J Breast Cancer.  2014 Dec;17(4):332-338. 10.4048/jbc.2014.17.4.332.

Transient Receptor Potential Vanilloid 1 Expression and Functionality in MCF-7 Cells: A Preliminary Investigation

Affiliations
  • 1Department of Veterinary Sciences, Section of Pharmacology and Toxicology, University of Turin, Grugliasco, Italy. cristina.vercelli@unito.it
  • 2Department of Clinical and Biological Sciences, Azienda Ospedaliero-Universitaria San Luigi Gonzaga, Orbassano, Italy.

Abstract

PURPOSE
Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel belonging to the transient receptor potential family, and it is expressed in different neoplastic tissues. Its activation is associated with regulation of cancer growth and progression. The aim of this research was to study the expression and pharmacological characteristics of TRPV1 in cells derived from human breast cancer MCF-7 cells.
METHODS
TRPV1 presence was assessed by binding studies and Western blotting. Receptor binding characteristics were evaluated through competition assays, while 3-(4,5-dimethylthiazol-2-yl)-2,5,-dipheyltetrazolium bromide reduction assays were performed to confirm an early hypothesis regarding the modulation of cancer cell proliferation. The functionality of TRPV1 was evaluated by measuring Ca2+ uptake in the presence of increasing concentrations of TRPV1 agonists and antagonists.
RESULTS
Binding studies identified a single class of TRPV1 (Bmax 1,492+/-192 fmol/mg protein), and Western blot showed a signal at 100 kDa corresponding to the molecular weight of human TRPV1. Among the different tested agonists and antagonists, anandamide (Ki: 2.8x10(-11) M) and 5-iodoresiniferatoxin (5-I-RTX) (Ki: 5.6x10(-11) M) showed the highest degrees of affinity for TRPV1, respectively. All tested TRPV1 agonists and antagonists caused a significant (p<0.05) decrease in cell growth rate in MCF-7 cells. For agonists and antagonists, the efficacy of tested compounds displayed the following rank order: resiniferatoxin>anandamide>capsaicin and 5-I-RTX=capsazepine, respectively.
CONCLUSION
These data indicate that both TRPV1 agonists and antagonists induce significant inhibition of MCF-7 cell growth. Even though the mechanisms involved in the antiproliferative effects of TRPV1 agonists and antagonists should be further investigated, it has been suggested that agonists cause desensitization of the receptor, leading to alteration in Ca2+-influx regulation. By contrast, antagonists cause a functional block of the receptor with consequent fatal dysregulation of cell homeostasis.

Keyword

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Competition assays; Functionality assays; MCF-7 cells; TRPV1 receptor

MeSH Terms

Blotting, Western
Breast Neoplasms
Cell Proliferation
Homeostasis
Humans
MCF-7 Cells*
Molecular Weight

Figure

  • Figure 1 Scatchard plot (A) and saturation curve of MCF-7 binding membranes (B). One hundred microliters of cell membrane suspension were incubated with increasing concentration (0.025-3.2 nM) of 3[H]RTX and in presence or in absence of 1 µM unlabelled RTX. The specific bound labeled ligand was determined by Scatchard analysis using GraphPad Prism Software (GraphPad Software) (n=8). RTX=resiniferatoxin.

  • Figure 2 The samples used for Western blot analysis were at different concentrations: 25, 50, and 100 µg of protein. The image shows a signal corresponding to the molecular weight around 100 kDa that is reported to correspond to transient receptor potential vanilloid 1 (TRPV1) receptor. The signal was also present in the positive control (first column, K+), composed by proteic lysate of rat urinary bladder.

  • Figure 3 During functional studies, the stimulation of 45Ca2+ uptake in MCF-7 membranes was induced by adding increasing concentrations (10-11-10-3) of selective agonists: anandamide, resiniferatoxin (RTX), and capsaicin. For each data point in each experiment, four wells were assayed (n=4). The error bars indicate SEM (GraphPad Prism Software; GraphPad Software).

  • Figure 4 Inhibition of resiniferatoxin (RTX) induced 45Ca2+ uptake by two selective transient receptor potential vanilloid 1 antagonists (5-iodoresiniferatoxin [5-I-RTX] and capsazepine) in MCF-7 cell membranes. For each data point in each experiment, four wells were assayed (n=4). The error bars indicate SEM (GraphPad Prism Software; GraphPad Software). The antagonists have been tested in the presence of RTX 2 nM for the measurement of the percentage of inhibition of 45Ca2+ uptake (IC50) (GraphPad Prism Software).

  • Figure 5 MTT assay (n=6) to evaluate the modulation of MCF-7 cells growth after the treatment with increasing concentration (10-10-10-4 M) of capsaicin at different experimental time points (2, 24, 48, and 72 hours). All the samples treated with increasing concentrations of capsaicin demonstrated a significant decrease (p<0.05) of the cell growth rate compared to the controls (K). The MCF-7 cells proliferation was statistically analyzed using Kruskal-Wallis test and Dunn post-test (mean±SEM). OD=optical density; a, b, c, d=p<0.05. *p<0.05.

  • Figure 6 MTT assay (n=6) to evaluate the modulation of MCF-7 cells growth after the treatment with increasing concentration (10-10-10-4 M) of capsazepine at different experimental time points (2, 24, 48, and 72 hours). All the samples treated with increasing concentrations of capsazepine demonstrated a significant decrease (p<0.05) of the cell growth rate compared to the controls (K). The MCF-7 cells proliferation was statistically analyzed using Kruskal-Wallis test and Dunn post-test (mean±SEM). OD=optical density; a, b, c, d=p<0.05. *p<0.05.

  • Figure 7 MTT assay (n=6) to evaluate the modulation of MCF-7 cells growth after the treatment with increasing concentration (10-10-10-6 M) of resiniferatoxin (RTX) at different experimental time points (2, 24, 48, and 72 hours). All the samples treated with increasing concentrations of RTX demonstrated a significant decrease (p<0.05) of the cell growth rate compared to the controls (K). The MCF-7 cells proliferation was statistically analyzed using Kruskal-Wallis test and Dunn post-test (mean±SEM). OD=optical density; a, b, c, d=p<0.05. *p<0.05.

  • Figure 8 MTT assay (n=6) to evaluate the modulation of MCF-7 cells growth after the treatment with increasing concentration (10-10-10-6 M) of 5-iodoresiniferatoxin (5-I-RTX) at different experimental time points (2, 24, 48, and 72 hours). All the samples treated with increasing concentrations of 5-I-RTX demonstrated a significant decrease (p<0.05) of the cell growth rate compared to the controls (K). The MCF-7 cells proliferation was statistically analyzed using Kruskal-Wallis test and Dunn post-test (mean±SEM). OD=optical density; a, b, c, d=p<0.05. *p<0.05.


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