The Study on the Radiosensitizing Effect of PARP-1 Inhibitor on A549 and Calu-6 Human Lung Cancer Cells
|School||Tianjin Medical University|
|Keywords||PARP-1 Inhibitors ABT-888 Radiosensitization Radiation therapy|
Objective: poly (ADP-ribose) polymerase -1 (PARP-1) is a widely present in the cells of a protein modification and nucleotide polymerization polymerase play in cellular DNA damage repair after a crucial role. The radiotherapy main cause DNA damage and repair DNA damage is an important reason leading to reduced radiotherapy. This study attempts to confirm the radiosensitization effect of novel PARP-1 inhibitor ABT-888 in human lung cancer cells, and further explore the mechanism. Method: in vitro culture of human lung cancer A549, Calu-6 cell line as the research object. Select the cells in the logarithmic growth phase, the following experimental groups: blank control group, the simple drug group, radiotherapy group, drug combination radiotherapy group. Thiazole blue (MTT) assay were used to detect single-agent ABT-888 inhibited the proliferation of both cells to calculate the IC10 value, low cytotoxicity IC10 values ??as a follow-up experiment reference concentration of drug intervention. According to the reference concentration, using Western blot were used to detect the different drug concentrations, PAR protein expression changes, understand the different concentrations of ABT-888 on the inhibition of PARP-1 activity. Thus confirming the role of the concentration and duration of action of the subsequent experimental drug. By colony formation experiments were used to detect the role of each group in the two cell radiosensitizer and draw cell survival curves and calculate radiosensitization ratio. Flow cytometry (FCM) to detect changes in each group of the cell cycle distribution. Western blot analysis of apoptosis proteins: Bcl-2, Bax; G2 / M phase checkpoint proteins: P21 and cell DNA repair proteins: RAD51 protein expression. Results: 1.MTT experimental results show that: the A549 cells 24 hours, 48 ??hours and 72 hours of IC10 values ??were (10.58 ± 0.37) μmol / L (8.15 ± 0.15) μmol / L (6.70 ± 0.01) μmol / L . Calu-6 cells in 24 hours, 48 ??hours and 72 hours of IC1o value, respectively: (5.01 ± 0.29) μmol / L (4.06 ± 0.57) μmol / L, μmol / L (3.56 ± 0.08). The cells in each group at 24 hours, 48 ??hours and 72 hours of cell growth inhibition rate there was no significant difference (P the value gt; 0.05). While in the same concentration and duration of action of Calu-6 cells, ABT-888 significantly (P = lt; 0.05). The drug reference concentration therefore IC10 two cells were selected for follow-up experiments, A549 10μtmol / L, Calu-6 for 5μmol / L. Cells in each group within 24 hours, the inhibition rate of 48 hours and 72 hours there was no significant difference (P the value gt; 0.05), select 24 hours of the time of the follow-up of drug action. 2 selected in accordance with the MTT results drug experimental concentration gradient: 0 μmol / L, 0.5μmol / L, 1μmol / L of 2μmol / L of 4μmol / L and 8μmol / L. Western bolt results show that: both cell the PAR expression exists, and there are differences in the expression levels. With the increase of the concentration of PARP-1 inhibitors ABT-888, the expression of PAR is also suppressed, i.e. also been speculated that the activity of PARP-1 in system. A549 cells 8μmol / L, and Calu-6 cells in 2μmol / L PAR expression has been affected significantly inhibited. Inhibition of PARP-1 activity was significantly due to the two concentrations are in their respective IC10 or less, and low cytotoxicity, so preferably the concentration of the subsequent experiments. 3. Clone formation assay revealed: drugs combined with radiotherapy group SER 1.50 A549 cells in the experimental group, Calu-6 cells in experimental group, the drug combination group SER 1.88. ABT-888 significantly increased radiosensitivity of two different cells, and calu-6 cells sensitizing effect is more obvious. Cell cycle results show: For A549 cells, ABT-888 alone can make the arrest at the G2 / M phase, radiation alone makes the G2 / M phase cells were increased, but the effect is not obvious (P gt; 0.05 ). After the combined effects of the drugs and radiation, cells in G2 / M phase arrest is particularly evident (P lt; 0.05). For Calu-6 cells, also saw a similar phenomenon, and the joint effect is more pronounced than the A549 cells. 5. Western blot test results: two groups of cell lines showed apoptosis-related protein drugs in combination with radiotherapy: To promote the highest expression of proapoptotic Bax protein, inhibition of apoptosis Bcl-2 protein expression lowest. G2 / M phase checkpoint protein P21 highest expression was also in the drug combination group. The observation of the DNA repair protein RAD51 are not aware of the change. Out of this we can see that the increase in the G2 / M phase arrest drug combination radiotherapy; increased apoptosis, and have not reduced the expression of the DNA repair protein RAD51. Conclusion: PARP-1 inhibitor ABT-888 radiosensitizer, is expected to become the low toxicity of radiotherapy sensitizers. The radiosensitizer mechanism may be related to the increase of cells in G2 / M phase arrest and inhibition of DNA repair induced apoptosis.