Combine5-Fu And IL-12-encoding Plasmid For Treatment Of The Mice With Malignant Ascites

Malignant ascites is a common complication in many types of cancer, resulting fromtumor invasion of the peritoneal cavity.5-fluorouracil (5-Fu) is widely used in clinicalcancer chemotherapy, especially most widely treatment in the digestive tract cancer.However, this agent is toxic to most cells and it can also inhibit the immune system. IL-12can furbish and promote the immunologic function of T-cell which are inhibited by5-Fu. Inthis study, combine5-Fu and IL-12-encoding plasmid were used to explore the antitumoractivity and the possiple mechanism.Methods：H22hepatoma cells were diluted at a concentration of1×106cells/200μl, tumor modelwas developed by direct injection of cells into the abdominal.3days after tumor injection,the animals were randomized into five groups. The pCMV-mIL-12(2.5μg/1.7ml) plasmidswere injected into mice by hydrodynamic injection.5-Fu(20mg/kg) was administrated byintraperitoneal injection daily for5consecutive days. The expression level of mIL-12andIFN-γ in serum were detected by ELISA. Some of the mice were sacrificed at different timepoints to record the ascites volume. HE staining was applied to analyse the tumor cells inascites. The splenic lymphocytes to analyes the T lymphocytes subsets distribution by flowcytometry. Peripheral blood were harvested to analyes the number of WBCs.Results：1Effect on survival and body weightAfter treatment，the mice in the groups of saline and pCMVβ had significant ascites andpoor status. The mice in the groups of saline and pCMVβ group began to die in8days aftertreatment, all the mice were die of the two control groups in13days after treatment. Butcompared to the control group, pCMV-m IL-12and5-Fu group as well as the combinedtreatment group were significantly longer in survival time, prolonged life rate were77.2%,104.4%,133.6%. Until the end of experiment (29days),the combined theatment group stillhave mice alive. The body weights of two control groups increased significantly，thepCMV-m IL-12group has a gentle tendency to rise, but the body weights of combinedtreatment group have been maintained steady until the end of experiment（29days aftertreatment）.2Amount of ascitesThe ascites volume in two control groups grew quickly. In contrast, the ascitesvolume in combined treatment mice was substantially reduced, of which60%(3/5) has no ascites was produced. HE staining showed, there were a lot of giant cells in5-Fu group andcombined treatment group. The nucleus boundary was not clear, chromatin was disordered,rich of cell plasma. These giant cells in a state of instability is crumbling.3Level of mIL-12and IFN-γ in serumThe mIL-12level only detected in the pCMV-mIL-12group and combined treatmentgroup. The average expression of m IL-12in pCMV-mIL-12group was more than incombined treatment group. No IFN-γ could be detected in saline group and pCMVβ. Thehighest level of IFN-γ appeared in pCMV-mIL-12group followed by combined group, andthe lowest level was in5-Fu group.4Cell cycle analysisAnalysis each phase tumor cells in control group is mainly distributed in the G2, Sphase, and after treatment for cancer cells are mainly distributed in the G1phase.5Expression of splenic T-lymphocyte subsetsOn8th day，the highest level of CD8+T appeared in control group. However, on12thday, the expression level of CD8+T cells in three treatment groups increased significantly,the highest level of CD8+T appeared in the pCMV-mIL-12group. The lowest level of CD4+T in5-Fu group. The highest level of CD4+T appeared in the pCMV-mIL-12group.Compared with the control group, the Treg expression level of the three treatment groupswere decreased, the combined treatment group had the most obvious decreased; on12th day,the expression level of Treg in all groups had a significantly decrease.6The count of the peripheral blood WBCsWBCs count in mice treated by5-Fu was the lowest in all the groups. However, addpCMV-mIL-12joint injections into mice, WBCs count rose up, which was similar to that ofthe normal mice.Conclusions：1Combine5-Fu and IL-12-encoding plasmid can inhibition the growth of asciteseffectively, prolonged the survival time.2Combine5-Fu and IL-12-encoding plasmid can better activate the immune system,such as CD8+T lymphocytes activation, and inhibit Treg generation.3IL-12-encoding plasmid can improve the decreasing WBC count suppress by5-Fu.