| BackgroundGlobally,prostate cancer is a challenging health problem for men.Traditional prostate cancer treatment methods include radical prostatectomy,prostate cancer radiotherapy,endocrine therapy,and chemotherapy.Prostate cancer chemotherapy includes neoadjuvant chemotherapy and postoperative salvage chemotherapy.Commonly used clinical chemotherapy drugs include gemcitabine,docetaxel and paclitaxel,but these drugs have the problem of weak targeting and strong side effects.In addition,because prostate cancer is a multi-gene abnormality,the survival of cancer cells is regulated by a variety of signal transduction pathways.Therefore,the chemotherapy of prostate cancer urgently needs a preparation containing multiple drugs(targeting multiple pathways)to improve targeting property,reduce toxic side effects and effectively kill cancer cells.COX-2 and GLUT-1 can promote the occurrence and development of tumors by regulating related signal transduction pathways.Celecoxib can inhibit the expression of COX-2 protein,leading to the decrease of intracellular prostaglandin E2,thereby inhibiting tumor proliferation and invasion.However,celecoxib used in large doses alone can cause serious adverse reactions to patients.Therefore,realizing the targeted delivery of drugs to tumor tissues is an effective strategy for using celecoxib to treat tumors.Genistein is a well-known GLUT-1 protein inhibitor,which can induce the formation of ROS and is related to the activation of AMPK signaling pathway.It is a new tumor chemotherapy agent.However,due to the low water solubility of the drug,it is difficult to achieve intravenous administration,and the bioavailability of oral administration is poor.Therefore,it is an urgent problem to realize the dissolution of the drug in body fluids and its transport to tumor tissues.Nanoliposomes are a new type of nano-drug delivery system based on natural or synthetic lipid carriers.It has high biocompatibility,excellent targeting and multi-drug loading.It can overcome the short half-life of the original drug in vivo,poor drug solubility,large adverse reactions,weak targeting and toxicity of injection excipients.Nanoliposomes can encapsulate a variety of anti-tumor drugs and deliver them to specific tumor sites,and have excellent effects in combined anti-tumor.The drug nanoliposome can be slowly released according to the designed ratio,and has a long circulation period in the blood,which further improves the efficacy of the drug.Since nanomaterials with a diameter of about 100 nm exhibit "enhanced permeability and retention" effects,drug delivery nanocarriers can provide better therapeutic efficacy.Different from normal healthy blood vessels,the leaky vasculature in tumor tissue has a gap of about 600-800 nm between adjacent endothelial cells,which promotes the passive targeting of nanomaterial drugs.This defective vascular system has poor lymphatic drainage,allowing nanomaterial drugs to penetrate into the extravascular space and accumulate in tumor tissues.ObjectiveIn this study,bioinformatics methods and immunohistochemical detection were used to clarify the transcription and expression of COX-2 and GLUT-1 in prostate cancer tissue and to explore the correlation between the mutation gene and common tumor.Multifunctional nanolipids encapsulating celecoxib and genistein were prepared to achieve precise delivery of drugs to tumor cells and reduce adverse drug reactions.Cell experiments were performed to clarified the inhibitory effect of celecoxib and genistein nanoliposomes on the proliferation of prostate cancer cells,and explore its mechanism of action.This study provides a new preparation and experimental basis for the application of multifunctional nano liposomes encapsulating celecoxib and genistein to treat prostate cancer,especially castration-resistant prostate cancer.Methods1.Transcription and expression of COX-2 and GLUT-1 in prostate cancerOncomine database was used to analyze the transcription of COX-2 and GLUT-1m RNA in prostate cancer tissue and normal prostate tissue.STRING database was used to analyze the association of COX-2 and GLUT-1 gene with other tumor genes.Immunohistochemical staining method was employed to detect the expression of COX-2 and GLUT-1 protein in prostate cancer tissue and benign prostate tissue.2.Preparation and physical and chemical analysis of multifunctional nanoliposomes encapsulating celecoxib and genisteinThe preparation of target drugs includes empty nanoliposomes,nanoliposomes encapsulating celecoxib,nanoliposomes encapsulating genistein,nanoliposomes encapsulating celecoxib and genistein.Synthesis of empty nanoliposomes:(1)L-?-phosphatidylcholine(egg PC)and1,2-dipalmitoyl-tin-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] Ammonium salt(DPPE-PEG-2000)was dissolved in chloroform at a ratio of 95:5 mol% to prepare a total lipid of 25 mg/ml;(2)Remove the chloroform in the lipid mixture by blowing in N2 gas;(3)The obtained lipid was suspended in sterile salt solution or water,and vortexed intermittently at 60? for 30 min;(4)30 min later ultrasonic treatment of the above solution;(5)Use Avanti Mini-Extruder to extrude through a 100 nm polycarbonate membrane at 60?.Preparation of celecoxib and genistein nanoliposomes: After the preparation of the above 25 mg/ml concentration of total lipids is completed,celecoxib(100 ?M)and genistein(10 ?M)are combined separately and 10 :1 is dissolved in the above lipid chloroform solution.The subsequent steps are the same as above.Physical and chemical analysis of celecoxib and genistein nano-liposomes:(1)Observe the imaging characteristics of drug nano-liposomes under TEM with scanning electron microscope;(2)Measure nano-lipids dispersed in PBS and water Body hydrodynamic diameter and zeta potential;(3)Monitor the absorbance at 255 nm and 382 nm to quantify the loading of celecoxib and genistein in the nanoliposomes;(4)Measure at 10 m M glutathione the release kinetic pattern in peptide media.3.Experimental study of drug nanoliposomes against prostate cancerProstate cancer cells PC-3,LNCa P,and fibroblasts were cultured in vitro.The cells were divided into the empty nanoliposome group,the celecoxib nanoliposome group,the genistein nanoliposome group,the celecoxib and the genistein nanoliposome group.(1)The MTT method was used to detect the changes in cell viability under the action of each group of drugs;(2)The cell scratch healing experiment was used to detect the inhibitory effect of each group of drugs on PC-3 cells,LNCa P cells,and fibroblast metastasis;(3)ROS levels in prostate cancer cells in each group were detected after acting;(4)GSH levels in prostate cancer cells after drug treatment were detected;(5)Flow cytometry was used to detect 2-NBDG in cells to obtain the results of glucose uptake in cells,and to analysis the glucose transport levels in PC-3 cells and LNCa P cells under the action;(6)Western blot was used to detect the protein expression of COX-2,GLUT-1,Trx R,Prx-6,Cleaved caspase-3 in PC-3 cells and LNCa P cells under the action of each group in;(7)Image-J density analyzer was used to quantitatively analyze protein bands;(8)RT-PCR was employed to detect the m RNA transcription of COX-2,GLUT-1,GLUT-1,GLUT-1,Trx R,Prx-6 and Caspase-3 in PC-3 cells and LNCa P cells under the action of each group.Result1.Increased transcription and expression of COX-2 and GLUT-1 in prostate cancer tissue(1)Bioinformatics analysis results show that,compared with normal prostate tissue,COX-2 and GLUT-1 m RNA were up-regulated in prostate cancer tissue.The results of immunohistochemical staining showed that,compared with benign prostate tissue,the expression of COX-2 and GLUT-1 proteins were up-regulated in prostate cancer tissue,and were positively correlated with Gleason score.(2)The hydrodynamic diameters of nanoliposomes dispersed in PBS and water X were 112 nm,108 nm,98 nm,and 92 nm,respectively;the zeta potential measurement of nanoliposomes dispersed in PBS ranged from-2.0 to Between-0.6m V,nanoliposomes dispersed in water showed a value of-50 to-46 m V.(3)Encapsulation quantitative results showed that about 91.7% of celecoxib was encapsulated in CL,about 68.9% of genistein was encapsulated in GL,and celecoxib and genistein in CGL were 78.7% and 63.5% respectively.(4)The drug release kinetics showed that celecoxib and genistein from CGL were in a 10 m M glutathione medium for 48 hrs,the release rate of celecoxib was about 82%,and genistein release rate was about 79%.3.Multifunctional nano liposomes inhibit the proliferation and invasion of prostate cancer cells(1)The cell viability test results showed that after 24 hrs,CGL acts on PC-3cells to reduce cell viability by approximately 75%,and 72 hrs results in cell viability reduction by approximately 85%.The effect of CGL on LNCa P cells resulted in a reduction of cell proliferation by approximately 60%.CGL showed a slight decrease in cell viability in fibroblasts(<10%),but the action time was prolonged(48 hrs and 72 hrs).(2)Scratch healing experiments showed that when CGL acts on scratches,it is observed that at all time points 0 hrs,24 hrs and 48 hrs,it is observed that the migration of PC-3 cells to the scratch area is significantly inhibited,and CGL acts on 48 hrs,you can clearly see that the cells are spherical.(3)Studies on the levels of cell markers after drug action showed that PC-3cells produced significant(3 times)ROS after the action of CGL,and LNCa P cells under CGL action produced about 1.6 times the amount of ROS,EL,CL,GL and CGL has no obvious effect on the production of ROS in fibroblasts;GL and CGL affect the GSH production in PC-3 cells to decrease by about 30% and 90%,and CGL leads to a decrease of about 90% in GSH production in LNCa P cells.(4)The detection of cellular glucose uptake capacity showed that GL and CGL inhibited about 60% of glucose transport in PC-3 cells and LNCa P cells.(5)Western blot results showed that the expression of Cleaved caspase-3protein in PC-3 cells increased by 23 times after CGL treatment;Trx R expression in PC-3 cells increased by about 8 times after CGL treatment;Prx increased after CGL treatment in PC-3 cells The expression level of-6 is significantly reduced;the effect of GL and CGL leads to a significant decrease in the expression of GLUT-1 protein,and the effect of CGL leads to a decrease of about 90% of the expression of GLUT-1 protein;after the effect of CL and CGL,the expression of COX-2 protein in PC-3 cells is significant increase.(6)RT-PCR test results showed that the expression of COX-2 m RNA was significantly reduced after CGL acted on PC-3 cells;GL and CGL acted on PC-3cells significantly inhibited GLUT-1 m RNA synthesis;CGL acted on PC-3 The m RNA synthesis of Prx-6 and Trx R increased significantly after the cells;the Caspase-3 m RNA synthesis of PC-3 cells treated by CGL increased significantly.Conclusion1.Compared with normal prostate cells and prostate benign diseased tissues,the transcription and expression of COX-2 and GLUT-1 in prostate cancer tissues are up-regulated,and there is a positive correlation with Gleason score.COX-2gene and STAT3 gene have strong correlation,GLUT-1 gene and TP53 gene have strong correlation,suggesting that COX-2 and GLUT-1 play an important role in the development of prostate cancer.2.In the study,we successfully prepared the nanoliposomes encapsulated celecoxib and genistein.The diameter of nanoliposomes is about 100 nm,which is the volume of tumor cells easy to internalize.The prepared nanoliposomes have good encapsulation efficiency and stable release rate.3.Nanoliposomes inhibit the proliferation and migration of prostate cancer cells through a synergistic effect.The mechanism is to induce ROS production,reduce cell GSH concentration,inhibit COX-2 signaling pathway,reduce GLUT-1protein,inhibit glucose uptake,and reduce Prx-6 protein and increase Trx R and Cleaved caspase-3 protein levels,promote cancer cell apoptosis. |
PDF Full Text Request