| During these years,lung cancer has become the most common and deadly malignancy all over the world.According to the data showed from the National Health planning committee,the current growing speed of lung cancer’s incidence in China is at a rate of 26.9% per year.In the past 50 years,the number of lung cancer patients has doubled every 10 to 15 years.According to the third survey of cause of residents’ death in China,the mortality rate of lung cancer has increased by 465% in the past three decades,thus replacing liver cancer to be the most common malignant tumor in China.Although the cause of lung cancer is still not completely clear,it deeply and seriously affects the quality of the patients’ life.Lung cancer can be divided into two categories by the different treatment methods: small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),the latter is much more common than the former,and it currently accounts for about 87% of lung cancer patients,of which about 75% of patients are found to be in the mid-late stage,the 5-year survival rate is extremely low,and do not has the chance to be cured by surgery.Therefore,the comprehensive treatment mode based on radiotherapy has occupied an important position in the treatment of NSCLC.According to the different aim of radiotherapy,radiotherapy can be divided into five types: radical radiotherapy,concurrent radiotherapy and chemotherapy,palliative radiotherapy,preoperative and postoperative radiotherapy.For the patients who cannot or refuse to do the surgery,radiotherapy is an effective local treatment;for the patients who have already received the surgery,radiotherapy is an important adjuvant therapy;for the advanced lung cancer patients,radiotherapy is an important palliative treatment.However,although there are about 60% of patients with non-small cell lung cancer(NSCLC)accept radiation therapy at different stages of treatment,with the progress of radiation therapy,the cancer cells show the increasing resistant to the radiation,they do not die after irradiation,most patients show radiation resistance,which ultimately leads to failure of radiotherapy.All in all,how to increase the radiation sensitivity of NSCLC and overcome its radiation resistant is the most important and direct research aim to enhance the effect of radiotherapy,and it is a scientific problem urgent to be solved deeply in the field of lung cancer radiotherapy.More and more studies have shown that transglutaminase 2(TG2)plays an important role in the development of lung cancer and may become a new target for radiosensitization of lung cancer.TG2 is a widely studied and extensive multifunctional protein in the transglutaminase family.Its main function is to catalyze Ca2+-dependent post-translational protein modification through amino acid covalent cross-linking mechanism.At the same time,TG2 has GTP/GDP binding activity,protein disulfide isomerase activity and protein kinase activity,thus participating in a variety of physiological and pathological processes,such as tissue fibrosis,apoptosis,autophagy,EMT etc.,and playing a key role in the process of tumor development,invasion and metastasis.Nowadays it has been found that TG2 is highly expressed in tumor cells such as lung cancer,ovarian cancer and breast cancer,and the survival rate of patients with lung cancer,which has high expression of TG2,is lower than the survival rate of patients with lung cancer,which has low expression of TG2.Recent studies have shown that TG2 may be involved in the regulation of DNA damage repair and play an important role in the resistance of chemotherapeutic drugs.In the previous work of this project,firstly we used the TG2 inhibitor Glucosamine to radiation sensitivity of the in situ tumor-bearing mice,then we found that the in situ tumor-bearing mice treating with Glucosamine group significantly prolonged the survival period after irradiation,and the tumors’ size was significantly reduced,radiation sensitivity increased significantly.On the second step,we used tissue clinical patient lung cancer samples and lung cancer patients’ database to further clarify the relationship between TG2 expression and the treatment of patients with NSCLC,which confirmed that high expression of TG2 seriously affects the treatment of patients.Then in vitro,we used Western blot method to detect TG2 levels in different NSCLC cells(A549,H1299 and H460)and human normal bronchial epithelial cells BEAS-2B.It was definitely confirmed that the TG2 content in lung cancer cells was significantly higher than that in normal bronchial epithelial cells.We also used Annexin V cell apoptosis flow analysis method,in which we found that the apoptosis rate of A549 was significantly increased after inhibiting TG2,but the normal bronchial epithelial cells BEAS-2B had no significant effect.Then we did the clonogenic survival assay on different NSCLC cells(A549,H1299,H460),and we found that the radiation sensitivity of all were significantly increased after inhibition of TG2.Subsequently,in order to clarify the definite mechanism of inhibiting TG2 plays the role of radiosensitivity in NSCLC,we used neutral comet assay and found that the DNA damage in NSCLC cells was significantly aggravated after inhibition of TG2.Furthermore,western blot and immunofluorescence assays showed that the level of γH2AX phosphorylation induced by TG2 inhibition was significantly delayed,the main proteins’ phosphorylation(DNA-PKcs、ATM、ATR、Rad51)in non-homologous recombinant end joining(NHEJ)and homologous recombination(HR)DNA damage repair pathway were greatly reduced.More interestingly,through Western blot and immunofluorescence experiments we found that ionizing radiation caused TG2 to enter the nucleus from the cytoplasm and reached the maximum in 30 min.After 8 h,TG2 gradually nucleated.Furthermore,we used the laser irradiation method to spot the nucleus and found that TG2 recruited DNA damage repair sites,which indicates that TG2 is likely to directly participate in the DNA damage repair process.Then we separated the DNA-binding protein and non-DNA-binding protein in the nucleus and found that TG2 began to bind to DNA at 10 min after irradiation,while TG2 gradually detached from DNA at 2 h after irradiation,and 4 h TG2 was the most in the nucleus.In order to find TG2-mediated DNA damage repair and radiation-tolerant direct interacting proteins,we used immunoprecipitation and protein profiling techniques to identify 134 proteins that interact with TG2 after radiation and conducted preliminary validation.Combined with literature research and experiments verification,we finally focus TOPOⅡα as the downstream effector molecule of TG2.The results of immunoprecipitation showed that TG2 was clearly bound to TOPOⅡα after irradiation,and in TOPOⅡα knockdown cells,we found that DNA damage was aggravated and repaired slowly after irradiation,while Glucosamine showed no effect on DNA damage in these cells.Rescue experiments showed that TOPOⅡα overexpression significantly activated the DNA damage repair pathway in TG2 knockdown cells,but TG2 overexpression did not affect the activation of DNA damage repair pathway in TOPOⅡα knockdown cells.In order to find the exact interaction domain of TG2 and TOPOⅡα,we constructed cell lines with TG2 functional domain deletion or core domain point mutation,and performed the immunoprecipitation experiments,and then we found that any functional domain deletion affected its binding to TOPOⅡα.TG2 W241 mutation may affect the interaction between TG2 and TOPOⅡα,suggesting that TGase activity may play a key role in the TG2-TOPOⅡα signaling pathway.In conclusion,this study firstly demonstrates the inhibition of TG2 can radiosensitive NSCLC cells.It is clear that TG2 can play a key role in the regulation of DNA damage repair in NSCLC cells,and the direct interaction protein of TG2 is TOPOⅡα,furthermore,TGase activity may play the key role in TG2-TOPOⅡα signaling pathway.These findings have found new molecular targets for lung cancer radiotherapy sensitization research,and providing new directions and means for increasing the sensitivity of lung cancer cells to radiotherapy,which would show great significance in clinically improving the therapeutic effect of radiotherapy. |
PDF Full Text Request