Study On The Mechanism Of Procaine Hydrochloride Inhibiting The Proliferation And DNA Methylation Of Human Colon Cancer Cells

Objective:DNA methylation is one of the molecular mechanisms of epigenetics,which plays an important role in the occurrence and development of colorectal cancer.Hypermethylation of CpG islands in the promoter region of tumor suppressor genes leading to transcriptional silencing is currently considered to be a common feature of tumorigenesis.Because DNA methylation is reversible,which is different from classical genetics,it is possible to reverse the process of DNA methylation by applying a DNA methyltransferase inhibitor to reactivate the silenced tumor suppressor genes,thereby to inhibit tumor growth.This is very significant for the treatment of tumors.Decitabine has the strongest demethylation effect among several nucleoside inhibitors,so it is used for the treatment of patients with hematological malignancies such as primary and refractory recurrent myelodysplastic syndromes.However,it can cause severe gastrointestinal reactions and bone marrow toxicity in patients,which limits its widespread clinical application.Procaine hydrochloride（PCA）,as a commonly used local anesthetic in clinical practice,has been found to be a DNA demethylating agent in recent years,and it has growth inhibitory effects on human breast cancer MCF-7 cells and gastric cancer SGC-7901 cells.However,the effects of procaine hydrochloride on the DNA methylation of colon cancer cells and their biological functions are rarely reported.In this study,human colon cancer DLD-1 and HCT116 cells were used as models to analyze the effects of procaine hydrochloride on the methylation status and expression level of tumor suppressor gene Septin9,and to explore the molecules that procaine hydrochloride inhibited tumor cell proliferation Mechanism,to provide theoretical basis and experimental basis for the clinical treatment of procaine hydrochloride in colorectal cancer.Methods:（1）Procaine hydrochloride was prepared at different concentrations（1.0,1.5,2.0 and 2.5mmol/L）for colon cancer DLD-1,HCT116 cells for 24,48 and 72 hours,respectively.Then MTS was used to detect the inhibitory effect of procaine hydrochloride on colon cancer DLD-1 and HCT116 cells.（2）Observed the morphological changes of colon cancer DLD-1 and HCT116 cells before and after treatment with procaine hydrochloride through an inverted microscope.（3）Flow cytometry was used to detect the effects of procaine hydrochloride on the apoptosis and cell cycle of colon cancer DLD-1 and HCT116 cells.（4）Transwell test was used to detect the effect of procaine hydrochloride on the migration ability of colon cancer DLD-1 and HCT116 cells.（5）Bisulfite genomic sequencing（BGS）was used to detect the methylation status of Septin9 gene before and after procaine hydrochloride affected colon cancer DLD-1 and HCT116 cells.（6）Realtime PCR（RT-PCR）was used to detect the expression level of Septin9 gene before and after procaine hydrochloride treated colon cancer DLD-1 and HCT116 cells.Results:（1）Procaine hydrochloride can inhibit the proliferation of colon cancer DLD-1and HCT116 cells,and its growth inhibitory effect has a concentration-dependent effect and a time-dependent effect.（2）After procaine hydrochloride treatment,the volume of DLD-1 cells shrinks and the growth density decreases;HCT116 cells shrink into clumps,appear cavitation phenomena and shrink from the surface on which they grow.（3）Compared with the blank control group,procaine hydrochloride treatment can significantly induce the apoptosis of DLD-1 and HCT116 cells,and block the growth of DLD-1 and HCT116 cells at G₂/M period.The differences were statistically significant（P<0.05）.（4）Transwell experiments showed that the number of transmembrane cells of DLD-1and HCT116 cells was significantly lower than that of the blank control group after procaine hydrochloride treatment.The differences were statistically significant（P<0.05）.（5）After procaine hydrochloride acts on DLD-1 and HCT116 cells,the degree of methylation of CpG islands in the promoter region of Septin9 gene decreased,and the mRNA expression level of Septin9 gene increased significantly.The differences before and after treatment were statistically significant（P<0.05）.Conclusion:Procaine hydrochloride can inhibit the proliferation of colon cancer DLD-1 and HCT116 cells by demethylating the Septin9 gene and up-regulating the expression level of Septin9 mRNA.This suggests that procaine hydrochloride is expected to be a safe candidate for colorectal cancer treatment based on epigenetics.