Fig 1.
Effect of MRS on TRVPV1-mediated Ca2+ signaling.
A) Schematic model of TRPV1 channel modulation by MRS in cancer cells. Homo-tetrameric TRPV1 is permeable to cations, notably Na+ and Ca2+. I) In the absence of TRPV1 agonist the channel is closed. II) Binding of a positive allosteric modulator (PAM) alone, e.g. MRS, does not activate channel opening. III) Endogenous TRPV1 agonists present in the tumor microenvironment are weak stimulators of TRPV1, possibly involved in tumor progression. IV) Exogenous agonists such as CAPS are potent TRPV1 activators. Resulting from TRPV1 hyper-activation, CAPS induces oxidative stress. V-VI) MRS amplifying the effect of both endogenous and exogenous agonists may evoke a more pronounced cytotoxic effect (oxidative stress). B-I) Acute effects of MRS on the intracellular Ca2+ regulation in various cell types. The different substances were added at the time points indicated by arrows and remained in the solution until the end of the experiments B) Fluorescence recordings from time-lapse videos show an increase in [Ca2+]i after CAPS administration in NIH-3T3 cells stably transfected with the TRPV1 channel. Traces represent mean + SD. More than 20 single cell recordings were evaluated (n > 20). MRS added at t = 5 min evoked a second long-lasting elevation in [Ca2+]i. (gray curve). In cells stimulated by CAPS only, [Ca2+]i continuously decreased after the initial peak at approximately 2 min (orange curve). Statistically significant differences between the two curves (Student t-test, p<0.05) are marked with asterisks. C-I) Single-cell (colored traces) and average fluorescence recordings of the entire cell population (grey traces) from time-lapse videos show changes in [Ca2+]i. Bars represent SD. All experiments were repeated two or more times with similar results. C) NIH-3T3TRPV1 cells responded to CAPS with an increase in [Ca2+]i, but not to MRS alone. D) Primary breast epithelial cells (prB) responded to CAPS with a brief increase in Ca2+]i often lasting for less than a min. E) Primary breast epithelial cells did not respond to MRS1477 alone, but responded to serum re-administration. F) MCF7 breast cancer cell line responded to CAPS alone and G) to MRS1477 alone with brief Ca2+ transients. H) Neither MCF7 cell extracts nor CAPS (50 μM) evoked an elevation in Ca2+]i in control (un-transfected) NIH-3T3 cells; a Ca2+ signal was induced by serum re-administration I) Application of a MCF7 cell extract increased [Ca2+]i in NIH-3T3TRPV1 cells; the Ca2+ signal was further increased by CAPS (50 μM). J-K) MRS augmented the endogenous agonist-evoked rises in [Ca2+]i. The initial rise in [Ca2+]i was evoked either by 13-HODE (1 μM) (J) or by 8,9-EET (1μM) (K).
Table 1.
Treatment groups.
Fig 2.
Effects of MRS treatment on TRPV1 channel activity in MCF7 cells.
The holding potential was set at -60 mV; W.C. denotes whole-cell patch clamp configuration. A) Recording from a control cell without stimulation. B) Recording from a cell of the control group: TRPV1 currents in the cells were activated by bath-perfusion of CAPS (10 μM) and subsequently inhibited by the TRPV1 antagonist CapZ (0.1 mM) perfused into the patch chamber. Currents were completely blocked in Na+-free solution (NMDG). C) Corresponding I/V-relation of currents recorded in B) at the indicated time points 1 and 2. D) Recording from a cell of the MRS group: cells were incubated with MRS (2 μM for 72 h) prior to the patch-clamp recording. Cells were stimulated by bath-perfusion of CAPS (10 μM) and were inhibited by CapZ (0.1 mM); currents were completely blocked at the end of the experiment by NMDG. E) Corresponding I/V-relation of currents recorded in D) at the indicated time points. F) Current densities were calculated by normalizing the current amplitudes by the cell membrane capacitance. Columns represent the mean + SD. The number of the measurements (n) is indicated within parentheses.
Fig 3.
Effects of MRS treatment on cell viability of cancer cell lines.
A. Cells were treated with MRS at different concentrations for 72 h. At this time point (72 h), MTT assays were performed; each dot represent mean + SD and n = 6 samples (2 independent experiments in triplicates). B. HODE levels in the extracts from different cells (n = 3). C. Cell viability in the presence of different CAPS concentrations in the absence (green curve) and presence of 2 μM MRS (red curve). MTT assays were performed; each dot represent mean + SD and n = 6 samples (2 independent experiments in triplicates) D. Cells were incubated with CAPS (10 μM), CapZ (0.1 mM) or both with and without MRS (2 μM) for 72 h and then MTT assays were performed. The columns represent mean + SD and n = 6 (2 independent experiments in triplicates). The letters denote the following: a—significant difference from control group, b—significant difference from CAPS group, c—significant difference from CapZ group, d—significant difference from CAPS+CapZ group, e—significant difference from MRS group, f—significant difference from MRS+CAPS group and g- significant difference from MRS+CapZ group.
Fig 4.
Effects of MRS treatment on apoptosis evidenced by either loss of lipid asymmetry (A) and caspase-3 and -9 activities (B) in MCF7 breast cancer cells (mean + SD; n = 6, 2 independent experiments in triplicates). Cells were incubated with CAPS (10 μM), CapZ (0.1 mM) or both with or without MRS (2 μM) for 72 h. Cells were then subjected to the APOPercentageTM assay (indicating the loss of plasma membrane lipid asymmetry) and to caspase activity assays. The letters denote the following: a—significant difference from control group, b—significant difference from CAPS group, c—significant difference from CapZ group, d—significant difference from CAPS+CapZ group, e—significant difference from MRS group, f—significant difference from MRS+CAPS group and g- significant difference from MRS+CapZ group.
Fig 5.
Effects of CAPS and MRS treatment on ROS production and mitochondrial membrane depolarization in MCF7 cancer cells (mean + SD, n = 6, 2 independent experiments in triplicates).
Cells were incubated with CAPS (10 μM), CapZ (0.1 mM) or both, with or without MRS (2 μM) for 72 h. Then they were subjected to the DHR 123 and JC-1 assays indicating the levels of ROS production and mitochondrial membrane potential, respectively. The letters denote the following: a—significant difference from control group, b—significant difference from CAPS group, c—significant difference from CapZ group, d—significant difference from CAPS+CapZ group, e—significant difference from MRS group, f—significant difference from MRS+CAPS group and g- significant difference from MRS+CapZ group.
Fig 6.
In vivo effect of MRS in NSG mice bearing MCF7 cell-derived tumors.
A) No significant difference in tumor size was observed between MRS-treated (10 mg/kg body weight (b.w.), i.p., twice a week) and vehicle-treated groups. Student t-tests were used for pairwise comparisons. Bars represent SD. B) Photographs taken from tumors derived from vehicle- and MRS-treated mice. No obvious structural differences were observed at the macroscopic level. Size bar represents 5 mm.