Study On The Mechanism Of The Effect Of Sulforaphane On EIF4F Translation Initiation Complex In Colorectal Cancer

Objective Colorectal cancer is a malignant tumor that occurs in the mucosal epithelium and glands of the colon and rectum,with high morbidity and mortality,with a cumulative liver metastasis of more than 15%,which brings heavy health pressure to many countries.At present,the main treatment for colorectal cancer is surgery plus radiotherapy and chemotherapy,but the search for new treatments has never stopped.The main purpose of this study is to study the effect of sulforaphane on eIF4F translation initiation complex in colorectal cancer cell line and to study the related mechanism in order to provide theoretical basis for the treatment of colorectal cancer.Methods（1）A total of 12 patients with colorectal cancer in the second affiliated Hospital of Anyi University from 2015 to 2017 were collected and made into tissue microarray.Immunohistochemical staining was used to detect the expression and optical density of eIF4E,P-eIF4E,eIF4G,4EBP1 and P-4EBP1 in tumor and paracancerous tissues.（2）Twelve healthy male nude mice were randomly divided into experimental group（n=6）and control group（n=6）.Both the experimental group and the control group were injected with cultured and well-mixed colorectal cancer cells（HT-29）0.1ml under the axilla.From the third day,the experimental group was intraperitoneally injected with sulforaphane(0.5mg kg^-1)dissolved in DMSO,and the control group was injected with solvent DMSO(0.5mg kg^-1)for 14 days.After 14 days,the nude mice were killed,the subcutaneous transplanted tumor was removed,the volume was measured and stored in refrigerator at-80℃.（3）HE staining was used to observe the morphology and structure of subcutaneous tumors in nude mice.（4）Immunohistochemistry was used to detect the expression of AKT,P-AKT,P-mTOR,4EBP1,P-4EBP1,eIF4E,P-eIF4E and eIF4G in subcutaneous transplanted tumor.（5）Culture of colorectal cancer cell line HT-29.（6）the effect of sulforaphane on HT-29activity was detected by MTT.（7）Westernblot method was used to detect the effect of sulforaphane on the protein expression of PI3K,AKT,P-AKT,mTOR,P-mTOR,4EBP1,P-4EBP1,eIF4E,P-eIF4E and eIF4G in HT-29 cells.Results（1）Immunohistochemical staining of human colorectal cancer microarray showed that eIF4E,P-eIF4E,eIF4G,4EBP1 and P-4EBP1 in tumor tissues were significantly higher than those in paracancerous tissues.（2）in the model of subcutaneous tumor formation in nude mice,sulforaphane can slow down the growth of subcutaneous transplanted tumor in nude mice.（3）HE staining showed that sulforaphane could increase the number of cell vacuoles in subcutaneously transplanted tumor.（4）Immunohistochemical staining showed that sulforaphane decreased the expression of AKT,4EBP1,P-4EBP1,eIF4E,P-eIF4E and eIF4G,but increased the expression of P-AKT and P-mTOR.（5）MTT results showed that sulforaphane treatment for 48 hours could significantly inhibit the activity of HT-29 cells in a dose-dependent manner.（6）sulforaphane(15,30μmol·L^-1)decreased the expression of AKT,mTOR,4EBP1,P-4EBP1,eIF4E and P-eIF4E protein and increased the expression of P-AKT and P-mTOR protein in HT-29 cells for 48 h,but had no significant effect on the expression of PI3K.Conclusions eIF4F translation initiation complex plays an important role in the occurrence and development of colorectal cancer.Sulforaphane can affect the up-regulation of eIF4F translation initiation complex in colorectal cancer cells through PI3K/AKT/mTOR/4EBP1 signal pathway,which related to the phosphorylation of 4EBP1.