Effect Of Exosomes On DNA Damage Induced By Cisplatin And Bleomycin

Background:as the most important and fundamental genetic material,the integrity and stability of DNA are regarded as the foundation of normal cellular activity.However,DNA is susceptible to a great number of endogenous and exogenous factors(e.g.,genotoxic substances)that result in various types of DNA damage,including the formation of protein-DNA adducts,intra-and inter-strand crosslinks,single-and double-strand breaks,etc.DNA damage is the key to the development of many diseases.To protect the integrity of DNA,cells are equipped with a complex DNA damage response(DDR)system to defend against various types of DNA damage and ensure the normal function of the body.Human embryonic stem cells-induced mesenchymal stem cells(hESC-MSCs)are mesenchymal stem cells(MSCs)that can proliferate indefinitely in vitro from a rich source,and it has a powerful DDR system to combat many types of DNA damage and prevent the accumulation of genetic mutation.An increasing number of studies have suggested that MSCs not only have a strong resistance to DNA damage,but can also transmit their anti-DNA damage effects to other cells through their paracrine action,and exosomes play a vital role in this process.Exosomes are a class of extracellular vesicles(EVs)with a bilayer of phospholipid structure and a diameter of 30～150 nm.Exosomes are capable of carrying nucleic acids,proteins,and other biomolecular components from cells,and play an important role in long-distance communication between cells.Previous studies suggested that exosomes of stem cells have a protective effect on the integrity of DNA,while the exact role of exosomes derived from MSCs on different types of DNA damage is not clear.Aim:this study aimed to explore the effect of hESC-MSC-Exos on different types of DNA damage and mechanism and to provide a new direction for the prevention and treatment of DNA damage-related diseases in clinical.Methods and results:this study contained two parts,we assessed the protective effect of hESC-MSC-Exos on two types of DNA damage,i.e.,inter-or intra-stand cross-links and DNA double-strand breaks,induced by two commonly used chemotherapeutic agents cisplatin(Pt)and bleomycin(BLM),respectively.We detected cell viability by cell counting kit,DNA single/double-strand breaks by comet assay,Immunofluorescence(IF)and Western blot(WB)assays were performed to detect the expression level of phosphorylated histone H2AX(γH2AX)to comprehensively reflect the degree of DNA damage.Moreover,the mechanism of the effect of hESC-MSC-Exos on DNA damage was explored by detecting intracellular reactive oxygen species(ROS)levels,mitochondrial membrane potential(MMP),apoptosis rate,and apoptosis-related proteins in each group.Part I:we first examined the influence of hESC-MSC conditioned medium on Pt-induced DNA damage.Exosome-free fetal bovine serum(FBS)culture medium was used to culture hESC-MSCs for 48 hours(h)and then the hESC-MSCs conditioned culture medium(hESC-MSC-M)was collected.He La cells were preincubated with different concentrations of hESC-MSC-M(0%,25%,50%,and 100%)for 6 h,followed by the addition of 1μMol/L Pt for another 24 h.The results of comet assay showed that hESC-MSC-M can effectively reduce Pt-induced DNA damage in a dose-dependent manner.When the hESC-MSC-M concentration reaches 100%,the tail DNA percentage(9.82±1.00 vs 20.50±1.78,p<0.01),tail moment(1.82±0.35 vs 9.84±1.39,p<0.01)and olive tail moment(2.31±0.23 vs 6.17±0.61,p<0.01)of hESC-MSC-M+Pt group were significantly lower than that of the Pt group,and there was no significant difference between hESC-MSC-M+Pt group and the untreated group in the tail DNA percentage(9.82±1.00 vs 7.91±0.50,p>0.05).The above results suggest that hESC-MSC-M can effectively reduce Pt-induced DNA damage.Next,we further explored the effect of hESC-MSC-Exos from hESC-MSC-M on Pt-induced DNA damage in cells.Firstly,we isolated exosomes from hESC-MSC-M,and cells were treated with various concentrations(0,0.5×10~6,1×10~6,and 2×10~6particles/m L)of hESC-MSC-Exos for 6 h,and then followed by 1?Mol/L Pt treatment for another 24 h.After that,we found hESC-MSC-Exos preincubation reduced the DNA fragmentation induced by Pt in a dose-dependent manner by comet assay.Specifically,when the concentration of hESC-MSC-Exos reached 2×10~6 particles/m L,the tail DNA percentage(10.40±1.34 vs 20.80±1.61,p<0.01),tail moment(2.37±0.64 vs 10.00±1.21,p<0.01)and Olive tail moment(2.06±0.28 vs 6.01±0.55,p<0.01)are significantly lower than of Pt-only group,and there was no significant difference between hESC-MSC-Exos treated group and the untreated group(10.40±1.34 vs 9.40±1.14,2.37±0.64 vs 2.08±0.51,2.06±0.28 vs 2.09±0.25,p>0.05).The above results indicated that exosomes are an important component in hESC-MSC-M and hESC-MSC-Exos can effectively reduce Pt-induced DNA damage in a dose-dependent manner.Reactive oxygen species(ROS)are regarded as important mediators in Pt-induced DNA damage.DCFH-DA staining indicated that hESC-MSC-Exos could inhibit the increase of intracellular ROS in Pt-treated cells in a dose-dependent manner.In particular,when the concentration of hESC-MSC-Exos reached 2×10~6 particles/m L,no significant difference was detected between hESC-MSC-Exos+Pt group and the untreated group in the intracellular ROS level(1.22±0.01 vs 1.00±0.01,p<0.01),demonstrating that hESC-MSC-Exos was effective in inhibiting the increase of intracellular ROS level induced by Pt.Part II:We further explore the effect of hESC-MSC-Exos on BLM-induced DNA damage in different concentrations(0,1×10~6,2×10~6,and 4×10~6 Particles/m L).Cell viability assays,comet assay,IF,and WB results showed that hESC-MSC-Exos could effectively reduce BLM-induced DNA damage in a dose-dependent manner.Especially,when the concentration of hESC-MSC-Exos reached 4×10~6 particles/m L,the"comet"tailing almost disappeared,and the tail DNA percentage(7.98±0.57 vs 21.80±1.31,p<0.01),tail moment(3.82±0.39 vs 12.00±0.81,p<0.01),and olive tail moment(2.19±0.18 vs 6.03±0.37,p<0.01)of hESC-MSC-Exos+BLM group were significantly lower than that of the BLM group and there was also no significant between hESC-MSC-Exos+BLM group and the untreated group in the tail DNA percentage(7.98±0.57 vs 6.70±0.60,p>0.05).In addition,DCFH-DA staining results showed that hESC-MSC-Exos could inhibit the increase of intracellular ROS in BLM-treated cells in a dose-dependent manner.When the concentration of hESC-MSC-Exos reached 4×10~6 particles/m L,there was no significant difference in the intracellular ROS level between hESC-MSC-Exos+BLM group and the untreated group(0.75±0.17 vs 1.00±0.28,p>0.05).To further explore the mechanism,we detected the relevant indicators of cell apoptosis in the effect of hESC-MSCs-Exos on BLM-induced DNA damage.The results showed that hESC-MSC-Exos could not mitigate the loss of mitochondrial membrane potential and increase in apoptosis induced by BLM,but also downregulate the expression of cleaved caspase3 and the pro-apoptotic protein Bax in a dose-dependent manner.Moreover,there was no significant difference in the expression of the apoptotic protein Bcl-2.Conclusion:in summary,this study demonstrated that hESC-MSC-Exos can reduce Pt-and BLM-induced DNA damage in a dose-dependent manner by reducing ROS accumulation in cells.hESC-MSC-Exos can also reduce apoptosis induced by BLM.