A Naturally Derived Glycosylated Oleanolic Acid Derivative Suppresses NF-κB Translocation and Induces Intrinsic Apoptosis in Lung Adenocarcinoma Cells

Abstract

This study investigates the molecular mechanism of action of F4B1, a novel oleanolic acid derivative in human lung adenocarcinoma cells. F4B1 is a naturally occurring oleanolic acid derivative that was isolated and purified from the leaves of Sesbania grandiflora. Structural analyses were carried out using 1D and 2D NMR, FT-IR, and mass spectrometric analyses. MTT assay was employed to evaluate antiproliferative effects in A549 (a model for lung cancer), MCF-7 (a model for breast cancer), HEP-2 (a model for head and neck cancer) and in MRC-5 (human lung fibroblast cells). Fluorescence staining, scanning electron microscopy, and flow cytometry were employed to study apoptosis and cell cycle. Western blotting, RT-PCR, and immunofluorescence techniques were followed to study the mechanisms of cell death. Schrodinger software was employed for docking studies. While preliminary screening was conducted in multiple cell lines, A549 cells were chosen for further mechanistic exploration based on their higher sensitivity to F4B1 treatment. F4B1 blocks the proliferation and causes intrinsic mode of cell death in lung adenocarcinoma cells. In particular, the above anticancer effect was mediated through a mechanism that is associated with the inactivation of NF-kappa B signaling and suppression of cyclin D1 expression leading to cell cycle arrest at the G1/S phase. Our study confirms that F4B1 induces apoptosis, as confirmed through Annexin V staining results. It is intrinsic apoptosis, as evidenced by upregulation of pro-apoptotic markers (BAX), downregulation of anti-apoptotic markers (BCL-2), cytochrome C release, activation of caspase-9, and caspase-3. These results establish the involvement of the mitochondrial-mediated apoptotic pathway. The pathway also involved the suppression of the proto-oncogene c-Myc both at the transcriptional and translational levels. Docking studies show that F4B1 has a high affinity binding towards CXCR4 and SRC kinase. Our findings specifically contribute to understanding the mechanism of F4B1, the isolated molecule from S. grandiflora, as an anticancer drug candidate and will hopefully pave the way toward further studies.