Bergenin inhibits growth of human cervical cancer cells by decreasing Galectin-3 and MMP-9 expression

Abstract

Cervical cancer is still the leading cause of cancer mortality worldwide even after introduction of vaccine against Human papillomavirus (HPV), due to low vaccine coverage, especially in the developing world. Cervical cancer is primarily treated by Chemo/Radiotherapy, depending on the disease stage, with Carboplatin/Cisplatin-based drug regime. These drugs being non-specific, target rapidly dividing cells, including normal cells, so safer options are needed for lower off-target toxicity. Natural products offer an attractive option compared to synthetic drugs due to their well-established safety profile and capacity to target multiple oncogenic hallmarks of cancer like inflammation, angiogenesis, etc. In the current study, we investigated the effect of Bergenin (C-glycoside of 4-O-methylgallic acid), a natural polyphenol compound that is isolated from medicinal plants such as Bergenia crassifolia, Caesalpinia digyna, and Flueggea leucopyrus. Bergenin has been shown to have anti-inflammatory, anti-ulcerogenic, and wound healing properties but its anticancer potential has been realized only recently. We performed a proteomic analysis of cervical carcinoma cells treated with bergenin and found it to influence multiple hallmarks of cancers, including apoptosis, angiogenesis, and tumor suppressor proteins. It was also involved in many different cellular processes unrelated to cancer, as shown by our proteomic analysis. Further analysis showed bergenin to be a potent-angiogenic agent by reducing key angiogenic proteins like Galectin 3 and MMP-9 (Matrix Metalloprotease 9) in cervical carcinoma cells. Further understanding of this interaction was carried out using molecular docking analysis, which indicated MMP-9 has more affinity for bergenin as compared to Galectin-3. Cumulatively, our data provide novel insight into the anti-angiogenic mechanism of bergenin in cervical carcinoma cells by modulation of multiple angiogenic proteins like Galectin-3 and MMP-9 which warrant its further development as an anticancer agent in cervical cancer.

Keywords: Bergenin; Cervical cancer; Galectin 3; HPV; Matrix metallo protease 9.

Figure 1

Bergenin reduces viability in cervical carcinoma cells (A) Chemical structure of Bergenin. (B) Cell viability was assessed in SiHa cells after treatment with varying concentrations of bergenin for (i) 24 h and (ii) 48 h through MTT assay. (C) Morphological changes in SiHa cells after treatment with bergenin (100 µM and 200 µM) for 24 h. (D) Representative dot plots of Annexin-V/7AAD of SiHa cells before and after bergenin(100 µM and 200 µM) treatment for 48 h.

Figure 2

Bergenin causes upregulation and downregulation of distinct set of proteins in cervical cancer cells. (A) Pictorial representation of dysregulated proteins after Label-free quantification (LFQ) of the SiHa cells before and after 100 μM of bergenin treatment for 48 h. (B) The heat map shows the dysregulated proteins with ≥  ± 1.5-fold change. The pink color represents the upregulation, whereas the blue represents the downregulation. (C) Graphical representation of the cellular compartmentalization of the dysregulated proteins (i) Downregulated. (ii) Upregulated.

Figure 3

Bergenin causes the alteration in different pathways in cervical cancer cells. Representative distribution of (A) downregulated pathways with proteins involved in different ways using Cytoscape software. (B) Upregulated pathways with proteins involved in different ways using Cytoscape software. (C) Distribution of downregulated proteins in various biological networks using CLUEgo. (D) Distribution of upregulated proteins in various biological networks using CLUEgo.

Figure 4

Bergenin influences migration and angiogenesis in cervical cancer cells. (A) Scratch assay was performed in SiHa cells with or without treatment of bergenin(100 µM and 200 µM) for 24 h. Pictures were taken at the start of the experiment (0 h) and 24 h later. Images of representative experiments are shown, and the means ± SD are illustrated as graphical representation. (B) Wound scratch assay was performed in C33A cells with or without treatment of bergenin (100 µM and 200 µM) for 24 h. Pictures were taken at the start of the experiment (0 h) and 24 h later. (C) Representative images from Transwell migration assay upon treatment of bergenin (100 μM and 200 μM) for 48 h in SiHa cells and means ± SD are illustrated as graphical representation. (D) Western blot of indicated markers of EMT and pathways after 100 μM and 200 μM bergenin treatment for 48 h in SiHa and C33A cell lines. The blots were cut before being subjected to hybridization with antibodies in the blotting procedure, and the original blots are provided in the supplementary information.

Figure 5

Molecular docking and molecular dynamic simulation of galectin-3 and bergenin complex. (A) The surface representation of galectin-3 and the docking/binding pocket of bergenin (ball-and-stick) are shown in the mesh. (B) Cartoon representation of bergenin binding site in galectin-3. (C) H-bonded interactions formed by docked bergenin compound with galectin-3. (D) The ligplot shows the hydrophobic and H-bonded interactions formed by bergenin. (E) Superimposition of galectin-3-bergenin complex at 0 and 100 ns of MD simulations. (F) RMSD plot of galectin-3-bergenin complex 100 ns MD simulations.

Figure 6

Molecular docking and molecular dynamic simulations of MMP-9 and bergenin complex. (A) The surface representation of MMP-9, along with the docking/binding pocket of bergenin (ball-and-stick) shown in the mesh. (B) Cartoon representation of bergenin binding site in MMP-9. (C) H-bonded interactions formed by docked bergenin compound with MMP-9. (D) The ligplot shows the hydrophobic and H-bonded interactions formed by bergenin (after docking). (E) Superimposition of MMP-9-bergenin complex at 0 and 100 ns of MD simulations. and (F) H-bond occupancy of the MMP-9-bergenin complex throughout 100 ns MD simulations.