Effects Of Shikonin On The Biological Behavior Of Fadu In Human Hypopharyngeal Carcinoma Cells

Background and Objectives: Head and neck tumors are more common in human tumors.As a rare malignant tumor,hypopharyngeal carcinoma accounts for about 3% to 5% of head and neck malignancies.The biological characteristics of hypopharyngeal cancer are poor,the concealed location is difficult to detect,and cervical lymph node metastasis is extremely easy to occur.The prognosis is poor and the survival rate is low.A large number of studies have shown that shikonin can induce necrotic apoptosis in esophageal cancer,glioma,liver cancer and other tumor cells.The advantage of this effect is that it can effectively avoid drug resistance during chemotherapy.problem.However,the effect of shikonin on hypopharyngeal cancer cells has not been explored.This experiment mainly explores the effect of shikonin on the death form of hypopharyngeal cancer cells(Fadu)and its possible mechanism,providing a new way for clinical treatment of hypopharyngeal cancer.Methods: Different concentrations of shikonin were used to treat Fadu cells for 24,48,and 72 h,respectively,and the cell survival rate was measured.The 0 μmol / L group was the blank control group.MTT assay was used to detect the effect of shikonin on Fadu cell proliferation;Transwell chamber method was used to detect the effect of shikonin on Fadu cell invasion and migration;wound healing experiments were performed to test the effect of shikonin on Fadu cells for 12 h,24h,48 h cell healing Ability change;JC-1 single staining kit to detect changes in mitochondrial membrane potential of shikonin on Fadu cells 18h;Annexin V-FITC / PI double staining method to detect different concentrations of shikonin(0,2,4,6 μmol / L)Apoptosis rate of Fadu cells for 24 h;Western blot was used to detect the relative expression levels of Bcl-2,Bax,RIP1,and RIP3 proteins in Fadu cells treated with shikonin.A model of xenograft tumor of nude mice with Fadu was established in the control group,high concentration group and low concentration group.influences.The tumor was dissected and immunohistochemical experiments were performed to detect the changes of RIP3 protein in nude mice after different shikonin concentration groups acted on tumors in nude mice.Results: 1.MTT results showed that shikonin inhibited the proliferation of Fadu cells in a time-and dose-dependent manner(P <0.05).IC50 at 24 h,48 h,and 72 h were 6.012,4.120,and 3.939 μmol / L,respectively.2.The results of the Transwell chamber method showed that Shikonin at 0,1,2,and 4 μmol / L acted on Fadu cells for 24 h.As the drug concentration increased,the number of transmembrane cells in Fadu cells gradually decreased(P <0.05);3.The results of wound healing experiments showed that Shikonin at 0,1,2,and 3 μmol / L acted on Fadu cells for 0,6,12 and 24 hours.The degree of scratch healing decreased as the concentration of shikonin increased.4.JC-1 single staining results showed that as the concentration of shikonin increased,the proportion of premature declining cells also gradually increased;5.Annexin V-FITC / PI double staining method showed that shikonin could induce Fadu cells in a concentration-dependent manner Apoptosis;6.Western blot results showed that shikonin(2,4,6 μmol / L)can down-regulate Bcl-2 and up-regulate Bax,induce Fadu cell apoptosis,and up-regulate RIP1 and RIP3 proteins to induce necrosis of hypopharyngeal cancer cells.Apoptosis;7.The model of hypopharyngeal cancer cell Fadu nude mice xenograft tumors showed that nude mice had tumors with different shikonin concentrations after tumor formation.After intraluminal injection,as the drug concentration increased,the tumor volume gradually decreased and the growth rate slowed.8.According to the results of immunohistochemistry,the expression of RIP3 in tumor sections increased with the increase of drug concentration.Conclusion: Shikonin induces Fadu cell apoptosis and necrotic apoptosis,inhibits Fadu cell invasion,migration and healing ability,thereby reducing tumor growth rate and weakening tumor metastasis ability,thereby achieving the purpose of anti-tumor.The mechanism of apoptosis may be to down-regulate the expression of apoptosis inhibitory protein Bcl-2 and up-regulate the expression of apoptosis-inducing protein Bax.The mechanism of necrotic apoptosis may be to up-regulate the expression of RIP1 and RIP3 proteins.