Studies On Anti-tumor Effect Of Brucine And Its Structural Analogues Targeting HERG Channels

HERG gene is one of the family members of the evolutional conservative voltage-gated extroversion potassium channel ether-a-go-go(eag)family.The HERG potassium channel encoded by the HERG is a special type of potassium channel,which has the introversion rectifying characteristics.Recently,tumor studies have begun to redefine the role of voltage-sensitive potassium channels in the malignant phenotype and progression of tumors.Studies have shown that the HERG potassium channel is selectively expressed in tumor cells from various tissues,and is involved in the regulation of tumor cell cycle,proliferation and apoptosis.The expression level of HERG protein may also become a biomarker which is associated with the malignant transformation of tumor.Therefore,the HERG potassium channel is expected to be a new therapeutic target for anti-tumor therapy.As a traditional Chinese medicine,Strychnos nux-vomica Linnis used to treat diseases such as cancer and rheumatic pain.Among them,strychnine and brucine are the two main alkaloids of seeds of Strychnos nux-vomica Linn.Our previous studies have shown that brucine and strychnine have a strong blocking effect on the HERG channel,and their IC50 values are 44.5 uM and 25.9 uM respectively.In this study,we discussed the anti-tumor effect of brucine and strychnine,and its possible mechanism.It would provide a new vision for anti-tumor therapy.We first examined the effects of brucine and strychnine on several tumor cells(HT29,PC9,A549)which express different HERG protein level.In addition,HT29 originates from human colon cancer cells,PC9 and A549 originates from human lung cancer cells.The expression level of HERG protein was A549<PC9<HT29.The result shows that brucine and strychnine has significant inhibitory effect against HT29,PC9 and A549 cell proliferation.Brucine inhibited the three cancer cell proliferation in a dose-dependent manner.with the IC50 value of 0.421mM,0.681mM,and 0.738mM on HT29,PC9,and A549 for 48h,respectively.And the the IC50 value for strychnine on HT29,PC9,and A549 for 48h were 0.363mM,0.712mM,and 0.661mM respectively.The results indicated that the inhibitory effect against cell proliferation was in accordance with the expression levels of HERG protein in the cell line,the higher level of HERG protein expression,the stronger inhibitory effect against cell proliferation.In order to investigate the mechanism of brucine and strychnine on the proliferation inhibition of tumor cells,the cell cycle distribution of HT29 and A549 were studied by flow cytometry.The results showed that compared with the control group,brucine could increase the proportion of G1 phase in HT29 cells and reduce the proportion of cell S and G2/M phase.At the same time,strychnine could decrease the ratio of S phase of HT29 cells,and the proportion of G1 and G2/M phase was not affected.Compared with the control,brucine induced a decrease in the proportion of G1 phase and an increase in the proportion of S phase on A549 with a high concentration of 0.75mM,but there was no difference in the proportion of cell G2/M phase.The results suggested that brucine and strychnine could cause Glcell cycle arrest in HT29 cells,but had no significant effect on the cell cycle of A549.To further explore the apoptotic mechanism of HT29,PC9,and A549 cells induced by brucine,western blot was performed to examine the protein expression level of HERG in brucine-treated cells.The result shows that brucine had no significant effect on protein expression level of HERG,suggesting that the anti-tumor effect of brucine and strychnine might be mediated by blocking the HERG channel current.Previous studies have found that the brucine N-oxide and strychnine N-oxide have no inhibitory effect on the HERG currents.We examined the anti-tumor effect of brucine N-oxide and strychnine N-oxide,and found that it had no significant anti-tumor effect on the above three tumor cells.Is the anti-tumor effect of brucine and strychnine induced by cell membrane depolarization?We further examined the effect of brucine and strychnine on the membrane potential of HEK293T cells which were transfected with HERG channel.It was found that the membrane potential was depolarized from-42mv to-23mv by brucine in the concentration of 200 uM,and the membrane potential restored to-42mV when washout with the extracellular solution.Similarly,the membrane potential was depolarized from-40mv to-21mv by strychnine in the concentration of 200 uM,and restored to-39.5mV when washout with the extracellular solution.In order to further confirm the anti-tumor effect of brucine and strychnine are mediated by HERG channels,we constructed five HERG channel mutant(HERG-S624A,HERG-V625A,HERG-G648A,HERG-Y652A,HERG-F656A).Alanine-mutagenesis identified Y652 is the most important residue for strychnine and brucine binding to HERG channel.Y652A mutation increased the IC50 for strychnine and brucine by 21.64-fold and 29.78-fold that of WT IHERG,respectively.Next step we proposed to build HERG-HEK293 and HERG-Y652A-HEK293 recombinant cell lines,and examine the inhibitory effect against HT29,PC9 and A549 cell proliferation and the results may provide further evidence of anti-tumor effect of brucine and its analogues targeting HERG channels.