Apoptotic induction mechanism of artonin E in 3D ovarian cancer cell lines

• Main Authors: Abd Rahman, Mashitoh, Mohd Hashim, Najihah
• Format: Article
• Published: Universitas Gadjah Mada - Faculty of Pharmacy 2022
• Subjects: RM Therapeutics. Pharmacology; RS Pharmacy and materia medica
• Online Access: http://eprints.um.edu.my/43548/
• Institution: Universiti Malaya

The three-dimensional culture model is gaining popularity because it is more physiologically relevant in vivo and predicts drug efficacy more accurately than conventional 2D culture. This study aimed to develop the 3D ovarian cells culture model and to investigate the cytotoxicity of artonin E in SKOV-3 cells in 2D and 3D cultures. The 3D cells culture was performed using BD™ Puramatrix™ Peptide Hydrogel. The Alamar blue assay and selectivity index analysis were conducted to examine the antiproliferative effects in both 2D and 3D cultures. The morphological apoptotic double staining and immunofluorescence studies were performed to investigate the possible apoptotic mechanism involved in treated 3D SKOV-3 spheroids. Result showed that the ovarian cell lines encapsulated in BD™ Puramatrix™ Peptide Hydrogel clearly demonstrated 3D-spheroids formation. The Alamar blue assay revealed that artonin E inhibited the growth of SKOV-3 cells in 2D and 3D culture, with IC50 values of 6.0 ± 0.8 μg/mL and 25.0 ± 0.8 μg/mL at 72 h treatment, respectively. This result indicated that the IC50 values of SKOV-3 cells treated with artonin E in 3D culture were four times higher than in 2D culture. Artonin E was found to be less toxic to normal human ovarian cell lines, T1074, with IC50 values at 72 h of 28.0 ± 0.8 μg/mL in 2D culture and 85.0 ± 0.5 μg/mL in 3D culture. The selectivity index analysis more than 2 suggested that artonin E was selective against cancer cells compared to normal cells. Artonin E treatment caused apoptotic morphological changes in 3D SKOV-3 spheroids. In a 3D immunofluorescence study, elevated levels of cleaved caspase-3, cleaved caspase-9, apoptotic proteins bax, and decreased levels of antiapoptotic proteins bcl-2, Hsp70, and survivin were observed. In conclusion, artonin E inhibits the growth of 3D SKOV-3 spheroids and induces cell death via a pro- and anti-apoptotic protein pathway. These findings demonstrate that 3D spheroids culture is an effective platform for testing artonin E therapeutic candidates in an in vivo mimic microenvironment. © 2022 by Indonesian Journal of Pharmacy (IJP).