Antitumor effect of Melaleuca alternifolia essential oil and its main component terpinen-4-ol in combination with target therapy in melanoma models

Abstract

Essential oils (EOs) have been recently emerging for their promising biological activities in preventing tumorigenesis or progression of different tumor histotypes, including melanoma. In this study, we investigated the antitumor activity of a panel of EOs in different tumor models. The ability of Melaleuca alternifolia (tea tree oil) and its main component, terpinen-4-ol, to sensitize the target therapy currently used for melanoma treatment was also assessed. Our results demonstrated that EOs differently affect the viability of human cancer cells and led us to select six EOs effective in melanoma and lung cancer cells, without toxic effects in human fibroblasts. When combined with dabrafenib and/or trametinib, Melaleuca alternifolia synergistically reduced the viability of melanoma cells by activating apoptosis. Through machine learning classification modeling, α-terpineol, tepinolene, and terpinen-4-ol, three components of Melaleuca alternifolia, were identified as the most likely relevant components responsible for the EO's antitumor effect. Among them, terpinen-4-ol was recognized as the Melaleuca alternifolia component responsible for its antitumor and proapoptotic activity. Overall, our study holds promise for further analysis of EOs as new anticancer agents and supports the rationale for their use to improve target therapy response in melanoma.

Fig. 1. M14 cells are differentially sensitive to a panel of EOs.

a Analysis of cell proliferation/viability by MTT assay of M14 cells treated with 61 essential oils (EOs, EO01-EO61, 50 μg/ml, 72 h). p-values were calculated between control (Ctrl) and EO-treated cells. *p < 0.05; **p < 0.01; ^#p < 0.001 after applying Student’s t test. Dotted columns represent the six EOs further investigated in this study. b MTT assay of M14 cells treated with the indicated EOs (10–50 μg/ml, 24–72 h). a, b Results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells × 100” and represent the average±standard deviation of at least three independent experiments. c Quantification of 50% inhibition of cell proliferation/viability (IC₅₀) of the indicated EOs calculated for M14 cells treated as reported in b. The median of IC₅₀ is shown. ***p < 0.001; ****p < 0.0001 after applying one-way ANOVA test.

Fig. 2. Six selected EOs affect melanoma cell proliferation/viability.

a–f Analysis of cell viability by MTT assay of six melanoma cell lines treated with the indicated EOs (10–35 μg/ml, 48 h). The results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells (Ctrl) × 100” and represent the average±standard deviation of at least three independent experiments. p-values were calculated between control and EOs treated cells. *p < 0.05; **p < 0.01 after applying Student’s t test.

Fig. 3. EO05 sensitizes M14 melanoma cells to dabrafenib treatment.

a Analysis of cell proliferation/viability by MTT assay (left) and relative isobologram (right) of M14 cells after treatment with dabrafenib (DAB) or EO05 alone or 24 h EO05 followed by 48 h dabrafenib (EO05-> DAB). The results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells (Ctrl) × 100”. b Quantification and c representative images of subG1 peak by propidium iodide staining of M14 cells treated with DAB (48 h, 0.2 μM), EO05 (24 h, 20 μg/ml) or with 24 h EO05 followed by 48 h dabrafenib (EO05- > DAB) in the presence or absence of zVAD (50 μM). The percentage of cells in the subG1 peak is reported. a, b The results represent the average±standard deviation of three independent experiments. Experiments with zVAD were repeated twice. b p-values were calculated between cells treated with combination and cells treated with single drugs, or between cells treated or not treated with zVAD. *p < 0.05; **p < 0.01 after applying Student’s t test. d Flow cytometric analysis of active caspase 3-PE staining in cells treated with DAB (48 h, 0.2 μM), EO05 (24 h, 20 μg/ml), or with 24 h EO05 followed by 48 h dabrafenib (EO05- > DAB). e Western Blot analysis of PARP cleavage in M14 cells treated as reported in d. HSP72/73 was used as loading and transferring control. Western blot representative of two blots with similar results is shown.

Fig. 4. EO05 sensitizes M14 melanoma cells to trametinib treatment.

a Analysis of cell proliferation/viability by MTT assay (left) and relative isobologram (right) of M14 cells treated with 48 h trametinib (TRAM) or 24 h EO05 alone or 24 h EO05 followed by 48 h trametinib (EO05- > TRAM). b Quantification and c representative images relative of subG1 peak by propidium iodide staining of M14 cells control (Ctrl) or treated with TRAM (48 h, 10 nM), EO05 (24 h, 20 μg/ml) or with 24 h EO05 followed by 48 h TRAM (EO05- > TRAM), in the presence or absence of zVAD (50 μM). The percentage of cells in the subG1 peak is reported. d Flow cytometric analysis of active caspase 3-PE staining in cells treated with TRAM (48 h, 10 nM), EO05 (24 h, 20 μg/ml), or with 24 h EO05 followed by 48 h TRAM (EO05- > TRAM). e Western blot analysis of PARP cleavage in M14 cells treated as reported in d. HSP72/73 was used as loading and transferring control. Western blot representative of two blots with similar results is shown. f MTT assay of M14 cells treated with dabrafenib (0.001 μM)+trametinib (0.1 nM) for 48 h, EO05 (20 μg/ml) for 24 h alone or 24 h EO05 followed by 48 h DAB + TRAM (EO05- > DAB + TRAM). a, f The results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells × 100”. a, b, f The results represent the average ± standard deviation of three independent experiments. Experiments with zVAD were repeated twice. b, f p-values were calculated between cells treated in combination and cells treated with single drugs, or between cells treated or not treated with zVAD. *p < 0.05; **p < 0.01 after applying Student’s t test.

Fig. 5. Terpinen-4-ol is responsible for EO05 antitumor activity in M14 cells.

a MTT assay of M14 cells treated for 72 h with eucalyptol (7 μg/ml), γ-terpinene (6 μg/ml), α-terpineol (4 μg/ml), terpinen-4-ol (18.5 μg/ml) or EO05 (50 μg/ml). b MTT assay of M14 cells treated with the indicated concentrations of EO05 or of terpinen-4-ol. c, d MTT assay (left) and relative isobologram (right) of M14 cells treated with c dabrafenib (DAB), d trametinib (TRAM), or terpinen-4-ol alone or in combination (24 h terpinen-4-ol followed by 48 h DAB or TRAM). a–d The results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells × 100”. The results represent the average±standard deviation of at least three independent experiments. p-values were calculated between control (Ctrl) and treated cells or cells treated in combination and cells treated with single drugs. *p < 0.05; **p < 0.01, after applying Student’s t test.

Fig. 6. Terpinen-4-ol induces apoptosis in combination with targeted therapy.

a Quantification and b representative images relative of subG1 peak by propidium iodide staining of M14 cells treated with 48 h dabrafenib (DAB, 0.2 μM) or trametinib (TRAM, 10 nM), 24 h terpinen-4-ol (7.4 μg/ml) alone or in combination (24 h terpinen-4ol->48 h DAB/TRAM), in the presence or absence of zVAD (50 μM). c Flow cytometric analysis of active caspase 3-PE staining in cells treated with 48 h dabrafenib (0.2 μM) or trametinib (10 nM), 24 h terpinen-4-ol (7.4 μg/ml) alone or in combination (24 h terpinen-4ol->48 h DAB/TRAM). d Western blot analysis of PARP cleavage in M14 cells treated as reported in c. HSP72/73 was used as loading and transferring control. Western blot representative of two blots is shown. e MTT assay of M14 cells treated with dabrafenib (0.001 μM)+trametinib (0.1 nM) for 48 h, terpinen-4-ol (7.4 μg/ml) for 24 h alone or 24 h terpinen-4-ol followed by 48 h DAB + TRAM (terpinen-4-ol->DAB + TRAM). The results are reported as “cell proliferation-viability of treated cells/cell proliferation-viability of control cells × 100”. a, e The results represent the average±standard deviation of three independent experiments. Experiments with zVAD were repeated twice. p-values were calculated between control (Ctrl) and treated cells, cells treated in combination and cells treated with single drugs, or between cells treated or not treated with zVAD. *p < 0.05; **p < 0.01, ***p < 0.001 after applying Student’s t test.