Securidaca–saponins are natural inhibitors of AKT, MCL-1, and BCL2L1 in cervical cancer cells

Abstract

Titus Chukwuemeka Obasi,1 Cornelia Braicu,2 Bogdan Cezar Iacob,1 Ede Bodoki,1 Ancuta Jurj,2 Lajos Raduly,2 Ilioara Oniga,3 Ioana Berindan-Neagoe,2,4,5 Radu Oprean1 1Department of Analytical Chemistry and Instrumental Analysis, Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Research Center for Functional Genomics, Biomedicine and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 3Department of Pharmacognosy, Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania; 4MEDFUTURE – Research Center for Advanced Medicine, University of Medicine and Pharmacy Iuliu-Hatieganu, Cluj-Napoca, Romania; 5Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof. Dr. Ion Chiricuta”, Cluj-Napoca, Romania Introduction: Scientific research is beginning to prove the connection between claims by African traditional medicine and the natural chemical specifics contained in medicinal plant Securidaca longipedunculata. Our previous studies showed that two natural saponin fractions (4A3 and 4A4) identified in the plant as triterpenoid glycosides are capable of activating apoptosis on cervical tumor cell lines. Considering this and some critical roles of human papillomavirus (HPV) E6 oncogene on cervical cells, by promoting carcinogenesis and cell survival, it became necessary to investigate the possible pathways for apoptosis transmission.Methods: Tests conducted on relevant cervical tumor cell lines such as Caski and Bu25TK included the following: MTT assay; scratch assay (to determine cell migration/invasion); fluorescence microscopy with Annexin V–fluorescein isothiocyanate, muscle progenitor cell) and propidium iodide staining; and finally reverse transcriptase quantitative PCR (RT-qPCR) for gene analysis.Results: Reduced cell proliferation was observed due to activities of 4A3 and 4A4 fractions, with half-maximal inhibitory concentration (IC50) of 7.03 and 16.39 μg/mL, respectively, on Caski cell line. A significant reduction in cell migration occurred within 48 and 72 hours, respectively, for Caski and Bu25TK cell lines. Late apoptosis was activated by 4A3, staining both Annexin V and PI, in contrast to 4A4’s early apoptosis. RT-qPCR data revealed a fold change (FC) inhibition of antiapoptotic proteins such as MCL-1 and BCL2L1, with diminished level of AKT-3, VEGFA, MALAT1, etc. The expression of p53, proapoptotic BAD, and caspase-8 was nonsignificant.Conclusion: The low expression of AKT-3 and antiapoptotic proteins (MCL-1 and BCL2L1), as well as VEGFA, could simply be an indication for possible suppression of cell survival mechanisms via multiple channels. We therefore conclude that 4A3 and 4A4 fractions mediate activity via the inhibition of phosphatidylinositol-3-OH kinase (PI3K)-AKT/mTOR/NF-kB-dependent antiapoptotic stimuli. Further studies are ongoing to reveal the chemical structures and compositions of these two fractions. Keywords: early apoptosis, RT-qPCR gene analysis, AKT-3, MCL-1 and BCL2L1 inhibition, triterpenoid saponins

Keywords

• Early apoptosis
• RT-qPCR gene analysis
• AKT-3
• MCL-1 and BCL2L1 inhibition
• triterpenoid saponins