Kemik Tümörlerinde Lipozomal Kannabinoidlerin Reseptör Aracılı Antiproliferatif ve Apoptotik Etkilerinin Araştırılması

Abstract

Primary osteosarcoma is the most common osteogenic tumor in children and adolescents, known with its rapid metastasis. The limitations of the treatment approaches applied in the clinic and the side effects observed in patients reveal the necessity of investigating novel treatment agents. Endocannabinoids are lipid-structured ligands that are known to be synthesized in the human musculoskeletal system and have been reported to regulate bone formation and resorption through CB1, CB2 and TRPV1 receptors in osteoblast, osteoclast and osteoprogenitor cells. Cannabinoids have been reported to reduce proliferation and induce apoptosis in osteosarcoma cells through CB2 or TRPV1 receptors. Synthetic specific CB2 receptor agonist CB65, which has been reported to have antiproliferative and apoptotic effect on hepatocarcinoma and endometrial cancer, may exert CB2 receptor-mediated antiproliferative and apoptotic effects on osteosarcoma cells. A biocompatible liposome delivery system can be used providing the controlled release and increasing the bioavailability of CB65 as a drug candidate for osteosarcoma. To test the hypothesis, an in vitro observational study with experimental and control groups was designed. In the thesis, human Saos-2 osteosarcoma cell line showed higher CB2 receptor expression when compared to positive control A549 cells by qRT-PCR. The presence of intracellular and membrane CB1 and CB2 receptors was demonstrated by flow cytometry in MG63 and Saos-2 cell lines in vitro. Accordingly, high CB2 immunolabeling was detected in MG63 and Saos-2 lines. When dose and time-dependent antiproliferative dose range of 10-12 –10-8 M CB65, by WST-1, was applied to MG63 and Saos-2 lines, ED50 of 1.11x10-11 M for MG63 and 4.95x10-11 M for Saos-2 cells were calculated by real-time proliferation analysis. The antiproliferative effect of CB65 on osteosarcoma lines was inhibited by CB2 antagonist AM630, showing its CB2 receptor-mediated effect. ED50 CB65 induced late apoptosis of 7.31% of MG63 cells at 48 hours and 19.06% of Saos-2 cells at 24 hours by Annexin/PI labelling. CB65 was loaded into liposomal system with a ratio of 51.12% by thin film formation method and 24 hour-controlled release was achieved. Liposomes releasing a dose of 1.11x10-11 M CB65 reduced proliferation of MG63 and Saos-2 cells from 16 56 hours. In conclusion, a new stable and early effective liposomal CB65 delivery system has been successfully generated and validated for its CB2 receptor-mediated agonistic antiproliferative and apoptotic effect on human osteosarcoma cells in vitro. The orthopaedic oncology clinics might translate the new formulation as a targeted and smart tool for bone tumors to cure paediatrics bone cancers following in vivo validation.