Fig. 4

Mix-EO treatment blocks tumor growth by targeting autocrine and paracrine MMP9 production. A Fold change in MMP9 expression in tumor-infiltrating mesenchymal stem cells (MSCs) isolated from mice treated with or without indicated EOs by qPCR. Expression was compared to bone-marrow-derived MSCs (WT) (n = 6/group). B Cell proliferation of B16F10 cells treated with/without rec. MMP9 in the presence/absence of 25 μg/ml Mix-EO (n = 6/group). C–G Experimental outline to generate MS-5 conditioned medium for the treatment of B16F10 cells: collection of conditioned medium of MS-5 cells treated with carrier/DMSO (Ctrl) or Mix-EO (25 μg/ml), MS-5 cells overexpressing hu MMP9WT (MMP9 OE) and cells, where mouse MMP9 had been gene-silenced (siMMP9) after 24 h (n = 6/group). D Fold change of MMP9 expression of MS-5 cells without (Ctrl) and with EO treatment (Mix-EO; 25 μg/ml), after MMP9 OE, and gene silencing of MMP9 (si-MMP9). Expression was compared to the expression in ctrl cells (n = 6/group). E–G B16F10 cell growth was determined in cultures supplemented with indicated conditioned medium and cotreated with or without MMP inhibitor (MMPI) by Trypan blue 24 h later (n = 6/group). F, G Fold change in P65 (F) and MMP9 (G) expression in B16F10 cells treated with indicated MS-5 conditioned media in the presence or absence of MMPI (n = 6/group). Expression was compared to the expression in control cultures. Data are expressed as mean ± SEM (unpaired Student's t test or One-way ANOVA *p < .05, **p < .01). H Proposed mode of action of fennel (Fe-EO) and M. fruticosa (Thy-EO) derived EOs: Fe-EO and Thy-EO as monotherapy, but more prominent in combination (Mix-EO) inhibited NFkB and MMP9 expression in tumor (autocrine action) and tumor stromal cells (paracrine action) suppressing tumor growth and migration. MMP9, matrix metalloproteinase-9; EO, essential oil; NFkB, nuclear factor kappa B