Exploring the Cytotoxic Effects of the Extracts and Bioactive Triterpenoids from Dillenia indica against Oral Squamous Cell Carcinoma: A Scientific Interpretation and Validation of Indigenous Knowledge

Abstract

Triterpenoids are ubiquitously distributed secondary metabolites, primarily scrutinized as a source of medication and preventive measures for various chronic diseases. The ease of isolation and excellent pharmacological properties of triterpenoids are notable reasons behind the exponential rise of extensive research on the bioactive triterpenoids over the past few decades. Herein, we attempted to explore the anticancer potential of the fruit extract of the ethnomedicinal plant Dillenia indica against oral squamous cell carcinoma (OSCC) and have exclusively attributed the efficacy of the extracts to the presence of two triterpenoids, namely, betulinic acid (BA) and koetjapic acid (KA). Preliminary in vitro screening of both BA and KA unveiled that the entities could impart cytotoxicity and induce apoptosis in OSCC cell lines, which were further well-supported by virtual screening based on ligand binding affinity and molecular dynamic simulations. Additionally, the aforementioned metabolites could significantly modulate the critical players such as Akt/mTOR, NF-κB, and JAK/STAT3 signaling pathways involved in the regulation of important hallmarks of cancer like cell survival, proliferation, invasion, angiogenesis, and metastasis. The present findings provide insight and immense scientific support and integrity to a piece of indigenous knowledge. However, in vivo validation is a requisite for moving to clinical trials and developing it as a commercial drug.

Scheme 1. Triterpenoids from DI Promotes Apoptosis and Inhibits Metastasis

Figure 1

Chemical structures of bioactive molecules (A) BA and (B) KA isolated from DI-ME Ext.

Figure 2

2D interaction diagrams of (A) BA and (B) KA.

Figure 3

RMSD plots of (A) 4JSP–BA and (B) 4JSP–KA.

Figure 4

P–L interaction histogram of (A) 4JSP–BA and (B) 4JSP–KA

Figure 5

Inhibition of clonogenic potential of SAS cells by (A) DI-H₂O Ext (B) DI-ME Ext (C) BA (D) KA. Untreated cells were kept as the control. Quantification of the number of colonies was done with the help of ImageJ software. Results presented are mean ± SD of three independent experiments; *, p < 0.05 vs control.

Figure 6

(1) Induction of cell death in SAS cells by (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA. Cells were treated with the indicated concentrations for 72 h, followed by PI staining and FACS analysis for the cell death profile. (2) Live and dead assay was performed to evaluate the cytotoxic effect of the indicated concentrations of (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA on SAS cells.

Figure 7

Induction of apoptosis in SAS cells upon treatment with (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA. Cells were treated with the indicated concentrations for 72 h, followed by Annexin V staining and FACS analysis.

Figure 8

Induction of cell cycle arrest in SAS cells by (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA. Percentages of each cell cycle phase were obtained using FCS Express software.

Figure 9

Inhibition of the migration of SAS cells upon treatment with (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA. Cells were scratch-wounded and then treated with the indicated concentrations, followed by the recording of wound areas at different timepoints.

Figure 10

Expression of various proteins in SAS cells upon treatment with (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA as examined by Western blot analysis. GADPH was used as housekeeping control.

Figure 11

Pathway deciphering the proposed mechanistic mode of action with (A) DI-H₂O Ext, (B) DI-ME Ext, (C) BA, and (D) KA treatment in SAS cells.