Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline
Aim: To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids. Methods: The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-08-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/13/16/2646 |
| _version_ | 1797585075091537920 |
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| author | Sergei Boichuk Firyuza Bikinieva Elena Valeeva Pavel Dunaev Maria Vasileva Pavel Kopnin Ekaterina Mikheeva Tatyana Ivoilova Ilshat Mustafin Aigul Galembikova |
| author_facet | Sergei Boichuk Firyuza Bikinieva Elena Valeeva Pavel Dunaev Maria Vasileva Pavel Kopnin Ekaterina Mikheeva Tatyana Ivoilova Ilshat Mustafin Aigul Galembikova |
| author_sort | Sergei Boichuk |
| collection | DOAJ |
| description | Aim: To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids. Methods: The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC<sub>50</sub>) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed. Results: An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC<sub>50</sub> for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model. Conclusions: An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies. |
| first_indexed | 2024-03-11T00:00:40Z |
| format | Article |
| id | doaj.art-de40a3537f9c47499e69fb4ae8f2a9d0 |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-11T00:00:40Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-de40a3537f9c47499e69fb4ae8f2a9d02023-11-19T00:48:05ZengMDPI AGDiagnostics2075-44182023-08-011316264610.3390/diagnostics13162646Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 SublineSergei Boichuk0Firyuza Bikinieva1Elena Valeeva2Pavel Dunaev3Maria Vasileva4Pavel Kopnin5Ekaterina Mikheeva6Tatyana Ivoilova7Ilshat Mustafin8Aigul Galembikova9Department of Pathology, Kazan State Medical University, Kazan 420012, RussiaDepartment of Pathology, Kazan State Medical University, Kazan 420012, RussiaCentral Research Laboratory, Kazan State Medical University, Kazan 420012, RussiaDepartment of Pathology, Kazan State Medical University, Kazan 420012, RussiaCytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, RussiaCytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, RussiaDepartment of Pathology, Kazan State Medical University, Kazan 420012, RussiaDepartment of Pathology, Kazan State Medical University, Kazan 420012, RussiaDepartment of Biochemistry, Kazan State Medical University, Kazan 420012, RussiaDepartment of Pathology, Kazan State Medical University, Kazan 420012, RussiaAim: To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids. Methods: The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC<sub>50</sub>) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed. Results: An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC<sub>50</sub> for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model. Conclusions: An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies.https://www.mdpi.com/2075-4418/13/16/2646Osteosarcoma (OS)ABC-transporterspaclitaxeldoxorubicinresistanceapoptosis |
| spellingShingle | Sergei Boichuk Firyuza Bikinieva Elena Valeeva Pavel Dunaev Maria Vasileva Pavel Kopnin Ekaterina Mikheeva Tatyana Ivoilova Ilshat Mustafin Aigul Galembikova Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline Diagnostics Osteosarcoma (OS) ABC-transporters paclitaxel doxorubicin resistance apoptosis |
| title | Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline |
| title_full | Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline |
| title_fullStr | Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline |
| title_full_unstemmed | Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline |
| title_short | Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline |
| title_sort | establishment and characterization of multi drug resistant p53 negative osteosarcoma saos 2 subline |
| topic | Osteosarcoma (OS) ABC-transporters paclitaxel doxorubicin resistance apoptosis |
| url | https://www.mdpi.com/2075-4418/13/16/2646 |
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