Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline.

Autor: Boichuk S; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia.; 'Biomarker' Research Laboratory, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russia.; Department of Radiotherapy and Radiology, Russian Medical Academy of Continuous Professional Education, Moscow 125993, Russia., Bikinieva F; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia., Valeeva E; Central Research Laboratory, Kazan State Medical University, Kazan 420012, Russia., Dunaev P; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia., Vasileva M; Cytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, Russia., Kopnin P; Cytogenetics Laboratory, Carcinogenesis Institute, N.N. Blokhin National Medical Research Center of Oncology, Moscow 115478, Russia., Mikheeva E; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia., Ivoilova T; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia., Mustafin I; Department of Biochemistry, Kazan State Medical University, Kazan 420012, Russia., Galembikova A; Department of Pathology, Kazan State Medical University, Kazan 420012, Russia.
Jazyk: angličtina
Zdroj: Diagnostics (Basel, Switzerland) [Diagnostics (Basel)] 2023 Aug 11; Vol. 13 (16). Date of Electronic Publication: 2023 Aug 11.
DOI: 10.3390/diagnostics13162646
Abstrakt: 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 50 ) 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 50 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.
Databáze: MEDLINE
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