Clinical And Experimental Study On The Mechanism Of Immune Escape And The Treatment Of CIK Cells For Advanced Ovarian Carcinoma With Multidrug Resistance

Objective: Ovarian carcinoma is leading cause of death in gynecologic malignancies, and its total 10-year-survival rate is about 4.0~20.0%. Peritoneal metastasis is a frequent type of recurrence after surgery and the reason for the poor prognosis of ovarian cancer. The survival rate cannot be improved after routine surgery, chemotherapy, and radiotherapy. Therefore biotherapy becomes the fourth treatment pattern for ovarian carcinoma. The modern medicial study and clinical research indicate that the multidrug resistance (MDR) plays an important role within chemotherapy. The ovarian cancer in advanced stage is often accompanied by MDR which results in chemotherapeutics such as Cisplatin (CDDP) can not get into nucleolus. The occurrence of MDR is related to the coexpression of MDR related genes, which reflects not only complexity of MDR but also the important cause of treatment failure for the past many years. Only by combination of multigenic detection, can it guide individualized treatment and clinical application. Recently, medical scientist pay close attention to the research concerned with immunological feature about multidrug resistance carcinoma. compared with sensitive cell strains, it is easier for MDR cell strains to escape host immune reaction. The depress expression of human leucocyte antigen (HLA) and costimulatory molecules such as B7 may be the primary cause of immune evasion on MDR tumor cells, and the expression of costimulatory molecules B7 on MDR cells is higher than sensitive cells. Adoptive immounotherapy is a potential choice for the treatment of MDR carcinoma that may enhance the immunogenicity and reverse immunotolerance of tumor. The study on treatment for peritoneal metastasis of human ovarian carcinoma with multidrug resistance has many problems which ought to be resolved. There were no study on the establishment and biologic characteristics of intraperitoneal transplantation model of human ovarian carcinoma cisplatin-resistant cell in SCID mice and the mechanism of anti-tumor action by CIK (cytokine-induced killer) cells against ovarian cancer cell lines SKOV3/CDDP in vitro and in vivo. There were few reports about the expression of multidrug resistance (MDR1, Topo-Ⅱβ) between primary and metastatic sites in patients with ovarian cancer also. In order to solve these problems, this research was designed to investigate the expression clinical significance of major histocompatibility complex (MHCⅠb,HLA-DR),B7 in human ovarian carcinoma tissues; To investigate the expression of multidrug resistance (MDR1, Topo-Ⅱβ) between primary and metastatic sites in patients with ovarian carcinoma. To evaluate the relationship between multidrug resistance and the clinical pathological types and provide the experimental data for the further biological treatment and prognosis of ovarian carcinoma. Meanwhile, base on the establishment of intraperitoneal transplantation model of human ovarian carcinoma SKOV3/CDDP cell in severe combined immunodeficiency (SCID) mouse, the anti-tumor affects against SKOV3/CDDP and the killing mechanisms of CIK cells combined with cisplatin in vitro and vivo. To provid immune evidence for further adoptive immunotherapy. Methods: 1 Detection of drug resistance-related genes expression between primary and metastatic sites in advanced ovarian carcinoma Co-expression of MDR1 and Topo-Ⅱβin 52 ovarian carcinoma patients including primary and metastatic sites were determined by immunohistochemistry and reverse transcription-PCR. 2 Detection of MHCⅠb, HLA-DR and hB7-1 expression in advanced ovarian carcinoma Fifty-two cases of advanced ovarian carcinoma received operation in ourhospital between 2002-09 to 2004-9 were selected. Adopting S-P immunocytochemistry technique to detect the expression of MHCⅠb,HLA-DR,hB7-1. 3 The establishment and biologic characteristics of intraperitoneal transplantation model of human ovarian carcinoma cisplatin-resistant cell in SCID mice Sixteen C.B17/SCID mice were randomly divided into 2 groups: Human ovarian carcinoma SKOV3 and SKOV3/CDDP cells(1×107/0.5 mL) were injected intraperitoneally into female SCID mouse respectively to establish a transplantation model of human ovarian carcinoma. Observe the behaviors of mice,tumor growth and the achievement ratio of transplantation. The histologic characteristics of this model were examined by pathomorphology. Cancer antigen 125(CA125) , the co-expression of MDR1, GST-πand Topo-Ⅱβwere examined by immunohistochemical staining. The content of IgG in serum was examined by ELISA. 4 The anti-tumor effects of CIK cells combined with cisplatin against advanced ovarian carcinoma cell lines SKOV3/CDDP in vitro and in vivo Peripheral blood monouclear cells (PBMC) from healthy donors were induced to become CIK cells by cytokine in vitro incubation. Compared with SKOV3 cells, MTT assay was employed to evaluate the cytotoxic activity of CDDP, CIK cells and both against SKOV3/CDDP cells in vitro. The antitumor activity of the CIK were evaluated in SCID mice bearing SKOV3/CDDP cells from ovarian carcinoma, and the content of CA125 in serum were detected by radioimmunity assay. 5 The observation of CIK cells action against advanced ovarian carcinoma cell lines SKOV3/CDDP The changes of ultramicrostructure, cell cycle, apoptosis, multidrug resistance-associated protein (MDR1, Topo-Ⅱβ) and immunogenicity (hB7-1,hB7-2,MHCⅠb,HLA-DR) were detected by electron microscope, MTT and FCM after treated with CIK cells. The changes of cytokine (IL-2,TNF-α,IFN-γ,GM-CSF) in serum in SCID mice bearing SKOV3/CDDP cells were detected by radioimmunit and ELISA. Results: 1 Comparison of drug resistance-related genes expression between primary and metastatic sites in ovarian carcinoma In primary sites,the high expression of MDR1 and Topo-Ⅱβin protein levels was 34.6%(18/52),38.5%(20/52)respectively. The coexpression of MDR1 and Topo-Ⅱβis 32.7%(17/52). The high expression of MDR1 and Topo-Ⅱβin mRNA levels of was 13.3%(7/52), 32.7%(17/52)respectively. The coexpression of MDR1 and Topo-Ⅱβis 13.3%(7/52). There were no significant relationship between MDR1, Topo-Ⅱβand the clinical pathological types (χ~2=1.978 or χ~2=1.277,P>0.05);In metastatic sites,the high expression of MDR1 and Topo-Ⅱβin protein levels was 53.8%(28/52),59.6%(31/52)respectively. The coexpression of MDR1 and Topo-Ⅱβis 50%(26/52). The high expression of MDR1 and Topo-Ⅱβin mRNA levels of was 21.1%(11/52),57.7%(30/52)respectively,the coexpression of MDR1 and Topo-Ⅱβis 21.1%(11/52). There were no significant relationship between MDR1, Topo-Ⅱβand the clinical pathological types (χ~2=3.171 or χ~2=2.151,P>0.05). It demonstrated that the expression of MDR1 and Topo-Ⅱβin protein levels almost coincided with their mRNA levels. There were no significant difference in expression of MDR1, Topo-Ⅱβbetween primary and metastatic sites in patients with ovarian carcinoma(P>0.05). 2 Expression and clinical significance of MHCⅠb, HLA-DR and hB7-1 antigens on advanced ovarian epithelial carcinoma The result showed that the expressions of MHCⅠb,HLA-DR and hB7-1 in protein levels was 13.5%, 59.6%, 19.2% respectively, and they had significant differences in ovarian carcinoma than those of in normal ovarian tissues and benign tumor (P<0.01 or P<0.05). The express of HLA-DR has significant differences related with clinical pathological types.(P<0.05). 3 The establishment and biologic characteristics observation ofintraperitoneal transplantation model in SCID mice bearing SKOV3/CDDP cells The achievement ratio of transplantation in the SKOV3 and SKOV3/CDDP groups were both 100%. Autopsy showed the tumors of the two models spread around the peritoneal cavity and mainly on the omentum, mesentery and peritoneum. And immunohistochemistry staining showed the ovarian associated antigen CA125 were both positive. The morphology, growth pattern and CA125 secretion of SKOV3/CDDP group remained as same as those of SKOV3 group. The content IgG (<0.33mg/L) detected is very small in SCID mice of SKOV3/CDDP group. Transmission electron microscope showed that the tumors of the two groups remained the characteristics of serous papillary adenocarcinoma of human ovary and there were no significant difference between them. Compared with SKOV3 group, the co-expression of MDR1,GST-πand Topo-Ⅱβin SKOV3/CDDP group were significantly higher (40.5%, P<0.05). 4 The anti-tumor effects of CIK cells combined with cisplatin against ovarian carcinoma cell lines SKOV3/CDDP in vitro and in vivo CIK cells possessed a higher antitumor cytotoxic activity on SKOV3/CDDP cells in vitro than SKOV3 cells; The killing activity of CIK cells combined with CDDP was 2.3 times more than that of separate use of CIK cells,and 5.8 times more than that of separate use of CDDP. In addition, CIK cells had a stronger suppressive effect on the tumor growth in SCID mice bearing SKOV3/CDDP tumor compared with SKOV3 cells in vivo (median inhibitory rates 89.7% VS 67.1%, P<0.05). The growth of tumors in CIK group and CIK+CDDP group were both inhibited significantly. All tumors were disapear. The tumor control rate in the two groups were both 100%. The content of CA125 in serum was different in different group. Compared with control group (2246士428 U/mL), it was 1029士485 U/mL in CIK+CDDP group and 1495 士472 U/mL in CIK group(P<0.05). It shows that CIK cells combined with CDDP can inhibit tumor growth, recurrence and transfusion through inhibiting the secretion ofCA125. 5 The mechanism of CIK cells action against ovarian carcinoma cell lines SKOV3/CDDP It showed that CIK cells could induce the SKOV3/CDDP cells to apoptosis by electron-microscopic observations. Compared with NS Group, the apoptosis rate in CIK group was 9.07%, and its cell cycle was arrested at S and G2/M phase (P<0.05). Compared with NS Group, the co-expression of MDR-1 and Topo-Ⅱβwas decreased significantly treated with CIK cells (P<0.05), and the expression of MHCⅠb, HLA-DR, hB7-1 and hB7-2 antigen were increased significantly (P<0.01). Compared with NS Group, the contents of IL-2,TNF-α,INF-γ,GM-CSF were increased significantly (P<0.01) in SCID mice of CIK group. Conclusion: 1 The co-expression of MDR1 and Topo-Ⅱβcan be detected in tumor specimens from patients with advanced ovarian carcinoma. There were no significant relationship between MDR1, Topo-Ⅱβand the clinical pathological types. The co-expression of MDR1 and Topo-Ⅱβin metastatic sites may not reflect an aggressive biologic behavior in ovarian cancer. It is very useful to reverse multidrug resistance. 2 The expressions of MHCⅠb,HLA-DR ,hB7-1 in human ovarian carcinoma tissues may play an important role in initiating auto-immunity against ovarian carcinoma. It must be very useful for the futher clinical theraphy and for the analysis of prognosis in patients with ovarian carcinoma. 3 An intraperitoneal transplantation model of human ovarian carcinoma SKOV3/CDDP in SCID mice has been developed successfully. It may be an ideal animal model for biotherapy research of ovarian carcinoma as it can simulate the biological behavior of peritoneal metastasis of advanced ovarian carcinoma and the drug tolerance is maintained. 4 CIK cells were highly efficient cytolytic effector cells which have a stronger significant suppression against the growth of MDR cells of ovarian carcinoma in vitro and in vivo. The result provides an experimental basis for...