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Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro
Published on: March 18, 2014
Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated
Yong Tao1, Yunsheng Ou1, Hang Yin1
1Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, P.R. China.
Abstract:
The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester‑mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved‑PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma cell lines which are valuable to explore the resistance-related mechanisms and the approaches to overcome resistance.
Insights
This study established new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT). These resistant cells exhibit altered proliferation, apoptosis, and drug resistance, offering a valuable model for studying MPPa-PDT resistance mechanisms.
Area of Science:
- Oncology
- Biochemistry
- Cell Biology
Background:
- Photodynamic therapy (PDT) is a promising cancer treatment.
- Developing resistance to PDT limits its clinical efficacy.
- Osteosarcoma is a challenging bone cancer with limited treatment options.
Purpose of the Study:
- To establish and characterize novel human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT).
- To investigate the mechanisms underlying MPPa-PDT resistance in osteosarcoma cells.
- To provide valuable models for exploring strategies to overcome PDT resistance.
Main Methods:
- Generation of MPPa-PDT-resistant cell lines (MG63/PDT, HOS/PDT) from parental MG63 and HOS osteosarcoma cells.
- Assessment of MPPa-PDT and cisplatin resistance, proliferation, apoptosis, cell cycle, invasion, and migration using assays like CCK-8, FCM, and western blotting.
- Analysis of intracellular reactive oxygen species (ROS) and expression of key proteins involved in apoptosis, drug resistance, and cell survival.
Main Results:
- Established MG63/PDT and HOS/PDT cell lines showed 1.67- and 1.61-fold resistance to MPPa-PDT, respectively, and cross-resistance to cisplatin.
- Resistant cells exhibited significantly lower apoptosis rates, reduced intracellular ROS, and altered expression of apoptosis-related proteins (e.g., cleaved-caspase 3, cleaved-PARP).
- Resistant cell lines displayed changes in proliferation, photosensitizer uptake, colony formation, invasion, migration, cell cycle distribution, and increased expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1, and ABCG2 compared to parental cells.
Conclusions:
- Successfully established two distinct human osteosarcoma cell lines with acquired resistance to MPPa-PDT.
- These cell lines are valuable tools for elucidating the complex mechanisms of MPPa-PDT resistance in osteosarcoma.
- The findings pave the way for developing novel therapeutic strategies to overcome resistance and improve treatment outcomes for osteosarcoma patients.
