| Background and objectivesGlioblastoma Mutiform(GBM)is the most common primary central nervous system tumor in the skull.It generally has a poor prognosis and often shows primary and secondary resistance to chemotherapeutics.Surgery is still the first clinical choice.Temozolomide(TMZ),an imidazole derivative,is a common antiglioma drug in clinical practice.Although temozolomide-based neurosurgery chemotherapy has been widely used in clinical practice,not all patients will benefit clinically because of the resistance to TMZ.The expression of O(6)-methylguanine-DNA methyltransferase(MGMT)protein induced by TMZ administration in drug-resistant patients is the most significant anti-TMZ determinant,and its expression can lead to GBM cells develop primary and secondary resistance,so new treatments are urgently needed to reverse this resistance.Combining previous research and the results of our research group,we found that resveratrol from natural plants showed obvious inhibitory effects on certain tumor cells and good safety at therapeutic doses,such as its effect on bladder cancer,breast cancer,prostate cancer,skin cancer,gastric cancer and other tumor cells.Resveratrol has a significant inhibitory effect on the tumor growth.There have been many reports of in vivo and in vitro experiments on resveratrol in combination with chemotherapeutics to treat tumors.Studies have shown that resveratrol can improve the sensitivity of many chemotherapeutics such as gemcitabine,vincristine,doxorubicin and paclitaxel against tumor cells.Besides,the low water-solubility,rapid metabolism and low bioavailability of resveratrol affect its various beneficial pharmacological activities,so its development and application are limited to a certain extent.Through synergistic administration,the bioavailability of resveratrol can be improved,promoting its early transformation from basic research to clinical application.Previous literatures have reported that the combined administration of resveratrol combining TMZ chemotherapy is used to treat gliomas,but whether the combined administration method has broad spectrum for all glioma cells,and whether the glioma cell line with dual drug resistance can be reversed after combined administration may need to be further studied in detail.Resverotral(Res)has anti-GBM activity,but also faces resistance problems,which is similar to TMZ.Since the compatibility of anticancer drugs is an effective way to overcome the resistance of tumor cells,we explore the possibility of improving the sensitivity of GBM chemotherapy by combining resveratrol with temozolomide.In addition,the limited in vivo bioavailability of resveratrol combined with the blockage of the blood-brain barrier make it difficult for conventional in vivo administration methods such as oral administration or intraperitoneal injection to reach intracranial glioma lesions.The lumbar puncture administration model has achieved certain effects in rats with orthotopic transplantation of glioma,but the invasive administration of lumbar puncture administration has some certain risks and easily makes patient discomfort.So,we continue to explore a new type of nasal administration of resveratrol to minimize damage while achieving efficacy.In this study,RG-2,which is sensitive to both resveratrol and temozolomide,and LN-18 and LN-428 GBM cell lines,which are not sensitive to both,were selected.A variety of experimental methods were used to determine the survival rate and apoptosis of cell phenotypic changes to mechanism studies,such as exploring changes in the STAT3 signaling pathway,and changes of Galectin-1 and drug resistance protein MGMT to evaluate the effect of combined administration of resveratrol combining TMZ chemotherapy on the impact of glioma cell growth and death.At the same time,an orthotopic glioma transplantation model of RG-2 cells in SD rats was constructed.Specimens of tumors treated with nasal administration of resveratrol were analyzed by detecting apoptosis to verify the feasibility of nasal administration and provide a basic experimental basis for further improving the bioavailability of resveratrol in the clinic.Materials and MethodsThe rat glioblastoma RG-2 cell line and human-derived glioblastoma LN-18 and LN-428 cell lines were provided by the Neurosurgery Laboratory of the University of Lausanne School of Medicine,Switzerland.SD rats were provided by the Laboratory Animal Center of Dalian Medical University.In this experiment,a cell culture method was used to establish an in vitro co-administration model of resveratrol and temozolomide,and with the help of the novel coverslip preparation dishes(Chinese invention patent number:ZL201520113833.9)developed in this experiment,different dishes with kinds of administration doses were collected at different detecting time points.The cell dishes were analyzed by MTT cell survival test and morphological staining for the sensitivity difference of glioblastoma to resveratrol and temozolomide and the inhibitory effect of different concentrations of the combined administration to determine the cells with different sensitivity.The effective concentration of the system is compatible with the dose.Furthermore,MTT(Methyl thiazolyl-tetrazolium,MTT)experiment,HE(Hematoxylin-eosin staining,HE)staining,flow cytometry combined with Annexin V-PI(Propidium iodide,PI),TUNEL(Terminal deoxynucleotidyl transferase d UTP nick end labeling assay,TUNEL)and other methods were used to verify the feasibility of synergistic sensitization through effective concentration of compatible administration.Immunocytochemistry(ICC)and western-blotting analysis were used to observe TMZ drug resistance protein MGMT,tumor-associated protein Galectin-1.Expression changes of STAT3(Signal transducer and activator of transcription 3,STAT3)and its phosphorylated protein p-STAT3 and their downstream anti-apoptotic protein Bcl-2,cell survival protein survivin protein were detected to explore the possible in vitro synergistic sensitization of combination of resveratrol and temozolomide.In the in vivo experiment,on the basis of constructing the orthotopic transplantation model of rat malignant glioma,we randomly divided the rats into a nasal solvent control group and resveratrol nasal administration group for nasal drip treatment twice a day.By recording survival time,using pathological sections to calculate the tumor area of the two groups for comparison,using immunohistochemistry(IHC)to analyze the change of tumor proliferative active antigen(Ki-67)after administration,and TUNEL staining to analyze the apoptosis of tumor cells after administration,we verified the authenticity and reliability of the treatment effect of the rat postoperative model after nasal resveratrol treatment.The internal molecular mechanism of anti-glioma effect of intranasal resveratrol at the molecular level through western-blotting,immunohistochemistry(IHC)and other methods were further discussed.Results1.The difference in sensitivity of human glioblastoma LN-18,LN-428 cells and rat glioblastoma RG-2 cells to resveratrol and TMZ and the feasibility of effective concentration of drugs for sensitization analysis1).MTT cell survival experiment and HE staining results showed that RG-2 cell growth was significantly inhibited and appeared apoptosis after treated with conventional doses of resveratrol and TMZ.While,they had no obvious growth inhibitory effect on LN-18 and LN-428 cells.2).Conventional dose of resveratrol could significantly reverse the resistance of RG-2 cell line to TMZ.The sensitivity of RG-2 cells to resveratrol was significantly higher than that of temozolomide.Temozolomide under the high concentration(T750,T1000,T1250,T1500)could exert the tumor killing effect;The above changes did not appear in RG-2 cells under the concentration of T250 and T500 alone.When R100 combined with temozolomide(T250,T500)were administered to RG-2 cells after 48h,the degree of cell growth inhibition was higher than that of high concentration temozolomide(T750,T1000,T1250,T1500)alone.3).The combination of low concentration resveratrol and TMZ effectively inhibited the growth of RG-2 cells,and the combination of high concentration resveratrol and TMZ inhibited the growth of LN-18 and LN-428 cells.MTT experiment and HE staining results showed that when R25,R50 and R75were administered in combination with T250 and T500 on RG-2 cells,the cell growth inhibitory effect was enhanced than the control group,and it was positively correlated with the increase in the compatible concentration dose and the passage of time.After treated with the combination of R50,R75,R100 and T500,T750 for 48h,the cell growth inhibitory of LN-18,LN-428 cells effect were stronger than that of the simple administration group,and it was positively correlated with the increase in the concentration concentration.There was mutual sensitization between them,and the sensitization effect in different cell lines was closely related to drug sensitivity.4).The effective dose of resveratrol/TMZ simultaneously reversed the resistance of LN-18,LN-428 cells and RG-2 cells.After R25/T250 treatment for 48h,MTT test showed that the RG-2 cell survival rate decreased,HE staining and TUNEL test results showed that the cell morphology changed significantly,and there appeared significant apoptosis morphological changes.The results of flow cytometry showed that the combination of R25/T250significantly induced the apoptosis of RG-2 cells,and the cell cycle was blocked in S phase.After 48 hours of co-administration of R75/T750 on the LN-18 and LN-428cells,the MTT test showed that the cell survival rate of the two cells was significantly lower than that of the simple administration group.HE staining and TUNEL results were consistent and showed that the cell morphological had apoptosis changes.The results of flow cytometry showed that the combination of T75/T750 significantly induced apoptosis in LN-18 and LN-428 cells,and the cell cycle were both blocked in G0/G1period.2.Research on the molecular mechanism of effective concentration combination administration to reverse the resistance of resveratrol and TMZ to LN-18,LN-428 and RG-2 cells1).Western-blotting and ICC staining results showed that the MGMT protein was expressed in RG-2 cells.Compared with TMZ group,the expression level of it was down-regulated by 44.9%in R25/T250 group,with statistical significance(P<0.01).MGMT protein was expressed in normal cultured LN-18 and LN-428 cells.Compared with TMZ group,the expression of it were down-regulated(LN18:38.7%,P<0.05;LN428:33.5%,P<0.01)in R75/T750 group for 48h.2).Western blotting and ICC results showed that RG-2 cells expressed Galectin-1 protein intracellularly,and after administration of R25/T250,the expression level of it was increased by 72.6%(P<0.05).Both LN-18 and LN-428expressed Galectin-1 protein.After 48 hours of administration with R75/T750,the expression level of Galectin-1 protein in LN-18 cells was up-regulated(LN18:79.3%,P<0.05;LN428:41.5%,P<0.05).3).Western blotting and ICC results showed that RG-2 cells expressed STAT3protein intracellularly.It was reduced by 25.4%(P<0.01)after administration of R25/T250.The expression of STAT3 protein in LN-18 and LN-428 were down-regulated after the combination of R75/T750(LN18:47.2%,P<0.05;LN428:47.4%,P<0.05)at 48h.Western blotting and ICC results showed that p-STAT3(Y705)protein was expressed in RG-2 cells.The expression level of it was significantly reduced by71.2%(P<0.01)after the combination of R25/T250.The expression of p-STAT3(Y705)protein in LN-18 and LN-428 cells were down-regulated after the combination of R75/T750(LN18:45.7%,P<0.05;LN428:32.6%,P<0.05)at 48h.4).Western blotting and ICC results showed that RG-2 cells expressed survivin protein,and the expression of it was down-regulated by 56.9%(P<0.05)after the treatment of R25/T250.The expression of survivin protein in LN-18 and LN-428were down-regulated 48 hours after the combination of R75/T750(LN18:43.5%,P<0.05;LN428:24.7%,P<0.05).RG-2 cells expressed Bcl-2 protein,and after combined administration of R25/T250,the expression level of it was reduced by 45.9%(P<0.05).The expression level of Bcl-2 protein in LN-18 and LN-428 cells were down-regulated after the combination of R75/T750(LN18:36.4%,P<0.05;LN428:34.2%,P<0.05)at 48h.3.Analysis of intranasal(IN)resveratrol therapy for orthotopic transplantation of RG-2 cell glioma in SD rats1).Nasal administration of resveratrol inhibited tumor growth and prolonged the survival time of model rats.The average survival time of the nasal solvent control group was 16.2±1.5 days,and it in the IN group was 20.4±2.8 days.The result of statistical analysis showed that the survival time of the rats in the IN group was longer than that of the control group,and the survival time is prolonged;the t-test analysis result showed that the P value of the IN group and the control group was less than 0.05,indicating that there was a statistical difference.The survival life chart also proved that the survival rate and time of the rats in the IN group were significantly better than those in the control group.2).Nasal administration of resveratrol inhibited the proliferation activity of glioma cells and induced apoptosis.The tissue sections of the tumor were dehydrated and embedded in paraffin.HE staining showed that the tumor tissue structure of the control group was complete,the tumor cells grew vigorously,and the clusters were densely clustered.A large number of died tumor cells and liquefied necrosis foci were seen in IN group,especially in the periphery of the blood vessels.The tumor tissues were relatively regular and there was no obvious invasive growth.Immunohistochemistry(IHC)analysis showed that Ki-67 expression was positive in the control group,and expression of it in the IN group was significantly reduced.TUNEL results showed that only a small number of cells were positive in the tumor tissue area of the control group,while a large number of dense positive cells were found in the tumor tissue area in the IN group.3).Intranasal resveratrol effectively inhibited the activation of STAT3 signal transduction pathway and down-regulated the expression of its downstream molecule.IHC analysis showed that there was STAT3 expression in the tumor cells of the control group,while the expression level of STAT3 in the IN group was significantly reduced.The phosphorylation of STAT3(p-STAT3)was positive in the control group,which is significantly weakened after treatment.Correspondingly,the expression level of Bcl-2,a downstream effector of STAT3,was also significantly down-regulated in the IN group.The results of western blotting grayscale analysis showed that resveratrol caused the expression level of STAT3(28%),p-STAT3(37%)and its downstream Bcl-2(32%)dropped significantly.4).Intranasal resveratrol significantly increased the expression of GFAP protein.IHC results showed that the GFAP protein had positive expression in the normal rat brain,while the control group showed negative expression and the IN group had significantly increased the expression of it.Western blotting results showed that the expression of GFAP protein was up-regulated in the IN group(P<0.05),suggesting that the expression of astrocyte markers had a tendency to be reversed.Conclusion:1.Conventional dose of 100?M resveratrol and 500?M TMZ effectively inhibited the growth of RG-2 cells,while LN-18 and LN-428 cells were less sensitive to both of them.The drug sensitivity was RG-2>LN-18>LN-428.2.TMZ induced RG-2 cell to differentiation and apoptosis in a dose-dependent manner.Low-dose TMZ combined with R100 significantly inhibited cell proliferation.3.Low-dose TMZ(T250,T500)combined with low-dose resveratrol(R25,R50,R75)had a more obvious killing effect on RG-2 cells than single drug,and this effect increased with time,suggesting that there was mutual sensitization between them.4.LN-18 and LN-428 cells could be induced to apoptosis and cell cycle arrest by combination of high concentration resveratrol/TMZ,suggesting that the glioma cells with different sensitivities needed to be treated with different effective concentrations and dosages in order to improve the individualized treatment effect and reduce the side effects of drugs.5.The high expression state of MGMT protein in vivo was directly related to the occurrence of resistance to TMZ in glioma patients,and may be one of the targets for reversing drug resistance in glioma patients.Effective combination of low concentration resveratrol and TMZ on RG-2 cells,high concentration resveratrol and TMZ on LN-18 and LN-428 cells downregulated the expression level of MGMT protein.6.The effective combination of resveratrol/TMZ in treating RG-2,LN-18,LN-428 cells up-regulated the expression level of Galectin-1 protein to varying degrees.7.The activation of STAT3 signaling pathway was positively correlated with the occurrence and development of glioblastoma.The effective combination of resveratrol/TMZ significantly inhibited the STAT3 signaling pathway.Bcl-2 and survivin,the expression of its downstream gene,all showed a downward trend.8.Targeted treatment of intracranial RG-2 glioblastoma of SD rats with resveratrol by nasal administration prolonged the life cycle of orthotopically transplanted glioma rats and inhibited the growth of gliomas.9.Intranasal resveratrol targeted gliomas by enhancing tumor apoptosis,down-regulating Ki67 expression to achieve the purpose of treatment.10.After intranasal administration of resveratrol to glioma tissue,the STAT3signaling pathway and its downstream Bcl-2 protein were inhibited,thereby exerting anti-tumor activity.11.After intranasal treatment of orthotopic rat glioblastomas with resveratrol,the expression of GFAP,the astrocytic marker of glioma tissue,increased. |
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