Research About The Effect And Its Mechanisms Of Curcumin On Proliferation,Apoptosis,Angiogenesis And Radiosensitization Of The Lung Cancer Cell In Vitro

Pulmonary carcinoma is one of the malignant tumors with high attack rate andmortality rate. For phaseâ… -â…¡of the non-small cell carcinoma, the main method fortreatment is operation. But the local recurrent or the distal metastasis may happen in atleast 50% of the patients after the operations, and 70% of these patients are not able totake the operation for the second time, then radiotherapy, the chemo, and the targetmolecule have to be the main treatment methods. But we know that when the patientstake these methods for a long time, there will be serious toxicity, large payment but badefferent. So now we are trying to get the drug with the better effect, lower toxicity,sensitization to the radiotherapy and can also inhibit the proliferation, indicate thenecrosis of the tumor cells from the traditional Chinese drugs, to find a new way for theclinical treatment of the pulmonary carcinoma.Curcumin (Cur) is a kind of phenols pigment which is extracted from thecurcumalonga of the curcuma category of the ginger family. Quite a lot of researches have indicated that the Cur has several biological functions such as anti-inflammation,antioxygen, anti-oncoma, and also, it is cheap-price, low-toxicity (LD₅₀ of mouses is2g/Kg). The researches at present mostly focus on the chemical prevention. Recently,the NCI of America has already studied it in clinic as the third generation of theanti-oncoma medicines. The researches on the Cur all over the world are most about the7 aspects below: 1. Anti mutation and anti-NO 2. Effecting on the pathway of the signaltransduction of the epithelial grow factor receptors EGFR, PKC. 3. Down-graduatingthe nuclear factor, inhibiting the activity of iNOS, COX-2.4. Graduating the expressionof the oncogenes and anti-oncogenes. 5. Inducing the block of the cell cycle. 6.Inhibiting the activity of uPA and the secretion of MMP-9. 7. Inhibiting the formationof blood vessels.The researches of the Cur on the anti-oncoma effection to the pulmonarycarcinoma have been limited within the regulation of the expression of the oncogeneand anti-oncogene to induce apoptosis, or inhibiting angiogenesis of pulmonarycarcinoma by reducing the expression of the angiogenesis factors. But nocorresponding reports in China. We observe the Cru of its functions to the A2pulmonary carcinoma cells in vitro in 3 aspects: reducing the apoptosis, inhibiting theangiogenesis of pulmonary carcinoma, and radiosensitization. Also, in this subject, wewill discus the mechanism on the molecular level by detecting the active changes of the protein expression of the Bcl-2, Bax, surviving, P53, C-myc, VEGF, MMP-9, cyclinB₁,P34^cdc2, phos-P34^cdc2, P21 and NF-kB, and the change of the Ang-1, Ang-2, and TSP.Part One The function and mechanism of the Curinhibiting the growth and reducing the apoptosisof the human pulmonary carcinoma cells (A2 cells)Method1,Cell cultureThe RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The cells withmonolayer adherence grew. We took the logarithm of the cells in the growing period inthe experiment.2,Drug sensitivity test in MTTThe cells in the growing period had been collected and digested, and regulated intothe 1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid 150μl, shaking the plates for 10 min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Analyzing the results by SPSS, and then calculated the IC₅₀ by the method of linearregession.3,etecting the cell apoptosis by Annexin-FITC marking methodThe cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5*10⁵/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10 min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.4,Morphology detection of the cell apoptosisThe cells had been fixed by the 2.5% glutaraldhyde, 1% osmium tetroxide,dehydrated by the gradient ethanol, and the acetone, cutted by the Epon812 resinembedding ultrathin section cutter (AO type), stained by natrium aceticum and Lead Citrate, then observed and photographed under the TME of JEM-1200EX and H-600.5,Total DNA abstraction of cells and agarose gel electrophoresisAfter collecting cells (5×10⁶-1×10⁸), we threw away the superstratum, used thecold PBS 1-10ml to resuspense the cells, 500-1000rpm, 5min, centrifugation, then gotrid of the superstratum, and repeated it again. The cells were resuspend with 500μlnucleus splitting fluid, 50℃aqueous bathed and shocked for 3-5 hours, then13000rpm/min centrifugated, extrated the aqueous phase into another centrifuge tube,added 1/10 volumed 3MNaCl and double volumed ethanol, reversed the mixture forseveral times. Centrifugated for 10min, 12000rpm/min, then deposit the DNA,abandoned the superstratum, and rinsed for 1-2 times by 70% etnanil, dried theremaining fluid. 50μl TE buffer with 100μg/ml RNAseA was added for 30min in 65℃.Get the fluid 8μl, added by the buffer sample, 1.8% agarose electrophoresis (voltage1-5V/cm), then we used automatic electrophoresis gel image analysis and tookphotographs.6,Western blotting detected GAP-associated protein Bcl-2, Bax, P53,survivin of A2 cellsTreated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample, boiling for 5 rain in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into Bcl-2, Bax, P53, and survivin, first antibody -4℃overnight. Incubated with the secondary antibody, then washed for 2 times, each time15min, transferred into the secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-Sheep IgG (2:5000) andincubated for 2 hours. Detection was carried out using ECL chemiluminescence system,X-ray sensitization. Mixed the A and B liquid according to the concentration of0.125ml/cm². Exposed the X-ray film in a dark room, the developed 5min and fixed for5min.Result1,MTT-medicine sensitivity testThe inhibition of the Cru to A2 cells was concentration dependent. The value of theIC50 for 48 hours was 40μmol/L. 2,Morphology of apoptosis cellsWe observed the cells under the TEM, founded the structures of nucleus,chondrosome and the endoplasmic reticulum in the A2 cells of the control group clearly,and also we could see the nucleus have taken the most part of the cell. After adding theCru into the cells, the nucleus crimpled, cracked without karyon membrane embedded,chromosome gathered to cell membrane, and the changes of the ultramicrostructure ofthe cell transformed to apoptosis bodies which were forming or have already formed.3,Annexin-FITC detected quantificationally apoptosis cellsWe collected the cells treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols, then marked Annexin-FITC and analyzed by flow cytometry to examine theapoptosis of the cells. We found that the proportion of A2 apoptosis cells were lowerafter Cru was added for 24h, 48h, (9.45%, 16.89%), while the proportion at 72h washigher (21.05%).4,DNA agarose gel electrophoresisWe collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous "membrane strip" appeared. However,because the molecular of DNA of normal cells was large and moved short distance, so they remained near the adding holes.5,Western blottingThe cells which were treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols were collected, then exacted the total protein, detected the expression of theapoptosis associated protein P53, Bcl-2, Bax, survivin, and we found that Bax and P53were increased gradually with time extending, while Bcl-2, surviving were decreased.Part Two Researches of the effection andmechanism of the Cru for the angiogenesisMethod1,Cell cultureThe RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The HUVEC,A2cells with monolayer adherence grew. We took the logarithm of the cells in thegrowing period in the experiment.2,Observing the effection of the Cru to the proliferation of HUVECsThe cells in the growing period had been collected and digested, and regulated into the1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3 replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid150μl, shaking the plates for 10min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Cell proliferation inhibited rate (%)=[1-(OD value of the experimental group-ODvalue of the blank group)/(OD value of the negative control group-OD value blankgroup)]×100%3,Annexin-FITC detected quantificationally apoptosis cellsThe cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5×10⁵/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.Cell cycle synchronization 4,Detecting the mRNA level of the Ang-1, Ang-2 and the TSP byusing the reverse transcription PCRAfter throwing the medium away, we added the Tripure in the bottles, and thenflew afloat the cells for several times, until the cells had dropped completely, thenmoved the fluid into the centrifuge tubes, placed for 5min. Added the chloroform as0.2ml per 1ml Tripure, shocked intensely for 15 seconds, then placed for 5min. Aftercentrifugating in 4℃, 12000rpm for 15rain, we took the upper colorless aqueous phase,added the -20℃dimethylcarbinol which had been colded as the ratio that 0.5ml perlml Tripure, placed upside down for several times, then placed in the environment of-20℃for 10min, centrifugated and threw the superstratum away. Then we added the-20℃, 75% ethanol which had been already colded as the ratio lml per 1ml Tripure,shocked turbulencely for 5min, then centriguagted for 5min. Waiting for the ethanolvolatilized spontaneously, we moved the total RNA into the 80ml DEPC, 55℃waterbath for 15min to help the dissolved to resuspense. Detected the purity of the total RNAusing the ultrabiolet apectra scanner. Reverse transcription: 2μl total RNA in each tubeto be taken as the template, with the reaction volume was 25μl. Added the up anddown-stream primers of Ang-1, Ang-2 and TSP separately, and the up anddown-stream primers ofβ-actin protein 1μl, 10 times density of the PCR buffer 25μl,TaqDNA polymerase 0.5μl in each tube, then added the water till 25μl. The conditions of amplification of the TSP were degeneration in 94℃for 30 seconds, regnaturation in60℃for 40 seconds, extension in 72℃for 30 seconds, took the process for 30 times.The conditions of amplification of the Ang-1, Ang-2 were degeneration in 94℃for 30seconds, regnaturation in 60℃for 30 seconds, extension in 72℃for lmin, also 30times. Analyzed the production of the PCR: taking the PCR production electrophoresisin the 2% agarose gel, then ratio of the result of the electrophoresis of the Ang-1,Ang-2 and the TSP to the absorbance of theβ-actin strap were used to indicate therelative expression intensities of the mRNA of the Aug-1, Ang-2 and TSP.5,Detecting the expression of the protein VEGF, MM-9 by usingWestern blotTreated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each time for 5min, then transferred into VEGF, MMP-9, first antibody -4℃ovemight. Incubatedwith the secondary antibody, then washed for 2 times, each time 15min, transferred intothe secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-RabbitIgG (1:5000), Horse anti-Sheep IgG (1:5000) and incubated for 2 hours. Detectionwas carried out using ECL chemiluminescence system, X-ray sensitization. Mixed theA and B liquid according to the concentration of 0.125ml/cm². Exposed the X-ray filmin a dark room, the developed 5min and fixed for 5min.Result1,Detect the result of HUVEC cells which had been treated by theCru by using MTTIt gave us a dose-effect relationship of the proliferation-inhibiting effection of the Cruto the HUVEC: there was significant inhibitory action of the Cru in different densitiesto the cultured-human vascular endothelial cells. With the increasing of the density andthe prolonging of the time, the inhibitory action was more obviously.2,The influence of the Cru to the apoptosis of HUVECsWe detected the effections of the Cru to the apoptosis of the HUVECs in 5, 10, 20, 40,80, 160μmol/L by flowing cytometry. It was shown that the apoptosis rate were 4.12%,9.44%, 15.91%, 17.98%, 21.05%, 22.21% of the HUVECs 24 hours later, much morehigher than the 1.64% apoptosis rate in the control group. 3,RT-PCRWe collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, and excreted the RNA, then detected the expression of themRNA of the Ang-1, Ang-2 and TSP. We could see that the longer the action time ofthe Cru, the lower expression the mRNA of the Ang-1, Ang-2 than the control group,while the higher expression of the mRNA of TSP.4,Western blottingWe collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, extracted the total protein, then detected, and we could seethat the expressions of VEGF and MMP-9 were observely lower than that of the controlgroup.In short, the Cru could inhibit the angiogenesis by many signal transductions: (1)decreasing the tumor cell angiogenesis factors (VEGF, Ang-1, Ang-2) and/orincreasing the expression of the inhibitory factors (TSP-1) to regulate the balance ofthe angiogenesis factors, and inhibit the phenotype transformation of angiogenesis. (2)inhibiting the proliferation of the vascular endothelium, and promoting the apoptosis ofthem. (3) reducing the expression of the matrix metalloproteinase (MMP-9) to inhibitthe degradation of the basal membrane and the extracellular matrix. Part Three The in vitro experimental researchof the radiotherapy sensitization of the humanpulmonary carcinoma cells (A2) effected by theCruMethod1,Colony forming testWe got the cells which grew in the log phase, made them into the suspension bythe digestion of 0.25%trypase and 0.02%EDTA, then according to the different exposedoses, we inoculated different number of cells (cell densities) with fresh culture fluidinto the culture capsules. 24 hours later, we changed the culture fluid into the Cru with-different densities (1, 5, 10μmol/L ) cultured for another 24 and 48 hours, then in theroom temperature, we used the ¹³⁷Cs radioactive source with the doses were 0Gy, 2Gy,4Gy, 6Gy, 8Gy, 10Gy to irradiate, then changed the culture fluid of the medicine-usedgroup into the fluid that without the medicine and cultured for another 9 hours togetherwith the irradiate-only group. After 9 days, the cells were fixed with the 95% ethanoland stained by the Giemsa, then≥50 cells were marked as a survival colony, for eachmeasuring point, we took 3 times experiments. The colony experiments were convertedinto the survival fraction, the survival fraction of the simple medicine-used groupmeant the rate of the colony-formation-rate of the group comparing with colony-formation-rate of the blank group, then we took the survival fraction (SF) asthe standard, and rectified the SF of the medicin-irradiation group to get theradiosensitization effection of the medicines, then we calculated the Dq values of theirradiation-only (control) group and the medicine-used group by the multi-targetssingle knocked mathematical model fitting cell survival curve. The effection ofradiosensitization was indicated by the SER_Dq.2,Detecting the cell cycle by FACScanThe cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 2 times, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum away, added the 70% (vol/vol) cold-ethanol, leaving them in -20℃, thenremoved the ethanol, washed for 2 times in PBS. Then, we added the 1 ml PBS withRNaseA (0.1mg/ml) in the cells, putting them into the 37℃incubatons for 30 min, andstained for 30 min in 1 ml PBS with 50μg/ml propidium iodie, 4℃, away from light. Atlast we analyzed the content of the DNA in the cells and the cell cycle by the method ofFACScan using the CELLQEST.3,Detecting the cell apoptosisWhen the cells were almost in the log phase, we digested the cells as a rule to makethem into the 0.5^*10⁶ cell suspensions and then inoculated into the 6 holes inoculatingplates. Then we took one hole in a plate to add the DNSO culture fluid as the blank (the simple-irradiation group), while 5, 10, 15μmol/L Cru as the experimental groups(medicine and irradiation groups). 24 hours later, irradiated by dose sources of 2, 4, 6,8Gy, the cells were cultured for another 24 hours, then washed by the PBS for 2 times,digested by the trypsin completely, 1000r/min centrifuged for 5min and then we got thedensity of the cells into 10⁶/ml by adding the PBS. Joined 200μl DNA-PREPTMLPRinto 100μl cell suspension, 30s later, added 2ml DNA-PREP-TM Stain and mixed.Avoided the light for 30min in room temperature, we detected the apoptosis of the cellsby the flowing cytometry, and deal with the results by the software of Systemâ…¡TMinvented by the Coulter Company. In each experiments, we took the negative andpositive controls.4,DNA segments analysisTook the method as we talked above, we culture the cells for another 24 hoursafter the A2 cells had been in the 10μmol/L Cru (medicine-irradiation group)or DMSO(simple irradiation group) for 24 hours and had been irradiated by the 4Gy. Then wedigested the cells by the trypsin and collected them, washed in PBS for 3 times,collected them after the centrifugalization, then extracted the genome DNA in thenormal way. Took 2μg sample DNA, then took the electrophoresis in the agarose gel(promeaga, USA) which containing 1.5% ethidium bromide, then took the photos andanalysed the DNA segments under the violet-perspection. 5,Detecting the cycle regulating factors and the expression of theNF-kB by the method of Western-blotTreated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rule thatgel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into cyclinB1,P34cdc2,phos-P34cdc2,P21,survivin andNF-kB, first antibody -4℃overnight. Incubated with the secondary antibody, thenwashed for 2 times, each time 15min, transferred into the secondary antibody of Sheepanti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-SheepIgG (1:5000) and incubated for 2 hours. Detection was carried out using ECLchemiluminescence system, X-ray sensitization. Mixed the A and B liquid according tothe concentration of 0.125ml/cm². Exposed the X-ray film in a dark room, thedeveloped 5min and fixed for 5min. We maked the A2 cells synchronizated in the G₀ phase by the serum starvationmethod. Then we diluted the log phase growth cells in the culture flask into 1^*10⁶/ml,adding the RPMI1640 medium without the serum, after 48 hours cultured in the 37℃,CO2 incubatons, the cells stayed in the G₀ phase. Then we threw the RPMI1640medium without the serum away, and got the medium with 10% fetal bovine serum in,then did the relative experiments after recovery phase.Result1,Cell cycle synchronizationAbandoned RPMI 1640 without serum, we obtained the cells synchronized at G₀ phrasethrough serum hungry, and cultured the cells with RPMI1640 containing 10% FBS. Weexamined the cells by flow cytometry every 3 hours. After 2 hours' convalescence, thecells reached the G₂ phase after 17 hours, and arrived at the end of the G₂ phase after 22hours. The whole cell cycle was about 23 hours.2,Cell cycle block40μmol/L Cru were added into each of 3 bottles of experimental groups of cellssynchronized at G₀ phase by serum hungry and convalesced for 2 hours. Then took 3bottles of cells 18 hours after the convalesced, added the Cru in all of them, getting thefinal concentration was 40μmol/L. A bottle of cells was collected every 3 hours,measured by flow cytometry. Cru acted on G1 phase inducing Glblocked slightly, acted on G2 phase causing G2 block, the cells staying at G2 no longer transiting G2/M.We could see the subdiploid peak of apoptosis at any period.3,The influences to the A2 cells from the different factorsThere were obviously subdiploid peaks of apoptosis before the G₀+G₁ peak in both themedicine-irradiation group and the simple irradiation group after the A2 cells had beentreated in these 2 groups, but in the medicine-irradiation group, the peak was moreobviously. So the intensities of the effection dealing with the apoptosis of A2 cells ofthese factors were: Cru+irradiation＞Cru or irradiation＞the contrast.4,DNA agarose gel electrophoresisWe collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous " membrane strip " appeared. However,because the molecular of DNA of normal cells was large and moved short distance, sothey remained near the adding holes.5,Western blottingTook the cells 18 hours after the synchronization recovery phase, added the Cru to getthe final concentration 10μmol/L, 3 hours later, we collected the cells 18 and 21 hoursafter the recovery phase, then detected the cyclinB₁,P34^cdc2, phos-P34^cdc2, P21, survivin, NF-kB, and Western blotting. We found that the there were no change of theexpressions of the P34^cdc2,phos-P34^cdc2, while the expressions of the cyclinB₁,survivin and NF-kB decreased and the P21 increased.In a word, the Cru could increase the radiosensitiveness of the pulmonary carcinomacells, with the mechanism was that blocking the G₂/M of the A2 cells, inhibiting theexpression of NF-kB. The block of the cell cycle had relationship with the decreasedexpressions of cyclinB₁, survivin and the increased expression of P21.Conclusion1,Cru could induce the apoptosis of A2 cells. The apoptosis had the relationshipwith the decreased expressions of c-myc,bcl-2,survivin, and the increased expressionsof bax,fas,p53,Caspase-3.2,Cru could inhibit the tumor angiogenesis by (1) decreasing the tumor cellangiogenesis factors (VEGF, Ang-1, Ang-2) and/or increasing the expression of theinhibitory factors (TSP-1) to regulate the balance of the angiogenesis factors, andinhibit the phenotype transformation of angiogenesis. (2) inhibiting the proliferation ofthe vascular endothelium, and promoting the apoptosis of them. (3) reducing theexpression of the matrix metalloproteinase (MMP-9) to inhibit the degradation of thebasal membrane and the extracellular matrix. 3,Cru could block the G₂/M of A2 cells specifically and decrease the expressionof NF-kB to increase the radiosensitiveness of the A2 cells.