The Investigation On The Effects And Mechanisms Of Interferon-γ In Immune Escape Of HCC

Purpose and BackgroundHepatocellular carcinoma is one of the most common primary malignant tumors in the human being. Its incidence takes the fifth place among all kinds of malignant tumors. Due to the difficulty in its early diagnosis and its poor response to the treatment, its mortality of 5-year is very high. In spite of the great achievements in its early diagnosis and treatment of small hepatocellular carcinoma, there are only limited therapy methods available for the disease.Surgical resection to remove a tumor is the most frequently adopted therapy to cure tumor patients. However, most of the clinically diagnosed patients of HCC are cases of middle or late cancer, and they have lost the opportunity to be cured with such an operation, after which the disease tends to recur. Liver transplantation is costly, and consequently, hard to popularize. Moreover, HCC resists the chemotherapeutic drugs that are currently used, and liver dysfunction affects the chemotherapy drug metabolism and the tolerance of the organism. Radiofrequency ablation therapy, percutaneous ethanol injection therapy, and transarterial chemoembolization are other therapies for HCC, but they are not effective—they are more or less palliative treatments. Therefore, it is of great significance to develop a new therapy and to improve the treatment for HCC.Biotherapy is one of the four categories of the HCC therapies. It and the other three conventional therapies, surgical resection, chemotherapy and radiotherapy, complement each other. The biotherapy in common use is immunotherapy. Immunotherapy helps and induces in the organism an immune response against HCC cells so that the tumor cells can be inhibited or killed. As a supplementary means to the conventional therapies, biotherapy can be used to kill the residual tumor cells after conventional treatments. It can prevent the tumor recurrence and metastasis. Studies show that HCC expresses such proteins as a-fetoprotein (AFP) and Melanoma-associated antigens (MAGE). It can be identified by the immune system, and become the target molecules for the cytotoxic T lymphocyte. The immunotherapy for HCC includes a variety of active immune therapy by vaccination against tumor, cytokine-induced killer cells (CIK), and antibody directed therapy, etc. In immunotherapy, the problem demanding urgent solution is immunologic escape.In liver tumorigenesis and growth, the imbalance between inflammatory reaction and anti-inflammatory reaction is the important mechanism that causes immune escape of HCC. It had been widely accepted that inflammation plays an important part in the development of tumor, for almost in all tumor microenvironments there are pro-inflammatory cytokines. They affect tumorigenesis and the organism's immune response to the tumor with complicated networks, and thus influence the tumor evolution.80%of HCC cases are related to the infection of hepatitis B virus (HBV) or/and hepatitis C virus (HCV). In China, HCC are usually associated with hepatitis and cirrhosis caused with HBV infection. During its immune response to the hepatitis virus infection, the organism generates a large number of pro-inflammatory cytokines such as tumor necrosis factor a (TNF-α), interferon-γ(IFN-γ) and interleukin 12 (IL-12). They activate lymphocytes and macrophages etc. through different mechanisms to destroy the liver cells infected by the virus, and meanwhile, they cause liver cell injury and liver dysfunction. Many cytokines, interleukin 10 (IL-10) and transforming growth factorβ(TGF-β) for example are anti-inflammatory. The imbalance between inflammatory reaction and anti-inflammatory reaction and interdependence induce immune dysfunction and cause the decrease in the immune function against the tumor. As a result the tumor cells cannot be cleared. The substances resulted from inflammation, like reactive oxygen species and NO, cause hepatocellular injury and gene mutation. Consequently, tumor cells are formed. IFN-γis a multifunctional cytokine, which plays an important part in the immune response of anti-infection and anti-tumor. Inducing IFN-y to improve the NK cell activity, the secretion of Thl type cytokines, and the generation of cellular immunity with anti-tumor activity is fundamental mechanism for the immunotherapy like vaccination against tumor to exert its anti-tumor effect. In the hepatitis virus infection, the organism generates IFN-γ, but inadequate to clear the virus. Patients with chronic hepatitis and HCC have IFN-y in their blood, yet tumor can still development and progression in their body, which means that they have dysfunction in secretion and regulation of IFN-γ. Studies revealed the low serum level of IFN-γis one of the mechanisms of the immune escape in liver cancer. But that fact that the immune escape cannot be reversed by adding IFN-γeven in large does hows that the function of IFN-γin the development and progression of liver tumor is complicated. HCC immune escape from the anti-tumor effect of IFN-γis still a problem to be conquered.The object of this study is patients with hepatocellular carcinoma or chronic hepatitis B,sound blood donors. The levels of IFN-y, other 6 kinds of cytokines and immune cell subsets, and the genotypes of the hepatitis B virus in the patients'blood serum are measured and compared with those of the sound blood donors. In combination with the treatment, the relationship between therapeutic effectiveness and changes in the serum level of IFN-y after percutaneous ethanol injection is analyzed. On the basis of clinical research, the effect of IFN-γon HCC antigen expression is studied with cultured heptatoma cell line of SMMC-7721. The study on the function and mechanism of IFN-y in the HCC immune escape is made on the molecule level.Material and Methods1. Collection of cases and samplesThe 54 cases are patients who were either in the in-patient department or hospitalized or in the out-patient department from April 2002 to May 2007.42 of them are male,12 female. They are aged from 33 to 73, with 58 as the average age. CT, MRI, AFP and/or liver pathology all confirm that they were HCC patients. They had not been treated before.2. Main experimental methods Double antibody sandwich ELISA method is used. Primary antibody is biotin-labeled antibody which resists IFN-γ, TNF-α, IL-2, IL-4, IL-10, and IL-12P70. The cytokine content is calculated according to according to the OD value of the sample, and the slope of the curve. The percentage of the lymphocytes of CD4+and CD8+T in the peripheral blood is measured with APAAP method. Argentation is adopted to measure the amount of argyrophilia protein in the nucleolus organizer of the T lymphocytes. The flow cytometry is employed to detect the expression of mononuclear cells of CD83 and HLA-DR adherent to culture dish of the HCC patients'peripheral blood. Real time PCR FQ was used to measure the HBV-DNA content in the serum and the HBV genotypes. MTT colormetry reveals the proliferative activity in the human hepatoma cells. Automatic biochemical detector together with the test reagent kits were used to detect the activity ofγ-GT, ALT, and AST in the lysate of the cultured human HCC cells SMMC-7721. Double antibody sandwich ELISA method is applied to detect the AFP expression of IFN-γ-treated SMMC-7721 cell line. indirect immunofluorescence method is applied to detect the expression of MAGE-1 in SMMC-7721 treated by IFN-γ. bisulfite-sequencing PCR is used to assay the methylation status of MAGE-1 gene promoter of SMMC-7721 treated by IFN-γ.Immunoprecipitation is used to assay the activity of the c-Jun N-terminal kinases in the HCC cell line of SMMC-7721 treated by IFN-y.3. Statistical MethodsThe measurement data is expressed through mean±standard deviation (x±s), and t test; the enumeration data is checked withχ2 (or method of continuity correction). The data is analyzed with SPSS Version 12, with p< 0.05 as statistically significant difference.Results1. Changes in cytokine type of the peripheral bloodIFN-γcontent in the serum of HCC patients is 4.92±2.15pg/ml, lower than that of the sound blood donors (19.16±2.34 pg/ml) and that of the patients who have chronic hepatitis B (11.42±6.86 pg/ml) (P< 0.05, and IL-2 and IL-12P70 content is also lower, but TNF-α, TGF-β1, IL-4 and IL-10 content is higher. The patients are divided into groups according to the size of the tumors. Comparison shows that the bigger tumor a patient has, the lower his IFN-γlevel is. And the changes in their regulation of IFN-γsecretion follow the same pattern. IFN-γ, however, can be found in every patient's blood. The untreated patients are divided into 2 groups according to the level ofα-fetoprotein. The comparative study shows that the IFN-γlevel declines as theα-fetoprotein content rises, but that the IL-12P70 level which regulates the IFN-y secretion has the same tendency with the IFN-γlevel.2. Changes in subsets of the peripheral blood mononuclear cells The percent of CD4+peripheral blood T lymphocytes, CD83+and HLA-DR+in HCC patients are lower than that of the chronic hepatitis B patients and the blood donors. AgNORs which represent the proliferative activity of the T lymphocytes was decreased in patients with HCC.3. The relation between the genotypes of hepatitis B virus and IFN-y concentrationThe real-time PCR is taken to classify the genotypes of the hepatitis B virus in the chronic hepatitis B virus carriers, chronic hepatitis B patients and HCC patients, and compare the IFN-γcontent in the serum. The result is that the IFN-γcontent in the serum of HCC patients who have been infected with the hepatitis B virus of genotype C is lower than that of the patients who have been infected with the hepatitis B virus of genotype B. The former is 6.69±2.11 pg/ml, and the latter 4.36±1.08pg/ml (P<0.05), The changes in hepatitis B patients are similar, but carriers of different hepatitis B virus genotypes have no difference in the IFN-γcontent.4. Percutaneous ethanol injection (PEI) can induce the hepatoma cells necrosis, and thus can reduce the number of the tumor cells and self-tumor vaccines help the organism generate immune responses against hepatocellular carcinoma. After PEI treatment, the tumor shrinks, AFP level dropped, and IFN-γlevel rised.5. IFN-γof different concentrations is applied to culture the human HCC cell line SMMC-7721. The concentration ofα-fetoprotein in the cell lysate of the cultured SMCC-7721 was measured with ELISA method. The result is that IFN-γcan obviously inhibit the proliferation of SMMC-7721, that the inhibition ratio is 19.4±2.7%at the concentration of 512U/ml, that the concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with IFN-γwas 0.18±0.03ng/ml, and that in the lysate was 0.73±0.04ng/ml. The concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with complete medium was 0.74±0.05 ng/ml, and that in the lysate was 0.79±0.07 ng/ml. The above data showed that the expression ofα-fetoprotein in SMMC-7721 declined under the action of IFN-γ. that the changes in the activity of ALT, AST andγ-GT is not significant. the methylation status of MAGE-1 gene promoter of SMMC-7721 was increased and the activity of JNK was decreased treated by IFN-y. Discussion and ConclusionThe immunologic escape of liver cancer is the serious barrier to break down in immunotherapy. It is caused by different mechanisms, including the chronic inflammatory environment of the body in the period of chronic virus hepatitis, the immune escape of liver cancer, anti-apoptotic effect, the immunosuppressive factor and antigenic modulation of the tumor cells. Based on research made in patients with HCC and the cultured human hepatoma cells of SMMC-7721, role of IFN-γin the immune escape of liver cancer was discussed. Main conclusions were followed:1. In HCC patients, the immune function is decreased, there was the immune shift from Thl to Th2, the IFN-γlevel in the serum is low than that of patients with chronic hepatitis and sound blood-donors, and the IFN-γlevel decreased as the size of the tumor increased.2. The patients with chronic hepatitis B virus of genotype C are more likely to develpe HCC, and they react poorly to interferon. The IFN-γlevel in their serum is discovered to be lower than that that in the patients with chronic hepatitis B virus of genotype B. That means that hepatitis B virus of genotype C may allow the infected cells to escape the antitumor and antivirus reactions induced by IFN-γ. That is one of pathogenesis of development and progression of HCC.3. IFN-y inhibits the secretion ofα-fetoprotein and the expression of MAGE-1, which contributed to the reduction of the tumor cell immunogenicity and the immune escape of liver cancer. IFN-y down-regulated the expression of MAGE-1, realized methylation status of MAGE-1 gene promoter partily, and habinted cellular proliferation of SMMC-7721 through JNK route.In the process of tumorigenesis and growth of HCC, there is reciprocal relationship between the tumor cells and IFN-γ. Hepatitis B virus caused immune response to the virus, and the secretion of IFN-γincreased so that the replication of the virus is inhibited. As a result, the cytotoxic T lymphocytes were activated and proliferated to kill the infected cells. The increased IFN-y promoted the generation of cytokines with immunosupressive and growth factor activity. Such reactions were helpful to the growth of mutant cells. IFN-y down-regulated the expression ofα-fetoprotein and MAGE-1, and promoted the antigenic modulation of the mutant cells leading to the cancer cells to escape the immune surveillance in the organism. The immunosuppressive factor secreted by the tumor cells further changed the balance of the cytokine network. But the exact mechanism is still to be investigated.