Akciğer Kanserinde Kannabinoidlerin CB Reseptörleri Aracılı Antiproliferatif ve Proapoptotik Etkisinin Araştırılması

Abstract

Lung cancer is the most common cause of death among all cancers in the world due to its rapid metastasis and late diagnosis. Non-small cell lung cancer (NSCLC) constitutes 80% of lung cancer cases. Endocannabinoids are endogenous cannabis ligands with lipid structure, which are synthesized in the body and generally act on classical cannabinoid receptors. It has been revealed that the endocannabinoid system is present in lung under physiological conditions and its components can play a mediating role in the pathogenesis of cancer via CB1 receptors. Arachidonoylcyclopropylamide (ACPA) is a lipid structured, synthetic-specific CB1 agonist that does not have a psychoactive effect and does not pass through the blood-brain barrier. ACPA can decrease proliferation and increase apoptosis via CB1 receptor agonism in NSCLC. A biocompatible polycaprolactone-based nanoparticular system with low biodegradation rate for controlled release of ACPA to the targeted lung tumor as a drug candidate can be used. An in vitro observative set up with experimental and control groups was designed in order to test the presumed hypothesis. In the thesis, firstly, the presence and levels of cannabinoid receptors were determined by in vitro qRT-PCR, flow cytometry and immunocytochemistry methods in human commercial, six non-small cell lung adenocarcinoma cell lines (A549, H1299, H358, H838, H1975 and SW-1573). Accordingly, high CB1 receptor expression was found in the A549, H1299, H358 and H838 lines. In the second step, the dose and time-dependent antiproliferative effect of ACPA has been tested by WST-1 and real-time impedance-based proliferation analysis in the A549, H1299, H358 and H838 cell lines that highly express the cannabinoid 1 receptor. The effect of ACPA on apoptosis has been quantified by flow cytometry technique. The antiproliferative dose range of ACPA in the cell lines was determined as 10-12 – 10-9 M by WST-1 technique. The effective dose (IC50) for the cells in the real-time proliferation assay was calculated as 1.39x10-12 M. In the third step, the stability studies of ACPA have been performed. The molecule alone in cell culture supernatants were not stable on days 1-3. Since ACPA is not stable, a new ACPA loaded polycaprolactone-based nanoparticular system was developed and optimized by nanoprecipitation method. Subsequently, controlled and prolonged release of ACPA with nanoparticles was achieved. In the final stage, the polycaprolactone-based nanoparticle system with ACPA was applied to NSCLC lines in impedance-based real-time proliferation analysis. As a result, polycaprolactone nanoparticles that release a dose of 1.39x10-12 M ACPA generally have antiproliferative effect. The long-acting and targeted formulation of the ACPA loaded polycaprolactone nanoparticular system may be a new drug candidate for NSCLC. In this context, an application was made to the Patent Office of Hacettepe Technopolis Technology Transfer Center with the outputs obtained within the scope of the thesis study.