| Abstract | Osteosarcoma is a rare malignant bone tumour that occurs primarily in adolescents and young adults. Prior to the adoption of chemotherapy in mid 1970s, more than 85% if post-surgery osteosarcoma patients developed metastasis. Nowadays, standard osteosarcoma treatment of osteosarcoma includes neo-adjuvant chemotherapy followed by surgical removal and adjuvant multi-drug chemotherapy. The combination of cisplatin, doxorubicin and high-dose methotrexate is the standard treatment for most patients. Surgery combined with chemotherapy has improved the survival rate for osteosarcoma patients to 60% - 70%. However, most of the patients with metastatic or recurrent osteosarcoma have poor prognosis due to the development of chemotherapeutic drug resistance.
Developing drug-resistant cancer cell models is one approach to study the mechanisms of chemoresistance in cancer cells. In this study, clinically relevant chemoresistant osteosarcoma cell models were developed from the cell lines MG-63 and HOS-143B. One of the strategies used the current study includes a multiple drugs combination approach, where cisplatin, doxorubicin, and methotrexate are combined in one treatment. The purpose of this method is to simulate a similar experience with osteosarcoma patients who are receiving clinical chemotherapy treatment and therefore, to establish a clinically relevant osteosarcoma-resistant model to study the mechanisms of drug resistance.
Cisplatin, doxorubicin, and methotrexate were used as single agents and in triple combination. The highest level of resistance to cisplatin was observed in MG-63/CISR8 (3.56 ± 0.43-fold; p=0.001), doxorubicin in HOS-143B/DOXR8 (1.99 ± 0.20; p=0.0002), and methotrexate in HOS-143B/MTXR8 (3.77 ± 0.90-fold; p=0.046). The MG-63/TRIR8 and HOS-143B/TRIR8 triple-resistance models showed lower levels of resistance, 2.28 ± 0.63-fold (p=0.032) and 2.17 ± 0.13-fold resistant (p=0.0004) to combination treatment; and were not resistant to the drugs individually. Apoptosis assays suggest that the resistance in MG-63/TRIR8 is mainly from cisplatin and methotrexate and not doxorubicin. In contrast, the resistance in HOS-143B/TRIR8 is mainly from doxorubicin and methotrexate instead of cisplatin. Upregulation of P-glycoprotein was seen in all resistant models except those developed with single-agent methotrexate. The P-glycoprotein inhibitor elacridar reversed the resistance of doxorubicin on MG-63/DOXR8 (0.36 ± 0.06-fold, p=0.003), MG-63/TRIR8 (0.72 ± 0.07-fold, p=0.04), HOS-143B/CISR8 (0.47 ± 0.09-fold, p=0.009), and HOS-143B/TRI (0.45 ± 0.03-fold, p=0.0005). The migration rate of the MG-63 resistant models was significantly increased by 2.12 – 2.46-fold, their invasion rate tended to increase, and RT-PCR showed a switch from epithelial to mesenchymal gene signalling. In contrast, a significant decrease in migration was seen in HOS-143B resistant models with 0.39 – 0.43-fold, their invasion rate tended to decrease and a switch from mesenchymal to epithelial gene signalling occurred.SPHK1 and HIF1A were upregulated in most of the resistant models from the PCR array analysis and SPHK1 protein level was also determined to increase in MG-63/CISR8 (2.03 ± 0.08-fold, p=0.034), MG-63/DOXR8 (1.77 ± 0.24-fold, p=0.02), and HOS-143B/CISR8 (3.55 ± 0.84-fold, p=0.0459). A strong correlation (r=0.726) was shown between the gene expression of SPHK1 and HIF1A in HOS-143B resistant sublines.
Currently, there is also a lack of effective treatments for patients who experience relapsed osteosarcoma. One treatment for relapsed patients is gemcitabine and docetaxel combination chemotherapy (GEMDOX). A systematic review was performed in this study to investigate the efficacy of combination therapy of gemcitabine and docetaxel on relapsed osteosarcoma patients. The results showed the age and gender of the patients would have a prognostic effect on the GEMDOX regimen as the second-line treatment for relapsed osteosarcoma, whereas the GEMDOX therapy was determined to have a higher efficacy on male patients and with age <18. There was no difference in toxicities between different doses (675 mg/m2 and 1,000 mg/m2) of the GEMDOX regimen, age, or gender of patients. A preclinical in vitro study was performed by investigating the sensitivity of GEMDOX therapy on the established resistant sublines. The established resistant osteosarcoma sublines were used to investigate the efficacy of the GEMDOX treatment in the relapsed setting. Out of 8 of the resistant models, MG-63/DOXR8 was significantly resistant to gemcitabine (2.44 ± 0.26-fold, p=0.001) compared to MG-63 and HOS-143B/MTXR8 was significantly resistant to docetaxel (2.32 ± 0.17-fold, p=0.005) compared to HOS-143B. These two resistant sublines were also significantly resistant to the combination of gemcitabine and docetaxel with 2.50 ± 0.53-fold (p=0.04) and 2.09 ± 0.32-fold (p=0.017) respectively. However, the rest of the 6 resistant sublines were not resistant to GEMDOX treatment, which indicates GEMDOX regimen as a potential therapeutic treatment for relapsed osteosarcoma.
This project is the first to develop chemoresistant osteosarcoma cell lines with a triple combination of drugs. The characteristics of these resistant models also provide a better understanding of the resistant mechanisms in osteosarcoma cells. Lastly, these developed single and multi-agents induced clinically-relevant osteosarcoma cell lines could act as an invaluable tool for future studies of drug resistant mechanisms in osteosarcoma cells. |
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