Enhancement Of Radiosensitivity By CpG ODN In Non Small Lung Cancer

Lung cancer worldwide each year, according to World Health Organization (WH O) more than 1.2 million new cases each year die from it up to about 1.1 million people, has become the first cancer killer. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer. Comprehensive treatment of radiotherapy in NSCLC occupies an important position, but the cancer cells themselves on the radiation resistance to radiation therapy often lead to failure. To find an effective radiosensitizer of lung cancer research over the years is a hot issue. Previous studies confirmed that contain unmethylated CpG oligodeoxynucleotides (CpG ODN) can activate the immune system, thereby enhancing the efficacy of various anti-cancer measures; but recent reports showed that CpG ODN may direc tly induce apoptosis, inhibit cancer cell growth and enhance the chemotherapy sensitivity of NSCLC or prostate cancer. Induction of apoptosis is a key mechanism of cytotoxicity for most antitumor agents, including radiation, and radiation-sensitive tumors undergo radiation-induced apoptosis more readily than do radiation-resistant tumors. Whether the CPG ODN can enhance the radio-sensitivity of NSCLC cells has not been reported.In the first part of this study, to explore the CpG ODN radiosensitization of human NSCLC and its possible mechanism, human lung adenocarcinoma A549 cell line was used. Firstly, MTS assay was used to determine the cell viability at 24 h,48 h after different concentrations of CpG ODN7909 (5,10,30,60μg/ml) treated. The results showed that CpG ODN7909 directly inhibited cell viability in a dose-and time-dependent manner. Sencondly, A549 cell randomly divided into four groups: control group, CpG ODN7909 group, irradiated group, CpG ODN7909+ irradiation group. Colony formation was tested in each group to further determine clonogenic cell survival; flow cytometry was used to detect cell apoptosis and cell cycle distribution; ELISA assay was used to measure tumor necrosis factorα(TNF-α) concentrations in cell culture medium. Decreased cell clonogenic survival, enhanced cell apoptotic index, accumulated pertentage of cells in G2/M phase and increased TNF-a secretion were showed in the combined treatments of CpG ODN7909 and irradiation as compared to either treatment alone (p<0.05). Thirdly, TLR9 mRNA was found express in A549 cells by RT-PCR. Therefore, we believe that CpG ODN7909 (10μg/ml) can effectively improve the radiosensitivity of A549 cells. Our group found in the previous studies in vivo, CPG ODN1826 (each mouse intraperitoneally 0.30 mg) can enhance the radiosensitivity of Lewis lung cancer by activating the immune system of C57BL/6 mice. To further explore the dose effect of CPG ODN1826 combined with irradiation, in the second part of this study, firstly, we successfully established model of Lewis lung cancer in C57BL/6J mice, which were randomly divided into 6 groups (8 mice in each group):control group (intraperitoneal injection of PBS solution), X-ray irradiation group (only for fractionated radiation, 250 cGY/fx, a total of 1250 cGY/5fx), CpG ODN group (intraperitoneal injection of each mouse CpG ODN1826, a total dose of 0.3 mg) and low, medium and high doses of CpG ODN (a total dose of 0.15,0.3,0.6 mg, respectively) combined with X-ray irradiation group (as above). Followed with tumor growth, mice were regularly viewed, like the general living conditions, the tumor growth, the immune function (spleen index), the apoptosis of tumor cells and so on. The results show that, CpG will help to improve the general living conditions of tumor-bearing mice in a dose-dependent manner; And mice in the treatment group compared with the control group, tumor growth delayed, the absolute tumor growth delay (AGD) extended; In high doses of CpG ODN combined with X-ray irradiation group radiosensitization ratio (SER) was 17.9; Compared with control group or irradiation alone group, tumor weight significantly reduced in CpG ODN and CpG ODN combined with X-ray irradiation group (p<0.05), and the inhibition rate increased gradually with the increasing concentration of CpG ODN; In CpG ODN group, medium and high doses of CpG ODN combined with X-ray irradiation group spleen index were (19.13±2.19) mg/g, (17.41±3.25) mg/g, (22.45±0.73) mg/g, increased compared with that in control group (11.97±3.73) mg/g or X-ray irradiation group (12.71±3.33) mg/g (p<0.05); Tumor cell apoptosis rates were (5.78±1.32)%, (14.12±2.89)%, (12.42±2.41)%, (20.89±4.87)%, (28.63±2.69)% in X-ray irradiation group, CpG ODN group and the low, medium and high doses of CpG ODN group combined with X-ray irradiation group, higher than that in the control group (2.67±2.14)%, respecitively (p<0.05). Therefore, CpG ODN1826 can significantly enhance radiosensitivity of Lewis lung cancer in a dose-dependent manner, by improving the general living conditions of mice, delaying tumor growth, increasing immune function and promoting tumor cell apoptosis.