compared with untreated tumor cells (Fig. 3a). However, decitabine-treated HepG2 cells induced very weak T cell proliferation. In particular, the combination treatment further significantly increased T cell proliferation compared with either treatment alone. In contrast, blockade of co-stimulatory molecules (CD40, CD80 or both) significantly decreased decitabine and IR-induced T cell proliferation (Fig. 3b). Thus, these results suggest that decitabine and IR-induced co-stimulatory molecules expression on tumor cells can directly enhance T cell stimulation. Also, we examined whether it can increase T cell-mediated cytotoxicity in the decitabine, IR or both treated tumor cells. A549, HCT-116, and HepG2 cells were treated with 5 μM decitabine for 24 h. Then, tumor cells were exposed to an 8 Gy radiation dose. T cells were co-cultured with the target tumor cells treated by decitabine, IR, or their combination for 4 h; the cytotoxicity assay was performed using flow cytometry. T cell-mediated cytotoxicity against target tumor cells significantly increased by treatment with decitabine and IR alone compared to that in untreated target tumor cells. Furthermore, the combination treatment significantly increased T cell-mediated cytotoxicity compared with either treatment alone (Fig. 3c). Although IR alone increased the cytotoxicity of T cells against target tumor cells than decitabine alone, there was no significant difference. To evaluate the effect of treatment with decitabine and IR-induced MHC class I and co-stimulatory molecules expression in the anti-tumor cytotoxicity of T cells, T cells were co-cultured with target tumor cells in the presence of blocking antibody against MHC class I, CD40 and CD80. As shown in Fig. 3d, blockade of MHC class I, CD40 or CD80 resulted in a substantial reduction of anti-tumor cytotoxicity against target tumor cells, although it was dependent on cell type. In particular, CD80 or MHC class I blockade strongly inhibited the anti-tumor cytotoxicity induced by combination treatment of decitabine with IR against all target tumor cells. Furthermore, the combination blockade of MHC class I and co-stimulatory molecules more significantly reduced T cell-mediated cytotoxicity compared with either blockade alone. These results suggest that decitabine and IR combination treatment further enhances the T cells-mediated cytotoxicity by increasing the expression of MHC class I and co-stimulatory molecules in tumor cells.

View Article: PubMed Central - PubMed

ABSTRACT

Decitabine has been found to have anti-metabolic and anti-tumor activities in various tumor cells. Recently, the use of decitabine in combination with other conventional therapies reportedly resulted in improved anti-tumor activity against various tumors. Ionizing radiation (IR) is widely used as a cancer treatment. Decitabine and IR improve immunogenicity and susceptibility of tumor cells to immune cells by up-regulating the expression of various molecules such as major histocompatibility complex (MHC) class I; natural-killer group 2, member D (NKG2D) ligands; and co-stimulatory molecules. However, the effects of combining decitabine and IR therapies are largely unknown. Our results indicate that decitabine or IR treatment upregulates MHC class I, along with various co-stimulatory molecules in target tumor cells. Furthermore, decitabine and IR combination treatment further upregulates MHC class I, along with the co-stimulatory molecules, when compared to the effect of each treatment alone. Importantly, decitabine treatment further enhanced T cell-mediated cytotoxicity and release of IFN- &gamma; against target tumor cells which is induced by IR. Interestingly, decitabine did not affect NKG2D ligand expression or NK cell-mediated cytotoxicity in target tumor cells. These observations suggest that decitabine may be used as a useful immunomodulator to sensitize tumor cells in combination with other tumor therapies.

No MeSH data available.


Related in: MedlinePlus