The inhibition of radiation repair by ranpirnase ± l-buthionine sulfoximide on lung cancer
Authors: Intae Lee, Daniel Martinez, Anna Kalota, Bruce Pawel, Alan Gerwitz, Kuslima Shogen
Journal: Cancer Research (2008)

433   Purpose: We recently reported that the cytotoxic RNase, ranpirnase (ONCONASE ® , ONC), significantly retarded the radiation-induced tumor growth of A549 NSCLC xenografts. One of physiological mechanisms was related to significant increases in K trans  values, monitored non-invasively by a dynamic contrast enhanced magnetic resonance imaging method, and tumor blood flow & SO 2 , by a near-infrared spectroscopy method (IN VIVO 21:721-728, 2007). We hypothesized that l-buthionine sulfoximide (l-BSO) facilitates the radiation sensitizing effect of ONC, mainly due to the l-BSO-induced reduction in GSH levels. The possible mechanisms of the radiation sensitizing effect of ONC ± l-BSO were studied on lung cancer.  Materials and methods: Cell culture;  3 human NSCLC cells (A549, HOP-62 & NCI-H1975) and 2 fibroblast cells (HLF-1 human lung fibroblast & 3T3) were cultured.  Clonogenic assays for sub-lethal damage repair (SLDR) and potentially lethal damage repair (PLDR);  SLDR by split dose assay and PLDR by assay of the delayed trypsin treatment post-irradiation were studied after l-BSO ± ONC on 3 NSCLC cell lines.  Apoptosis in vitro analysis;  Tumor cells were plated on chamber slides and incubated for 3 days prior to ONC ± l-BSO treatment. Apoptosis was measured by using the fluorescein  in situ  cell death detection kit with fluorescence/ phase contrast microscopy.  Apoptosis in vivo analysis;  10-week-old, female athymic nude mice bearing human NSCLC xenografts were utilized. 2-5 x 10 6  viable cells suspended in 50 µl of RPMI1640 medium were injected into the right thighs of mice prior to treatment with ONC ± l-BSO. H. & E. staining, TUNEL labeling, and caspase-3-antibody labeling were used for  in vivo  apoptosis detection.  Results: All 3 NSCLC cells were sensitive to ONC. NCI-H1975 & HOP-62 cells were sensitive to l-BSO, but not A549 cells. ONC or l-BSO alone significantly enhanced radio-sensitivity on the x-irradiated A549 & NCI-H1975 cells. However, PLDR was expressed when x-irradiated A549 cells were delayed trypsinization for 4 h with or without l-BSO. Recovery factor was  ~ 2.4 for the control and  ~ 2.9 for the l-BSO treated group when the cell dissociation was delayed in the x-irradiated A549 tumor cells (p=0.08). l-BSO alone did not inhibit PLDR in A549 cells. In contrast, ONC alone and ONC + l-BSO significantly inhibited PLDR in the x-irradiated A549 cells. To obtain insight into the cellular mechanisms of radiation sensitization by ONC, l-BSO, and/or ONC + l-BSO, we determined the induction of apoptosis in 3 NSCLC cells. ONC significantly increased apoptosis in all 3 NSCLC cell lines. However, l-BSO did not increase occurrence of apoptosis in A549 cells. Our preliminary results showed enhanced apoptosis in NCI-H1975 & HOP-62 (needed for confirmation). We will provide detailed data in our upcoming AACR meeting.  Conclusion: This study suggests important potential clinical uses of ONC ± l-BSO for the treatment of NSCLC cancer patients.