| Objective：Resveratrol (RSV;3,4’,5-trihydroxystilbene) is a kind ofnatural phytoalexin, has the function of inhibiting tumor cell proliferation, andplays the antitumor function mainly by inducing tumor cell autophagy andapoptosis. Reports indicate that tumor suppressor gene p53can regulate cellapoptosis and autophagy, but p53has different effect on the cell apoptosis andautophagy due to the condition differences. In this experiment,I take humancolorectal cancer cell lines HCT-116p53+/+and HCT-116p53-/-cells on as theobject of the research, through a variety of experimental methods, research therole of p53in resveratrol inducing colon cancer cells in the process ofautophagy and apoptosis, thus provide a new theoretical basis for themechanism of autophagy and apoptosis in colon cancer cells.Methods:1MTT assay was used to detect the variation of cell survival rate afterHCT-116p53+/+and HCT-116p53-/-cells were treated with resveratrol （0μM,25μM,50μM,100μM,150μM,200μM）for24,48and72hours.2ChanDan sulfonyl e diamine (MDC) staining was used to observe thevariation of the structure and quantity of acidic autophagy bubble afterHCT-116p53+/+and HCT-116p53-/-cells were treated with differentconcentrations of resveratrol（0μM,50μM,100μM,150μM）for24,48and72hours.3HCT-116p53+/+cells were treated with resveratrol（0μM,50μM,100μM,150μM）for24,48hours, then immunofluorescence was used to analyse thedistribution and expression of autophagy marker-LC3.44’,6-diamidino-2-phenylindole (DAPI) staining was used to observe thenucleus change and apoptosis phenomenon after HCT-116p53+/+andHCT-116p53-/-cells were treated with RSV100μM for0,24,48and72hours. 5Western Blot was used to analyse the expression of autophagymarker-Beclin1,LC3and apoptosis correlated proteinum Bax,Bcl-2,C-beclin1after HCT-116p53+/+and HCT-116p53-/-cells were treated with RSV（0μM,50μM,100μM,150μM）for24,48and72hours and HCT-116p53+/+andHCT-116p53-/-cells were treated with RSV100μM for0,24,48and72hours.Analysis the influence of p53on cell autophagy and apoptosis.6Western Blot was used to analyse the expression of Beclin1,LC3afterHCT-116p53+/+cells were treated with p53agonist Nutlin-3and resveratroltogether for24hours, to further confirm the relationship between the p53andautophagy.7Caspase-3enzyme activity was analyzed by caspase-3kits afterHCT-116p53+/+and HCT-116p53-/-cells were treated with differentconcentrations of resveratrol（0μM,50μM,100μM,150μM）for48and72hours，and HCT-116p53+/+and HCT-116p53-/-cells were treated with the sameconcentration of resveratrol100μM for0,24,48and72hours，to furtherconfirm the relationship between the p53and apoptosis.8HCT-116p53+/+and HCT-116p53-/-cells were treated with resveratrol(0μM,100μM) for24hours, and rabbit R IgG and rat M IgG was used aspositive control, Beclin1was used as precipitation antibody, then detectwhether Beclin1related to Bcl-2.9Experimental data was analysed by SPSS13.0statistical software,andthe experimental group and control group were compared with one-wayanalysis of variance and t-test. The results are expressed as x±SD.Differences were considered to be significant at a level of P <0.05.Results:1The effect of resveratrol on cell survival rate in HCT-116p53+/+andHCT-116p53-/-cellsDetermined by MTT results showed that under the influence ofresveratrol, the survival rate of HCT-116p53+/+and HCT-116p53-/-cells weresuppressed, and the inhibition to HCT-116p53-/-was more obvious.2The effect of resveratrol on cell autophagy in HCT-116p53+/+and HCT-116p53-/-cellsMDC staining results showed that under the influence of resveratrol, inHCT-116p53+/+cells, autophagy bubble of positive cells in MDC stainingincreased and strengthened gradually, autophagy was obvious; in HCT-116p53-/-cells, autophagy bubble of positive cells in MDC staining reduced graduallyand had the trend of weakening, autophagy was not obvious.Immunofluorescence showed that LC3mainly locate in the cytoplasm ofHCT-116p53+/+cells, and the expression of LC3increased with increasingconcentration of resveratrol.Western Blot showed that in HCT-116p53+/+cells, the expression ofBeclin1, LC3â…¡ increased with the increase of concentration of resveratroland enhanced gradually; Beclin1increased gradually with the extension ofduration of resveratrol,the expression of LC3â…¡increased with the extension ofduration of resveratrol at0-48h, but the expression of LC3â…¡ weakened withthe extension of duration of resveratrol at48-72h..Western Blot showed in HCT-116p53-/-cells, the expression of Beclin1reduced gradually with the increase of the concentration of resveratrol, and theexpression of Beclin1decreased gradually with the extension of duration ofresveratrol.3The influence of p53on resveratrol inducing autophagy after HCT-116p53+/+cells were treated with p53agonist Nutlin-3.Western Blot showed that after HCT-116p53+/+cells were treated with p53agonist Nutlin-3, the expression of Beclin1,LC3â…¡ increased with the increaseof p53, and the expression of MDM2increased corresponding. Compared thetreatment with p53agonist Nutlin-3and resveratrol together, the expression ofMDM2decreased along with the increase of p53ï¼›Compared the treatmentwith the resveratrol，the expression of MDM2decreased along with theincrease of p53.4The effect of resveratrol on cell apoptosis in HCT-116p53+/+and HCT-116p53-/-cellsDAPI staining results showed that with the extension of resveratrol effect time, both HCT-116p53+/+and HCT-116p53-/-cell occur apoptosis，but theamount and degree of apoptosis cells in HCT-116p53-/-cell was significantlygreater than HCT-116p53+/+cells.Results showed that Caspase-3enzyme activity of HCT-116p53+/+andHCT-116p53-/-cells increased with the increase of concentration and theextension of duration of resveratrol，but the Caspase-3enzyme activity inHCT-116p53-/-cell was significantly higher than HCT-116p53+/+cells.Western Blot showed that, the expression of Bax and Bcl-2increasedwith the increase of concentration of resveratrol and the extension of durationof resveratrol in HCT-116p53+/+cells. In HCT-116p53-/-cells, the expression ofN-Beclin1decreased gradually with the extension of duration of resveratrol,the expression of C-Beclin1increased gradually with the extension of durationof resveratrol.5The influence of Beclin1interaction with the Bcl-2to resveratrol inducedHCT-116p53+/+and HCT-116p53-/-cells autophagyThe results of immune precipitation showed that，Beclin1is associatedwith the Bcl-2in HCT-116p53+/+å'ŒHCT-116p53-/-cells.Conclusions:1Resveratrol induced HCT-116p53+/+and HCT-116p53-/-cell autophagyand apoptosis together.Resveratrol inhibited the growth of HCT-116p53+/+cellsthrough the way of autophagic cell death, inhibited the growth of HCT-116p53-/-cells through through the way of apoptosis.2p53played an important role in the transformation regulation ofautophagy and apoptosis. p53induced cells autophagy, delayed the occurrenceof apoptosis; the lack of p53,thus due to the lack of the ability of inducingautophagy, HCT-116cells entered into apoptosis state quickly. Resveratrolcould be effective drugs of p53mutations in the tumor.3p53regulate the transformation of cell autophagy and apoptosis byadjusting the cutting of Beclin1and the interaction with the Bcl-2, but thespecific mechanism is unclear. |
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