1, 6-O, O-Diacetylbritannilactone from Inula britannica Induces Anti-Tumor Effect on Oral Squamous Cell Carcinoma via miR-1247-3p/LXRα/ABCA1 Signaling

Abstract

Introduction: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy affecting the oral cavity and is associated with severe morbidity and high mortality. 1, 6-O, O-Diacetylbritannilactone (OODBL) isolated from the medicinal herb of Inula britannica has various biological activities such as anti-inflammation and anti-cancer. However, the effect of OODBL on OSCC progression remains unclear. Here, we were interested in the function of OODBL in the development of OSCC.

Methods: The effect of OODBL on OSCC progression was analyzed by MTT assays, colony formation assays, transwell assays, apoptosis analysis, cell cycle analysis, and in vivo tumorigenicity analysis. The mechanism investigation was performed by qPCR assays, Western blot analysis, and luciferase reporter gene assays.

Results: We found that OODBL inhibits the proliferation of OSCC cells in vitro. Moreover, the migration and invasion were attenuated by OODBL treatment in the OSCC cells. OODBL arrested cells at the G0/G1 phase and induced cell apoptosis. OODBL was able to up-regulate the expression of LXRα, ABCA1, and ABCG1 in the system. In addition, OODBL activated LXRα/ABCA1 signaling by targeting miR-1247-3p. Furthermore, the expression levels of cytochrome c in the cytoplasm, cleaved caspase-9, and cleaved caspase-3 were dose-dependently reduced by OODBL. Besides, OODBL increased the expression ratio of Bax to Bcl-2. Moreover, OODBL repressed tumor growth of OSCC cells in vivo.

Discussion: Thus, we conclude that OODBL inhibits OSCC progression by modulating miR-1247-3p/LXRα/ABCA1 signaling. Our finding provides new insights into the mechanism by which OODBL exerts potent anti-tumor activity against OSCC. OODBL may be a potential anti-tumor candidate, providing a novel clinical treatment strategy of OSCC.

Keywords: LXRα/ABCA1 signaling; OODBL; OSCC; anti-tumor; miR-1247-3p.

Figure 1

The structural formula of OODBL.

Figure 2

OODBL inhibits the proliferation of OSCC cells. (A and B) The effect of OODBL on cell viability was measured by MTT assays in the CAL27 and SCC15 cells treated with OODBL of indicated dose, respectively. (C and D) The effect of OODBL on cell proliferation was analyzed by colony formation assays in the CAL27 and SCC15 cells treated with OODBL of indicated dose, respectively.

Figure 3

OODBL impairs the migration and invasion of OSCC cells. (A) The effect of OODBL on cell migration was tested by Transwell assays in the CAL27 and SCC15 cells treated with OODBL of indicated dose. (B) The effect of OODBL on cell invasion was assessed by Transwell assays in the CAL27 and SCC15 cells treated with OODBL of indicated dose.

Figure 4

OODBL modulates cell cycle and apoptosis in OSCC cells. (A) The effect of OODBL on cell cycle was measured by flow cytometry in the CAL27 and SCC15 cells treated with OODBL of indicated dose. The percentages of cells in the G1, S, and G2/M phases were calculated using the Multi-cycle software, respectively. (B) The effect of OODBL on cell apoptosis was analyzed by flow cytometry in the CAL27 and SCC15 cells treated with OODBL of indicated dose.

Figure 5

OODBL activates LXRα/ABCA1 signaling. (A) The expression levels of LXRα, ABCA1, and ABCG1 were evaluated by Western blot analysis in the CAL27 cells treated with OODBL of indicated dose, in which β-actin was used as an equal loading control. (B) The results of Western blot analysis were quantified by ImageJ software.

Figure 6

OODBL activates LXRα/ABCA1 signaling by targeting miR-1247-3p. (A) The putative targeting sites for LXRα and miR-1247-3p were shown. (B) The luciferase activity of wild type LXRα (LXRα-WT) and LXRα with miR-1247-3p binding site mutant (LXRα-Mut) were analyzed by luciferase reporter gene assays in cells treated with control miRNA (miR-NC) or miR-1247-3p mimic. (C) The intuitive expression of mir-1247-3p was analyzed by qPCR assays in the cells. (D) The protein expression levels of LXRα were measured by Western blot analysis in the CAL27 cells treated with miR-1247-3p mimic or miR-1247-3p inhibitor. The results were quantified by ImageJ software. (E) The mRNA expression levels of LXRα were assessed by RT-qPCR in the CAL27 cells treated with miR-1247-3p mimic or miR-1247-3p inhibitor. (F) The expression levels of miR-1247-3p was tested by qPCR in the CAL27 cells treated with OODBL of indicated dose.

Figure 7

OODBL affects cytochrome C and caspases activities in OSCC cells. (A) The expression levels of cytochrome c in the cytoplasm, cleaved caspase-9 (c-caspase-9), and cleaved caspase-3 (c-caspase-3) were analyzed by Western blot analysis in the CAL27 cells treated with OODBL of indicated dose, in which β-actin was used as an equal loading control. The results were quantified by ImageJ software. (B) The expression levels of Bcl-2 and Bax were measured by Western blot analysis in the CAL27 cells treated with OODBL of indicated dose, in which β-actin was used as an equal loading control. The results were quantified by ImageJ software.

Figure 8

OODBL represses tumor growth of OSCC cells in vivo. (A–E) The effect of OODBL in tumor growth of OSCC cells in vivo was analyzed by nude mice tumorigenicity assay. (A) Images of dissected tumors from nude mice of the model group and OODBL treatment group (0.1mL/10g). (B) The average tumor weight was calculated are shown. (C) The average tumor volume was calculated are shown. (D) The expression levels of Ki-67 of the tumor tissues were measured by immunohistochemical staining. (E) The protein expression levels of LXRα, ABCA1, and ABCG1 were examined by Western blot analysis in the tumor tissues, in which β-actin was used as an equal loading control. The results were quantified by ImageJ software.