Expression And Function Of Temperature-sensitive TRP Ion Channels In Lung Cancer Cells

Lung cancer is one of the most fatal and common cancers all over the world.In the past 50 years,the incidence and mortality of lung cancer in male patients have been ranked the first place among malignant tumorsTransient receptor potential channels(TRPs)are a class of non-selective cation channels that on the surface of the cell membrane and can penetrate various cations such as Ca2+,Mg2+,Na+and K+.TRPs are known to be widely distributed,and 28 TRP channel proteins present in mammals can be divided into 6 families according to the amino acid sequence homology:TRPA(Ankyrin),TRPC(Canonical),TRPM(Melastatin),TRPML(Mucolipin),TRPP(Polycystin),TRPV(Vanilloid).Each subfamilies also have their members such as TRPV(Vanilloid)harboring 6 members of TRPV1,TRPV2,TRPV3,TRPV4,TRPV5 and TRPV6.These TRPs can be activated by a variety of exogenous and endogenous physical or chemical stimuli which make these ion channels as multifunctional cellular sensors.Temperature-sensitive TRP ion channels can be activated by temperatures and that are collectively referred to as Thermo-TRPs.Current research on Thermo-TRPs focuses on six temperature-sensitive TRP ion channels:TRPA1,TRPM8,TRPV1,TRPV2,TRPV3,and TRPV4.It has been reported that TRPA1、TRPM8 are abnormally expressed in lung cancer cells and are closely related to the physiological activities of cells.However,studies on Thermo-TRPs and lung cancer are still in their infancy.The present study attempts to screen and detect the differential expression of Thermo-TRPs in different lung cancer cells.Then the role of Thermo-TRPs in the development of lung tumor cells and its corresponding molecular mechanism will be further investigated.We hope the present study will provide a potential clinical therapeutic strategy in human lung cancer treatment.Objective:Determination of the expression profiles of Thermo-TRPs in lung tumor cells and fibroblasts;and investigation of the cell proliferation and cell cycle of lung tumor cells by TRPV2 channel activator 2-aminoethyl ester diphenylboronic acid(2-APB)and exploration of the role of Thermo-TRPs in the development of lung tumor cells and related molecular mechanisms.Methods:(1)Western Blot,real-time quantitative PCR combined with digital PCR experiments to detect the expression profiles of TRPA1,TRPM8,TRPV1,TRPV2,TRPV3,TRPV4 channel in human lung fibroblast epithelial cells(MRC5)and lung tumor cells(A549,H358,H460,H1299,H2170)from protein and mRNA levels.(2)Calcium imaging experiments verify the functional TRP ion channel in lung cancer cells.(3)Cell morphology analysis,cell proliferation assay(MTT)and colony formation assay to detect the effect of activation of TRPs in lung cancer cells.(4)Flow cytometry combined with western blotting to detect the effect of TRP ion channel activator on the cell cycle of lung cancer cells,and to find the mechanism underlying the activation of TRP ion channel to regulate cell cycle of lung cancer cells.Results:(1)Data from Western blot,real-time PCR and digital PCR showed that the expression of TRPA1 and TRPM8 ion channels were consistent with those reported by previous researchers;TRPV3 ion channel was not detected;The expression levels of TRPV4 ion channel in H1299 and H358 were higher than those in MRC5 cells;TRPV1 were highly expressed in A549,H358,H1299 and H2170 lung cancer cells;For TRPV2 ion channels,mRNA levels was only highly expressed in H1299 cells than normal lung fibroblasts and other lung cancer cells.(2)Calcium imaging experiments showed that 2-APB against H1299 cells dominated by TRPV2 ion channel function.(3)MTT tests showed that the concentration of 50 μM of 2-APB was sufficient to inhibit the cell proliferation of lung tumor cells H1299(P<0.05),and the inhibition was increased with dependent of drug concentration(IC50=50 μM);cell morphology analysis and colony formation experiments verify this conclusion.(4)Cell cycle was detected by flow cytometry.After 24 h treatment,2-APB arrested the cell cycle of H1299 cells in G1/S phase.Simultaneous detection of cell cycle-related cyclin was also confirmed the conclusion;after 24 h treatment,western blotting results showed that 2-APB regulates the cell cycle of H1299 by activating the ErK-AKT signaling pathwayConclusions:(1)Thermo-TRPs exhibited ectopic distribution in lung cancer cells.(2)TRPV2 is functional in H1299 lung cancer cells.(3)2-APB inhibits cell proliferation of H1299 cells via activation of TRPV2 ion channel.(4)2-APB-TRPV2 regulates the cell cycle of H1299 cells by activating the ErK-AKT signaling pathway.