Multimodal effects of cilengitide on circadian clock gene expression in SH-SY5Y cells

Circadian rhythms, governed by a network of core clock genes such as PER1, RORA, NR1D1, CRY1, CLOCK, and ARNTL, play a pivotal role in regulating cellular and physiological processes. This study investigates the effects of cilengitide, a cyclic RGD pentapeptide and selective integrin inhibitor, on the expression of these circadian genes in SH-SY5Y human neuroblastoma cells. Using quantitative real-time PCR (qRT-PCR), we assessed gene expression following cilengitide treatment at varying concentrations, with 50 µg/mL identified as the optimal dose based on cytotoxicity assays (IC50 ≈ 52.3 µg/mL). Our results revealed significant upregulation of PER1 and RORA, alongside downregulation of NR1D1, CRY1, CLOCK, while ARNTL exhibited decreased gene and increased protein expression. PER1 and PER2 were markedly increased, whereas CRY1 and NR1D1 were reduced in a dose-dependent manner. Flow cytometry analysis revealed cilengitide-induced apoptosis; 17.56% of cells underwent early (9.13%) and late (8.43%) apoptosis. These findings suggest that cilengitide modulates circadian gene expression, potentially disrupting the transcription-translation feedback loop (TTFL) critical for cellular rhythmicity. Our study highlights cilengitide’s dual impact on integrin-mediated signaling and circadian clock machinery. These insights open new avenues for exploring cilengitide as a chronotherapeutic agent in neuroblastoma and neurodegenerative diseases, warranting further investigation into its mechanisms and in vivo applicability.