Effects Of CpG ODNs On Lung Cancer Cells And Lymphoma Cell

This study includes two parts, the first part is the in vitro and in vivo study of CpG ODN alone in the treatment of lung cancer; the second part is the in vitro study of CpG ODN on human B cell lymphoma and acute T cell leukemia with or without VCR (vincristine).Part 1. Effects of CpG ODNs on A549 cell and Lewis Lung carcinoma xenotransplant in miceLung cancer is a leading cause of death in all cancers world-wide, non-small cell lung cancer (NSCLC) takes the most part (about 80%) of it. The cause of lung cancer is still not clear, but datas have indicated that smoking cigarettes is the most common cause, the more years a person smokes, the greater the risk. Besides that, it also has relations with air pollution, individual's immune states and hereditary factors.Currently, treatments of non-small cell lung cancer are maily focused on surgery, chemotherapy and radiotherapy, combined with biological therapy, targeted therapy and Chinese therapy. Although surgery is the first choice, most NSCLC patients (>70%) lost the opportunity who are diagnosed in the late stage. For these patients, chemotherapy could prolong survival and improve life quality. But as a result of the non-selectivity of chemo drugs, patients who are frail and elderly could not tolerate the side effects. For these reasons, many biological therapy and targeted therapy are developed in recent years.Targeted therapy of NSCLC concludes Endothelial growth factor receptor (EGFR) inhibitors, Angiogenesis inhibitor, Signal transduction inhibitors and Matrix metalloproteinase inhibitors. Currently these targeted therapy drugs in clinical trials are mostly used in combination with chemotherapy to enhance the effect of chemotherapeutic agents. However, most of these targeted therapy drugs only have a single target, which means they can not fully activate the immune system to inhibit the tumor growth. In addition, a number of targeted therapy drugs are still in clinical trials, and the effects are not very satisfactory. Synthetic non-methylated CpG-oligodeoxynucleotides (CpG ODNs) are agonists of Toll-like receptor 9 (TLR9) that have been shown to be potent activators of immune system, which can provoke various immune cells and induce the production of a wide variety of T helper-1 (Th1)-promoting cytokines, such as interleukin 12 (IL-12), interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α) and IL-6. As a strong immunostimulatory agent, CpG ODN is able to protect the body from bacterial, viral and parasitic infections, and the administration of CpG ODN alone is also capable of triggering potent anti-tumor immune responses against various experimental tumors, for example, lymphoma, leukemia, glioma, breast cancer, colon carcinoma, fibrosarcoma, melanoma, lung cancer, uterine cervix cancer, cervical cancer, neuroblastoma. CpG ODN also enhances the anti-tumor efficacy of monoclonal antibodies or cancer vaccines when used as immune adjuvants in animal tumor models.In this study, two CpG ODNs that were designed by ourselves were tested : B-type CpG ODN (ODN 01) and C-type CpG ODN (ODN 02). We detected the direct inhibitory effects of these two ODNs on human lung cancer cell A549 in vitro and mice Lewis lung cancer in vivo.1. Direct effect of CpG ODNs on A549 cellWe adopted cell counting and MTT assay to detect the direct inhibitory effect of ODN 01 on A549 cell incubated in 96 well plate, conODN (without CG motif) as negative control.The result shows that ODN 01 has inhibitory effect on A549 cell growth compared to medium and conODN group though there is some difference between cell count and MTT assay result. The inhibitory effect is related to cell density and the time of ODN 01 administration. The higher cell density put in the well, the stronger inhibitory effect of ODN 01 exhibits. Especially when A549 cells grew up to fill up the well, means the cells could contact with each other, the inhibitory effect of ODN 01 is obviously detected. As this result, we speculate that ODN 01 probably induced A549 cell to have the nature of contact inhibition again.2. Effects of CpG ODNs in mice bearing Lewis lung cancer xenotransplant Female C57BL/6 mice were inoculated subcutaneously right posterior on the back with Lewis lung cancer cell 2×106 per mouse at day 0. When the transplanted tumor could be seen at about day 10, we considered tumors are successfully transplanted. Mice bearing Lewis lung cancer were randomly divided into 3 groups: PBS, ODN 01 and ODN 02. Administration with ODN 25μg per mouse began at day 10, subcutaneously injected at right groin every other day, 6 times all together. By measuring the tumor volume, we found the growth rate of tumors on mice of group ODN 02 was the lowest compared with ODN 01 and PBS group. We could draw the conclusion that ODN 02 had inhibitory effect on Lewis lung cancer bearing mice.Part two: Effects of CpG ODN on lymphoma/leukemia cellsNon Hodgkin's lymphoma (NHL) is the most common disease in lymphatic system malignant tumor, B cell derived NHL takes about 85 percents. The morbidity and mortality trends are increasing year by year. NHL is sensitive to the first time chemotherapy, but recurrence and drug resistance is common. The therapy effect will significantly decrease when these patients receive secondary chemotherapy, so NHL is considered as difficult to cure.Burkitt's lymphoma (BL) is a kind of high invasion NHL that often develops outside the lymph node or develops to acute leukemia. BL is most happens on African children, more males than females. In our country it often happens on children and youths.Vincristine (VCR) is a alkaloid extracted from oleander plant Vinca. Because of the good anti-tumor activity, it is widely use in acute lymphocytic leukemia, Hodgkin's and non- Hodgkin's lymphoma, and also breast cancer, bronchiogenic cancer, soft tissue sarcoma and neuroblastoma. The anti-tumor mechanism of VCR is to binding to microtubulin, inhibiting microtubule polymerization, and spindle can not be formed so that cells stop at metaphase of cell division. Besides that, VCR interferes protein synthesis and RNA polymerase so that it can kill G1 phase cell as well.The target of VCR is the cells in the dividing phase, and B-type CpG ODN can stimulate the proliferation of B cells, which means taking the cells into dividing phase. On account of this two mechanism, we tried to use ODN 01 combined with VCR on Raji cells to enhance the anti-tumor effect of VCR.1. Effects of CpG ODN on human malignant B cell lymphoma Raji cell with or without VCRFirst we investigated the effect of ODN 01 alone on Raji cells. Cell counting and MTT assay result showed that ODN 01, as a B-type CpG ODN, could significantly stimulate malignant B cell lymphoma proliferation compared to medium group.Next, we investigated the effect of ODN 01 combined with VCR on Raji cells. We found a suitable concentration of VCR, under which VCR had almost no lethal effect on Raji cells (Invalid maximum dose , 10ng/mL). We use ODN 01 combined with this concentration of VCR administrated on Raji cells. By cell counting and MTT assay we found ODN 01+VCR (10ng/mL) had stronger inhibitory effect on Raji cells compared to VCR (10ng/mL) alone. According to these results, we can draw a conclusion that ODN 01 could enhance the anti-tumor activity of VCR.2. Effects of CpG ODN on human acute T cell leukemia Jurkat cells with or without VCRWe used same methods as above to investigate the effect of CpG ODN on human T cell leukemia Jurkat cells. On one hand, neither stimulatory nor inhibitory effect can be seen of ODN 01 on Jurkat cells. On the other hand, Jurkat cell is not that sensitive to VCR as Raji cell. Consequently, there was no enhancement of VCR with ODN 01 can be seen when they administrated together on Jurkat cells.The innovation of this study is the utilization of B-type CpG ODN that can stimulate malignant B cell proliferation as an enhancer of VCR on B-cell lymphoma/leukemia therapy.