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Objective: To investigate the Effects of entinostat on the expression of NKG2D ligands in the non-small cell lung cancer (NSCLC) cell lines, A549 and HCC-827, and to detect the effect of entinostat-mediated NK cell killing of A549 and HCC-827 cells. Meth-ods: The effect of entinostat on A549 and HCC-827 cell proliferation was measured by MTT assay. Flow cytometry was used to detect the expression of NKG2D ligands. mRNA levels of the ligands were detected by RT-PCR . The level of soluble MICA in cell culture super-natant was evaluated by ELISA. The cytotoxicity of NK cells against A549 and HCC-827 cell lines (treated with entinostat) was assessed using lactate dehydrogenase release assay. Results: Entinostat showed a time-and dose-dependent inhibition effect on the prolifera-tion of A549 and HCC-827 cell lines. The expression of NKG2D ligands and mRNA transcription levels of MICA and MICB were en-hanced after treatment with 0.5, 1μmol/L entinostat for 48 h. The soluble MICA level in A549 cell culture supernatant was increased by 1μmol/L entinostat. The sensitivity of HCC-827 cells to NK cells was enhanced upon treatment with 0.5, 1μmol/L entinostat. Con-clusions: entinostat enhanced the killing effect of NK cells on non-small cell lung cancer cells by up-regulating the expression of NKG2D ligands. This provides a new method and theory for the treatment of NSCLC.
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Objective To investigate the effect of ionizing radiation on the expression of NKG2D ligand on the surface of oral squamous cell carcinoma cell line SCC25 and its cytotoxicity to tumor cells.Methods When SCC25 cells were cultured into logarithmic growth phase,they were randomly designed as control (without treatment) and experimental group (2 Gy ionizing radiation treatment) by drawing lots.Flow cytometry was used to detect the expressions of NKG2D ligands major histocompatibility complex class Ⅰ chainrelated molecule (MIC)A,MICB,UL16 binding protein (ULBP)1 on the surface of SCC25 in the control group and the experimental group cultured for 24 h.Real-time fluorescence quantification polymerase chain reaction (RT-PCR) was used to detect the changes of NKG2D ligand mRNA expression on SCC25 cell surface after 24 h culture in the experimental group and the control group.The cells were prepared and divided into blank control group (NC),2 Gy ionizing radiation group (R),NK1 group (target ratio was 5 ∶ 1),NK2 group (target ratio was 20 ∶ 1),NK1 + R group (target ratio was 5 ∶ 1,2 Gy ionizing radiation),NK2 + R group (target ratio was 20 ∶ 1,2 Gy ionizing radiation).After each group was cultured for 24 h,the killing abilities of ionizing radiation and natural killer (NK) cells to oral squamous cell carcinoma SCC25 cells were detected by CCK8.Results Flow cytometry experiment showed that,among the NKG2D ligands,the MICA fluorescence values of experimental group and control group were respectively 21.04 ± 0.39,22.90 ± 0.40 (t =2.465,P =0.069),MICB fluorescence values were 27.58 ± 0.50,29.83 ± 1.05 (t =1.936,P =0.125),and ULBP1 fluorescence values were 21.04 ± 0.40,21.78 ± 0.50 (t =1.154,P =0.313).This indicated that after ionizing radiation on SCC25,the NKG2D ligand MICA,MICB,ULBP1 expression increased slightly,but the differences were not statistically significant.RT-PCR indicated that mRNA expressions of MICB,ULBP1 were significantly different between the control group and the experimental group (t =18.334,P =0.000;t =6.381,P =0.008).The expressions of the experimental group were respectively 6.49,1.64 times as those of the control group.The results of CCK8 showed that,there was a significant difference in cell killing ability among NK1 group,NK2 group and NC group (F =344.600,P =0.000),suggesting that NK cells could kill tumor cells,and the higher ratio of NK cells and SCC25,the stronger killing effect.The comparison between R group and NC group showed that the difference in cell killing ability was not statistically significant (P =0.567).NK1 + R group and NK1 group were compared and the difference was not statistically significant (P =0.915).There was no significant difference between NK2 + R group and NK2 group (P =0.678).This showed that the killing effect of ionizing radiation was weak.Conclusion Ionizing radiation can increase the mRNA expression of NKG2D ligands MICB and ULBP1.This may provide a new way for tumor immunotherapy.The killing effect of ionizing radiation on cells is not obvious.It may be related to low radiation dose and only 24 h for cell culture.
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Objective To investigate the effect of ionizing radiation on the expression of NKG2D ligand on the surface of oral squamous cell carcinoma cell line SCC25 and its cytotoxicity to tumor cells.Methods When SCC25 cells were cultured into logarithmic growth phase,they were randomly designed as control (without treatment) and experimental group (2 Gy ionizing radiation treatment) by drawing lots.Flow cytometry was used to detect the expressions of NKG2D ligands major histocompatibility complex class Ⅰ chainrelated molecule (MIC)A,MICB,UL16 binding protein (ULBP)1 on the surface of SCC25 in the control group and the experimental group cultured for 24 h.Real-time fluorescence quantification polymerase chain reaction (RT-PCR) was used to detect the changes of NKG2D ligand mRNA expression on SCC25 cell surface after 24 h culture in the experimental group and the control group.The cells were prepared and divided into blank control group (NC),2 Gy ionizing radiation group (R),NK1 group (target ratio was 5 ∶ 1),NK2 group (target ratio was 20 ∶ 1),NK1 + R group (target ratio was 5 ∶ 1,2 Gy ionizing radiation),NK2 + R group (target ratio was 20 ∶ 1,2 Gy ionizing radiation).After each group was cultured for 24 h,the killing abilities of ionizing radiation and natural killer (NK) cells to oral squamous cell carcinoma SCC25 cells were detected by CCK8.Results Flow cytometry experiment showed that,among the NKG2D ligands,the MICA fluorescence values of experimental group and control group were respectively 21.04 ± 0.39,22.90 ± 0.40 (t =2.465,P =0.069),MICB fluorescence values were 27.58 ± 0.50,29.83 ± 1.05 (t =1.936,P =0.125),and ULBP1 fluorescence values were 21.04 ± 0.40,21.78 ± 0.50 (t =1.154,P =0.313).This indicated that after ionizing radiation on SCC25,the NKG2D ligand MICA,MICB,ULBP1 expression increased slightly,but the differences were not statistically significant.RT-PCR indicated that mRNA expressions of MICB,ULBP1 were significantly different between the control group and the experimental group (t =18.334,P =0.000;t =6.381,P =0.008).The expressions of the experimental group were respectively 6.49,1.64 times as those of the control group.The results of CCK8 showed that,there was a significant difference in cell killing ability among NK1 group,NK2 group and NC group (F =344.600,P =0.000),suggesting that NK cells could kill tumor cells,and the higher ratio of NK cells and SCC25,the stronger killing effect.The comparison between R group and NC group showed that the difference in cell killing ability was not statistically significant (P =0.567).NK1 + R group and NK1 group were compared and the difference was not statistically significant (P =0.915).There was no significant difference between NK2 + R group and NK2 group (P =0.678).This showed that the killing effect of ionizing radiation was weak.Conclusion Ionizing radiation can increase the mRNA expression of NKG2D ligands MICB and ULBP1.This may provide a new way for tumor immunotherapy.The killing effect of ionizing radiation on cells is not obvious.It may be related to low radiation dose and only 24 h for cell culture.
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Objective To assess the efficacy of combination of Jiehe pellet and the standardized anti-tuberculosis therapeutic regimen for pulmonary tuberculosis complicating cervical lymph node tuberculosis in the aged. Methods A total of 103 aged patients with pulmonary tuberculosis complicating cervical lymph node tuberculosis were enrolled and randomly allocated to either a standardized anti-tuberculosis therapeutic regimen group (control group with 51 patients) or a standardized anti-tuberculosis therapeutic regimen plus Jiehe pellet group (treatment group with 52 patients). The patients in the control group and the treatment group received the treatment with 2HRZE/4HR and 2HRZE/4HR plus Jiehe pellet for 6 months, respectively. The abscessed lymph nodes were treated by either total excision or incision and drainage after 4 weeks of medicine treatment in both groups. Sputum smear was examined for acid-fast bacilli. The CD8 cells expressing natural killer T cells receptors NKG2A, NKG2D in peripheral blood were detected by flow cytometry. The treatment outcome was measured at the end of treatment. Results The rates of lesion resolution (78.85%vs. 58.82%;χ2=4.439, P<0.05) and cavity closure (62.86% vs. 35.48%;χ2=3.893, P<0.05) in the treatment group were significantly higher than those in the control group. In the end of 2, 4 and 6 months of treatment, cumulative rates of sputum conversion from positive to negative in the treatment group were significantly higher than those in the control group (χ2 were 5.343, 5.067 and 4.118,all P<0.05). The CD8 cells expressing NKG2A after treatment in the treatment group were significantly lower than those before treatment in the treatment group (t=9.510, P<0.01) and after treatment in the control group (t=9.832, P<0.01);the CD8 cells expressing NKG2D after treatment in the treatment group were significantly higher than those before treatment in the treatment group (t=10.622, P<0.01) and after treatment in the control group (t=10.433, P<0.01). The serum levels of IL-6 and TNF-αafter treatment were significantly lower than those before treatment in both groups (t were 17.344 and 21.142 in the treatment group, 10.984 and 12.203 in the control group;all P<0.01 );the serum levels of IL-6 and TNF-α after treatment in the treatment group were significantly lower than those after treatment in the control group (t were 7.832 and 5.478,all P<0.01). The serum IL-10 levels after treatment were significantly higher than those before treatment in both groups (t were 12.454 in the treatment group, 7.934 in the control group; all P<0.01 ); and the serum IL-10 level after treatment in the treatment group was significantly higher than that after treatment in the control group (t=4.720, P<0.01). The effective rate for cervical lymph node tuberculosis in the treatment was significantly higher than that in the control group (88.5%vs. 64.7%;χ2=6.855, P<0.01). Conclusion Combination of Jiehe pellet and the standardized anti-tuberculosis therapeutic regimen may improve immune function, increase the rate of sputum conversion from positive to negative, and facilitate lesion resolution in aged patients with pulmonary tuberculosis complicating cervical lymph node tuberculosis.
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Objective:To explore the effect of paclitaxel (PTX) and cisplatin (DDP) on the expression of NKG2D ligands of hu-man esophagus carcinoma cell EC9706 and on the cytotoxicity of cytokine-induced killer (CIK) cells, as well as to discuss its molecu-lar mechanisms. Methods: The half maximal inhibitory concentration (IC50) values of PTX and DDP against EC9706 cells for 24 h were measured by MTT assay. The expression levels of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, and ULBP3) on the EC9706 cell surface before and after 24 h culture with 1/2 IC50 of PTX or DDP were assayed by flow cytometry. Cytotoxicity of CIK cells against EC9706 cells before and after 24 h culture with 1/2 IC50 PTX or DDP was analyzed by lactate dehydrogenase release assay at an effector to target cell ratio (E:T) of 20:1 and 30:1, respectively. The expression levels of DNA damage repair genes (ATM, ATR, CHK1, CHK2, and p53) of EC9706 cells before and after 24 h incubation with 1/2 IC50 PTX or DDP were detected by quantitative fluorescent PCR. Results:The IC50 values of PTX and DDP were 10 and 5μg/mL, respectively. MICB, ULBP2, and ULBP3 on EC9706 cells were upregulated after 24 h culture with 1/2 IC50 PTX (P0.05), whereas ATM, ATR, CHK1, and CHK2 were over-expressed after 24 h treatment with 1/2 IC50 DDP (P<0.05). Conclusion:PTX or DDP can enhance the susceptibility of EC9706 cells to CIK cell-mediated lysis by upregulating the expression of NKG2D ligands through activating DNA damage repair genes.
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Objective To identify high affinity ligand MULT1-transgenic mice,analyze its elementary phenotype,and to perform primary functional study in MULT1.Methods We identified the genotype of transgenic mice by PCR and detected transcription level of MULT1 in tails by real-time PCR.The flowcytometry and immunohisto-chemistry were used to analyze the expression of MULT1 in various tissues.Results 25 3'S-strain and 13 4'S-strain MULT1-transgenic mice were obtained and which contain high level of MULT1 transcripts.MULT1 protein is mainly expressed in intestinal intraepithelial lymphocytes (iIELs) and thymocytes but not on splenocytes.Moreover,the levels of MULT1 expression in iIELs and thymocytes are associated with age.Within iIELs,the percentage of CD8~+ Tcells decreased,while CD4~+ CD25~+ Treg cells increased,when compared with wild-type mouse.Conclu-sion We successfully produced MULT1-transgenic mice.MULT1 is possibly associated with thymic development and aging,as well as immunoregulation in mice.
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Objective To study the relationship between the expression of NKG2D ligands and oxidative stress,and to analyze the effect of oxidative stress on the function of NK cells. Methods Tumor cells were cultured and exposed to hydrogen peroxide to develope an oxidative stress model. Then to detect the expression of NKG2D ligands in cells by Real-time PCR and Flow Cytometry. The cytotoxicity of NK cells to tumor cells was detected and compared by CCK-8 kit before and after oxidative stress. Results The expression of NKG2D ligands was induced by oxidative stress,however the NKG2D ligands induced was variable. The up-regulation of NKG2D ligands increased the cytotoxicity of NK cells efficiently,and this effect was blocked by anti-NKG2D antibody. Conclusion The expression of NKG2D ligands can be selectively induced by oxidative stress on tumor cells,and the improvement of the cytotoxicity of NK cells may enhance the immune responses accordingly.