{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T21:34:24Z","timestamp":1790112864519,"version":"4.0.1"},"reference-count":38,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2023,8,11]],"date-time":"2023-08-11T00:00:00Z","timestamp":1691712000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["20-15-00001"],"award-info":[{"award-number":["20-15-00001"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>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 (IC50) 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 IC50 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.<\/jats:p>","DOI":"10.3390\/diagnostics13162646","type":"journal-article","created":{"date-parts":[[2023,8,11]],"date-time":"2023-08-11T10:33:23Z","timestamp":1691750003000},"page":"2646","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2415-1084","authenticated-orcid":false,"given":"Sergei","family":"Boichuk","sequence":"first","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"},{"name":"\u201dBiomarker\u201d Research Laboratory, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia"},{"name":"Department of Radiotherapy and Radiology, Russian Medical Academy of Continuous Professional Education, Moscow 125993, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9012-6525","authenticated-orcid":false,"given":"Firyuza","family":"Bikinieva","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Valeeva","sequence":"additional","affiliation":[{"name":"Central Research Laboratory, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pavel","family":"Dunaev","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Vasileva","sequence":"additional","affiliation":[{"name":"Cytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2078-4274","authenticated-orcid":false,"given":"Pavel","family":"Kopnin","sequence":"additional","affiliation":[{"name":"Cytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ekaterina","family":"Mikheeva","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatyana","family":"Ivoilova","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilshat","family":"Mustafin","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0293-2974","authenticated-orcid":false,"given":"Aigul","family":"Galembikova","sequence":"additional","affiliation":[{"name":"Department of Pathology, Kazan State Medical University, Kazan 420012, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,11]]},"reference":[{"key":"ref_1","first-page":"115","article-title":"Bone sarcomas","volume":"14","author":"Rutkowski","year":"2018","journal-title":"Oncol. 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