The Mechanistic Study On The Action Of Two Small Molecule Drugs In Targeted Cancer Therapy

Part I: The mechanism of FGF19 gene in drug resistance of hepatocellular carcinoma cells.Hepatocellular carcinoma(HCC)is a tumor with high mortality and strong malignancy.In China,patients with liver cancer account for about half the world's mortality.Such a severe situation has brought a heavy burden to China's economic development.Now,sorafenib as the clinical treatment of advanced liver cancer has the ideal effect,but with the extension of the time of medication,the patient will have the problem of sorafenib resistance.The emergence of drug-resistant problem also caused the poor prognosis of liver cancer patients,this problem seriously hindered the sorafenib in clinical application.Therefore,it is of great significance to study the resistance mechanism of sorafenib to improve its anti-tumor effect.Fibroblast growth factor 19(FGF19)is a member of the FGF family,which is located in the chromosome 11q13.1,and its encoded protein has 216 amino acids.The high expression of FGF19 gene is closely related to the development of multiple tumors,such as liver cancer,lung cancer,colon cancer,breast cancer,thyroid cancer,prostate cancer and cholangiocarcinoma.Relevant studies have shown that the expression of FGF19 is associated with the development of liver cancer and poor prognosis.In the previous research of this group,FGF19 was found to be an oncogene in liver cancer,and its expression level was closely related to the proliferation,migration and invasion of liver cancer.On the basis of this study,this work first revealed the important role of oncogene FGF19 in the drug resistance of sorafenib.The main results are as follows:1.FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib.When FGF19 was overexpressed,sorafenib could inhibit the production of reactive oxygen species and apoptosis in HCC.In contrast,when FGF19 or its receptor FGFR4 expression was absent,sorafenib induced increased levels of ROS production and enhanced apoptosis rate in hepatocellular carcinoma cells.In addition,knockdown of FGF19 in sorafenib-resistant HCC cells significantly enhanced the sensitivity to sorafenib.The results of this work suggest for the first time that inhibition of FGF19/FGFR4 signaling pathway may serve as a new measure to address the resistance of sorafenib to liver cancer.Importantly,ponatinib as the third generation of chronicmyelogenous leukemia inhibitor block FGF19 / FGFR4 signaling pathway to overcome sorafenib hepatocellular carcinoma drug-resistant problem provides a new method of treatment,this mainly by sorafenib treatment liver cancer cells increased ROS related to apoptosis.Co-treatment of ponatinib and sorafinib may represent an effective therapeutic approach for eradicating HCC.2.Implications of FGF19 on sorafenib-mediated nitric oxide production in hepatocellular carcinoma cells.Hepatocellular carcinoma is a tumor that is mainly derived from liver cells,and its mortality rate ranks third in the world.Previous work has shown that FGF19,an oncogenic driver,acts as a negative regulator of the therapeutic efficacy of the tyrosine kinase inhibitor sorafenib in HCC cells.The FGF19-mediated mechanism affecting sorafenib treatment,however,still remains to be resolved.Here,we hypothesize that the FGF19/FGFR4 axis may affect the effectiveness of sorafenib in the treatment of HCC.When overexpression FGF19 can reduce the NO level of sorafenib induced cell production,and can reduce the proliferation ability of hepatocellular carcinoma cells.In contrast,either FGF19 silencing or knockout of its receptor FGFR4 sensitized HCC cells to sorafenib through the induction of NO generation.At the same time,the study found that inactivation of FGFR4 by BLU9931 enhanced the sensitivity of HCC cells to sorafenib.The experiment data shown that the FGF19/FGFR4 axis may play a critical role in the effects elicited by sorafenib in HCC cells.Blocking the FGF19/FGFR4 axis may provide novel opportunities to improve the efficacy of sorafenib in the treatment of patients with HCC.Part ?: The new anticancer mechanism of small molecular compound CYT997.Head and neck squamous cell carcinoma(HNSCC)is the sixth most common cancer worldwide with 650,000 new cases and 350,000 deaths every year.More than 90 percent of head and neck tumors are squamous cell carcinoma,so they are also known as head and neck squamous cell carcinoma.It comes from epithelial cells in the digestive tract and respiratory tract.Head and neck cancer is closely related to environmental and lifestyle factors,including smoking,drinking,UV exposure,especially chemical exposure and human papillomavirus(HPV)infection.The main pathogenic factors are smoking and excessive alcohol consumption.This type of cancer is often invasive,and most patients have advanced and secondary tumors whendiagnosed.Head and neck cancer research mainly involves the mouth,mouth swallow,swallow,throat and paranasal sinuses squamous cell carcinomas,smoking and alcohol are the main pathogenic factors.Now,the main treatment for head and neck cancer is the use of radiotherapy,chemotherapy,targeted therapy and surgery,but the patient's survival rate has not significantly improved.Therefore,it is of great significance to find new chemotherapy drugs for the treatment of head and neck cancer.This study demonstrated for the first time that use of the small molecule compound CYT997 in combination with an autophagy inhibitor can inhibit the growth of head and neck cancers.The results of the study are as follows:1.Small molecular compound CYT997 induces apoptosis in head and neck cancer cells.The small molecular compound CYT997 can inhibit the growth of cells when it acts on head and neck cancer,and the specific induction pathway is unknown in this process.Therefore,we mainly analyzed the molecular mechanism of CYT997 in the induction of apoptosis of head and neck cancer cells.In this work,cell proliferation assay was used to evaluate the proliferation ability of cells.At the same time,the apoptosis rate of the cells was detected by means of crystal violet staining and DAPI staining,and Western Blot was used to evaluate the apoptosis of the cells.Electrochemical biosensor and cell ROS fluorescence staining were used to test the changes of ROS levels after treatment.Through the above experiment,it was found that different head and neck cancer cell lines shown a strong sensitivity under CYT997 treatment.The cell proliferation assay shown that the median lethal concentration of the drug to the cell was 100 n M,and the HN4 has a more sensitive than HN12 and HN30 under the CYT997 100 n M treatment.Further experiments showed that the ROS level and apoptosis of cells were increased under drug treatment.Thus,CYT997 induced apoptosis was achieved by increasing the cell production of ROS.2.The role of autophagy in CYT997-induced cell apoptosis.The functional relationship between apoptosis and autophagy in anticancer drug treatment is extremely complex,and the molecular machinery is obscure.This study aims to investigate the efficacy of CTY997,a novel microtubule-disrupting agent,in head and neck squamous cell carcinomas(HNSCC)and explore the autophagyapoptosis puzzle involved in drug action.In order to study the effect of CYT997 in head and neck cancer,the cell proliferation experiment,DCFH-DA fluorescence staining experiment,electrochemical biosensors,flow cytometry and Western Blot wereassessed the levels of cell oxidative stress and apoptosis under drug treatment.CYT997 triggered autophagy was performed autophagy fluorescent staining,transmission electron microscopy(TEM)and Western Blot experiments to complete.At the same time,the transplanted tumor mouse model constructed with HNSCC cells was used to evaluate the effect of drugs on tumor growth.CYT997 triggered oxidative stress-associated apoptosis and the m TOR-dependent autophagy in HNSCC cells.CYT997-induced autophagy appears to have a protective role against apoptosis by inhibiting induction of excessively high reactive oxygen species(ROS).Blockade of autophagy by ATG7 depletion or addiction of hydroxychloroquine(HCQ)sensitized HNSCC cells to CYT997,as evidenced by enhanced ROS-associated apoptosis.Moreover,HCQ exhibited a certain degree of synergism with CYT997 on induction of apoptosis in HNSCC xenografts without cytotoxicity.Therefore,the combination of CYT997 and autophagy inhibitors can enhance the anti-tumor effect of CYT997.At the same time,this study further reveals the importance of ROS balance between autophagy and apoptosis in the treatment of CYT997 and the related molecular mechanism,which provides a theoretical basis for the design of new strategies for the treatment of head and neck cancer.