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

Abstract

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: AKT-3; MCL-1 and BCL2L1 inhibition; RT-qPCR gene analysis; early apoptosis; triterpenoid saponins.

Figure 1

Cell viability measured by MTT assay. Notes: (A, B) Caski cell line and (C, D) Bu25TK cell line after 24- and 48-hour treatment, respectively. Data are expressed as the percentage of control after 24 and 48 hours, in incubation with different concentrations of the most effective fraction (4A3, 4A4, and 4A5) on Bu25TK and Caski cell lines; log(concentration, μg/mL) = log(concentration of bioactive fraction, μg/mL) (mean ± SD, n=3).

Figure 2

Autophagy and apoptosis assessment by fluorescence microscopy. Notes: Cells were treated for 48 hours with a single dose of 4A3 and 4A4 on Bu25TK and Caski cell lines. 4A3 shows the late apoptosis staining, both Annexin V (green) and PI (red), while 4A4 shows the preferentially stained Annexin V–FITC (20× magnification). Abbreviations: C, control; FITC, fluorescein isothiocyanate; MDC, monodansylcadaverine; PI, propidium iodide.

Figure 3

In vitro scratch assay. Notes: Cell migration evaluated on Bu25TK and Caski cell lines treated with 4A3 and 4A4, respectively, and different time points (4× magnification).

Figure 4

Gene expression. Notes: (A) Evaluation of response due to treatment with 4A3 and 4A4 at 24 hours. (B) Gas5 and MALAT1 expression level for same treatment scenarios. (C) String network for the transcripts with altered expression level. Data are generated using default settings (medium confidence of 0.4). *P≤0.05.

Figure 5

BCL-2 and VEGF protein expression quantification. Notes: (A) Confocal microscopy for BCL-2 immunostaining for control and 4A3- and 4A4-treated CASKI and Bu25TK cells for 48 hours post treatment in eight-well chamber slides (20× magnification). (B) VEGF quantification from cell culture medium was performed using ELISA for same experimental groups at 48 hours post treatment. *P≤0.05; **P≤0.01; ***P≤0.001.

Figure 6

Schematic diagram of molecular network and proposed mechanism of activity. Notes: (A) The predicted mechanism for the reversal of cell survival, angiogenesis, and inhibition of apoptosis. (B) Proposed molecular network of interaction as response to treatment. Data supported the activity of 4A3- and 4A4-induced apoptosis, resulting from the inhibition of AKT and antiapoptotic proteins (Mcl-1 and Bcl-2) as well as the VEGFA known for angiogenesis. This implies a possible role by PTEN tumor suppressor against PI3K-AKT pathway. In cervical cancer, PTEN is negatively regulated by miR-21 the expression of which is dependent on HPV E6 oncoprotein for the activation of cell survival, tumorigenesis, angiogenesis, and diminished apoptosis. The data in green and gray shadings indicate the level of expression of genes due to treatment. Data determined by RT-qPCR. Abbreviations: HPV, human papillomavirus; PI-3K, phosphatidylinositol-3-OH kinase; PTEN, phosphatase and tensin homolog; RT-qPCR, reverse transcriptase quantitative PCR.