The role of TRPV1 ion channels in the suppression of gastric cancer development

Abstract

Background: Although the aberrant expression and function of most Ca²⁺-permeable channels are known to promote gastrointestinal tumors, the association between transient receptor potential vanilloid receptor 1 (TRPV1) channels and gastric cancer (GC) has not yet been explored. Herein, we sought to determine the role of TRPV1 channels in the development of GC and to elucidate the underlying molecular mechanisms involved therein.

Methods: Immunohistochemistry, qPCR, Western blot, immunofluorescence assays were used to detect the mRNA and protein expression of TRPV1 in GC cells and tissues, and the clinical significance of TRPV1 in GC was also studied by clinicopathologic analysis. CCK8, colony formation, flow cytometry assays were used to detect the proliferation and survival of GC cells, while transwell assay was used to detect migration and invasion of GC cells in vitro. Tumor xenograft and peritoneal dissemination assays in nude mice were used to examine the role of TRPV1 in GC development in vivo.

Results: TRPV1 expression was significantly downregulated in human primary GC tissues compared to their adjacent tissues. The decreased expression of TRPV1 proteins in GC tissues was positively correlated with tumor size, histological grade, lymphatic metastasis, clinical stage, and was strongly correlated with poor prognosis of GC patients. Moreover, the expression of TRPV1 was closely correlated with Ki67, VEGFR, and E-cadherin, all of which are the well-known cancer markers for proliferation and metastasis. TRPV1 proteins were predominately expressed on the plasma membrane in several GC cell lines. TRPV1 overexpression blocked cell cycle at G1 phase to inhibit GC cell proliferation and attenuated migration and invasion of GC cells in vitro, but TRPV1 knockdown increased these parameters. TRPV1 significantly reduced gastric tumor size, number and peritoneal dissemination in vivo. Mechanistically, TRPV1 overexpression in GC cells increased [Ca²⁺]_i, activated CaMKKβ and AMPK phosphorylation, and decreased expression of cyclin D1 and MMP2, while TRPV1 knockdown induced the opposite effects.

Conclusions: TRPV1 uniquely suppresses GC development through a novel Ca²⁺/CaMKKβ/AMPK pathway and its downregulation is correlated with poor survival of human GC patients. Thus, TRPV1 upregulation and its downstream signaling may represent a promising target for GC prevention and therapy.

Keywords: Calcium signaling; Gastric cancer; Invasion; Metastasis; Proliferation; TRPV1 channel.

Fig. 1

TRPV1 expression in gastric cancer and its correlation with clinical progression. a Transcript levels of TRPV1 detected by qPCR in human primary GC tissues and their adjacent tissues (*P < 0.05, n = 20 patients). b Representative images of immunohistological staining on TRPV1 proteins in GC tissues and adjacent tissues. c Summary data of immunohistological staining of TRPV1 proteins (***P < 0.001, n = 80 patients). d Analysis of survival rates in GC patients with low and high TRPV1 expression levels (**P < 0.01, n = 100 patients). Correlation between TRPV1 and Ki67 (e), VEGFR (f), and E-cadherin (g) expression (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 100 patients). h Western blot analysis of TRPV1 protein levels in a normal human gastric epithelial cell line (GES-1) and five GC cell lines (**P < 0.01 vs. GES-1, n = 3). i QPCR analysis of TRPV1 mRNA levels in GES-1 and nine GC cell lines (**P < 0.01, ***P < 0.001, ****P < 0.0001 vs. GES-1, n = 3). j Immunofluoresence staining images of TRPV1 proteins in GES-1, MKN45 and BGC823 cells. Negative control was not treated with primary antibody against TRPV1. Nuclei were stained in blue with DAPI. The white scale bars on the lower right are 7.5 μm. These images are representative of three independent experiments

Fig. 2

Effects of TRPV1 overexpression and knockdown on cell proliferation and clonogenicity. a Representative images of TRPV1 and Ki67 proteins in TRPV1-overexpressed BGC823 cells and TRPV1-knockeddown MKN45 cells. b, c The summary data of TRPV1 and Ki67 protein expression in GC cells (*P < 0.05, **P < 0.01, vs. NC, n = 3). d mRNA levels of TRPV1 detected by qPCR in TRPV1-overexpressed BGC823 cells and TRPV1-knockeddown MKN45 cells (**P < 0.01, ****P < 0.0001, vs. NC, n = 3). e GC cells proliferation in TRPV1-overexpressed BGC823 cells (left) and in TRPV1-knockeddown MKN45 cells (right) (shRNA-2 ****P < 0.0001, shRNA-1 ^####P < 0.0001 vs. NC, n = 3). f Effects of TRPV1 overexpression or capsaicin (50 μM) alone and in combination on BGC823 cells proliferation (*P < 0.05, **P < 0.01, n = 3). NS: no significant difference. g Effects of TRPV1 knockdown or capsaicin (50 μM) alone and in combination on MKN45 cell proliferation (*P < 0.05, n = 3). h, i Effects of TRPV1 knockdown on clonogenicity of GES-1 and MKN45 cells (*P < 0.05, **P < 0.01 vs. NC, n = 3). j Effects of TRPV1 overexpression on clonogenicity of BGC823 cells (**P < 0.01 vs. NC, n = 3)

Fig. 3

Effects of TRPV1 on GC growth in nude mice and GC cell cycle. a Images of subcutaneously xenografted gastric tumors in 7 nude mice. TRPV1-overexpressed BGC823 cells were implanted on the right side of mouse armpits, and NC cells were implanted on the left side. b Summary data of tumor weight (left) and volume (right) (***P < 0.001 vs. NC, n = 7 mice). Cell cycle analysis by flow cytometry in TRPV1-overexpressed BGC823 and NC cells (c), and summary data (d) (***P < 0.001, ****P<0.0001 vs. NC, n = 3). Cell cycle analysis by flow cytometry in TRPV1-knockeddown MKN45 and NC cells (e), and summary data (f) (****P < 0.0001 vs. NC, n = 3)

Fig. 4

Effects of TRPV1 genetic manipulation on GC cell migration, invasion and metastasis. GC cell migration presented as original images and presented as summary data in TRPV1-overexpressed BGC823 cells (a) and TRPV1-knockeddown MKN45 cells (b) (*P < 0.05, **P < 0.01 vs. NC, n = 3). GC cell invasion presented as original images in and presented as summary data in TRPV1-overexpressed BGC823 cells (c) and TRPV1-knockeddown MKN45 cells (d) (*P < 0.05, **P < 0.01 vs. NC, n = 3). e Images of nude mice peritoneally injected with NC cells (left) and TRPV1-overexpressed BGC823 cells (right). f Summary data of tumor numbers from each groups of abdominal transplantation mouse model (**P < 0.01 vs. NC, n = 7 mice)

Fig. 5

Changes in tumor-related signaling molecules after genetic manipulation of TRPV1 in GC cells. a Screening of phosphorylation of 40 key cancer-related signaling molecules analyzed by signaling pathway phosphorylation microarray after TRPV1 overexpression in BGC823 cells (*P < 0.05, n = 3). b ERK1/2 phosphorylation in TRPV1-overexpressed BGC823 cells and TRPV1-knockeddown MKN45 cells. Representative images are shown on the left and summary data on the right (*P < 0.05 vs. NC, n = 3). c, d Phosphorylation of β-catenin and AKT in TRPV1-overexpressed BGC823 cells and TRPV1-knockeddown MKN45 cells (NS: no significant difference, vs. NC, n = 3). e AMPK mRNA levels in TRPV1-overexpressed BGC823 or TRPV1-knockeddown MKN45 cells (NS: no significant difference, vs. NC, n = 3). f AMPK phosphorylation after TRPV1 overexpression in BGC823 cells and AMPK knockdown in TRPV1-overexpressed BGC823 cells (*P < 0.05, n = 3). g AMPK phosphorylation after TRPV1 knockdown in MKN45 cells and AMPK overexpression in TRPV1-knockeddown MKN45 cells (*P < 0.05, n = 3)

Fig. 6

Effects of genetic manipulation of AMPK on GC cell proliferation, migration and invasion. a Effects of AMPK on cell proliferation in TRPV1-overexpressed BGC823 cells and in TRPV1-knockeddown MKN45 cells (***P < 0.001, ****P < 0.0001, n = 3). b Representative images of protein expression of cyclin D1 and MMP2 in TRPV1-overexpressed BGC823 cells transfected with AMPK-siRNA or TRPV1-knockeddown MKN45 cells transfected with AMPK-overexpression as well as their NC cells. c, d Summary data of cyclin D1 and MMP2 proteins in GC cells generated from the original data as in b (*P < 0.05, **P < 0.01, n = 3). e, f Effects of genetic manipulation of AMPK on cell migration and invasion in TRPV1-overexpressed BGC823 cells and TRPV1-knockeddown MKN45 cells. Representative images are shown on the left and summary data on the right (*P < 0.05, **P < 0.01, n = 3)

Fig. 7

TRPV1/Ca²⁺-mediated GC suppression through activation of CaMKKβ/AMPK phosphorylation. a Representative time course of 5 mM CaCl₂-induced [Ca²⁺]_i signaling in TRPV1-overexpressed BGC823 cells (middle) vs. NC (left) and TRPV1-overexpressed BGC823 cells treated with SB-705498 (50 μM) (right). Summary data are shown as a bar graph (**P < 0.01, ****P < 0.0001, n = 20 cells). b Representative time course of 5 mM CaCl₂-induced [Ca²⁺]_i signaling in TRPV1-knockeddown MKN45 cells (middle) vs. NC (left). Summary data are shown as a bar graph (right) (***P < 0.001 vs. NC, n = 20 cells). c Expression levels of CaMKKβ proteins after TRPV1 overexpression in BGC823 cells or TRPV1 knockdown in MKN45 cells. Representative images are shown on the left and summary data on the right (*P < 0.05 vs. NC, n = 3). d Effects of CaMKKβ knockdown on CaMKKβ expression in TRPV1-overexpressed BGC823 or NC cells (*P < 0.05, n = 3). e Effects of BAPTA-AM (2 μM) on CaMKKβ expression in TRPV1-overexpressed BGC823 or NC cells (*P < 0.05, n = 3). f Effects of CaMKKβ knockdown on AMPK phosphorylation in TRPV1-overexpressed BGC823cells or NC cells (*P < 0.05, **P < 0.01, n = 3). g Proposed mechanisms of TRPV1-mediated GC suppression. The Ca²⁺ entry through TRPV1 channels causes CaMKKβ activation and AMPK phosphorylation that inhibits cyclin D1 and MMP2, leading to suppression of GC cell proliferation, migration and invasion. OE: overexpression, KD: knockdown, CaMKKβ: calcium/calmodulin-dependent protein kinase kinase β, AMPK: adenosine mono phosphate activated protein kinase, MMP2: matrix metalloproteinase-2