Abstract:
Mutations in p53 and KRAS genes are present in many human cancers. Both genes play important roles in tumor initiation and in the acquiring of oncogenic properties that enable the tumor to increase its proliferation, become invasive, and metastatic. Although the role of these genes in tumorigenesis and tumor invasiveness is well established, their effect on drug sensitivity are still unclear. Herein we evaluated the response of MN minipigs and Crossbred transgenic swine cells containing KRASG12D and p53R167H mutations to the chemotherapeutic drugs doxorubicin and 5-fluorouracil. Transgenic pigs were engineered to contain oncogenic KRASG12D and dominant-negative p53R167H, downstream of a LoxP-polyA(STOP)-LoxP sequence (LSL) and CAG promoter. We have generated fibroblast cell lines from purebred MN minipigs and Minipig x domestic cross transgenic animals. Oncogene expression (KRASG12D and p53R167H) was induced in the fibroblasts when recombination was triggered by Cre-recombinase (CRE) in the cell lines. MTT colorimetric assay was used to evaluate the effect of KRASG12D and p53R167H expression on the cells response to doxorubicin and 5-fluororacil anticancer drugs. Transgenic cells (CRE-treated and their corresponding normal controls) were seeded at a density of 2 x 104 cell per well and then treated with doxorubicin or 5-fluorouracil at concentrations of 0.25, 0.5, 1, 2 and 4 μg/mL for 24, 48 and 72 hours. Following incubation, 20 μl of MTT (5 mg/mL) was added to each well, and the cells were incubated for an additional 3 hours at 37°C. Differences in total cellular metabolism were detected at a wavelength of 492 nm using a microplate reader. Doxorubicin treatment reduced the number of viable cells in both KRASG12D/p53R167H expressing lines (CRE) to less than 50% at concentrations from 1 μg/mL after 48 hours of treatment and upon concentrations from 0.5 μg/mL after 72 hours of treatment. In the controls lines doxorubicin just was able to reduce significantly the cell viability upon doses from 2 μg/mL in 72 hours. Both purebred minipigs and crossbred cells showed the same drug response pattern to doxorubicin. On the other hand, the effectiveness of 5-fluoruoracil treatment was distinct between purebred minipigs and crossbred cells. At 72 hours of treatment 5-fluorouracil was able to reduce the viability of CRE purebred minipigs cells up to 50% at all treatments doses. The crossbred cells showed a resistance to 5-fluorouracil at all doses and treatment times both in CRE and control cells, with the number of viable cells equal or higher than 75%. Although there are a few reports in the literature about doxorubicin chemotherapy resistance in the presence of oncogenic KRASG12D or dominant-negative p53R167H alone; our data suggests that the combination of these two point mutations does not affect the cell response to doxorubicin. However, CRE treated minipig cells were more sensitive than control cells to 5-fluorouracil at the doses tested, and the response in crossbred cells did not differ between CRE and control treatments. The overall results suggest that the expression of KRASG12D and p53R167H mutated proteins does not promote chemotherapy resistance to doxorubicin and may increase the sensitivity to 5-fluoruoracil in transgenic minipig cells. There may be genotypic differences in drug metabolism.
Acknowledgments: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)