Study On The Mechanism Of Cetrimonium Bromide-Induced Autophagy And Apotosis In Hepatocellular Carcinoma Cells

Cetrimonium Bromide(CTAB)belongs to a group of quaternary ammonium compounds,which has an antibacterial effect.It has been reported that CTAB also has a potential antitumor role to head and neck cancer.However,the molecular mechanisms of CTAB in antitumor activity have not been widely studied.Autophagy and apoptosis are closely related to the occurrence and development of cancer.Inhibiting autophagy or promoting apoptosis could achieve the purpose of anti-tumor treatment.In this study,the effects of CTAB on autophagy and apoptosis in hepatocellular carcinoma(HCC)cells were studied by using molecular cell biology technique,which will reveal the molecular mechanism of CTAB anti-cancer effect.The results are as follows:For autophagy study:(1)The effect of CTAB on autophagy was studied using the stable cell line expressing EGFP-LC3 and Western blot.Accumulation of autophagosomes in CTAB treated cell was verified by detecting the GFP-LC3 puncts,and CTAB treatement significantly promoted the expression of LC3-? in a time dependent manner,indicating that CTAB increased the number of autophagosome.(2)CTAB also promoted the degradation of autophagic substrate p62,and in the presence of the lysosomal inhibitor Bafilomycin A1(BAF A1),CTAB further increased LC3B-? levels.The results indicate that CTAB enhances the autophagic flux in HCC cells.(3)To test whether CTAB activates TFEB,the effect of CTAB on the translocation of TFEB was detected using HeLa cells stably expressing TFEB-GFP,subcellular fractionation and Western blot.The results showed that CTAB treatment induced the nuclear translocation of TFEB-GFP,and increased the levels of endogenous TFEB in cell nucleus.(4)The effect of CTAB on the mTORC1 pathway was determinated by Western blot.The results showed that CTAB significantly inhibited the phosphorylation of mTOR and p70S6K.These data indicate that CTAB activates transcription factor TFEB by the inhibition of mTORC1 pathway.For apoptosis study:(1)The effects of CTAB on the cell viability and apoptosis were detected in HepG2 and 7721 hepatocellular carcinoma cells by MTT,Western blot and flow cytometry.The results showed that CTAB inhibited the viability of the two cancer cells in a dose dependent manner after 24 h and 48 h treatment.In contrast,CTAB treatment had little effect on the cell viability of normal liver cells(L02 cells).Concurrently,CTAB treatment induced apoptosis in HCC cells by activating caspase 9 and caspase 3.(2)Western blot showed that CTAB induced endoplasmic reticulum(ER)stress by increasing the protein level of p-eIF2a and ATF4,and activated caspase 12 to induce apoptosis.However,the levels of CHOP and BIM were not changed.These data indicate that CTAB treatment induces apoptosis by inducing ER stress and activating caspase 12.In conclusion,CTAB promotes autophagy in HCC cells by inhibiting mTORC1 and inducing the nuclear translocation of TFEB.Concurrently,CTAB induces apoptosis by inducing ER stress and activating caspase 12.This study clarifies the molecular mechanism of CTAB-induced autophagy and apoptosis,and provides a theoretical basis for the development of anti-tumor drugs with CTAB as the lead compound.