Butylidenephthalide Abrogates the Snail-Induced Cancer Stemness in Oral Carcinomas

Abstract

Oral cancer is one of the most common cancers worldwide, especially in South Central Asia. It has been suggested that cancer stem cells (CSC) play crucial roles in tumor relapse and metastasis, and approaches to target CSC may lead to promising results. Here, aldehyde dehydrogenase 1 (ALDH1) and CD44 were utilized to isolate CSCs of oral cancer. Butylidenephthalide, a bioactive phthalide compound from Angelica sinensis, was tested for its anti-CSC effects. MTT assay showed that a lower concentration of butylidenephthalide was sufficient to inhibit the proliferation of patient-derived ALDH1⁺/CD44⁺ cells without affecting normal cells. Administration of butylidenephthalide not only reduced ALDH1 activity and CD44 expression, it also suppressed the migration, invasion, and colony formation abilities of ALDH1⁺/CD44⁺ cells using a transwell system and clonogenic assay. A patient-derived xenograft mouse model supported our in vitro findings that butylidenephthalide possessed the capacity to retard tumor development. We found that butylidenephthalide dose-dependently downregulated the gene and protein expression of Sox2 and Snail. Our results demonstrated that overexpression of Snail in ALDH1^-/CD44^- (non-CSCs) cells induced the CSC phenotypes, whereas butylidenephthalide treatment successfully diminished the enhanced self-renewal and propagating properties. In summary, this study showed that butylidenephthalide may serve as an adjunctive for oral cancer therapy.

Keywords: ALDH1; CD44; Snail; butylidenephthalide; cancer stem cells; oral cancer.

Figure 1

The cytotoxic effect of butylidenephthalide on the viability of normal human oral keratinocyte (NHOK) and patient-derived ALDH1⁺/CD44⁺ cells. (a) Chemical structures of butylidenephthalide; MTT assay was utilized to examine the cell viability of (b) NHOK and two patient-derived ALDH1⁺/CD44⁺ cells in response to butylidenephthalide at 12.5, 25, 50, 100, and 200 μg/mL. Results are presented as means ± SD of three independent experiments; *** p < 0.001 compared to NHOK. Butylidenephthalide (BP).

Figure 2

Butylidenephthalide suppresses the expression of CSC markers in two patient-derived ALDH1⁺/CD44⁺ cells. (a) Flow cytometry analyses of two patient-derived ALDH1⁺/CD44⁺ cells treated with various concentrations of butylidenephthalide for 48 h followed by incubation with ALDEFLUOR kit (to measure an ALDH1 enzymatic activity) in the presence of 4-diethylaminobenzaldehyde (DEAB, a specific ALDH1 inhibitor) as a control or (b) anti-CD44 antibody. Results are presented as means ± SD of three independent experiments; * p < 0.05 compared to Ctrl (control group containing 0.1% DMSO without butylidenephthalide). Butylidenephthalide (BP).

Figure 3

Butylidenephthalide eliminates the phenotypes of CSCs. The transwell culture system was employed to assess the (a) migration and (b) invasion capacities of two patient-derived ALDH1⁺/CD44⁺ cells in response to butylidenephthalide treatment. (c) Colony forming ability of ALDH1⁺/CD44⁺ cells was examined by clonogenic assay. Results are presented as means ± SD of three independent experiments; * p < 0.05 compared to Ctrl (control group containing 0.1% DMSO without butylidenephthalide). Butylidenephthalide (BP). Scale bar indicate 200 μm.

Figure 4

Butylidenephthalide inhibits in vivo tumorigenicity in the ALDH1⁺/CD44⁺-1-transplanted immunocompromised mice. After implantation of ALDH1⁺/CD44⁺ cells subcutaneously, BALB/c nude mice receiving various concentrations of butylidenephthalide were analyzed for the bioluminescence signal using IVIS imaging system. (a) The representative image and (b) quantitative analysis of signal intensity at day 22 are shown; (c) tumor volume; (d) body weight. The emitted signal by the implanted cells was monitored for 22 days (each group, n = 5). Results are means ± SD; * p < 0.05 compared to Ctrl (control group; administration with normal saline without butylidenephthalide). Butylidenephthalide (BP).

Figure 5

Administration of butylidenephthalide represses the expression of Sox2 and Snail. (a) Gene and (b) protein expression levels of stemness marker Sox2 and EMT marker Snail were both downregulated in the ALDH1⁺/CD44⁺ cells following treatment at various concentrations of butylidenephthalide. (c) The relative level of indicated protein expression was measured by densitometer and normalized against GAPDH. The control was set as 100%. Optical density values represent the mean ± SD of three independent experiments; * p < 0.05 compared to Ctrl (control group containing 0.1% DMSO without butylidenephthalide).

Figure 6

Butylidenephthalide diminishes numerous characteristics of CSCs through downregulation of Snail. (a) Overexpression of Snail in ALDH1⁻/CD44⁻ cells followed by treatment with or without butylidenephthalide. The enhanced (b) self-renewal, (c) migration, and (d) invasion abilities in the Snail-overexpressing ALDH1⁻/CD44⁻ cells were all inhibited after butylidenephthalide treatment. Data are shown as the mean ± SD of three independent experiments; * p < 0.05 compared to Ctrl (control group; transfection with control vector); # p < 0.05 compared to the Snail-overexpressing ALDH1⁻/CD44⁻ cells without-treatment group. Butylidenephthalide (BP). Scale bar indicate 200 μm.

Figure 7

The graphic abstract of the present study illustrates the mechanism of butylidenephthalide to eliminate oral CSCs by suppressing the Snail-induced cancer stemness properties. The administration of butylidenephthalide suppressed the Snail expression, resulting in the downregulation of SOX2 expression, thereby abolishing the stemness markers’ expression, self-renewal, proliferation, migration, and invasion of oral CSCs. Red, solid line with vertical head indicates the inhibiting effect; gray, dotted line with arrowhead indicates the weakened increasing effect; red arrowhead indicates the downregulation.