Honokiol Inhibits Colorectal Cancer By Targeting TMEM16A Ca²⁺-activated Cl^- Channels

Objective:Colorectal cancer is one of the most common malignancies in the world.According to cancer statistics in the United States,colorectal cancer accounts for more than 100,000 new cases every year,ranking the third among all malignancies in the world,and the second among all cancers in terms of mortality.TMEM16A calcium activated chlorine channel is highly expressed in many tumors,which promotes the occurrence and development of tumors.Recent studies have found that the overexpression of TMEM16a can promote the proliferation,migration and invasion of colorectal cancer.Targeting TMEM16A provides a novel and promising therapeutic strategy for the treatment of colorectal cancer.Therefore,the discovery of new TMEM16A inhibitors is of great significance for the treatment of colorectal cancer.Honokiol is one of the main active components isolated from Magnolia officinalis,which has many pharmacological effects.Although it has been found that honokiol can inhibit the proliferation and tumor growth of colorectal cancer cells,the molecular target and anti-proliferation mechanism of honokiol remain unclear.The purpose of this study was to investigate the effect of honokiol on TMEM16A calcium-activated chlorine channel and the effect of honokiol on the proliferation of colorectal cancer cells by targeting TMEM16A channel.Methods:1.Transfect the TMEM16A overexpression plasmid into HEK293 cells,and record the currents of HEK293 cells at 0.2μM and 1μM Ca²⁺after transfection in the whole-cell patch clamp recording mode.Honokiol of different concentrations（0.1-50μM）was added to detect the inhibitory effect of honokiol on TMEM16A current.The Inside-out patch clamp recording mode was used to record the current of HEK293 cells transfected with TMEM16A overexpression plasmid at 1μM Ca²⁺.10μM honokiol was added to detect the direct inhibitory effect of honokiol on TMEM16A current.The whole cell patch clamp recording mode was used to record the current of HEK293 cells transfected with TMEM16A overexpression plasmid at0.2μM Ca²⁺.Add 10μM honokiol,wash after the inhibition is complete,and observe whether the effect of honokiol on TMEM16A is reversible.The TMEM16B overexpression plasmid was transfected into HEK293 cells,and the whole cell patch clamp recording mode was used to record the current of HEK293 cells at 1μM Ca²⁺after transfection.Different concentrations（10-400μM）of honokiol were added to detect the inhibitory effect of honokiol on TMEM16B current.2.Compare the amino acids of each transmembrane region of TMEM16A and TMEM16B,find out the amino acids with large differences in chemical properties,and select some amino acids from them to use site-directed mutagenesis to mutate into the corresponding amino acids of TMEM16B.The TMEM16A mutant plasmid was transfected into HEK293 cells,10μM honokiol was added,and the whole cell patch clamp recording mode was used to detect the inhibitory effect of honokiol on the currents of different TMEM16A mutants at 0.2μM Ca²⁺.Using site-directed mutagenesis,the amino acids different from TMEM16B in the second transmembrane region of TMEM16A were mutated into amino acids corresponding to TMEM16B,and the positively charged amino acids in the second transmembrane region of TMEM16A were mutated to alanine.The TMEM16A mutant plasmid was transfected into HEK293 cells,10μM honokiol was added,and the whole cell patch clamp recording mode was used to detect the inhibitory effect of honokiol on the current of different TMEM16A mutants at 0.2μM Ca²⁺.The three-point mutation of amino acids R429,K430 and N435 on TMEM16A was carried out by site-directed mutagenesis.The three-point mutation of TMEM16A R429A/K430L/N435G was transfected into HEK293 cells,and the inhibition of R429A/K430L/N435G by different concentrations（1-400μM）of honokiol at 0.2μM Ca²⁺was detected by whole-cell patch clamp recording mode.The two-point mutation of amino acids L372 and G377on TMEM16B was carried out by site-directed mutagenesis.The two-point mutation of TMEM16B,L372K/G377N,was transfected into HEK293 cells.The inhibitory effect of different concentrations（1-100μM）of honokiol on L372K/G377N current at1μM Ca²⁺was detected by whole-cell patchpin recording mode.3.Whole-cell patch clamp was used to record the calcium-activated chloride current of SW620 cells under different Ca²⁺（0-1μM）.The relative expression of m RNA in SW620 cells transfected with TMEM16A-sh RNA was detected by q RT-PCR technology.Western blot technology was used to detect the expression of TMEM16A protein after SW620 cells were transfected with Scrambled sh RNA and TMEM16A-sh RNA.TMEM16A-sh RNA was used to knock down the TMEM16A protein,and the whole-cell patch clamp recording mode was used to record the current of SW620 cells transfected with Scrambled sh RNA and TMEM16A-sh RNA at1μM Ca²⁺.The whole cell patch clamp recording mode was used to record the current of SW620 cells at 1μM Ca²⁺,and 10μM T16Ainh-A01 or Ani9 was added to detect the inhibitory effect of T16Ainh-A01 and Ani9 on the calcium-activated chloride current of SW620 cells.Western blot was used to detect the expression of TMEM16A protein in NCM460 and SW620 cells and the expression of TMEM16A protein in NCM460 cells transfected with TMEM16A-Vector,WT TMEM16A and the reduced channel functionΔ₄₄₄EEEEEAVKD₄₅₂plasmids for 48 h.CCK-8 was used to detect the cell viability of NCM460 cells transfected with TMEM16A-Vector,WT TMEM16A andΔ₄₄₄EEEEEAVKD₄₅₂plasmids for 48 h,the cell viability of SW620cells transfected with Scrambled sh RNA and TMEM16A-sh RNA for 48 h,and the cell viability of SW620 cells with 10μM T16Ainh-A01 or Ani9 after 48 h.The whole-cell patch clamp recording mode was used to record the calcium-activated chloride current of SW620 cells at 1μM Ca²⁺,and different concentrations（0.1-50μM）of honokiol were added to detect the inhibition of honokiol on SW620endogenous TMEM16A current effect.CCK-8 was used to detect the inhibitory effect of honokiol at different concentrations（1-20μM）on the proliferation of SW620 cells.CCK-8 was used to detect the inhibitory effect of 10μM honokiol on the proliferation of SW620 cells transfected with Scrambled sh RNA and TMEM16A-sh RNA.CCK-8was used to detect the inhibitory effect of honokiol at different concentrations（1-20μM）on the proliferation of NCM460 cells.Western blot was used to detect the expression of TMEM16A protein in NCM460 cells transfected with TMEM16A-Vector,WT TMEM16A and R429A/K430L/N435G plasmids for 48 h.CCK-8 was used to detect the inhibitory effect of 10μM honokiol on the proliferation of NCM460 cells transfected with TMEM16A-Vector,WT TMEM16A and R429A/K430L/N435G plasmids.Results:1.In whole-cell mode,Honokiol inhibits TMEM16A current heterologously expressed in HEK293 cells transfected with TMEM16A overexpressing plasmid in a concentration-dependent manner.IC₅₀values at 0.2μM and 1μM Ca²⁺are 5.29±0.95μM and 5.83±0.37μM,respectively.In Inside-out mode,10μM honokiol at 1μM Ca²⁺can significantly inhibit the TMEM16A current heterologously expressed by HEK293 cells.In whole-cell mode,adding 10μM honokiol at 0.2μM Ca²⁺can significantly inhibit the TMEM16A current heterologously expressed by HEK293cells,and the current can be restored to about 80%of the previous value after washing.In whole-cell mode,honokiol has a weak inhibitory effect on the heterologous TMEM16B current expressed by HEK293 cells at 1μM Ca²⁺,with an IC₅₀value of68.71±2.05μM.2.At 0.2μM Ca²⁺,the inhibitory effect of 10μM honokiol on the single-point mutation TMEM16A current of R429A,K430L and N435G was significantly weaker than that of WT TMEM16A.At 0.2μM Ca²⁺,the inhibitory effect of honokiol on R429A\K430L\N435G current was significantly weaker than that of WT TMEM16A,with an IC₅₀value of 40.01±3.33μM.Compared with WT TMEM16B,the inhibitory effect of honokiol on L372K\G377N current at 1μM Ca²⁺was significantly enhanced,with an IC₅₀value of 16.53±1.23μM.3.Calcium-activated chloride current was detected in SW620 cells at 1μM Ca²⁺in whole-cell mode.After knocking down the TMEM16A protein with TMEM16A-sh RNA,the expression of TMEM16A m RNA and protein were significantly reduced.After knocking down TMEM16A in SW620 cells,the detected calcium-activated chloride current was significantly reduced.In whole-cell mode,the TMEM16A specific inhibitors T16Ainh-A01 and Ani9 of 10μM can significantly inhibit the calcium-activated chloride current in SW620 cells at 1μM Ca²⁺.The protein expression of TMEM16A in SW620 cells was significantly higher than that in NCM460.The expression of TMEM16A protein in NCM460 cells transfected with WT TMEM16A and the reduced channel functionΔ₄₄₄EEEEEAVKD₄₅₂plasmids was basically the same.Compared with TMEM16A-Vector,the proliferation ability of NCM460 cells transfected with WT TMEM16A plasmid was significantly enhanced,while the proliferation ability of NCM460 cells transfected withΔ₄₄₄EEEEEAVKD₄₅₂plasmid did not change significantly.The proliferation ability of SW620 cells knocked down by TMEM16A-sh RNA was significantly weakened,and the proliferation ability of SW620 cells was significantly reduced by the application of TMEM16A specific inhibitors T16Ainh-A01 and Ani9.In whole-cell mode,Honokiol inhibits the endogenous TMEM16A current in SW620 cells in a concentration-dependent manner at 1μM Ca²⁺,with an IC₅₀value of 4.78±0.47μM.Honokiol inhibited the proliferation of SW620 cells in a concentration-dependent manner.After knocking down the TMEM16A protein with TMEM16A-sh RNA,the inhibitory ability of 10μM honokiol on the proliferation of SW620 cells was significantly weaker than that of Scrambled sh RNA.Honokiol’s ability to inhibit the proliferation of NCM460 is weak.The expression of TMEM16A protein in NCM460cells transfected with WT TMEM16A and R429A\K430L\N435G plasmids was basically the same.After transfection with WT TMEM16A plasmid,the inhibitory ability of 10μM honokiol on the proliferation of NCM460 cells was significantly stronger than that of TMEM16A-Vector,while after transfection with R429A/K430L/N435G plasmid,honokiol’s inhibitory effect on the proliferation of NCM460 cells is weaker.Conclusion:1.Honokiol inhibits TMEM16A calcium-activated chloride channel current;2.R429、K430 and N435 are the critical amino acids that honokiol inhibits TMEM16A;3.Honokiol inhibits the proliferation of colorectal cancer cells by targeting TMEM16A.