Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels

Abstract

There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 μM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 μM) either alone or together with CAPS (10 μM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 μM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required.

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-3T3^TRPV1 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-3T3^TRPV1 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).

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 APOPercentage^TM 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.