Abstract 93P
Background
Methotrexate (MTX)-resistance of osteosarcoma results in poor prognosis. Further understanding of the basis and ramifications of the MTX-resistance is therefore needed. The present study aimed to determine, in a super MTX-resistant osteosarcoma cell line, its degree of malignancy and the expression level of malignancy-related genes, PI3K/AKT/mTOR and c-MYC.
Methods
Super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from 143B parental osteosarcoma cells (143B-P) by culturing the cells with stepwise increasing concentrations of MTX (0.04 μM to 100 μM) for 12 months. Colony formation capacity on plastic and in soft agar was examined to compare the malignancy of 143B-P and 143B-MTXSR in vitro. Orthotopic xenograft mouse models of 143B-P and 143B-MTXSR, in which 2.5 × 105 cells were implanted in the tibia of nude mice, were established to compare malignancy in vivo. Expression of dihydrofolate reductase (DHFR), PI3K/AKT/mTOR, and c-MYC was determined by Western immunoblotting. The mouse studies were approved by Institutional Animal Care and Use Committee of AntiCancer Inc.
Results
143B-MTXSR gained 5500-fold resistance to MTX (IC50: 147.5 μM), compared to 143B-P (0.027 μM), due to a 9.8-fold increase in DHFR. 143B-MTXSR had reduced colony formation capacity on plastic (P = 0.032) and in soft agar (P < 0.01), indicating the reduction of malignancy. 143B-MTXSR formed significantly smaller tumors than 143B-P (P < 0.001), further indicating that 143B-MTXSR lost malignancy. 143B-MTXSR showed an increased expression of PI3K (P < 0.01), phosphorylated AKT (activated AKT) (p-AKT) (P = 0.031), phosphorylated mTOR (activated mTOR) (p-mTOR) (P = 0.043), and c-MYC (P = 0.024), compared to 143B-P, along with their decreased malignancy.
Conclusions
The present study demonstrated that 143B-MTXSR results in loss of malignancy, and for the first time, indicates that the increased expression of PI3K/AKT/mTOR and c-MYC are linked to MTX-resistance and malignancy. This new insight into MTX resistance of osteosarcoma should lead to more effective strategies to treat this recalcitrant disease.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.