1.Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle.
Yang DONG ; Hai-Lian SHI ; Jian-Rong SHI ; Da-Zheng WU
Acta Physiologica Sinica 2010;62(4):349-356
Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.
Animals
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Calcium Channels, L-Type
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metabolism
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Cold Temperature
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Colon
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metabolism
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physiology
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In Vitro Techniques
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Male
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Muscle Contraction
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physiology
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Muscle, Smooth
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metabolism
;
physiology
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Physical Stimulation
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RNA, Messenger
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genetics
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metabolism
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Rats
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Rats, Wistar
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TRPA1 Cation Channel
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TRPC Cation Channels
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genetics
;
metabolism
2.Expression of transient receptor potential canonical channel proteins in human non-small cell lung cancer.
Qi ZHANG ; Jianxing HE ; Wenju LU ; Weiqiang YIN ; Haihong YANG ; Xiaoming XU ; Daoyuan WANG
Chinese Journal of Lung Cancer 2010;13(6):612-616
BACKGROUND AND OBJECTIVETransient receptor potential canonical (TRPC) proteins, a group of Ca2' permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC) and mediate store-operated calcium entry (SOCE) in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC).
METHODSReal-time quantitative PCRwas performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot.
RESULTSAmong the seven family members of TRPC so far identified (TRPC1-7), we detected the expression ofTRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1 approximately equal TRPC6 > TRPC3 > TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue.
CONCLUSIONOut of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.
Adult ; Aged ; Blotting, Western ; Calcium ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Female ; Humans ; Lung Neoplasms ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; analysis ; TRPC Cation Channels ; genetics ; physiology
3.TRPC6 mediates the enhancements of pulmonary arterial tone and intracellular Ca2+ concentration of pulmonary arterial smooth muscle cells in pulmonary hypertension rats.
Ming-Fang ZHANG ; Xiao-Ru LIU ; Na YANG ; Mo-Jun LIN
Acta Physiologica Sinica 2010;62(1):55-62
Pulmonary arterial hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that canonical transient receptor potential channel 6 (TRPC6) genes, which encode receptor-operated cation channels (ROCC) in PASMCs, play an important role in Ca2+ regulation and cell proliferation. The aim of the present study was to investigate the role of TRPC6 in monocrotaline (MCT)-induced pulmonary artery hypertension. Sprague-Dawley rats were randomly divided into normal control group and MCT group. In MCT group, pulmonary arterial hypertension was induced by a single intraperitoneal injection of MCT at a dose of 60 mg/kg. After 3 weeks, the right ventricular systolic pressure (RVSP) and the right ventricular mass index (RVMI) were measured. The lung sections were stained by HE and observed under light microscope. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot were performed to detect the expression of TRPC6 in rat pulmonary arteries. The 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced contractile tension of pulmonary arteries were measured by vascular ring tension analysis and the intracellular Ca2+ concentration ([Ca2+](i))of PASMCs was monitored using Fluo3-AM assay. The results showed that RVSP and RVMI markedly elevated in MCT group (P<0.01) in comparison to CON group. The thickness of pulmonary vascular smooth muscles was increased and the inner diameter of pulmonary arteries was diminished in MCT group. Though there was no significant difference in the levels of mRNA and protein of TRPC6 between CON and MCT groups, the application of OAG, which can directly activate ROCC, induced greater contraction tension of pulmonary arteries (P<0.01) and more Ca2+ entries in PASMCs (P<0.05) in MCT group compared to those in control group. These results indicate that MCT induces pulmonary artery hypertension and thus remodeling of the right ventricle and pulmonary arteries in rats. The expression of mRNA and protein of TRPC6 is not potentiated by MCT, but the TRPC6/ROCC-mediated Ca2+ entry in PASMCs and vascular tone of pulmonary arteries are significantly increased with MCT treatment.
Animals
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Calcium
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metabolism
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Hypertension, Pulmonary
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chemically induced
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metabolism
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physiopathology
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Male
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Monocrotaline
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pharmacology
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Muscle, Smooth, Vascular
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cytology
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metabolism
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Myocytes, Smooth Muscle
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cytology
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metabolism
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Pulmonary Artery
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cytology
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metabolism
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physiopathology
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RNA, Messenger
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genetics
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metabolism
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Rats
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Rats, Sprague-Dawley
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TRPC Cation Channels
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genetics
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metabolism
4.TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts.
Jin Seok WOO ; Chung Hyun CHO ; Do Han KIM ; Eun Hui LEE
Experimental & Molecular Medicine 2010;42(9):614-627
During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.
Animals
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Calcium/metabolism
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Calcium Channels/metabolism
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Calcium Channels, L-Type/genetics/metabolism
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Cations/metabolism
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*Cell Differentiation
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*Cell Proliferation
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Cells, Cultured
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Excitation Contraction Coupling
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Gene Knockdown Techniques
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Membrane Potentials
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Mice
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Muscle Fibers, Skeletal/*metabolism
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Muscle Proteins/metabolism
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Myoblasts, Skeletal/*metabolism
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Ryanodine Receptor Calcium Release Channel/metabolism
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Sarcoplasmic Reticulum/*physiology
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Synaptophysin/metabolism
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TRPC Cation Channels/genetics/*metabolism
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Transient Receptor Potential Channels/metabolism
5.Effect of TRPC6 knockdown on puromycin aminonucleoside-induced podocyte injury.
Xifeng SUN ; Yongli CHU ; Chun ZHANG ; Xiyun DU ; Fangfang HE ; Shan CHEN ; Pan GAO ; Jianshe LIU ; Zhonghua ZHU ; Xianfang MENG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(3):340-345
This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.
Animals
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Cell Membrane Permeability
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drug effects
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physiology
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Cell Survival
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drug effects
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physiology
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Cells, Cultured
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Gene Knockdown Techniques
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Mice
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Mice, Knockout
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Podocytes
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drug effects
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physiology
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Puromycin Aminonucleoside
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pharmacology
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TRPC Cation Channels
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genetics
;
metabolism
6.Recent advances in the research on mechanisms underlying podocyte-specific gene mutation-related steroid-resistant nephrotic syndrome.
Chinese Journal of Contemporary Pediatrics 2014;16(1):99- following 102
Steroid-resistant nephrotic syndrome poses a significant clinical challenge. Its pathogenesis has not been fully elucidated. In recent years, numerous studies have shown that podocyte-specific gene mutations may play important roles in the development of steroid-resistant nephrotic syndrome. Among the identified genes mutated in podocytes include NPHS2, NPHS1, WT1, TRPC6, MDR1, PLCE1, LMX1B, and LAMB2. This review aims to summarize the characteristics of these mutated genes in podocytes. The putative role for these podocyte-specific mutated genes in the pathogenesis, diagnosis, treatment and prognosis of steroid-resistant nephrotic syndrome is also discussed.
ATP Binding Cassette Transporter, Sub-Family B
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ATP-Binding Cassette, Sub-Family B, Member 1
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genetics
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Genes, Wilms Tumor
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Humans
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Intracellular Signaling Peptides and Proteins
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genetics
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LIM-Homeodomain Proteins
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genetics
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Membrane Proteins
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genetics
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Mutation
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Nephrotic Syndrome
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congenital
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genetics
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Podocytes
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metabolism
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TRPC Cation Channels
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genetics
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TRPC6 Cation Channel
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Transcription Factors
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genetics
7.Essential role of TRPC6 in the proliferation of gastric cancer and its mechanism.
Rong CAI ; Gang REN ; Xia DING ; Yi-zheng WANG ; Ye-ning JIN
Chinese Journal of Oncology 2012;34(8):577-581
OBJECTIVETo investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer.
METHODSThe expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 (DNC6). The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided.
RESULTSThe TRPC6 protein in the tumor tissues and para-tumor tissues was (21.60 ± 8.32)% versus (7.14 ± 2.24)%. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were (36.90 ± 1.13)%, (44.06 ± 2.17)%, (52.12 ± 2.76)% and (50.89 ± 1.97)%, respectively. The clone formation rates of control group and DNC6 group were (14.70 ± 3.00)% versus (43.80 ± 7.00)%. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the G(2)/M phase arrest was (20.34 ± 1.98)%, (24.31 ± 2.37)%, (27.70 ± 2.36)%, (35.10 ± 3.0)% in the DNC6 group and (18.40 ± 2.01)%, (18.0% ± 1.72)%, (17.50 ± 1.74)%, (16.80 ± 1.71)% in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors (P < 0.05).
CONCLUSIONIn the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G(2)/M phase transition.
Adenoviridae ; genetics ; Animals ; CDC2 Protein Kinase ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin B ; metabolism ; Cyclin-Dependent Kinases ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Recombinant Proteins ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; TRPC Cation Channels ; metabolism ; TRPC6 Cation Channel ; Transfection ; Tumor Burden