| Objective:Poly (ADP-ribose) polymerase -1 (PARP-1) is a widely present in the cells of a protein modified with polymerization and nucleotide polymerase, involved in cellular DNA repair process after injury. Radiation can cause DNA damage, PARP-1 observation of the new inhibitor ABT-888 on lung cancer cells have increased sensitivity effects, and to explore its causes.Methods:Cultured human lung cancer A549, Calu-6 cells for the study. Select the cells in the logarithmic phase, each cell is divided into four experimental treatment their groups:control group, drug group, radiotherapy group, drug combination radiotherapy group. Thiazolyl blue (MTT) assay were used to detect single-agent ABT-888 inhibited the proliferation of two cells to calculate their IC10 value, select the IC10 value as a reference for follow-up test the concentration of drug intervention. Based reference concentration, Western blot were used to detect different drug concentrations, PAR protein expression; follow-up tests confirm drug concentration and time. Colony formation assay were used to detect two kinds of cells in each group the role of radiosensitization and the mapping of cell survival curve, and calculate the ratio of radiation sensitization. Flow cytometry (FCM) to detect the changes in cell cycle distribution. Apoptosis was detected by Western blot protein:Bcl-2, Bax; cell cycle checkpoint proteins:P21 and cellular DNA repair proteins:RAD51 protein expression.Results:1. MTT results showed that:A549 cells for 24 hours,48 hours and 72 hours of the IC10 values were (10.58±0.37)μmol/L, (8.15±0.15)μmol/L, (6.70±0.01)μmol/L. Calu-6 cells for 24 hours,48 hours and 72 hours of the IC10 values were: (5.01±0.29)μmol/L, (4.06±0.57)μmol/L (3.56±0.08)μmol/L. Cells in each group at 24 hours,48 hours and 72 hours have no significant difference in inhibition rates (P values> 0.05).2. According to the results of the selected drug experiments MTT concentration gradient:0.5μmol/L, 1μmol/L,2μmol/L,4μmol/L and 8μmol/L. Western bolt results showed that:there are two kinds of cells the expression of PAR, and there are differences in expression levels. With the PARP inhibitor ABT-888 increased the concentration along, PAR inhibited the expression of that PARP activity was inhibited. And A549 cells 8μmol/L and Calu-6 cells in 2μmol/L PAR expression has been affected significantly inhibited. Because the two are in their respective IC10 concentrations below the cytotoxic activity was low and inhibition of PARP, it is chosen as the concentration of the follow-up experiments.3. Colony formation assay showed that:A549 cells in the experimental group, the drug was combined with radiotherapy group SER 1.50, Calu-6 cells in the experimental group, the drug combined with radiotherapy group SER 1.88. ABT-888 significantly increased the radiosensitivity of two different cells, and Calu-6 cells, sensitizing effect more visible.4. The cell cycle showed that:For the A549 cells, ABT-888 alone can make arrest in G2/M phase, radiation alone can make the G2/M phase cells increased in proportion. Combined cffects of drugs and radiation, the cell G2/M phase arrest in particular. For Calu-6 cells, we also have seen a similar phenomenon, and the joint effect is more obvious than the A549 cells.5. Western blot test results:two cell lines are indicative of the drug combination of apoptosis-related proteins after radiotherapy:to promote the highest apoptotic Bax protein, inhibition of apoptosis Bcl-2 protein expression in the lowest. And G2/M phase checkpoint protein P21 expression in the drug combined with radiotherapy in the highest. Observe and participate in DNA repair proteins RAD51, did not find changes. It can be seen after radiation therapy drug combination increased G2/M phase arrest; cell apoptosis increased, and not the DNA repair protein RAD51 reduced expression.Conclusions:PARP inhibitor ABT-888 on lung cancer cells can increase sensitivity to radiation therapy, the mechanism may be associated with increased cell G2/M arrest and inhibition of DNA repair induced apoptosis. |
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