Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo

Abstract

Background: Melanoma is one of the most invasive and aggressive types of cancer with a very poor prognosis. Surgery remains the most efficient treatment prior melanoma invasion and metastasis formation. However, therapy becomes a challenge once the cancer cells colonized other tissues. At present, there are two main classes of therapies acting with a certain efficiency on metastatic melanoma: immune check point inhibitors (anti-PD1/PDL1) and targeted therapy such as Vemurafenib. Unfortunately, these therapies are not fully responsive, induce resistance and/or generate unwanted side effects. In this respect, it is important to continue to discover new cancer therapeutics. Here, we show that daphnane diterpenes type of compounds can prevent melanoma metastasis by inhibiting metastasis-associated matrix metalloproteinases expression without cytotoxicity.

Methods: Evaluation of the anti-metastasis effect of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) and its bioactive component gnidilatidin was carried out in vitro using B16 murine melanoma cells and in vivo using male C57BL/6 J mice. Global gene expression in B16 cells was done using DNA microarray, validated using real-time PCR, to further understand the effect of daphnane diterpenes, specifically daphnane diterpenoid gnidilatidin.

Results: Oral administration of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) suppressed MMP2 and MMP9 expression, decreasing lung tumor in mice injected with B16 murine melanoma cells. Validation of these observations in vitro showed reduced B16 cells migration, adhesion, and invasion. Results of microarray analysis of B16 cells treated with daphnane diterpenoid gnidilatidin from TH revealed an upregulation of tumor suppressor Egr1 while inhibiting metastasis-associated genes Id2 and Sytl2 expression. A downregulation of the melanoma oncogene microphthalmia-associated transcription factor (Mitf) was observed, and most likely caused by the inhibition of Id2, a gene that regulated HLH transcription factors such as MITF and also reported to promote tumor cell migration and invasion.

Conclusions: Daphnane diterpenes have inhibitory effect on the metastatic potential of B16 melanoma cells, and the results of this study provided evidence for their potential for use in the prevention and inhibition of melanoma metastasis.

Keywords: Daphnane diterpenes; Gnidilatidin; Id2; MMPs; Melanoma; Mitf.

Fig. 1

Effect of daphnane diterpenes on mice lung colonization by B16F10 cells. a Photographs of lungs of mice injected without (−)B16F10 or with (+) B16F10, and treated with dacarbazine (DTIC) or 1/1000 v/v T. hirsuta extract (TH). Arrows point to the lung nodules or tumors; b Plot of the number of mice lungs nodules (tumor) in mice injected without (−)B16F10 or with (+) B16F10, and treated with dacarbazine (DTIC) or TH. **Statistically significant (P ≤ 0.01) difference between treated cells and control

Fig. 2

Effect of daphnane diterpenes on the expression of MMP2 and CD44 in vivo. The expression of (a) CD44 (b) and metalloproteinases MMP9 and MMP2 in mice injected without (−) B16F10 or with (+) B16F10, and treated with dacarbazine (DTIC) or 1/1000 v/v T. hirsuta extract (TH). Total proteins were then extracted and resolved by SDS-PAGE; the resolved proteins were then blotted onto a PVDF membrane. CD44, MMP2, and MMP9 were detected by immunoblotting with an anti-CD44, anti-MMP2, or anti-MMP9, polyclonal antibodies. The signals were visualized using LiCor Odyssey Infrared Imaging System after reaction with goat anti-mouse IRDye 680LT, or goat anti-rabbit IRDye 800CW (LI-COR). CD44, MMP2, and MMP9 levels were normalized to β-actin expression

Fig. 3

In vitro evaluation of the effect of daphnane diterpenes on B16F10 cells metastasis. a Cell proliferation evaluated using the MTT assay; b Viability of B16F10 cells treated with 1/000 v/v T. hirsuta extract (TH) evaluated using flow cytometry (Guava ViaCount assay); c Scratched cell monolayer images for the wound healing assay results (cell migration assay); d Wound healing assay results; e Cell invasion results obtained using the matrigel invasion assay; f Cell adhesion assay results evaluated by performing the MTT assay on B16 cells cultured in fibronectin-coated 96-well plates. Results represent the mean ± SD of triplicate samples. *Statistically significant (P ≤ 0.05) difference between treated cells and control. **Statistically significant (P ≤ 0.01) difference between treated cells and control

Fig. 4

Quantitative real-time PCR of cancer metastasis-associated genes expression in 0.1 μM gnidilatidin-treated B16F10 melanoma cell lines. Relative gene expression levels of (a) Early growth response 1 (Egr1), (b) Inhibitor of DNA binding (Id2), and (c) Synaptogamin-like 2 (Sytl2) genes. Results represent the mean ± SD of triplicate samples. *Statistically significant (P ≤ 0.05) difference between treated cells and control. **Statistically significant (P ≤ 0.01) difference between treated cells and control

Fig. 5

Effect of gnidilatidin (0.1 μM) on wound healing, cell adhesion, and Mitf expression in B16F10 melanoma cell lines. a Scratched cell monolayer images for the wound healing assay results (wound healing assay results; b Cell adhesion assay results evaluated by performing the MTT assay on B16 cells cultured in fibronectin-coated 96-well plates; c Relative gene expression of Mmp2, Mmp9, and Cd44 genes; d Relative gene expression of Mitf; e Relative gene expression of tyrosinase (Tyr), c tyrosinase-related protein 1 (Trp1), dopachrome tautomerase (Dct), and (f) Ras-related protein (Rab27a), determined using TaqMan real-time quantitative PCR; g Melanin content of B16 cells. Results represent the mean ± SD of triplicate samples. *Statistically significant (P ≤ 0.05) difference between treated cells and control. **Statistically significant (P ≤ 0.01) difference between treated cells and control