Chemical Modification Of Cellular Sensitivity In Tumor By No Radicals

Nitric oxide is a ubiquitous molecule capable of inducing a multitude of biological effects, and also plays an extremely critical role in the regulation of tumor evolution and progression. Nitric oxide (NO) is concerned with multiplicative signaling mechanisms which lead to the modifications of proteins. These protein changes are concentration-dependent. Sustained production of NO modulate apoptosis by activating caspases, whereas low or physiological concentrations of NO prevent cells from entering apoptosis. Our previous research has led to a signaling pathway model which attempts to describe the induction of radioresistance and the depression of chromosome aberrations via the action of p53 and NO radicals. However, these initial observations are very recent, and the detailed mechanisms which are responsible for the induction of cellular sensitivity to NO radicals are still not fully understood. The aim of the study described here was to clarify the effect of different doses of NO radicals on the induction of cellular sensitivity in cancer cells, and their possible molecular mechanisms。In the first study, we chose human lung cancer cells as the research target, to focus on the effects of nitric oxide (NO) on radiation-induced cell killing and chromosomal aberrations in these cells with a different p53 gene status, and the possible molecular mechanisms related to the action or involvement of p53. Wild-type (wt) p53 and mutated (m) p53 cell lines were used which were derived from the human lung cancer H1299 cell line which is p53-null. The wtp53 and mp53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pre-treated with different concentrations of isosorbide dinitrate (ISDN, an NO donor) and/or 1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl) tetrahydroimidazole (c-PTIO, an NO scavenger), and then exposed to X-rays. The stable expression of the vectors in the cells was analyzed by RT-PCR and restriction fragment length polymorphism (RFLP). Cellular sensitivity, apoptosis, chromosomal aberrations andγH2AX were detected using a colony-forming assay, Hoechst33342 staining assay, TUNEL assay, chromosomal banding techniques and flowcytometry, respectively. And the expression of the proteins relative to apoptosis was analyzed by Western blot. The results are as follows. The bands of 110 base pairs (bp) obtained by RT-PCR in exon 7 of the p53 gene could be clearly observed in H1299/wtp53 and H1299/mp53 cells. And after exon 7 was digested specifically with MspI or BsrI restriction enzymes, bands of 70 base pairs (bp) could be clearly observed in H1299/wtp53 cells or in H1299/mp53 cells, respectively. These data show this experimental model is successful and valid. In wtp53 cells, the induction of radioresistance and the inhibition of apoptosis, DNA double strand breaks and dicentric chromosomal aberrations were observed in the presence of ISDN (5μM) at a low concentration before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. However, at high concentrations of ISDN (500μM) clear evidence for radiosensitization, improvement of apoptosis, DNA double strand breaks and dicentric chromosomal aberrations was detected in wtp53 cells. In addition, the data of the protein expression of Bax and Caspase-3 as an indicator of apoptosis after radiation in H1299/wtp53 cells show that Bax-mediated apoptosis through Caspase-3 induced by X-irradiation was dually affected by NO radicals, when p53 is functionally normal. Accordingly, biphasic effects of NO radicals on p53-dependent X-ray-induced apoptosis seems to be an important determiner of cellular sensitivity in human lung cancer cells. However, All of these phenomena were not observed in mp53 cells at either concentration range with ISDN.Due to these observations above, we only chose human lung cancer cells with a wild-type (wt) p53 genotype as the research target in next research. The purpose of the second study was to examine the dual effects of different doses of NO radicals on radiation-induced cell killing and chromosome aberrations as a function of the cell cycle position in H1299/wtp53 cells. Cells were pre-treated with different concentrations of ISDN or a 0.02 Gy priming irradiation, and then exposed to acute X-rays 6 h after the pre-treatment. Cell synchronization was achieved with serum starvation. Floating mitotic cells in the medium were collected by centrifugation and were cultured in fresh medium. Cellular sensitivity, cell cycle distributions, and chromosomal aberrations were detected using colony-forming assays, flow cytometry and chromosome banding techniques, respectively. The results are as follows. In the cells, the radioadaptive response, as measured by the induction of radioresistance and the inhibition of chromosome aberrations, was observed in the presence of low concentrations (5μM) of ISDN, or after a 0.02 Gy priming irradiation before an acute X-irradiation mainly at 17.5 h after mitosis. Radio-adaptation responses induced by ISDN or by a low dose priming irradiation was observed during a clearly shortened G2/M phase (P<0.01 when compared to the control group) and during a slightly prolonged S phase (P<0.05 when compared to the control group). In contrast, in the presence of a high concentration of ISDN (500μM), radio-sensitization and the enhancement of chromosome aberrations was detected principally at 17.5 h after mitosis, and this radio-sensitization was observed during a significantly prolonged G2/M phase (P<0.001 when compared to the control group) and slightly shortened S phase (P<0.05 when compared to the control group). Cells in the G2/M phase were most sensitive to ISDN-induced cell lethality and chromosomal aberrations, and the effects of ISDN on cell survival and chromosomal aberrations clearly depended on the cell cycle position.Because the sensitizing effects of NO radicals in tumor cells were affected by p53 gene status, the aim of next research is find a new target to improve the effects of sensitizing treatment through improving DSBs induced by NO radicals in tumor cells. Cultured mouse embryonic fibroblasts used here are sufficient and deficient in DNA DSB-repair genes involved in homologous recombination (HR) repair (X-ray repair cross-complementing group 2 (XRCC2) and radiation sensitive mutant 54 (Rad54)), and in non-homologous end joining (NHEJ) repair (DNA Ligase IV (Lig4) and Ku80) and sufficient and deficient in DNA base excision repair (BER) gene DNA polymeraseβ(polβ). Cultured Chinese hamster ovary cells (CHO) used here are sufficient deficient in HR repair gene breast cancer susceptibility gene 2 (BRCA2). After ISDN treatment, cellular sensitivity andγH2AX were detected using a colony-forming assay and flowcytometry, respectively; down regulation of BRCA2 gene was accomplished by siRNA treatment. The results are as follows. The relative D50 values listed sequentially in the order in which they increase (reflecting decreasing sensitivities to NO) are: BRCA2mt cells <<< polβ-/- cells < XRCC2-/- cells < Lig4-/- cells < Rad54-/- cells < Ku80-/- cells. This research indicates the most effective molecular target which correlated with cellular sensitivity to NO was BRCA2. Furthermore, relativeγH2AX levels after NO treatment for 8, 12, 24 and 48 h in defective (BRCA2mt) cells were higher significantly than in proficient (BRCA2wt and rev) cells. At 24 h after NO treatment for 24 h, there were also significant differences in relativeγH2AX levels between the BRCA2wt, BRCA2rev cells and BRCA2mt cells. These results of histone H2AX phosphorylated at serine 139 (γH2AX) with flow cytometry suggest that BRCA2 can generate cellular resistance to NO by repairing DSBs induced by it. In addition, it was found that BRCA2 small interference RNA (siRNA) efficiently enhanced sensitivity to NO in human lung cancer H1299 cells.In conclusion, these results reported above indicate low and high concentrations of NO radicals can choreograph inverse radiosensitivity, Bax and Caspase-3 mediated apoptosis, DNA double strand breaks and dicentric chromosomal aberrations in human lung cancer cells, and NO radicals can affect the fate of cells through the regulation of p53 gene expression. These differing effects of NO radicals in wtp53 cells depend on the cell cycle position and are G2/M phase dependent. In addition, it is proposed that HR repair gene BRCA2 contributes significantly towards the repair of NO-induced DSBs, and down regulation of BRCA2 possibly be a powerful tool during NO chemo-sensitization therapy for some kinds of cancer patients such as with p53 null.