Characterization of multidrug-resistant osteosarcoma sublines and the molecular mechanisms of resistance

Abstract

Multidrug resistance (MDR) is a challenge for the treatment of cancer and the underlying molecular mechanisms remain elusive. The current study exposed MG63 osteosarcoma cells to increasing concentrations of vincristine (VCR) to establish four VCR‑resistant MG63/VCR cell sublines (MG63/VCR1, 2, 3 and 4). The drug resistance indices (RI) of these sublines was detected with the CCK‑8 assay and determined to be163, 476, 1,247, and 2,707‑fold higher than that of parental cells, respectively. These sublines also exhibited cross‑resistance to doxorubicin, paclitaxel and pirarubicin. With increased RI, the proliferative capacity of these sublines was gradually reduced and cell morphology was also altered, characterized by increased formation of pseudopodia and long cytoplasmic processes at opposite poles. However, the migration capacity and expression of certain drug resistance‑associated genes were not in accordance with the increased RI; multidrug resistance protein 1 (MDR1) expression was significantly increased in these sublines compared with parental cells. However, in the highly resistant MG63/VCR3 and MG63/VCR4 cells, MDR‑associated protein 1, topoisomerase II and LIM domain kinase 1 levels were significantly reduced compared with the moderately resistant MG63/VCR2 cells. Expression of glutathione S‑transferase‑π mRNA was determined using reverse transcription‑quantitative polymerase chain reaction and determined that it was not changed between MG63 and MG63/VCR cells. The data of the present study demonstrated that the molecular alterations of drug resistance may change with the degree of drug resistance. Taking cell morphology into consideration, the intratumor clonal and phenotypic heterogeneity may be responsible for drug resistance. These MG63/VCR sublines may be a valuable tool to assess drug resistance and the underlying mechanisms, and to identify novel drug resistance‑associated genes or strategies to overcome MDR in human osteosarcoma.

Identification of differentially expressed genes in MG63 osteosarcoma cells with drug‑resistance by microarray analysis

“…In the present study, the roles of differentially expressed genes in VCR resistance of osteosarcoma cells were investigated by microarray analysis. Numerous genes determined as differentially expressed in VCR-resistant osteosarcoma cells may also be associated with VCR resistance in the present study, such as MDR1 (11), which was upregulated in MG63/VCR cells. Previous studies have indicated that MDR1 serves a key role in the proliferation and survival of epithelial and malignant cells during tumorigenesis, as well as in acquired drug resistance (15,16).…”

“…The human VCR-resistant osteosarcoma cell line MG63/VCR and its parental cell line MG63 were obtained from the Scientific Research Center, China-Japan Union Hospital of Jilin University (Jilin, China) (11). The cells were cultured in high-glucose Dulbecco's modified Eagle's medium (H-DMEM; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in a humidified atmosphere with 5% CO 2 at 37°C; three biological replicates were conducted using the MG63 and MG63/VCR cells.…”

“…MG63 cells were treated with the following concentrations: 64, 16, 4, 1, 0.25 and 0.0625 ng/ml. The half-maximal inhibitory concentration (IC 50 ) of VCR in MG63/VCR and MG63 cells was reported to be 453.4 and 0.952 ng/ml, respectively (11). A total of two groups of cells continued to culture 48 h in drug medium at 37°C.…”

LIM kinase 1 serves an important role in the multidrug resistance of osteosarcoma cells

“…2B ) ( 1 , 21 ). These drugs included an anti-microtubule agent (PTX), an antimetabolite (MTX) and two topoisomerase II inhibitors (DOX and THP) ( 22 ). Notably, the mRNA and protein expression levels of LIMK1 and MDR1/P-gp were higher in MG63/VCR cells than in MG63 cells ( Fig.…”

Mechanisms of Resistance to Conventional Therapies for Osteosarcoma

“…There are two isoforms of topoisomerase II: Topoisomerase IIα and topoisomerase IIβ [90,91]. OS cell lines (143B, MG63, and HOS) showed a reduction of topoisomerase II expression in DOX-resistant cells compared to sensitive cells [85,92,93], suggesting that a decrease of DOX target is one of the mechanisms of drug resistance.…”