Inhibition Of Rac1 Attenuates Radiation-induced Lung Injury While Sensitizes Lung Cancer To Radiotherapy

Backgrounds:Since invented,radiation therapy?RT?has still been the cornerstone of treatments to many malignant tumors.The goal of RT is to complete eliminate tumor or reduce tumor size,and in the meantime,retain normal tissues.About 40%of tumor patients received RT,either to cure disease,control local tumor or to relieve symptoms.The lung is one of the most sensitive tissues to ionizing radiation,thus,radiation-induced lung injury?RILI?stays a key dose-limiting factor for thoracic radiotherapy and is an inevitable companion of thoracic radiotherapy.With the development of high-precision radiotherapy technology,in-depth understanding of the molecular mechanisms of RILI,advances in animal models and the screening of drug candidates for the prevention and treatment of RILI,the incidence of RILI has been lowered.However,there is still little progress in the safe and effective treatment of RILI,which significantly affects the treatment of patients with lung cancer,breast cancer,lymphoma or bone marrow transplantation,reduces tumor control probability?TCP?and leads to dyspnea,pulmonary fibrosis,also,affects patients'quality of life.Ideally,radio-protectors should have minimal side effects,and above all,do not exert protective effects on tumor cells,in other words,do not affect the killing effects of radiation on tumor cells.Therefore,seeking such a radio-protective agent or molecular target that can mediate such a bidirectional effect has become the key to improving tumor radiotherapy.Ras-related C3 botulinum toxin substrate1,Rac1,is a small guanosine triphosphatase?GTPase?.Rac1 is involved in oxidative stress and DNA damage process,and is closely related to TGF-?and the process of epithelial-mesenchymal transition?EMT?.These biological processes are all important mechanisms of radiation-induced tissue damage.Thus,Rac1 may be an important molecule that mediates radiation damage,inhibition of which may produce a protective effect on RILI.In addition,Rac1 is over-expressed or mutated in various tumors.The gain-of-function?GOF?mutation of Rac1 can lead to cancer-related phenotype.Rac1 not only participates in the formation,progression,invasiveness and angiogenesis of tumors,but also mediates tumor cell resistance to radiotherapy,chemotherapy and immunotherapy.Inhibition of Rac1 can inhibit tumor growth and restore the sensitivity of patients to cancer therapy.Therefore,inhibiting Rac1 has the potential of protecting normal tissues from radiation-induced injury,and at the same time,inhibiting tumor growth and sensitizing tumor to radiation therapy,which makes it a promising ideal molecular target for clinical radiation protection.Objectives:To investigate the protective effects and mechanisms of Rac1 inhibition on RILI,verify the differential effects of Rac1 inhibition on normal tissue and tumor cells,and explore the possible molecular mechanisms that mediate this differential effects of Rac1 inhibition.Methods:⁶⁰Co radioactive source was used for ionizing radiation.Radiation-induced lung injury mouse model was constructed.The effects of Rac1 inhibition on pneumonitis and pulmonary fibrosis after irradiation were studied by H&E staining and Masson staining through the application of Rac1-specific inhibitor,NSC23766.The effects of Rac1inhibition on the expression levels of phosphorylated H₂AX??-H₂AX?,TGF-?and vimentin,which are markers of DNA damage,radiation damage and EMT,respectively,were studied by immunohistochemical staining.Normal mouse lung epithelial cell line,MLE-12,was used and Rac1-knockout MLE-12 cells were constructed to verify the protective effects of Rac1 inhibition on the cellular level.The expression changes of apoptosis-related proteins,cell cycle regulating proteins and DNA damage related proteins after radiation were studied through Western Blot.Annexin V/PI staining for apoptosis ratio detection,PI/RNase staining for cell cycle detection and Ed U methods for detection of cell proliferation activity were conducted with flow cytometry instrument.RNA-seq was used for the screening of differential gene expression after Rac1 inhibition,thereby finding the possible molecular pathway by which Rac1 mediates its protective effects.q PCR was used to verify the results of RNA-seq.The molecular mechanisms of Rac1 inhibition was studied at the cellular level.Mouse lung cancer cell line,LLC,was used to study the sensitizing effects of Rac1 inhibition on tumor cells to irradiation.Finally,a subcutaneous tumor-bearing nude mouse model and an orthotopic lung tumor-bearing mouse model were constructed to further verify the bidirectional effects of Rac1 inhibition on normal lung tissue and tumor cells.Results:?1?Radiation-induced pneumonitis and pulmonary fibrosis in mice were reduced by the Rac1 inhibitor,NSC23766;?2?The radiation-induced apoptosis of MILE-12was reduced,the G2/M cell cycle arrest reversed and the DNA damaged alleviated,while the proliferation activity of MLE-12 was not significantly affected by inhibition or knockdown of Rac1;?3?The expression level of tumor protein p53-inducible nuclear protein 1,Trp53inp1,was down-regulated by Rac1 inhibition,and over-expression of Trp53inp1 partially reversed the protective effects of Rac1 inhibition on MLE-12;?4?The radiation-induced apoptosis of LLC was increased,the proliferation activity significantly inhibited,and the proportion of cell cycle phases not influenced by inhibition or knockdown of Rac1;?5?Over-expression of Trp53inp1 produced an synergistic pro-apoptotic effect with Rac1 inhibition on LLC cells,and had little influence on the cell cycle distribution and proliferation ability of LLC cells;?6?Rac1 was significantly over-expressed in LLC cells compared with that of MLE-12,and there existed insertion mutation of Rac1 in LLC cells;?7?The expression of Trp53inp1 in LLC cells was significantly decreased compared with that of MLE-12;?8?Nude mouse subcutaneous tumor-bearing model and orthotopic lung tumor-bearing model further proved that inhibition of Rac1 could significantly inhibit tumor growth,and at the same time,exert a protective effect on the radiation damage of normal lung tissue.Conclusion:Radiation-induced pneumonitis and pulmonary fibrosis in a mouse model of RILI were alleviated through Rac1 inhibition.Rac1 inhibition produced its protective effects by reducing the apoptosis ratio,reversing the cell cycle arrest and alleviating the DNA damage of normal lung epithelial cells induced by ionizing radiation.The above effects were partially mediated by down-regulating the expression of Trp53inp1.Over-expression of Trp53inp1 could partially reverse the protective effects of Rac1inhibition on normal lung epithelial cells.In addition,Rac1 inhibition significantly increased the sensitivity of mouse lung cancer cells to radiation damage and inhibited the growth of tumor cells.The over-expression and mutation of Rac1,and the significant decrease of Trp53inp1 in tumor cells,may be the reasons for the differential effects of Rac1 inhibition on normal cells and tumor cells.