FEN1 Regulates The Mechanism Of Cisplatin Tolerance In Non-small Cell Lung Cancer

Lung cancer is one of the leading causes of worldwide cancer deaths.The rate of Lung cancer survival is very low,because of its metastatic and recurrence nature.Lung cancers are of two types,small cell lung cancer(15%)(SCLC),non-small cell lung cancer(85%)(NSCLC),Metastatic NSCLC will generate intrinsic resistance to chemotherapy and is generally incurable.Since the discovery of its anticancer activity in 1970s,cisplatin and its analogs have become widely used in clinical practice.Cisplatin represents successful landmark in the history of cancer treatment.Advanced treatment for lung cancer is,cisplatin in combination with other drugs.Considerable efforts have been dedicated to improve the therapeutic efficacy but the effect was not satisfied,due to the resistance of cancer cells to the therapeutic drugs.Studies showed that cisplatin had lost the activity after entered cancer cells,the deactivation of cisplatin in the form of platinum nanoparticles.This is just one of these resistance mechanisms,and our work is to find a new mechanism of cisplatin resistance.Once taken it into the cell,cisplatin will intercalate DNA and form crosslink on DNA.This leads cancer cells to triggering apoptosis or necrosis.We speculate if the resistance of cancer cells associated with DNA damage repair.There are many kinds of DNA damage repair pathways,such as base excision repair,nucleotide excision repair,recombination repair,mismatch repair and so on.These repair mechanisms can repair the DNA damage and make cells resistance.A protein called FEN1 plays an important role in a variety of repair pathways,which associated to the Rad2 nuclease family.FEN1 is especially well-known for its critical roles in Okazaki fragment maturation,long-patch base excision repair(BER)and telomere maintenance.Recent investigation reported that FEN1 had a certain relationship with the development of tumor,but the relationship between the FEN1 resistances to tumor cells was extremely rare.So we speculate that the tumor cell resistance to chemotherapeutic drugs may be due to FEN1 protein.Studies have shown that cisplatin entered the cancer cells,before reaching the target nuclei have lose activity,the deactivation of cisplatin drugs can't be kill cancer cells and DNA complexation,the deactivation of cisplatin in the form of platinum nanoparticles,including glutathione transferase played a key role.This is just one of these resistant resistance mechanism,is to find a new mechanism of cisplatin resistance produce resistance of cancer cells to cisplatin drugs associated with DNA damage repairA protein called FEN1 plays an important role in a variety of repair pathwaysIn order to solve these problems,we have a series of experiments,include the following sections:At first,we analyzed the mRNA level of FEN1 in lung cancer tissue cells from the database(TCGA)and find the cancer tissue cell have much higher.Immuno-histochemical experiments further showed that FEN1 protein is highly expressed in lung cancer and the survival rate of high expression of FEN1 in lung cancer patients is significantly lower than that of low expression FEN1.Then,we detected the expression of FEN1 between NSCLC and normal lung fibroblasts(HELF)from the cellular level,and the results showed that A549 cells expressed the highest.Next,we want to explore the relationship between A549 lung cancer cell growth,apoptosis and FEN1.We used lentivirus vectors to infect A549 cells to construct the FEN1 knockdown and overexpression stable cell lines.Using Western Blot,3D-Agar,TUNEL and other experimental techniques was carried out.It was found that FEN1 knockdown in A549 cells grow lower than wild type A549 cells and the colony forming ability also decreased.After Cisplatin treatment,FEN1 knockdown cells have a significant reduction in drug resistance,apoptosis is also more obvious.In contrast,over expression of FEN1 is very different from the knockdown.In general,if DNA damage happens,the repair systems will be activated.We found that in A549 cells,FEN1 protein expression is higher,which need more FEN1 involved in DNA damage repair.If the FEN1 gene is knocked down,DNA damage will not be the corresponding repair,then the apoptosis will appear.We used immunofluorescence assay and chromosome display experiment showed that the DNA damage of FEN1 knockdown cells was significantly increased,and there was a significant increase in chromosome breakage.But in case of overexpression FEN1 it is completely contraries,we also used Western Blot to detect the expression of relevant apoptotic protein in P53 pathway.In addition,we found that FEN1 inhibitors may be an effective treatment for NSCLC.We design novel small molecule inhibitors of FEN1 and to detect its therapeutic effect.Through data analysis,FEN1 inhibitors have a certain role in A549 cells,but not in HELF cells,which can become a new target for the treatment of tumor cell resistance.So our study will provide an efficient tumor-targeting therapy for lung cancer.The animal experiments also showed combining FEN1 inhibitor and Cisplatin can effectively inhibited cancer cells growth.So we consider FEN1 is suppressed,DNA damage cannot be effectively repaired,p53 pathway will be activated,which leads to cell apoptosis.It explains FEN1 is an important part of BER pathway,and also explains the mechanisms of tumor resistance to chemotherapy drugs.