| Background:Lung cancer is the second most common cancer with a high fatality rate.Non-small cell lung cancer(NSCLC)accounts for about 80-85%of all lung cancers.The 5-year survival rate is only 23%,and there is no effective clinical therapy.At present,the efficient and safe combination therapy are facing great challenges in lung cancer.Hydrogen sulfide(H2S)is a new endogenous gas signaling molecule involved in physiological and pathological regulation,which can inhibit tumor.However,the rapid release of H2S from existing hydrogen sulfide donors,lack of stability and tumor targeting are extremely limited in clinical application.Objective:In view of the above scientific issues,we prepared nano-drug(CL2/3)co-assembled an amphiphilic pentapeptide(RGDFF)with a novel mercaptan activated hydrogen sulfide donor,which inhibits tumor growth and increased sensitivity to radiation therapy in NSCLC.To confirm the safety and efficacy of CL2/3 in NSCLC,we are obtained the data at the cellular and animal levels,and explored the mechanism of therapy further.Methods:(1)Preparation and characterization of Two thiol-activated H2S donors(CL2/3):CL2/3 were co-assembled with RGDFF pentapeptides,respectively.CL2/3 were identified by NMR and mass spectrometry,and its functional groups were analyzed by infrared spectroscopy.The particle size was determined by atomic force microscopy,transmission electron microscopy and dynamic light scattering.The optical properties were analyzed by ultraviolet absorption and fluorescence spectrum.The release of H2S was detected by methylene blue method.(2)In vitro Data:The safety of CL2/3 was evaluated by MTT and cytoskeleton in L929 cells.In addition,the efficiency was measured by MTT,cell scratches,transwell migration,cytoskeleton,activity of SOD,CAT and LDH,content of GSH and MDA,ROS,mitochondrial membrane potential changes,apoptosis,cell cycle,the content of H2S in H1299 cells.Finally,the protein expressions of oxidative stress,cell cycle,apoptosis and H2S-related protease with irradiation and without irradiation were detected by WB to preliminarily explore the mechanism of therapy.(3)In vivo Data:Balb/c mice were used to construct the animal model of H1299 subcutaneous tumor transplantation.They were randomly divided into 4 groups:negative control group,CL3 group,irradiated(IR)group and CL3+ irradiated(IR)group.The body weight,tumor size,Kaplan Meier survival curve and oxidative stress index were measured in tumor tissue.The protein expressions of Bcl-2,BAX,STAT3,Nrf2,Hif-1α,CBS and CSE were detected by WB and immunohistochemistryon the treatment mechanism of CL3.The biocompatibility was analyzed by H&E staining and immunohistochemistry in the liver.Results:(1)Characterization:The RGDFF were co-assembled with sulfhydryl activated H2S donors to form a nano-carriers(CL2/3).The synthesis CL2/3 was identified by NMR,mass spectrometry and FT-IR spectra.The data of AFM,TME and DLS showed that CL2/3 had good dispersion.The particle size distribution of CL2 and CL3 ranges from 37nm-175nm and 50nm-190nm,respectively.The absorption peak was shifted to more than 300nm in the ultraviolet absorption spectrum,which confirmed CL2/3 were successfully co-assembled.The fluorescence spectra showed blue fluorescence of CL2 and green fluorescence of CL3.The methylene blue method confirmed the slow release of H2S.The encapsulation rate of CL2/3 were(31.1±5.4,33.02±3.5)%,and the load rate of CL2/3 was(8.57±6.2,11.71±4.7)%,respectively.(2)In vitro,50μg/mL CL2/3 had satisfactory biocompatibilty by the activity and cytoskeleton on L929 cells.The activity of H1299 cells were decreased with dosedependent change.It inhibited cell migration,changed the microfilaments of cytoskeleton,increased the content of MDA,ROS,LDH,the activity of CAT,and decreased the content of GSH.When mitochondrial membrane potential was reduced,the apoptosis was increased by the increase of mitochondrial membrane potential.the ratio of G0/G1 phase of cell cycle was decreased with the dose-dependentchange,while that of G2/M phase was increased.What’s more,H2S can be produced in cells slowly.WB results showed that CL2/3 inhibited the protein expression of Nrf2,NFκB,Hif-1α,Bcl-2,STAT3,CyclinE1,CyclinB 1,CBS,CSE and MPST;but promote the expression of Bax in H1299 cells.The regulation of related protein expression was more obvious after irradiation.CL3 is more effective than CL2.(3)In vivo:both CL3 and CL3+ irradiation(IR)groups could inhibit tumor growth.In CL3 group,the activities of MDA,LDH and CAT increased,while the activities of GSH and SOD decreased.The protein expression of Nrf2,Hif-1α,Bcl-2,STAT3,CSE were down-regulated,the protein expression of Bax was up-regulated,and CBS had no significant change.In addition,immunohistochemical results showed that the positive rate of Bax increased,while that of Nrf2,Hif-1α,Bcl-2,CSE and CBS decreased,and STAT3 had no significant change.HE and immunohistochemical results showed that CL3 had no obvious toxic on liver.Conclusion:We have successfully prepared two kinds of RGDFF pentapeptides co-assembled new hydrogen sulfide donors CL2/3 nanodrugs,which have good dispersion and physicochemical properties.CL2/3 has good biosafety and efficacy.CL2/3 can inhibit the viability and migration of H1299 cells,reduce the activity of antioxidant enzymes,promote intracellular ROS accumulation,inhibit the expression of Nrf2 and Hif-1α protein,induce oxidative stress,improve the oxygen-poor state in cells,regulate ROS/mitochondrial pathway,reduce mitochondrial membrane potential,and down-regulate the expression of Bcl-2 and STAT3 proteins.The protein expression of pro-apoptotic protein Bax was up-regulated.Down-regulating the expression of CyclinB1 and CyclinE1,the cell cycle was arrested in G2/M phase.The expression of endogenous H2S synthetase proteins CBS,CSE and MPST were inhibited,reduce the production of endogenous H2S,and inhibit tumor growth by regulating ROS/mitochondrial pathway with chain-react. |
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