| Cancer is seriously endangering human life and health,especially lung cancer,ranking the top cause of death in China's malignant tumors,and the incidence is still increasing year by year.Non-small cell lung cancer(NSCLC)is the most common,accounting for 80%to 85%of the total number of lung cancer.About 70%of patients had been in the moderate and advanced stages when diagnosed,making the treatment of lung cancer more difficult.At present,the study of radiotherapy and chemotherapy is increasingly thorough,but for the treatment effect of NSCLC is still not optimistic,while strong side effects and drug resistance brings great pain to patients.In recent years,with the deepening study of tumor molecular biology,angiogenesis as a target to explore the anti-tumor pathways and research and development of related drug has also become a hot topic in the treatment of NSCLC,and VEGF/VEGFR2,a key connection point in the process of tumor/tumor angiogenesis,is one of the most important molecular targets for targeted therapy.However,the inhibition of tumor angiogenesis has shown a certain resistance caused by mutations in directly targeting VEGF and its receptors or other regulatory factors in the angiogenesis pathway,and the researchers are attempting to aim at multiple targets or other ways to solve these clinical problems.Tumor and tumor blood vessel are interdependent,the cell membrane of tumor cells and tumor-derived vascular endothelial cells can not be separated from cholesterol.From a large number of related literature found that,glycoside alkaloids have the characteristics of binding to cholesterol(including cell membrane lipid raft cholesterol)to form insoluble complexes,combined with the latest research progress in the regulation of cholesterol efflux destroys cell membrane lipid raft structure and blocks VEGF/VEGFR2 binding to inhibit angiogenesis,as well as tumor angiogenesis theory for association analysis,imagining that by agglutinating tumor vascular endothelial cell membrane cholesterol to destroy cell membrane lipid raft structure whether can achieve the role of inhibiting angiogenesis,either,thereby cutting off the nutrition supply of the tumor,so that the growth can't continue,and then achieve the anti-tumor effect.That is to say,we propose the hypothesis of anti-tumor mechanism,"glycoside alkaloids inhibit the angiogenesis by directly agglutinating lipid raft cholesterol."Solanum lyratum,as a kind of traditional Chinese medicine,is the dried whole plant of Solanum lyratum Thunb(Solanaceae),and has more than two thousand years of application history,which was first recorded in the "Shen Nong's herbal classic",and was listed in the top grade.As an anti-cancer drug,Solanum lyratum can be used for the treatment of a variety of tumors,including lung cancer,esophageal cancer,cervical cancer,capillary hemangioma.The previous study proved alkaloids were anti-tumor active components of Solanum lyratum.Total alkaloids,which were extracted from dried whole plant of Solanum lyratum,were separated by traditional column chromatography and high performance liquid chromatography-mass spectroscopy(HLPC-MS),then,four kinds of glycoside alkaloid monomers were obtained.The acute toxicity of total alkaloids from Solanum lyratum and the inhibitory effect on three types of non-small cell lung cancer were investigated.Then,based on the angiogenesis,the mechanism of anti-tumor effect of the alkaloids from Solanum lyratum inhibited the angiogenesis via agglutinating lipid raft cholesterol was investigated.The main research contents are as follows:1.First,70%ethanol was refluxed to extract the dried whole plant of Sol anum lyratum,the extracting solution was enriched by D151 ion exchange mac roporous adsorption resin,and desalted by AB-8 macroporous resin to obtain t he total alkaloids of Solanum lyratum(recorded as STA).Then,the total alkalo ids Solanum lyratum were prefractionated by column chromatography with elue nt ethyl acetate-95%ethanol(2.5:1)as eluant to obtain A and B parts.Then,with the separation and purification by HLPC-MS,SA1 and SA2 were obtain ed from part A,SA3 and SA4 were obtained from part B.The structures of f our compounds were identified by nuclear magnetic resonance(including one-di mensional NMR:1H-NMR and 13C-NMR,two-dimensional NMR:1H-1H COSY,HMQC,HMBC,TOCSY and NOESY).They are(3?,22?,25R)-spirosolan-5-en-3-yl O-?-D-glucopyranosyl-(1?2)-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyrano side(SA1)?(3?,5?,22?,25R)-spirosolan-3-yl O-?-D-glucopyranosyl-(1?2)-O?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(SA2)?(3?,22?,25R)-spirosol-5-en-3-yl O-?-D-glucopyranosyl-(1?2)-O-[?-D-xylopyranosyl-(1?3)]-O-?-D-glucopyrano syl-(1?4)-?-D-galactopyranoside(SA3)?(3?,5?,22?,25R)-spirosolan-3-yl O-?-D-g lucopyranosyl-(1?2)-O-[?-D-xylopyranosyl-(1?3)]-O-?-D-glucopyranosyl-(1?4)-?-D-galactopyranoside(SA4).Four compounds are all steroidal glycoalkaloids wi th spiroslane as the aglycone.SA2 was used as the reference substance in the determination of total alkali content by acid-dye colorimetry.The total alkali c ontent determination method with good precision/stability/reproducibility,and hi gh recovery rate were selected by methodology examination,total alkali content of three samples of STA were more than 90%detected by the method.2.Acute toxicity was tested in mice in order to study the oral toxicity of STA,and effect of STA on the survival time of Lewis lung carcinoma xenograft in mice was studied.The nude mice xenograft models were established by inoculating three different cell subtypes of human NSCLC to study the antitumor effect of total alkaloids in vivo.Hematoxylin and eosin(HE)staining was used to observe the pathological changes of tumor tissues in A549 xenogragt nude mice,microvessel density(MVD)in tumor tissues was assessed by CD31 immunohistochemical analysis.Protein expression levels of VEGF/VEGFR2,CyclinA and CyclinBl in tumor tissues were detected by immunohistochemistry,either.Finally,cell apoptosis in tumor tissue was detected by TUNEL method.Results of acute toxicity test showed no animals died at doses of 10000 mg/kg.BW,and no animals abnormal in maximum tolerance experiments,so safe dose range of STA in oral were gotten,STA had no oral toxicity to some extent due to the large safe dose range.Results of life extension test of Lewis lung carcinoma xenograft in mice showed STA could significantly prolong the survival time(P<0.05,vs model group),The life extension rate of STA high dose group was similar to taxol group(P>0.05),which used was as the positive control,and first death time longer in STA high dose group.STA showed a dose dependent inhibition in nude mice inoculated with three different cell subtypes of human NSCLC,tumor volume and weight were significantly reduced,and body weight increased gradually,while weight of animals in taxol group increased relatively slow,which showed STA had advantage with anti-tumor activity while few side effects.Pathological results of tumor tissues by HE staining showed cell growth activity decreased,and the tissue necrosis area enlarged in treatment groups,compared with model group,especially significant necrosis in STA high-dose group.Blood vessels decreased with the increase of STA,and the blood vessels were hardly observed in high-dose group.The proliferation of fibrous tissue was also decreased after drug administration.Results of MVD test in tumor tissues,assessed by CD31 immunohistochemical analysis,showed that compared with model group,with the increase of the concentration STA,the total number of tumor tissues decreased gradually.The results of immunohistochemistry showed that the inhibition of STA was also reflected in the expression of CyclinA and CyclinB1(P<0.05,vs model group),which indicated that STA could regulate the cell cycle of NSCLC.Finally,the inhibition of NSCLC by STA as STA increased the apoptosis index of the tumor cells.3.HUVECs and A549 cells were co-cultured in Transwell chamber to construct a co-culture system to prepare the tumor-derived vascular endothelial cells(Td-ECs).The expressions of TEM1 and TEM8 in Td-ECs,which were specific antigens of tumor-derived vascular endothelial cells,were detected by RT-PCR.The ultrastructure of Td-ECs was observed by transmission electron microscopy and scanning electron microscopy.MTT assay was used to detect the proliferation of Td-ECs in vitro.Cell scratching assay and Transwell migration assay were used to detect the migration ability of Td-ECs,and Transwell invasion assay were used to detect the invasion ability of Td-ECs.Experiments of tube formation in vitro,sprouting of rat arterial ring,angiogenesis of chick chorioallantoic membrane and zebrafish were used to detect anti-angiogenesis of alkaloids from Solanum lyratum from cell,tissue and the over-all level.The results showed that the co-culture system was successfully constructed by Transwell chamber,the polycarbonate membrane between the upper chamber A549 cells and the lower chamber HUVECs,so that tumor cells secreted related factors to induce HUVECs into tumor-derived vascular endothelial cells.Td-ECs prepared by co-culture can expand the bands of 316 bp(TEM8)and 349 bp(TEM1),which were specific antigens of tumor-derived vascular endothelial cells,were detected by RT-PCR.The Td-ECs obtained by co-culture had a series of changes in ultrastructural activity compared with HUVECs.Under the transmission electron microscope,the mitochondrial morphological changes were observed,and more vesicle structure appeared in the Td-ECs cells.Compared with the HUVECs alone,the mitochondria vacuolization was obvious.Under the scanning electron microscope,cell elongation reduced,cell terminal filopodia became less and shorter.These changes made Td-ECs easier to proliferate and migrate.Alkaloids of Solanum lyratum inhibited proliferation of Td-ECs in a dose-dependent manner,the inhibition of SA1 was the weakest,and the half inhibitory concentration(IC50)was not reached within 100?M.The half inhibitory concentration of SA2,SA3,SA4,STA were 81.334?M,75.265 ?M,2.950?M,49.548 ?g/mL,respectively.The IC50 and structural formulas of four glycoside alkaloids showed the difference between sugar moieties and saturation of the 5,6-bond of aglycone might affect the inhibition of glycoside alkaloids on cell proliferation.Scratch and Transwell chamber assay proved that alkaloids of Solanum lyratum could inhibit the migration and invasion of Td-ECs from the horizontal and longitudinal,as well as the tube formation of Td-ECs.In addition,the inhibition of SA2 on sprouting of rat arterial ring in dose-dependent and time-dependent manners,the inhibition of SA2 on CAM and SIV of zebrafish embryo in adose-dependent manner.4.The combination of S A2 and cholesterol in vitro was tested by test tube method,integrity and permeability of Td-ECs cell membrane were determined by extracellular fluid LDH activity and PI staining,intracellular and extracellular cholesterol content of Td-ECs cells was measured by cholesterol kit,then,lipid rafts were extracted for the detection of cholesterol levels in raft areas,and fluorescent immunoassay was used for the observation of co-localization of cholesterol and lipid rafts,the expression of ABCA1,ABCG1 and AIBP protein was detected by Western blot.The structure and number of cellular membrane caveolae were observed by transmission electron microscopy.The content of extracellular fluid VEGF was detected by ELISA,and the phosphorylation-levels of VEGFR2,AKT,SRC,FAK and ERK were detected by Western blot.The co-localization of VEGFR2/CAV1 and VEGFR2/EEA1 were detected by fluorescence immunochromatography to investigated the endocytosis of VEGFR2.Finally,the anti-tumor effect of SA2 was evaluated by A549 lung cancer zebrafish xenograft model.The results showed that SA2 could combine with cholesterol directly,then,the integrity of cytomembrane was destructed,which leading to the leakage of intracellular LDH.After treatment of SA2 on Td-ECs,the intracellular and extracellular cholesterol levels did not show good dose and time dependence.After treatment with low/middle-dose SA2,the total cholesterol content of the cells decreased firstly and then increased.While,Under treatment with middle/high-dose SA2,the total cholesterol level of the cells decreased from the elevated to the increasing trend,and gradually decreased to no significant difference with the control group(P>0.05,vs control group).Free cholesterol level of the cells showed a general trend of decline after firstly rising,free cholesterol level of the culture medium showed a decreasing trend as a whole.The expression of ABCA1,ABCGl and AIBP,which were cholesterol-related proteins,decreased in a dose-dependent manner.With the treatment of SA2(40 ?M),the cholesterol content in lipid raft area increased significantly.Fluorescence double labeling showed that cholesterol and lipid raft co-localized,the fluorescence intensity changes,which indicated cholesterol,were basically same to the result of cholesterol content of the cells detected by kit.The above results consider that,SA2 agglutinated the cholesterol of lipid raft,and then cholesterol feedback regulation of the cell was stimulated,which induced intracellular cholesterol synthesis,resulting accumulation of intracellular cholesterol,while the constraint of glycoside alkaloids,cholesterol was wrapped in cells which was difficult to flow out,with the increase of SA2 concentration and treated time,eventually leading to the fragmentation of cytomembrane.Transmission electron microscopy observation showed that the number of cellular membrane caveolae was significantly decreased after treatment of SA2,and it was difficult to find the structure of caveolae.SA2 inhibited the secretion of VEGF from tumor cells in a dose-dependent manner,and blocked the phosphorylation levels of VEGFR2,AKT,SRC,FAK and ERK in a dose-dependent manner.Fluorescence immunization double labeling result showed that VEGFR2 and CAV1,VEGFR2 and EEA1 co-localized.With the treatment of SA2,the Pearson correlation coefficient decreased,which affected the endocytosis of VEGFR2,and increased the expression of tumor suppressor gene-CAV1.Finally,SA2 showed significant anti-tumor effects in vivo using A549 lung cancer zebrafish xenograft model.In conclusion,four glycoside alkaloids with spiroslane as the aglycone were extracted and isolated from Solanum lyratum by column chromatography and high performance liquid chromatography-mass spectroscopy in this study.STA showed inhibition in nude mice inoculated with three different cell subtypes of human NSCLC.The inhibition of alkaloids from Solanum lyratum on angiogenesis was demonstrated by the proliferation/migration/invasion,and tube formation of Td-ECs,the sprouting of rat arterial ring,the angiogenesis of chick chorioallantoic membrane and zebrafish.S A2 could bind directly to cholesterol was proved,and agglutinated cholesterol of lipid raft,so that,cholesterol feedback regulation of the cell was stimulated.With the increase of SA2 concentration and treated time,eventually leading to the fragmentation of cytomembrane.SA2 disrupted the structure of caveolae,and inhibited the normal operation of VEGF/VEGFR2 upstream and downstream related signaling pathways of angiogenesis.Suggesting that the hypothesis of anti-tumor mechanism,"glycoside alkaloids inhibit the angiogenesis by directly agglutinating lipid raft cholesterol",was initially proved.But still requires validation of in vivo experiments,as well as more comprehensive experimental data support,including cholesterol feedback regulation,in order to provide a more direct basis for the hypotheses. |
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