Cytostatic effect of ricobendazole on primary cultures of soft tissue sarcomas in vitro

Soft tissue sarcomas (STS) are often resistant to treatment. The search for new antitumor compounds against STS remains an urgent task. Purpose of the study. To assess the sensitivity of primary STS cultures of various histological subtypes to albendazole sulfoxide (ricobendazole) and doxorubicin, the primary metabolite of albendazole. Materials and methods . STS tumor samples were used. The enzymatic dissociation method was used using 300 units/ml collagenase I (Thermo Fisher Scientific, USA). Ricasol® (NITA-PHARM, Russia) and Doxorubicin-­LENS® (VEROPHARM, Russia) were used as test substances. Sensitivity to ricobendazole and doxorubicin was tested using the MTT test. The cultures were seeded in a 96‑well plate at 7,000 cells in DMEM medium with 10 % FSC added. After 24 h, the medium was replaced with PPS with ricobendazole in a series of two-fold dilutions from 35.5 μmol/l to 0.0347 μmol/l or doxorubicin from 10 μmol/l to 0.009 μmol/l. After 72 hours of incubation, the MTT test was performed. The cells were seeded in a 24‑well plate and cultured in PPS with 2 μmol/l ricabendazole for 72 h. Hoechst 33342 dye (Life Technologies, USA) was added to the culture at a concentration of 1 μg/ml, and photographs were taken using a LionHeart FX digital automatic microscope (BioTek Instruments Inc., USA). Results. Four primary sarcoma cultures were obtained: SAR‑1, SAR‑2, SAR‑3, and SAR‑4. SAR-1 and SAR-4. Cultures demonstrated the most rapid growth, with doubling times of 38 and 27 hours, respectively. The slowest proliferation was observed in the SAR-2 culture (doubling time 156 hours), while SAR-3 showed a doubling time of 45 hours. According to the MTT assay, the IC50 values for ricobendazole were 4.54 ± 1.2 μmol/L for SAR-1, 3.31 ± 0.7 μmol/L for SAR-3, and 1.51 ± 0.2 μmol/L for SAR-4, whereas the slowly dividing SAR-2 culture proved to be insensitive to ricobendazole. The cytostatic activity of doxorubicin was higher than that of ricobendazole. The SAR‑2 culture was the least sensitive (IC50 could not be determined), and SAR‑4 (IC50 SAR‑4 = 0.16 ± 0.01 μmol/l) was the most sensitive to the action of doxorubicin. The IC50 value of SAR‑1 = 0.64 ± 0.02 μmol/l and IC50 SAR‑3 = 1.8 ± 0.1 μmol/l. The effect of ricobendazole caused pronounced disturbances in the nuclei of SAR‑1 and SAR‑4 cultures, in SAR‑2 and SAR‑3 they were less pronounced. Conclusion. Ricobenzale had a cytostatic effect on primary STS cultures characterized by rapid cell growth, but the activity was lower than that of doxorubicin. Changes in the morphology of cells and nuclei indicated probable disturbances in the functioning of the spindle and cytoskeleton occurring under the action of this compound. Of particular interest for further research is the combination of ricobendazole with taxanes and other tubulin inhibitors.