Effects Of CXCR1/2 Antagonism By G31P,Combination With Gefitinib On Lung Injury Induced By Bleomycin In Mice

Objective:CXC chemokines play an important role in the development of inflammation,angiogenesis,invasion and metastasis of tumor cells.IL-8,one of CXC chemokines,is involved in chemotactic effect of neutrophils,T cells and other inflammatory cells by binding to its receptor CXCR1 and CXCR2.Role of IL-8 was also proved in invasion,angiogenesis and metastasis processes of different tumors.G31 P,a chemokine receptor CXCR1/2 antagonist,can combine with CXCR1/CXCR2 with high affinity and block many chemokines which bind with CXCR1 and CXCR2 receptors,including IL-8,and act as anti-inflammatory and antitumor agent.Gefitinib,an inhibitor of epidermal growth factor receptor(EGFR)tyrosine kinase,is an effective treatment for epidermal tumors,including non-small cell lung cancer(NSCLC),and is generally well tolerated.Transforming growth factor and epidermal growth factor stimulate fibroblast proliferation and play an important role in pathogenesis of pulmonary fibrosis.However,some studies reveal that inhibition of epidermal growth factor signal by epidermal growth factor receptor inhibitors(EGFR-TKI)may prevent pulmonary fibrosis.Bleomycin is an antitumor antibiotic that was isolated from a strain of strptomyces verticillus.It has been used successfully to treat a variety of malignancies,including squamous cell carcinoma of the head and neck,cervix and esophagus,germ cell tumors and both Hodgkin and non-Hodgkin lymphoma.The major limitation of bleomycin therapy is the potential for life-threatning interstitial pulmonary fibrosis(also called fibrosing alveolitis)in up to 10 percent of patients receiving the drug.Objectives:In this research,we study the effects of combination therapy of G31 P and Gefitinib on A549 cell inhibition.At the same time,the effects of combination therapy of G31 P and Gefitinib on mice lung injury.This study provides new ideas for the treatment of cancer in clinical.Methods:(1)In vivo,We generated a mouse model of lung injury induced by bleomycin.C57BL/6 mice divided into 5 groups,Control group,Bleomycin group,Gefitinib group,Gefitinib+G31P group,G31 P group.All groups were injected intratraceally with bleomycin(0.5mg/kg)or saline,with intragastric administration of gefitinib(100mg/kg body weight)or saline and subcutaneous injection of G31P(500ug/kg)for 3 weeks.After 3 weeks mice were sacrificed and lung tissue were harvested and investigated for pathogenesis.During whole study body weight and other physical activities check every day.Mice lung were also fixed into 10% formaldehyde and did hematoxyline and eosin staining to find out pathogenesis in lung tissue of bleomycin and gefitinib and G31 P and control group.RNA was extracted from the tissue of mice lungs from all five groups and RNA converted into cDNA and by PCR find out the difference in inflammatory proteins.MPO and i NOS content was found out in mice lung tissue by standard protocol.(2)In vitro: A549 Cells processing and grouping,and proliferation inhibition assay and migration experiment methods was used to detect the effect of G31 P combine with Gefitinib on the proliferation of A549 cells.Results:(1)In vivo: After 21 days of drug treatment mice were sacrificed and lung tissue harvested and take photograph of mice lung which shows that mice treated with bleomycin without gefitinib or G31 P are worse than mice treated with gefitinib and G31 P when compare to control which prove that bleomycin cause severe lung injury in mice and gefitinib with G31 P treatment reverse that or prevent that injury.The histopathological results showed that bleomycin successfully induced lung injury in mice,and gefitinib with G31 P prevented lung injury and suppressed the proliferation of fibroblast cells.Mice with gefitinib and G31 P survived more than mice without gefitinib or G31 P after intratraceal administration of bleomycin.Results also showed that mice treated with gefitinib and G31 P have high body weight than mice treated alone with gefitinib or alone with G31 P.Body weights of gefitinib and G31 P treated mice are nearly growth of mice in control group.RT-PCR results showed that inflammatory marker TNF-?,IL-1,IL-5 and IL-6 are more significantly reduced in gefitinib and G31 P treated mice lung.Bleomycin group showed high level of TNF-?,IL-1,IL-5 and IL-6 mRNA level as compare to the control group while Gefitinib and G31 P treatment after bleomycin intra-tracheal administration these inflammatory marker reduce their expression.Western blot results showed that the inflammatory protein NF-?B is reduced in Bleomycin group,Bleomycin group showed low level of NF-?B level as compare to the control group while Gefitinib and G31 P treatment after bleomycin intra-tracheal administration this protein increase this expression.MPO and iNOS determination results show that the G31 P combine with gefitinib can obviously decrease the content of iNOS and MPO.(2)In vitro: Cell inhibition and cell migration assay to detect G31 P and gefitinibim atinib on A549 cell proliferation and cytotoxicity by.Used alone G31 P group results sh ow on A549 cells had no obvious inhibitory effect;G31P combined low concentration of Gefitinib group results show on A549 cells have obvious cell inhibition;The inhibiti on effect on A549 cells is not so obvious in G31 P with high concentrations of Gefitini b group.Conclusion:(1)The combination of G31 P and Gefitinib can obviously inhibit the growth and migration of A549 cells.(2)These results reveal that gefitinib and G31 P reduces Lung injury by bleomycin in mice.