| Objective:Oncolytic adenovirus(OA)is a kind of genetically modified adenovirus with typical diameter ranging from 80nm to 100nm.It is becoming one of the most important biologic therapies of tumor and attracting broad attention among scientists.However,the immunogenicity from OA and lower the expression of the receptors on tumor surface reduce its ability to kill the tumor,hence limit its clinical application.Microparticles(MPs)are vesicles formed during cell apoptosis under external stimuli including ultraviolet,high temperature,etc.In response to stimuli,cells change their cytoskeleton,leading to the encapsulation of cytosolic contents within cellular membrane to form vesicles with diameters ranging from 100nm to 1000nm,which will be released subsequently to become MPs.Studies have shown that tumor cell produced MPs can work not only as tumor vaccine to prevent and cure tumors but also as nanomaterials to encapsulate chemotherapeutics for tumor therapy.In this section,we focus on the feasibility and advantages of MP encapsulated OA(OA-MP)for tumor treatment.Furthermore,we try to explore the mechanism through which OA-MPs enter and interact with the tumor cell.Methods:(1)To prove whether MP can encapsulate the OA,we turned to transmission electron microscope to observe the relative position of OA and MP in OA-MP.To evaluate the uptake of OA by unit MP,we conducted real time qPCR to analyze the amount of viral genome DNA in both OA and OA-MPs.(2)To evaluate their biological activity,OA-MPs,OA or MPs were co-cultured with A549 tumor cells.The proteins including hexon,fiber,E1A were analyzed by western blot.(3)To compare their efficiency of tumor treatment,OA-MPs,OA and MPs were added to A549 and co-incubated for 48h before observing under microscope.To test the influence of MPs' origin on their ability of tumor treatment,A549 were co-incubated with OA-MPs derived from A549,HEK293 and K562.The cell apoptosis were then analyzed by flow cytometry.(4)To study the impact of OA-MPs on mice liver and kidney function,PBS,OA,OA-MPs,MPs were i.p injected into mice once a day for five days and the eyeball blood was extracted for analysis of ALT and CRE.To study the impact of OA-MPs on normal cells,peripheral blood mononuclear cell separated from blood of volunteers were incubated with OA-MPs that are stained by PKH26.The fluorescent expression of PKH26 in PBMCs was analyzed by flow cytometry at different time points(6 h,12 h and 24 h).Moreover,OA-MPs,OA and MPs were added to normal human PBMCs and incubated for 12h before analyzing the viability of PBMC with flow cytometry.(5)To study the replication of OA-MPs in tumor,OA-MPs or naked OA were added in A549 cells and co-cultured.Viral production in the tumor cells was determined by real time qPCR at different time points.In addition,to describe the pharmacodynamics of OA-MPs in tumor tissue,OA-MPs were injected into A549 tumor mass of nude mice and tumor tissues were cut off at different time points(6h,12h,24h,36h,72h and 96h)to determine the viral DNA amount by real time qPCR.(6)Various factors were studied to examine their influence on the uptake of MPs by tumor cells.These factors included heparin,EDTA,temperature,MPs size,trypsin and pH.(7)The relative positions of MP membrane and tumor cell membrane were observed with the aid of laser confocal microscopy.(8)Molecular dynamics simulation was also conducted to understand the microscopic morphology during the process of MPs' entering tumor cell.Results:(1)Under the transmission electron microscope,we observed that there were OA that existed in MPs.The results of real time qPCR showed that one MP could encapsulate about 5 VP OA.(2)When co-cultured with OA-MPs or OA,A549 tumor cells could express proteins including hexon,fiber and E1A,while no trace of these three proteins was found in MP or PBS treated cells.(3)After incubation with OA-MPs,A549 tumor cell detached from the petri dish and became spherical,which was the worst condition.After OA co-incubation,the cells detached from the petri dish and showed a poor condition.In control and pure MPs case,the cells were in good condition.Based on flow cytometry,the possibility of inducing tumor apoptosis of OA-MPs from A549,HEK293 and K562 were 31%,29%and 22%,respectively.(4)The ALT level in the blood of group OA,OA-MPs and MPs were about 13U/L,which was close to the control group.Similarly,the CRE level in the all the four groups were 18 umol/L.The relative uptakes of OA-MPs by PBMC after 6h,12h and 24h were 50%,59%and 63%.The apoptosis rate of PBMC after 12h incubation with OA-MPs,OA,MPs and PBS were 18.3%?21.4%?24.3%?19.2%,respectively.(5)According to RT qPCR,at all the studied time points,the virus titer in A549 cells were obviously higher when incubated with the presence of OA-MPs than with OA.We injected OA-MPs into A549 tumor mass in nude mice and cut off tumor tissue at different time points to determine the viral DNA by RT qPCR.The kinetics of viral burst showed that the peak of viral load was reached after 36 h.(6)The relative uptakes of MPs by tumor cells after 2h and 4h were 20.2%and 28.1%at 4?,87.6%and 92.3%at 37?.The relative uptakes of MPs by tumor cells pretreated with PBS or trypsin were 85.6%and 18.1%respectively at 2h;94.8%and 28.4%at 4h.With EDTA concentration of 0?M,100?M,500?M,1000?M,the relative uptake of MPs were 88.4%,83.7%,47.2%,40.4%.The relative uptake of MPs at pH values 6.4,6.8,7.2,7.5,8.0,8.5,9.0 were 76.4%,76.2%,71.5%,64.8%,65.4%,14.8%,5.93%,respectively.The relative uptakes of MPs with size ranges 100-220nm,220nm-600nm and 600-1000nm were 63.1%,21.6%and 14.2%,respectively.The relative uptakes of MPs were 95%,37.2%,36.1%,35%and 32.5%with heparin concentration of Oug/ml,lOug/ml,20ug/ml,50ug/ml and 100ug/ml.(7)The tumor cell membrane was stained with red by PKH26 and MPs with green by PKH67.After 20min co-incubation,MPs appeared in the cell membrane or inside the cell.When only MPs were stained with green by PKH67 and the incubation time was extended to 6h,laser confocal microscopy gave that more green regions could be identified inside the cell while some green markers were still found on the tumor cell membrane.PKH67-MP,dextran-red,A549 tumor cells were cultured together,and then were observed in laser scanning confocal microscope.Green fluorescence from PKH67-MP appeared in the cell surface or inside the cell while red fluorescence from dextran appeared inside the cell.However,the regions of MPs and dextran did not coincide with each other.(8)Coarse grained MD simulation showed that MPs vesicles with raft structures would fuse with cell.As the size of either MPs or cell increase,the fusion between MP and cell become less readily.When the size of the cell is orders larger than the MP,it is possible that MP goes through the cell membrane directly by punching a hole in the membrane.Conclusion:MPs can encapsulate OA,forming OA-MP.Compared with nude OA,OA-MP has the following advantages:First,OA-MPs are fatal to tumor cells and have minimal side effect on normal cells.Second,OAs from OA-MPs can replicate inside the tumor cells more effectively.Third,OA-MPs are independent on the CAR receptors when they enter tumor cells.The fusion or punching a hole may be involved in the process of MPs entering cell.All these three merits underlie a promising future of OA-MPs for tumor therapy in clinic.Objective:6-aminonicotinamide(6AN)is metabolized to 6-amino-NAD(P+),which is a competitive inhibitor of NAD(P')-requiring processes,especially the pentose phosphate pathway(PPP)involving glucose-6-phosphate dehydrogenase(G6PD),leading to an increase in the reactive oxygen species(ROS)level in cytoplasm.6AN is often regarded as an adjuvant drug for tumor treatment due to its sensibilization ability to chemotherapy and radiotherapy.In recent years,tumor immunotherapy has become a hot research topic and tumor associated macrophage(TAM)has attracted wide attention among scientists.Studies show that the ROS level in the cytoplasm plays an important role on the regulation of macrophages phenotypes.In this study we try to discuss the possibility of whether 6AN can activate the macrophages and explain its relevant mechanism in the activation process in order to bring more insights into the function of 6AN on tumor therapy.Methods:(1)To study whether 6AN can activate macrophages in vitro,we exposed MO macrophages to either 6AN or PBS as control.The cellular morphology was observed under inverted microscope and the surface markers(including CD80,CD86 and MHC-II)and intracellular factors(including IL-12,IFN-y)were observed by flow cytometry.To study whether 6AN can activate tumor associated macrophages in vivo,Balb/c mice were i.p.injected with H22 cells,and from the second day,were i.p.injected with 2.0mg/kg 6AN once per day for another 7 days,using PBS buffer as control.On the 8th day,the mice were killed and the peritoneal TAM was isolated to analyze the expression of relevant surface markers and intracellular factors by flow cytometry.(2)To investigate 6AN's regulation pathway in macrophage activation,macrophage cells were first pretreated with 6AN.After that,GSH/GSSG level in cell lysates were examined by enzyme mark color meter with GSH/GSSG kit,ROS level were examined by flow cytometry with ROS sensitive kit,phosphorylated AKT/MAPK pathway protein expression were examined by western blot,and NF-?B's position were observed with immunofluorescence with anti-NF-?B antibody as marker.To further determine the role of macrophage in the treatment of tumor by 6AN,macrophages depleted mice tumor model was also used.(3)To investigate the influence of 6AN on tumor stem cell in vitro,we directly used 6AN to treat stem-like tumor-repopulating cells in 3D fibrin gels and then observed their change of number and size.To investigate the influence of 6AN on tumor stem cell in vivo,we used 6AN to treat H22 tumor bearing mice.Then we isolated H22 cells to cultivate them in 3D and observed their change of number and size.(4)To further examine the universality of 6AN on tumor therapy,we used 6AN to treat three different kinds of tumor on mice including CMT-93 colon cancer,H22 liver cancer,and B16F1 melanoma.The involved survival time and tumor sizes were assessed.(5)In order to verify that 6AN sensitizes tumor cells to cisplatin in vivo,we tried to combine both 6AN and cisplatin on three tumor bearing mice models including CMT-93 colon cancer,H22 liver cancer,and B16F1 melanoma.The involved survival time and tumor sizes were observed.Results:(1)In vitro,exposed to 6AN,MO macrophages transformed from flat diamond to long fusiform,with long corner angle on both sides.For macrophages exposed to PBS or 6AN,the relative expression of surface markers CD80 were 48.8%and 91.1%,CD86 were 16.1%and 56.1%,MHCII were 36.7%and 71.5%.The relative expression of intracellular factors IFN-? were 1.17%and 5.01%,IL-12 were 0.93%and 3.02%.In vivo,macrophages from tumor mice i.p.injected with PBS or 6AN,the relative expression ratios of surface markers CD80 were 11.5%and 23.1%,CD86 were 5.96%and 32.0%,MHCII were 4.56%and 10.2%.The relative expression of intracellular factors IFN-? were 70.6%and 85.2%,IL-12 were 9.08%and 35.4%.(2)Pretreated with 6AN,the ROS level of 6AN treated macrophages were about five times that in PBS and the GSH/GSSG level of 6AN group was about 1/3 of that in PBS control group.6AN treated group had a higher phosphorylated AKT protein level than blank at 30min?1h?2h?6h?8h time points,especially at the 30min point.The phosphorylated ERK protein level showed similar behavior.Phosphorylated JNK level was higher at 30 time and 1 hour points,whereas phosphorylated p38 level was higher only at the 30 min point.After being treated by 6AN for 20h,macrophage cells sample was stained with anti-NF-KB antibody and its secondary anti-mouse IgG(red),followed by the blue cell nucleus dye DAPI.NF-?B could be found in the macrophage cell nuclei in 6AN treated group,comparing sharply with that in control.The size or weight of tumor in 6AN treated group were smaller than both PBS treated group and 6AN with macrophage depleted group.(3)In vitro,the colony size of TRC in PBS group was three times and colony number was two times that of 6AN group.In vivo,both the colony size and number of TRC in PBS group were about two times that in 6AN group.(4)6AN could inhibit tumor growth in vivo.The survival curves of C57BL/6 mice with CMT-93 tumor and Balb/c mice with H22 cells showed significant prolongation of survival in 6AN treated mice compared with PBS treated group.For B16F1 cells on C57BL/6 mice,tumor size or weight were smaller in the 6AN treated group.(5)The number of the tumor on lung tissue in 6AN-cisplatin combination treatment group was smaller than the cisplatin treated group.6AN-cisplatin combination treated mice including rectal tumor bearing mouse and hepatocarcinoma bearing mouse had a longer survival time than those with only cisplatin treatment.Conclusion:6AN can polarize macrophages towards M1 phenotype through ROS/Akt/MAPKs/NF-?B signaling pathway,resulting in the increase of surface markers such as CD80,CD86,MHCII and intracellular factors such as IL12,IFN-?,which are beneficial for tumor immunotherapy.Besides,we also corroborated that 6AN could directly inhibit tumor stem cell growth and sensitize tumor to cisplatin.Therefore,6AN can be a potential cancer therapy drugs by virtue of its abilities of polarizing macrophages towards M1 phenotype,directly inhibiting tumor stem cell growth and sensitizing tumor to chemotherapy and radiotherapy. |
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