Cengiz, Celal; Vejselova Sezer, Canan; Gür, Bahri; Kutlu, Hatice Mehtap; Ayhanci, Adnan; Bakan, Nuri; Bayrakdar, Alpaslan; Cengiz, Mustafa

The current study aims to present a novel approach to alleviate the aggressiveness of ovarian cancer by pharmacological disruption of the Yes-Associated Protein (YAP) − 1/Transcriptional Enhancer Factor domain (TEAD) heterodimer structure using Carmofur. The Ovcar-3 cell line was incubated with progressively diluted Carmofur solutions. Proliferation inhibition was assessed using the MTT assay. Flow cytometry was employed to determine the pro-apoptotic properties. Cellular morphological alterations were assessed with confocal microscopy. The migration inhibition status was assessed via the scratch assay, whilst the colony formation inhibition level was evaluated using the colony assay. The western blotting technique was employed to determine YAP-1 expression levels. The disruptive impact of Ceramide and Carmofur on the YAP-1/TEAD heterodimer structure was elucidated via molecular modeling, considering three interfaces: the β-strand, the α-helix, and the Ω-loop. Furthermore, validation was conducted by assessing the disruptive impact of Verteporfin on the YAP-1/TEAD heterodimer. The findings indicated that Carmofur diminished cell viability in a concentration- and time-dependent manner, with IC50 values of 17.26 µM at 24 h, 10.15 µM at 48 h, and 9.22 µM at 72 h, respectively. Carmofur showed a 26.65% apoptotic effect at the IC50 value over 24 h. Additionally, Carmofur diminished the relative YAP-1 levels from 9.72 to 2.98 in the Ovcar-3 cell line and exhibited an anti-metastatic effect by suppressing colony formation and migratory capabilities. This is the first study to provide in silico and in vitro evidence that Carmofur induces apoptosis by disrupting the YAP-1/TEAD heterodimer, thereby inhibiting the proliferation and migration of Ovcar-3 cells.

Erden-Karaoğlan, Fidan; Karaoğlan, Mert

Pichia pastoris is a successful expression system that is frequently preferred in the secretion of proteins for both basic research and industrial purposes. In this study, recombinant Rhizomucor miehei (RmASNase) L-asparaginase was produced in Pichia pastoris. The impact of gene copy number on increasing protein production was examined with six clones harboring various gene copy numbers (1–5 and 5 +). The results demonstrated that the clone with three copies of the expression cassette integrated had the highest production level. Also, biochemical characterization of the enzyme was performed. It was determined that the optimum pH and temperature values of the purified enzyme were pH 7.0 and 50 °C, respectively. Stability analyses of the enzyme showed that it maintains its activity of 80% in the pH range of 5–9 and 67% in the temperature range of 20–50 °C. Ca+2 and Mn+2 ions increased the enzyme activity to 121% and 138%, respectively. In future studies, it is also possible to improve the activity and stability values of the enzyme with advanced molecular techniques and to increase production efficiency by producing at fermenter scale and under optimum conditions.