Cinnamolide Sesquiterpene Lactone Suppresses in Vitro and in Vivo Cancer Cell Growth in Cisplatin-Resistant Human Cervical Carcinoma Cells by Inducing Mitochondrial Mediated Apoptosis, Caspase Activation, Loss of MMP and Targeting Akt/β-Catenin Signaling Pathway

J BUON. Mar-Apr 2020;25(2):709-715.

Abstract

Purpose: This study was designed to examine the in vitro and in vivo antitumor effects of Cinnamolide against cisplatin-resistant human cervical cancer cells (HeLa cells).

Methods: Cell viability was examined by WST-1 cell viability assay. Cinnamolide-induced apoptosis was examined by fluorescent microscopy using acridine orange (ΑΟ) /ethidium bromide (EB) staining and flow cytometry in combination with annexin-V/propidium iodide (PI) staining. Western blot was used to study the effects of Cinnamolide on apoptosis-related protein expressions including Bax and Bcl-2 as well as to study effects on numerous caspases and Akt/β-Catenin signaling pathway. Effects on mitochondrial membrane potential (MMP) were evaluated by flow cytometry. In vivo studies using xenograft mouse model were carried out to evaluate the efficacy of Cinnamolide under in vivo conditions.

Results: Cinnamolide decreased the viability of the HeLa human cervical cancer cells and exhibited an IC50 of 16.5 µM. The cytoxicity of Cinnamolide was also investigated on the MDCK normal cervical cells which showed that Cinnamolide exerted very low toxic effects on these cells. Cinnamolide also caused remarkable changes in the morphology of the HeLa cancer cells and suppressed their colony forming potential. The AO/EB staining showed that this molecule inhibits the viability of cancer cells via induction of apoptotic cell death which was associated with increase in Bax and decrease in Bcl-2 levels. The apoptotic cells increased from 3.5% in control to around 59% in HeLa cells at 50 µM concentration. Cinnamolide treatment also led to activation of caspase-3 and caspase-9. It was also seen that Cinnamolide treatment led to a significant and dose-dependent loss of MMP in HeLa cancer cells. It also significantly inhibited the Akt/β-catenin signalling pathway by reducing the levels of phosphorylated Akt and GSK-3β. The results also showed that Cinnamolide suppressed the tumor volume and the tumor weight of the xenografted tumors.

Conclusion: The results of this study indicate that Cinnamolide natural product has the potential to be developed as a promising anticancer agent against human cervical carcinoma.