Background
Gastric cancer (GC) is the second most common human cancer worldwide and is difficult to diagnose in its early stage [
1]. GC is extremely difficult to cure once it metastasizes [
2,
3]. Although the occurrence and progression of cancer are complex, numerous findings indicate that aberrant intracellular Ca
2+ ([Ca
2+]
i) signaling is involved in the development of several types of gastrointestinal (GI) cancers, including GC and colon cancer [
4]. Since plasma membrane Ca
2+-permeable channels play important roles in the regulation of [Ca
2+]
i, their aberrant expression and function are positively associated with the occurrence and development of GI tumors [
5,
6]. Consistently, we revealed that activation of G protein-coupled receptors (GPCRs), such as Ca
2+ sensing receptors (CaSR) and vasoactive intestinal polypeptide (VIP) receptors, promotes GC progression via transient receptor potential vanilloid receptor 4 (TRPV4) channels and the Ca
2+ signaling [
7,
8]. Therefore, the Ca
2+-permeable TRPV channels deserve further intensive investigation since they could be novel potential drug targets for GI tumor therapy [
9].
The TRPV1 channel belongs to the Ca
2+ permeable TRPV channel family and responds to noxious heat (> 43 °C), low pH value (< 5), capsaicin and so on [
10‐
12]. The TRPV1 channel plays an important role in several physiological and pathological processes, such as nerve conduction, visceral pain sensing [
9,
13,
14], and activation of immunity [
15]. Furthermore, a few studies previously shown that TRPV1 was likely involved in tumor progression [
16], and its activation reduced cell proliferation, migration and invasion in breast cancer [
17], urothelial cancer [
18] and papillary thyroid carcinoma [
19]. However, little is currently known about the role of TRPV1 channel in GI tumorigenesis, except for Amaya G. et al., who reported that TRPV1 regulates neurogenic inflammation in the colon to presumably protect mice from colon cancer [
20]. We also revealed that the TPRV1 channel inhibited EGFR-induced epithelial cell proliferation to prevent mice from developing colon polyps [
21]. Although the expression of TRPV1 channel has been detected in rat gastric epithelial cells [
22], almost nothing is known about its functional role in the upper GI epithelial cells, let alone its potential involvement in the pathogenesis of gastric disease. Importantly, the role of TRPV1 channel in gastric tumorigenesis has not been explored so far.
Aberrant [Ca
2+]
i signaling contributes to multiple aspects of tumor progression such as cell proliferation, migration, invasion, apoptosis and autophagy [
23,
24], and calmodulin (CaM) is one of the key proteins that triggers various signaling events in response to an increase in [Ca
2+]
i. Upon binding with Ca
2+, CaM activates downstream calcium/calmodulin-dependent protein kinase kinases (CaMKK), including CaMKKα and CaMKKβ to further regulate adenosine mono phosphate activated protein kinase (AMPK). AMPK, a heterotrimeric Ser/Thr kinase, is well known to be involved in tumor progression [
25]. Thr-172, as one of the important sites for AMPK activation, can be phosphorylated by CaMKKβ [
25]. Several studies previously reported that AMPK inhibits proliferation and induces apoptosis in GC cells [
26‐
28]. Although CaMKK has a well-established connection between Ca
2+ signaling and cancer pathogenesis [
29,
30], the role of aberrant Ca
2+/CaMKKβ/AMPK signaling in GC progression and the underlying molecular mechanisms remain unexplored.
In the present study, we focused on the role of TRPV1 channels in GC progression and the underlying molecular mechanisms therein. Herein, we demonstrate for the first time that TRPV1 expression was decreased in primary human GC tissues, which was closely correlated with poor prognosis of GC patients. Moreover, TRPV1 could increase AMPK phosphorylation through Ca2+/CaMKKβ to downregulate the expression of cyclin D1 and matrix metalloproteinase-2 (MMP2), leading to the inhibition of GC cell proliferation, migration and invasion. Our results not only reveal the role of TRPV1 channel in GC suppression, but provide a novel insight to GC prevention and treatment.
Materials and methods
Ethics statement and human tissue samples
Twenty pairs of GC and adjacent tissues from the surgical patients in Xinqiao Hospital of Third Military Medical University (during 2016 and 2017) were used for real-time quantitative PCR, and all resected specimens were confirmed by pathological examination. Informed consent was obtained from all patients. All clinical studies were approved by the Clinical Research Ethics Committee of Third Military Medical University and were performed in accordance with approved guidelines. GC and adjacent tissue microarray for immunostaining were purchased from SHANGHAI OUTDO BIOTECH CO., LTD (Shanghai, China).
Cell culture
MKN45, SGC7901, AGS, MGC803, BGC823 human gastric cancer cell lines and the human gastric normal epithelial mucosa cell line (GES-1) were purchased from Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 or DMEM-HIGH GLUCOSE medium (HyClone, USA) supplemented with 10% fetal bovine serum (HyClone, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin (Invitrogen, USA). All cells were grown in a 37 °C humidified atmosphere containing 5% CO2.
Preparation and infection of lentiviruses
Lentiviruses were purchased from Genechem Co., Ltd. (Shanghai, China). A lentivirus containing the full-length coding sequence (CDS) of TRPV1 (NM_080704) was designed to increase its expression in BGC823 cells, and lentiviral-based shRNA was used to silence the expression of TRPV1 in MKN45 cells. Sequences for TRPV1 shRNA and control were as follows: shRNA-1 (5′-GCATCTTCTACTTCAACTTCC-3′), shRNA-2 (5′-GGCCGACAACACGAAGTTTGT-3′) and control (5′-GTTCTCCGAACGTGTCACGT-3′). All shRNA groups that do not have a designed number used shRNA-1. Cells were infected with lentiviruses according to the protocol of the manufacturer. Briefly, cells were plated in 24-well plates at 1 × 105 cells/well, lentiviruses were added into culture medium separately (the volume of lentiviruses was calculated as a MOI of 20), and medium were refreshed after 8 h. Puromycin was used to screen the stable cells after 72 h of lentivirus infection.
Preparation and transfection of plasmids
The full length CDS of AMPK (NM_006251) was cloned into pcDNA3.1 to prepare overexpression plasmids; AMPK-siRNA sequence 5′-CTGCTTGATGCACACATGAAT-3′; CaMKKβ-siRNA sequence 5′- GTCAAGTTGGCCTACAATG-3′ were cloned into the GV102 vector separately to prepare siRNA-knocked down plasmids. Transfection of plasmids into cells was performed according to the protocol of FuGENE@ HD Transfection Reagent (Cat. No. E2311, Promega, USA). In brief, 1 × 105 cells were cultured in each well of 24-well plates, and 1.5 μL of transfection reagent with 0.5 μg of plasmids were mixed and added to 500 μL culture medium. Cells were incubated with the mixture for 72 h.
RNA extraction and real-time quantitative PCR (qPCR)
Total RNA was extracted from each group using the RNAiso Plus reagent (Cat. No. 9109, Takara, Japan). cDNA was synthesized using PrimeScript® RT-polymerase (Cat. No. R050A, Takara, Japan). Next, 50 ng of each cDNA was amplified as a template, and qPCR was performed using a SteponePlus device (Art. No. 272008342, Life Technologies, USA) with a SYBR® Premix Ex TaqTM II kit (Cat. No. RR820A, Takara, Japan). All samples were run in triplicate, and β-actin was used as an internal control. Data were quantified using the 2−ΔΔCt relative quantitative method, normalized to β-actin expression, and expressed as the ratio of TRPV1 to β-actin mRNA levels. Primers were designed as follows:
TRPV1: 5′-TGGTATTCTCCCTGGCCTTG-3′ (forward)
5′- CTTCCCGTCTTCAATCAGCG-3′ (reverse)
AMPK: 5′-TGGTAGGAAAAATCCGCAGA-3′ (forward)
5′-CGACTTTCTTTTTCATCCAGC-3′ (reverse)
β-actin: 5′-GGCATCCACGAAACTACCTT-3′ (forward)
5′-CGGACTCGTCATACTCCTGCT-3′ (reverse)
Immunohistochemistry (IHC)
Tissue samples were paraffin embedded and cut into 5 mm slices. After dewaxing and rehydration, tissue samples were incubated with anti-TRPV1 (Cat. No. ab3487, Abcam, UK) overnight at 4 °C after blocking. TRPV1 was detected using HRP-conjugated anti-rabbit secondary antibody (Cat. No. ZB-2301, ZSGB-BIO, China) and visualized with DAB. The negative control contained secondary antibody only. The gastric cancer tissue microarray we purchased also contains pathological score data for Ki67, VEGFR, E-cadherin and other proteins. The degree of staining in the TRPV1 sections was observed and scored by a pathologist. According to previously defined criteria [
31,
32], the percentage of TRPV1 positivity was scored from 0 to 3 as follows: 0, < 10%; 1, 10–30%; 2, 30–50%; 3, > 50%. Staining intensity was scored according to a 4-point scale as follows: 0 (no staining); 1 (weak staining, light yellow); 2 (moderate staining, yellowish brown); and 3 (strong staining, brown). Subsequently, TRPV1 expression was calculated by multiplication of the percent positivity score and staining intensity score, resulting in a final score ranging from 0 to 9. Since TRPV1 was expressed as 0 in a large number of gastric cancer samples, expression of 0 was set as low expression and 1–9 as high expression. Ki67 was positively localized in the nucleus, VEGFR was positively localized in the cytoplasm and E-cadherin was positively localized in the plasma membrane. The scoring standard was the same as that described above.
Immunofluorescence assay
Cells of each group were plated onto coverslips in 35 mm dishes and fixed in 4% polyformaldehyde for 15 min at room temperature. Coverslips were washed in PBS three times for 5 min. Cells were blocked in goat serum for 1 h at room temperature and then incubated with anti-TRPV1 antibody overnight at 4 °C. After three washes in PBS, cells were incubated with Cy3 labeled anti-rabbit (Cat. No. A0516, Beyotime, China) secondary antibody for 1 h at room temperature. Finally, nuclei were stained with DAPI for 10 min. Images were captured on a confocal microscope (Leica SP5, Germany).
Western blot analysis
Whole-cell lysates were separated by SDS-PAGE on denaturing 10% or 12% gels and transferred to polyvinylidene fluoride membranes (Cat. No. ISEQ00010, Millipore, USA). Blots were blocked in 5% milk for 1 h at room temperature and then separately incubated at 4 °C overnight with the following specific primary antibodies: anti-TRPV1, anti-Ki67 (Cat. No. ab15580, Abcam, UK), anti-AMPK (Cat. No.5832, Cell Signaling Technology, USA), anti-phospho-AMPK (Cat. No.2535, Cell Signaling Technology, USA), anti-CaMKKβ (Cat. No. ab168818, Abcam, UK), anti-cyclin D1 (Cat. No. ab16663, Abcam, UK), anti-MMP2 (Cat. No.87809S, Cell Signaling Technology, USA), anti-β-catenin (Cat. No.8480, Cell Signaling Technology, USA), anti-phospho-β-catenin (Cat. No.9567, Cell Signaling Technology, USA), anti-AKT (Cat. No.9272, Cell Signaling Technology, USA), anti-phospho-AKT (Cat. No.4060, Cell Signaling Technology, USA), anti-ERK1/2 (Cat. No. ab184699, Abcam, UK), anti-phospho-ERK1/2 (Cat. No. ab214362, Abcam, UK), and anti-GAPDH (Cat. No. TA-08, ZSGB-BIO, China). All primary antibodies were diluted 1:1000. After rinsing, blots were incubated in HRP-conjugated anti-rabbit or anti-mouse (Cat. No. A0239 and A0216, Beyotime, China) secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence (Cat. No. 34094, Thermo, USA) was used to detect immunoreactive bands. A human phospho-kinase array was used to detect changes in tumor-related signaling pathways (Cat. No. ARY003B, R&D Systems, USA), and 2 μM BAPTA-AM (Cas No. 126150–97-8, MedChemExpress, USA) was used to chelate intracellular calcium with cells being treated with BAPTA-AM for 2 h. Each experiment was performed in triplicate and repeated three times. The gray value of the bands was measured by ImageJ software for statistics.
Cell proliferation assay
Cell viability and proliferation were measured by CCK8 assay (Cat. No.C0038, Beyotime Biotechnology, China). Cells were plated in 96-well plates (3000 cells/well) in triplicate and cultured for 0, 24, 48, 72 h or 96 h followed by addition of 100 μl of medium/CCK8 mixture (medium: CCK8, 9:1) 1–2 h before the endpoint of incubation. A Multiskan EX plate reader (Thermo Fisher Scientific, Germany) was used to quantify viable cells by measuring the absorbance at 450 nm, which estimates relative cell numbers rather than counting cells.
Cell cycle analysis
Cells were digested, centrifuged and washed twice in cold PBS. The supernatant was subsequently discarded, and pre-cooled 70% ethanol was slowly added to the cell pellets that were kept at 4 °C overnight. After centrifugation the next day, the ethanol was removed, cells were washed once with PBS and were then incubated in a solution containing 0.2% Tween 20, 100 U/mL RNase, and 50 μg/mL propidium iodide for 20 min at 37 °C. Cell cycle analysis was performed using flow cytometry in which samples were gated on live cells with an excitation wavelength of 488 nm and an emission wavelength of 620 nm. LMD files were further analyzed using ModFit LT (Verity Software House, Topsham, ME).
Long-term survival of cells was assessed by their ability to form colonies. Cells were plated in 6-well plates with 3 mL culture medium with 500 cells/well. After 10–12 days, the cell culture medium was removed, and cell clones were washed in PBS and fixed in 4% polyformaldehyde. Clone numbers were quantified after staining with crystal violet (Relative clonogenicity = Clone numbers/Average clone numbers of control group).
Transwell migration and invasion assays
Twenty-four-well transwell chambers (Corning, USA) were used for this assay. 5 × 104 cells were plated into each upper chamber with 8-μm pores and cultured in 200 μL serum-free RPMI-1640. The lower chambers were filled with 500 μL complete RPMI-1640 medium. After 24 h of incubation, cells that had migrated onto the lower surface were stained with crystal violet, and counted under a microscope (Olympus Corporation, Japan). The average value of three randomly selected fields was recorded as the number of migrated cells. Then, the upper surface of the polycarbonate filter was coated with 10% Matrigel (Collaborative Biomedical, USA), and 1 × 105 cells were added to detect cell invasion. The other conditions were the same as those in the migration assay.
Calcium measurement
Plating 1 × 104 cells on coverslips, cells were loaded with 5 μM Fura-2 AM (Cat. No. F1221, Invitrogen, USA) in physiological salt solution (PSS) at 37 °C for 60 min and then washed in PSS or PSS with and 50 μM of the TRPV1 inhibitor SB-705498 (Cas No.501951–42-4, MCE, USA). Next, cells on coverslips were mounted in a standard perfusion chamber on a Nikon microscope stage. The ratio of Fura-2 fluorescence at 340 or 380 nm excitation (F340/380) was followed over time and captured with an intensified CCD camera (ICCD200) and a MetaFluor Imaging System (Universal Imaging, Downingtown, PA). PSS used in Ca2+ measurement contained the following: 140 mM Na+, 5 mM K+, 2 mM Ca2+, 147 mM Cl−, 10 mM HEPES, and 10 mM glucose at pH 7.4.
Tumor xenograft and peritoneal dissemination assay in nude mice
The animal use protocol was approved by the Third Military Medical University Committee on Investigations Involving Animal Subjects. All animal care and experimental studies were conducted in accordance with the guidelines of the Animal Ethical Committee of Third Military Medical University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.8023, revised 1978). Animal studies were reported in compliance with the ARRIVE guidelines. First, 1 × 106 TRPV1-overexpressing BGC823 cells and negative control (NC) cells were injected into the armpits of seven 4-week old male nude mice for tumor xenograft assay. TRPV1-overexpressing BGC823 cells were injected into the right armpit, and NC cells were injected into the left side. After 30 days of implantation, mice were sacrificed and tumor volumes in mm3 were calculated using the formula 1/2(length× width2). For the peritoneal dissemination assay, 1 × 106 TRPV1-overexpression BGC823 cells and the NC cells were injected into the abdominal cavity of nude mice. Five weeks later, mice were sacrificed, and nodules were observed and quantified.
Statistical analysis
SPSS Statistics 26.0 (IBM, USA) and Prism 8.0 (GraphPad, USA) software was used to analyze the data. All data are shown as MEANS ± SD. Pearson Chi-Square test was used to compare the correlations of TRPV1 expression and clinicopathological features. The relationship between TRPV1 expression and patient survival was examined by the log-rank test using the Kaplan-Meier method. Student’s t-test was used to analyze differences between two groups. One-way ANOVA was used to compare three or more groups. Significant differences are expressed in the figures and figure legends as *P < 0.05. All experiments were repeated using three biological replicates.
Discussion
In the present study, we revealed that: (a) the expression of TRPV1 channel at the levels of mRNA and proteins was significantly decreased in human primary GC tissues; (b) the downregulation of TRPV1 expression was closely correlated with poor GC progression; (c) TRPV1 inhibited proliferation, migration and invasion of GC cells in vitro, and reduced gastric tumor size, number and peritoneal dissemination in vivo; and (d) mechanistically, TRPV1 suppresses GC progression through Ca2+/CaMKKβ/AMPK signaling pathway to reduce cyclin D1 and MMP2 expression.
We have verified that TRPV1 expression is downregulated in human primary GC tissues compared to their adjacent tissues, which is consistent with the prediction from the Oncomine tumor database. Importantly, we demonstrate for the first time that down-regulation of TRPV1 expression in GC tissues was correlated with large tumor size, high histological grade, lymphatic metastasis, advanced clinical stage and poor prognosis. Our clinicopathologic data strongly suggest a role for TRPV1 in the progression and development of GC. Moreover, TRPV1 expression in GC was positively correlated with E-cadherin expression, better prognosis and survival ratio of GC patients, but was negatively correlated with Ki67 and VEGFR expression. Since Ki67 is a well-known marker of cell proliferation, the faster tumor grows, the more sensitive to Ki67 [
44]. While VEGFR promotes tumor angiogenesis [
45], E-cadherin is closely related to tumor metastasis since the metastasis of tumor losses E-cadherin expression [
46]. Therefore, TRPV1 could be a potential marker for GC prognosis due to its close association with GC progression.
We have provided further experimental data to support our notion that TRPV1 channel plays an important role in the progression and development of GC. The upregulation of TRPV1 expression attenuated GC cell proliferation, invasion and metastasis both in vitro and in vivo. In contrast, downregulation of TRPV1 expression promoted GC cell proliferation, invasion and metastasis. Therefore, TRPV1 channel seems to play a special role as GC suppressor because the aberrant expression and function of most Ca
2+-permeable TRP channels are usually associated with GI tumor promotion [
35,
47,
48]. It is well known that [Ca
2+]
i is an important second messenger to regulate a wide range of cellular functions. The opening of TRP channels can promote Ca
2+ entry which activates downstream signaling pathways, such as Ca
2+/calmodulin kinase II (CaMKII), mitogen-activated protein kinase (MAPK), AMPK and so on to control cell proliferation, apoptosis and migration [
49,
50]. Over the past two decades, several research groups including ours have identified six TRP channels (TRPC6, TRPM2, 5, 7, TRPV4, 6) that play an important role in GC development [
7,
8,
47,
51‐
54]. However, all six of these TRP channels have been suggested as oncogene and tumor promoter in GC. Intriguingly, in contrast to the enhanced expression of these six TRP channels, TRPV1 channel expression is down-regulated in human GC and plays a suppressive role in GC. Therefore, it is important to elucidate the underlying molecular mechanisms for how TRPV1 as a Ca
2+-permeable channel suppresses rather than promotes GC development.
Using phosphorylation chip screening for signaling pathway, we screened 40 key molecules that are closely related to cancer development. Interestingly, we found that phosphorylation level of AMPK was increased most significantly after overexpression of TRPV1 in GC cells, while ERK1/2 was decreased. Although TRPV1 regulation of AMPK is well studied in endothelial cells [
55], smooth muscle cells [
56], cardiomyocytes [
57], and immune cells [
58,
59], this has not been reported in digestive cancer cells. In the present study, we have provided sufficient evidence to demonstrate for the first time that TRPV1 is a Ca
2+-permeable channel that uniquely suppresses GC development through activation of a novel CaMKKβ/AMPK pathway. The interesting role of TRPV1 channels in GC suppression needs further investigation, but it is not surprising since different Ca
2+-permeable channels mediate cellular Ca
2+ signals with various temporal and spatial precision, which may play different roles of anti-tumor or pro-tumor.
We revealed that cyclin D1 and MMP2 are the downstream molecules of AMPK activation to finally inhibit both proliferation and migration of GC cells. Cyclin D1 has been recognized as a proto-oncogene to promote cell cycle from G1 phase to S phase by activating CDK4, a cyclin-dependent kinase specific to G1 phase. Some studies have reported that AMPK inhibits GC growth via cyclin D1 suppression [
41,
60]. As is known, the metastasis is a crucial characteristic for the late stages of cancer [
42,
61]. MMP2 belongs to the zinc-dependent metalloproteinase gene family and plays a critical role in cancer metastasis [
62]. AMPK, an upstream regulator of MMP2 [
63], could decrease the migration and invasion of colorectal cancer through inhibition of MMP2 [
64].
Capsaicin has been recognized as an anti-cancer agent in variety of cancers due to its apoptotic effect and inhibitory effect on cancer cell growth, metastasis and tumor angiogenesis [
65]. Although capsaicin is a commonly used TRPV1 agonist in neurons used to induce Ca
2+ influx, its mechanism of action in tumorigenesis is complex. Capsaicin directly activates PI3K/AKT and PKA in a TRPV1/Ca
2+-independent manner [
66,
67]. Moreover, capsaicin activates TRPV6 instead of TRPV1 to induce apoptosis in GC and lung cancer cells [
35,
36]. In the present study, we found that capsaicin could not induce Ca
2+ signaling in GC cells expressing functional TRPV1 channels at high concentration of 50 uM (comparing the IC
50 of 0.5 uM in many other cell types) [
36]. One possibility is loss of TRPV1 channel function in GC cells. However, this is not the case since: 1) pharmacological blocker and genetic manipulation of TRPV1 could alter [Ca
2+]
i in GC cells, 2) genetic manipulation of TRPV1 could alter GC cell proliferation, migration and invasion both in vitro and in vivo, and 3) [Ca
2+]
i chelator BAPTA-AM efficiently prevented TRPV1-mediated CaMKKβ activation and AMPK phosphorylation in GC cells. Another possibility is an aberrant functional alteration/mutation of TRPV1 channel in GC cells that causes a loss of TRPV1 sensitivity to capsaicin, which needs further investigation. Due to the fact that TRPV1 can be stimulated by a variety of substances [
10,
11], TRPV1 channels in GC cells could still be activated by the other non-capsaicin agents, such as gastric acid and heating diets.
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