טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIfergan Ilan
SubjectThe Role of the Methotrexate Transporter in the Chemotherapy
of the Osteosarcoma
DepartmentDepartment of Biology
Supervisors Professor Yehuda Assaraf
Professor Yoram Reiter


Abstract

High-dose methotrexate (MTX) continues to be a major component of combination chemotherapy regimens for the treatment of osteosarcoma (OS). MTX uptake proceeds primarily through the reduced folate carrier (RFC), whereas MTX efflux occurs also via multidrug resistance protein 1 (MRP1). Here we determined RFC and MRP1 protein levels (normalized to β-actin) in two OS cell lines MG63 and U2OS which are resistant to 1 mM MTX and in twenty human OS specimens, nine of which were at diagnosis and eleven at relapse. The OS specimens contained three matched specimen pairs obtained at diagnosis and relapse. Western blot analysis revealed that the OS cell lines MG63 and U2OS express 2- and 4- fold less RFC levels and 7- and 20- fold higher MRP1 levels respectively, relative to the established leukemia cell line CEM-w.t. The finding that the OS cell lines express low RFC levels (the transporter that enables the entrance of MTX to the cell) and high MRP1 levels (the exporter that pumps MTX out of the cells) may explain the resistance of these OS cell lines to the millimolar concentrations of MTX. Examination of the levels of these transporters in the OS specimens revealed that the average RFC level in the relapse tumors that are absolutely resistant to MTX-containing chemotherapy was significantly higher than at diagnosis (P = 0.0005). Furthermore, in all three matched specimen pairs, RFC levels in the relapse individuals were higher than in their diagnosis counterparts. The relationship between RFC as well as MRP1 expression and the histological response to preoperative chemotherapy was examined. Tumors with poor histological response (≤ 90% necrosis) had significantly lower RFC levels than those with a favorable response to chemotherapy (P=0.0016). In contrast, there was no correlation between MRP1 levels at diagnosis and the histological response to chemotherapy (P=0.8764). Although the average MRP1 level in tumor specimens was higher at relapse than at diagnosis, this was not statistically significant (P = 0.2056).

Hence, the significant correlation between low RFC protein levels at diagnosis and the poor histological response to preoperative chemotherapy suggests that RFC protein levels at diagnosis may be a useful predictor of chemosensitivity. Furthermore, the post-chemotherapy progression from diagnosis to relapse appears to be associated with a significant increase in RFC protein levels.