Objective: Propranolol (PRO), a non-selective beta-adrenergic receptor inhibitor, has been recently discovered to possess anti-tumorigenic effects in cancer patients. There-fore, we aimed to investigate the in vitro effects of PRO in A549-derived lung cancer spheroids in terms of cell viability, spheroid formation, cell cycle regulation, cell differen-tiation, and apoptosis.
Materials and Methods: The effect of 24-hour PRO treatment on A549 cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A sub-cytotoxic PRO concentration (125 µM) was employed to evaluate its impact on the clonogenicity of A549-derived cancer spheroids after seven days of incubation. Messenger Ribonucleic Acid (mRNA) levels of cell cycle regulators including cyclin-dependent kinase inhibitor 1A (p21) and G2 checkpoint kinase (WEE1), apoptotic markers such as caspases 3, 8, 9 (CASP3, CASP8, CASP9), and stem cell differentiation markers, namely POU class 5 ho-meobox 1 (octamer-binding transcription factor 4 (OCT4)), prominin 1 (CD133), and adenos-ine triphosphate (ATP) binding cassette subfamily G member 2 (ABCG2) were measured us-ing reverse transcription quantitative polymerase chain reaction (RT-qPCR) after a 24-hour treatment of cancer spheroids with PRO.
Results: PRO treatment reduced cell viability and inhibited the clonogenicity of cancer spheroids by activating intrinsic apoptotic markers CASP3 and CASP9, leading to cell cy-cle arrest via increased p21 expression. PRO did not significantly alter stem cell differen-tiation markers.
Conclusion: The proliferation and clonogenic activity of lung cancer spheroids can be ef-fectively suppressed with PRO, primarily through inducing intrinsic apoptosis following p21-mediated cell cycle arrest. While short-term PRO exposure did not affect the gene ex-pression levels of stem cell differentiation markers, the notable decrease in both cell viability and spheroid formation efficiency suggests the potential of PRO as a therapeutic drug in lung cancer treatment.