Deciphering the effects of bixin on pulmonary alveolar adenocarcinoma migration and proliferation via targeting BAX/BCL-2 and Cyclin D1

Abstract

There is a tremendous upsurge in lung cancer incidences due to changing lifestyles and other environmental risk factors. Unfortunately, the use of clinical therapeutics is causing serious side effects and drug-resistant tumors. Taking account of the severity of lung cancer malignancy and the pressing need for natural therapeutics, we investigated the anticancer potential of bixin in A549, pulmonary alveolar adenocarcinoma cell lines meticulously for the first time. Bixin is an apocarotenoid present in the seed arils of Bixa orellana known for its remarkable coloring utilities and high medicinal value. Here, we identified the cytotoxic and anti-migratory nature of bixin through MTT and scratch assay. Bixin also induced characteristic apoptotic morphological changes in cells which were distinguished through 4',6-diamidino-2-phenylindole (DAPI), and Acridine orange/Ethidium bromide (AO/EB) labeling. Bixin induced the mitochondrion-associated intrinsic apoptosis in A549 cells as evidenced in mitochondrial membrane potential assay, apoptosis assay, cell cycle analysis, and caspase assays. The relative gene expression studies proved that the bixin upregulated BAX, and downregulated BCL-2 and Cyclin D1. The in-silico analyses, molecular docking and molecular dynamics simulation underlined the interaction features of bixin and targeted proteins.

Keywords: A549; Apoptosis; BAX; BCL-2; Bixin; Cyclin D1; Lung cancer.

Fig. 1

Bixin inhibited the proliferation of A549 lung cancer cell line and HEK-293 cell lines. (A) Microscopic images of A549 treated with gradient concentration of bixin (0–1200 µM) at 24 h. Graph showing the MTT results of A549 treated with gradient concentrations of bixin (0–1200 µM) at 24 h. (B) Microscopic images of A549 treated with gradient concentration of bixin (0–1200 µM) at 48 h. Graph showing the MTT results of A549 results treated with gradient concentration of bixin (0–1200 µM) at 48 h. (C) Microscopic images of HEK-293 treated with gradient concentration of bixin (0–1200 µM) at 24 h. Graph showing the MTT results of HEK-293 treated with gradient concentration of bixin (0–1200 µM) at 24 h. Scale for all the images is 100 µm. Data are presented as mean ± SD, n = 3, ***p < 0.001 compared to control.

Fig. 2

(A) Effect of bixin on cell migration of A549 at different concentrations of 400 μM, 800 μM and 1200 μM. Scale bar is 100 µm. (B) Wound closure percentage in the time period of 48 h. Data are presented as mean ± SD, n = 3, *p ≤ 0.05, **p < 0.01, ***p < 0.001 compared to control.

Fig. 3

Effect of bixin on A549 at different concentrations of 400 μM, 800 μM and 1200 μM. (A) Fluorescent images representing apoptotic morphology evaluated by DAPI. Scale for images is 75 µm. (B) Relative fluorescence indicating the reduction in the number of cells at different concentration. Data are presented as mean ± SD, n = 3, **p ≤ 0.01, ***p < 0.001 compared to control. (C) Fluorescent images representing apoptotic morphology evaluated by Acridine Orange-Ethidium bromide staining. Scale for images is 75 µm.

Fig. 4

(A) Images representing decrease in fluorescence as indicative of reduction in MMP evaluated by Rhodamine 123 at 488/535 nm. Scale for images is 75 µm. (B) Relative fluorescence indicating the reduction of MMP. Data are presented as mean ± SD, n = 3, **p ≤ 0.01, ***p < 0.001 compared to control. (C) Effect of bixin on mitochondrial membrane potential of A549 at different concentrations of 400 μM, 800 μM and 1200 μM analyzed through flow cytometry using Rhodamine 123. The reduction in the percentage of fluorescence emitted by the cells is an indicative of decrease in mitochondrial membrane potential as seen in apoptotic conditions. (D) Images representing decrease in fluorescence as indicative of reduction in ROS evaluated by DCFH-DA. Scale for images is 75 µm. (E) Relative fluorescence indicating the reduction in the ROS. Data are presented as mean ± SD, n = 3, **p ≤ 0.01, ***p < 0.001 compared to control.

Fig. 5

(A) The Effect of bixin on apoptosis of A549 lung cancer cell line at different concentrations of 400 μM, 800 μM and 1200 μM analyzed through flow cytometry using annexin V-FITC/ propidium iodide. (B) Impact of bixin on cell cycle progression of A549 lung cancer cell line at different concentrations of 400 μM, 800 μM and 1200 μM analyzed through flow cytometry.

Fig. 6

(A) Bixin enhanced the activity of caspase 3/7 and Caspase 9 of A549 lung cancer cell line at different concentrations of 400 μM, 800 μM and 1200 μM. (B) Effect of bixin on expression of B. BCL-2 (C) BAX and (D) Cyclin D1/CCND1 of A549 lung cancer cell line at different concentrations of 400 μM, 800 μM and 1200 μM analyzed through RT-PCR. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 compared to control. (E) Gel images showing the expression of genes in control and varying concentrations of bixin. Lane 1, lane 2, lane 3 and lane 4 are control, 400 μM, 800 μM and 1200 μM respectively.

Fig. 7

The docking poses and the 2D images of the interaction of bixin with (A) BCL-2, (B) BAX and (C) Cyclin D1.

Fig. 8

The RMSD profiling of the interaction of bixin with (A) BCL-2, (C) BAX and (E) Cyclin D1. The RMSF analysis of interaction of bixin with (B) BCL-2, (D) BAX and (F) Cyclin D1.