| ObjectiveNatural killer(NK) cells, which are crucial components of the innate immune system, are considered the first line of defense against infected and malignant cells. NK cells are enriched in the hepatic tissues, showing high cytotoxicity against tumor cells compared to spleen and peripheral NK cells. NK cell activation is initiated by a balance between activation and inhibition of signals involving the receptors on NK cells and ligands on target cells. Receptor-natural killer group 2 member D(NKG2D) is a well characterized activating receptor expressed in NK cells, and plays a key role in immune mediated disease. Similarly, MHC class I chain-related proteins A(MICA) is a major ligand for NKG2 D expressed on the thymus and gastrointestinal epithelium in normal cells. It acts as a signal during early immune response by binding to NKG2 D and activating cytolytic responses of NK cells against infection or spontaneously creating tumor. Thus, the MICA-NKG2 D pathway is a critical mechanism by which the host immune system recognizes and kills tumor cells. Impairment of natural killer(NK) cell activity is regarded as an important mechanism of tumor immunoevasion and one of the important targets to treat refractory tumors.In a previous study, we showed that polypeptides extracted from scorpion venom(PESV), which is from the Buthus martensii Karsch scorpion, inhibited the proliferation of tumor cells. However, effect of PESV on dysfunctional and exhausted natural killer cells which contribute to tumor escape from immune surveillance remain to be elucidated. In this study, we treated HepG2 cells and H22 cells orthotopic transplantation tumors with PESV to observe the effect of PESV on the tumor-infiltration of NK cells in vitro and in vivo. Our res?lts indicate that PESV has inhibitory effects on hepatic carcinoma that are likely mediated by the up-regulation of NK activity via the MICA-NKG2 D pathway.Methods1. Effect of PESV on cell viabilities of HepG2 cells and PBLs: The cell viabilities of HepG2 cells and PBLs under the treatment of different concentrations of PESV were analyzed by MTT; The cell cycle distribution of PESV-treated HepG2 cells were tested using Flow cytometry.2. The expression of m MICA on the different group HepG2 cells were measured by Flow cytometry.3. Isolation of NK cells and LDH cytotoxicity assays: Human NK cells were obtained and the cytotoxicity analysis was performed with the Cytotox 96 Non-Radioactive LDH Cytotoxicity Assay.4. A H22 cells orthotopic transplantation tumor mouse model was established by injecting H22 cells into the left liver in C57BL/6 mice. The experimental mice were then randomly divided into control group, PESV low-dose group(PESV-L) and PESV high-dose group(PESV-H). Three days after surgery, each group received intragastric administration of PESV or NS once every day for 14 days. The PESV-H and PESV-L groups received gavage at the dose of 40 mg/kg and 10 mg/kg, respectively. Then, mice were sacrificed to collect the liver, spleen, and tumor. Tumor weight and volume were measured, the rates of tumor inhibition were calculated, and the effects of PESV on survival time were measured.5. The pathological changes on liver tumor tissues were observed with HE staining.6. Flow cytometry was performed to determine the prevalence of NK cells and NKG2 D expression in the liver and spleen simultaneously.7. The detection of NK1.1+ cells in liver tumors was performed by immunohistochemical.8. The expression of perforin and granzyme B m RNA in liver tumors were measured by real-time PCR Results1. The cell viability of HepG2 cells was decreased by PESV in a dose-dependent manner, while there were no significant differences in the PBLs between PESV-treated cells and control. PESV influenced the cell cycle distribution, and the G1 phase cell population was increased.2. Flow cytometry showed that the expression of mMICA in Hep G2 cells treated with PESV was significantly up-regulated with maximum effects observed at the 60?g/ml PESV concentration(**P < 0.01). PESV treatment increased NK cytoxicity on HepG2 cells, meanwhile using a blocking anti–MICA mAb which mask the cell-surface MICA decrease activation showed that cytolytic activity were reduced, suggesting that the up-regulation of MICA expression by PESV increase of cytotoxicity was dependent on MICA interaction.3. Tumor volumes and tumor weights in the PESV-high group(PESV-H) were significantly lower than that of the control group(*P < 0.05, **P < 0.01).In contrast to control group, the tumor inhibition rate was 15.38% and 30.77% in PESV-L group and PESV-H group respectively. The survival analysis showed that PESV-H could significantly prolong the survival time of mice, and life extension rate was 34.06%,(P<0.05).Histological analysis revealed significant pleomorphism of the neoplastic cells and invasive extendion in control group, while there were more necrosis and less degree of atypia in PESV-L and PESV-H.4. The level of tumor-infiltrating and spleen NK cell was significantly higher in PESV-H than in tumor-bearing control group,(P<0.05). The expression of NKG2 D on NK cells are up-regualted by PESV controled with tumor-bearing control group(P<0.05). Immunohistochemical results showed that the NK cells were mainly infiltrated in peritumoral lesions.5. The mRNA of perforin and granzyme B in PESV-H were respectively 3.62 and 5.82 times than control group(P < 0.05) Conclusion1. The cell viability of HepG2 cells was decreased by PESV in a dose-dependent manner, and the G1 phase cell propulation was increased with the treatment of PESV.2. The expression of MICA protein in HepG2 cells was increased by PESV, and the cytotoxic activities of NK cells on HepG2 cells were enhanced by PESV through improving the expression of MICA.3. Tumor growth in mice was significantly inhibited by PESV and the survival time of tumor-bearing mice treated with PESV was significantly prolonged.4. Levels of tumor-infiltrating NK cells, NKG2 D protein, and perforin and granzyme B mRNA were significantly increased in the group treated with PESV compared with the tumor-bearing control group.5. The inhibitory effects of PESV on hepatic carcinoma are likely mediated by up-regulation of NK cell activity via the MICA-NKG2 D pathway. |
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