Establishment and Characterization of Single and Triple‐Agent Resistant Osteosarcoma Cell Lines

Abstract

surgical removal and adjuvant multi-drug chemotherapy. [5,6] The combination of cisplatin (CIS), doxorubicin (DOX), and highdose methotrexate (MTX) is the standard treatment for most patients. Surgery combined with chemotherapy has improved the survival rate for osteosarcoma patients to 60-70%. [7] However, most patients with metastatic or recurrent osteosarcoma have poor prognosis due to the development of chemotherapeutic drug resistance. [8,9] One of the methods to improve the survival rate of osteosarcoma patients is by overcoming chemotherapy resistance. [10] The development of drug resistance in osteosarcoma has been studied and several mechanisms demonstrated, including genetic alterations, [11] drug-target mutation and amplification, [12] altered drug accumulation, [13] and autophagy. [14] The expression of the gene MDR1, which is responsible for producing P-glycoprotein (P-gp), has been widely studied in osteosarcoma. [15,16] P-gp is a membrane-bound protein which transports doxorubicin and methotrexate out from the cells, leading to chemoresistance. [17,18] Developing drug-resistant cancer cell models is a useful approach to study the mechanisms of chemoresistance in cancer cells. Previous resistant models have been established by using osteosarcoma cell lines such as SOSP-9607, [19] Saos-2, [20] MG-63, [21] and U-2OS. [22] These established osteosarcoma resistant models have increased fold resistance ranges from 6 to 120-fold compared to their parental cell lines. [19][20][21][22] They also exhibit cross-resistance to other chemotherapeutic drugs including ifosfamide, epidoxorubicin, pirarubicin, and paclitaxel. [19][20][21][22] In the present study, clinically relevant chemo-resistant osteosarcoma cell models were developed from the cell lines MG-63 and HOS-143B. MG-63 was established from a 14-year old male with marginally metastatic osteosarcoma; [23] and HOS-143B originally derived from HOS, was established from a 13-year old Caucasian female with significant metastatic characteristics. [24] Chemo-resistant models of MG-63 and HOS-143B were developed by using a pulsed-selection strategy where cells were incubated with constant concentration of chemotherapeutic drugs for 72 h and then allowed to recover in drug-free media. The purpose of this method is to simulate a similar experience with osteosarcoma patients who undertake clinical chemotherapy treatment; and therefore, this is a clinically Two human osteosarcoma cell lines (MG-63 and HOS-143B) are developed into drug-resistant models using a short-term drug exposure and recovery in drug-free media. Cisplatin, doxorubicin, and methotrexate are used as single agents and in triple combination. The highest level of resistance to cisplatin is observed in MG-63/CISR8, doxorubicin in HOS-143B/DOXR8, and methotrexate in HOS-143B/MTXR8. The MG-63/TRIR8 and HOS-143B/TRIR8 tripleresistance models show lower levels of resistance to combination treatment and are not resistant to the drugs individually. Apoptosis assays suggest that the resistance in MG-63/TRI...