Study On The Autophagy Of NRK Cells And Human Colon Cancer Cells Induced By Harmine

Objective: Colon cancer is one of the most common malignant tumors in digestive system.There are many studies on the pathogenesis of colon cancer,but the pathogenesis is still not clear.Therefore,further research is needed to find new treatments.In recent years,the role of autophagy in the development and progression of colon cancer has received increasing attention and the research on the anti-tumor effect of natural drug monomer components through autophagy pathway is also more and more in-depth.p62 is a key protein involved in autophagy.To investigate the relationship between p62 protein,autophagy and colon cancer,has a positive meaning for elucidating pathogenesis of colon cancer and finding potential therapeutic strategies.Peganum harmala is the traditional medicinal herb of Uygur medicine in Xinjiang.Total alkaloids of harmaline and harmine extracted from Peganum harmala are rich in biological activity.To explore their molecular mechanism of autophagy induction of cells,which is of great significance for the development of new anti-colon cancer treatment drugs.Method: 1)Clinical data of 60 patients with sporadic colon cancer were collected,including their gender,age,ethnicity,primary tumor site,tumor size,histological type and degree of differentiation of tumor,tumor invasion depth and lymph node metastasis.Their surgical specimens were also collected.The expression of p62 protein in tumor tissues and adjacent normal tissues was detected by immunohistochemistry,RT-qPCR and western blot,and combined with the clinical data of patients for statistical analysis.We investigated the level changes of p62 in human colon cancer and their effects on prognosis.2)The GFP-LC3 plasmid was transfected into normal rat kidney cells(NRK cells)to construct NRK cells stably expressing GFP-LC3 fusion protein,then,the GFP-LC3-expressed NRK cells were treated with different concentrations of total alkaloids of harmaline or harmine and the number of autophagosomes formed in the cells was observed under a laser confocal microscope.The expression of LC3 and p62 in NRK cells which were treated with total alkaloids of harmaline or harmine was detected by western blot.And the expression of p62 in NRKcells which were treated with the combination of harmine and autophagy inhibitor bafA1 or 3-MA was also investigated.The autophagic flux was observed in both GFP-LC3 and Lamp-1-mCherry expressed NRK cells which were treated with total alkaloids of harmaline or harmine.Transmission electron microscope was used to observe the autophagosomes formed in NRK cells induced by total alkaloids of harmaline and harmine.The expression of p-p70s6 k was examined through western blot to verify the signal pathway of NRK cells autophagy induced by Harmonia.3)The expression of LC3 in SW480 cells which were treated with different concentrations of harmine was detected by western blot.And the expression of LC3 in SW480 cells which were treated with the combination of harmine and bafA1 or 3-MA was also investigated.The inhibitory effect of harmine on the proliferation of SW480 cells was investigated by clone formation inhibition experiment,and the effect of autophagy in this process was investigated by using bafA1.Results: 1)The transcription and expression level of p62 in colon cancer tissues were higher than that in adjacent normal tissues(P < 0.05).The expression of p62 in colon cancer tissue had no significant correlation with the patient's gender,age,ethnicity,primary tumor site,tumor size,histological type and degree of differentiation of tumor,tumor invasion depth,but related to lymph node metastasis(P < 0.05).In patients with high expression of p62,their lymph node metastases increased.2)The number of autophagosomes increased in NRK cells which were treated with the total alkaloids of harmaline or harmine,and the number increased with the increase of drug concentration.The fusion of autophagosomes with lysosomes was not inhibited,and autophagic flux within the cells was unobstructed.The expression of LC3-? was increased and the expression of p62 was decreased.The autophagy induced by harmine could be inhibited by the bafA1 or 3-MA.The expression of p-p70s6 k did not decrease,suggesting that mTOR was not inhibited by total alkaloids of harmaline or harmine.3)The expression of LC3-? increased in SW480 cells which were treated with harmine.This autophagy can be inhibited by bafA1.The harmine had a significant inhibitory effect on the clonal formation of SW480 cells compared with the control group,and the combination of bafA1 attenuated the inhibitory effect.It is suggest that inhibition of autophagy weakened Harmine's anti-tumor effect.Conclusion: 1)The abnormal transcription and expression levels of p62 protein in colon cancer tissues were associated with lymph node metastasis,suggesting that autophagy may be involved in the progression of colon cancer.2)From the two aspects of morphology and function,it was confirmed that the total alkaloids of harmaline and harmine could induce the autophagy activity of NRK cells.Both totalalkaloids of harmaline and harmine-induced cell autophagy may be non-mTOR-dependent.3)Harmine can induce autophagy in SW480 cells,and the autophagy can be inhibited by bafA1.Harmine has obvious inhibitory effect on the proliferation of SW480 cells,and autophagy may be one of its anti-tumor mechanisms.