Cytotoxic Effects of Thymus serpyllum L. and Mentha × piperita L. Essential Oils on Basal Cell Carcinoma-An In Vitro Study

Abstract

This study investigated the potential of Thymus serpyllum L. and Mentha × piperita L. essential oils (EOs), known for their bioactive properties, as adjunctive treatments targeting Basal cell carcinoma cancer stem cells (BCC CSCs). Primary cultures were established from ten BCC tumor samples and their distant resection margins as controls. The chemical composition of the EOs was analyzed by gas chromatography-mass spectroscopy (GC-MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The biological effects were evaluated via colony and spheroid formation, scratch assays, MTT and neutral red cytotoxicity assays, and qRT-PCR for Hh (SHH, PTCH1, SMO, and GLI1) and Notch (Notch1 and JAG1) gene expression. GC analysis identified thymol, p-cymene, and linalool as the main components of the EO of T. serpyllum L., and menthone and menthol in the EO of M. × piperita L. IC50 values were 262 µg/mL for T. serpyllum L. and 556 µg/mL for M. × piperita L. and were applied in all experiments. Both EOs significantly reduced CSC clonogenicity and migration (p < 0.05). The EO of T. serpyllum L. downregulated SMO and GLI1, while the EO of M. × piperita L. upregulated PTCH1, Notch1, and JAG1 (p < 0.05). These findings suggest that both EOs exhibit anticancer effects in BCC CSCs by modulating key oncogenic pathways, supporting their potential in BCC therapy.

Keywords: Mentha × piperita L.; Thymus serpyllum L.; basal cell carcinoma; cancer stem cells; essential oil.

Figure 1

GC-FID chromatograms of (A) T. serpyllum L. and (B) M. × piperita L. essential oils. In T. serpyllum EO, the predominant compound was thymol (50.47%), followed by p-cymene (23.56%), linalool (5.84%), 1,8-cineole (4.98%), carvacrol (3.18%), camphene (2.33%), and caryophyllene oxide (2.28%). In M. × piperita EO, the main constituents were menthone (53.66%), menthol (13.52%), iso-menthone (6.64%), menthofuran (6.53%), and 1,8-cineole (6.13%).

Figure 2

Average FT-IR spectrum of (A) T. serpyllum L. and (B) M. × piperita L. essential oils.

Figure 3

Dose- and time-dependent inhibition of BCC stem cells viability exerted by essential oils of T. serpyllum L. (A) and M. × piperita L. (B). Cytotoxicity was determined by the MTT (A,B) and Neutral Red (C,D) assays. The results are expressed as the mean of triplicate (±SD).

Figure 4

Colony forming, Spheroid formation, and Scratch wound healing assay with quantitative analysis (representative images at higher magnification 100×, scale bar: 200 µm). Treated and untreated CSCs showed higher clonogenic ability (A) and the ability to form tumorsphere (B) compared to margin cells. There was a significantly decreased capacity of tumor cells to form a colony (A) and spheres (B) after treatment with T. serpyllum L. and M. × piperita L. Representative images of migratory potential. There is a statistically significant difference in cell speed between tumor cells (CSCs and CSCs treated with T. serpyllum L. and M. × piperita L.) and margin cells (C). In the quantitative analysis, error bars represent standard deviation calculated from experiments, while asterisks * and ** designate p-values lower than 0.05 and 0.01, respectively.

Figure 5

The gene expression analysis of the Sonic Hedgehog and Notch signaling pathway in BCC CSCs after treatment with T. serpyllum L. and M. × piperita L. The mRNA levels of Sonic Hedgehog markers SHH, SMO, and GLI1 were significantly higher in tumor cells compared to the healthy control (A,C,D). There was a significant decrease in SMO and GLI1 levels after treatment with T. serpyllum L. compared to untreated cells (C,D). PTCH1 was significantly lower in tumor cells compared to the healthy control and there was a significant increase in the expression of this gene after the treatment with M. × piperita L. (B). The level of Notch 1 and JAG1 genes was lower in CSCs compared to margin cells (E,F). After treatment with M. × piperita L. there was a significant increase in the expression of these genes, while after treatment with T. serpyllum L., there was no difference (E,F). The error bars represent the standard error calculated from experiments. Asterisks * and ** designate p-values lower than 0.05 and 0.01, respectively. Abbreviation: SHH—Sonic Hedgehog Signaling Molecule; PTCH1—Patched 1 Receptor; SMO—Smoothened, Frizzled Class G protein-coupled Receptor; GLI1—glioma-associated oncogene 1.