| Objective:In recent years,the new discipline of chemical biology has developed rapidly.In this subject,the existing large number of small molecule compound libraries with diverse chemical structures are used to screen the biological activities of the compounds needed by researchers,and the related mechanisms are studied on the basis of these libraries.The selected drugs in this study are chrysanthemum lactone and emodin,which are small molecular compounds extracted from natural plants.Its biological activity has obvious anti-cancer effect,especially in intestinal adenocarcinoma,which has been gradually increased in recent years.Therefore,to continue our previous studies on the anti-intestinal adenocarcinoma biological activity of chrysanthemum lactone and emodin,we used in vitro experiments to study the mechanism targets of chrysanthemum Lactone-induced apoptosis and drug resistance reversal of HCT-8/VCR cells,and the mechanism targets of emodin-induced apoptosis of CACO-2 cells and regulation of PI3K/AKT signaling pathway.Points were studied.The anticancer activity and mechanism of chrysanthemum lactone and emodin were studied in order to provide a basis for the anticancer effect of small molecular compounds derived from natural plant drugs,and to open up a market for the use of natural plant drugs in the treatment of clinical tumors.In the future,we will analyze the correlation between the biological activity and the structure of the two small molecule compounds,modify the unsatisfactory structure,and contribute to the research and development of new anti-cancer small molecule drugs.Method:? ? Study on apoptosis and resistance reversal of HCT-8/VCR cells induced by chrysanthemum lactone1.Assay the cell proliferation inhibition effect on HCT-8?HCT-8/VCR cells by MTT.We cultured colon cancer cell line HCT-8,HCT-8/VCR cell line,added in the 96-well plates,the experimental group with different concentrations of PTL,the control group with equal amount of medium without drugs,treated for 12,24,48 hours.We assayed theabsorbance(OD)value by MTT and calculated rate of cell proliferation inhibiton rate2.Detection the effect of PTL on the HCT-8,HCT-8/VCR cells apoptosis by flow cytometry(Annexin V-FITC/PI staining)We digested and collected HCT-8,HCT-8/VCR cells of control group,PTL5?mol/L,10?mol/L,20?mol/L treated for 24 h group.Then we conducted Annexin V-FITC/PI staining and analyzed the percentage of cell apoptosis by flow cytometry.3.Detection the effect of PTL on the HCT-8,HCT-8/VCR apoptosis by acridine orange(AO)/ ethidium bromide(EB)fluorescent stainingHCT-8 and HCT-8/VCR cells in logarithmic growth phase were inoculated into 6-well plates with 1 *104/ml and 2 ml per hole,and covered glass was placed in the hole.A total of 4groups of pyrethroids(5 mol/L,10 mol/L,20 mol/L)and the control group were set up.Each group had 3 compound holes.After 24 hours of administration,the cover slides were taken out,fixed,AO/EB stained,sealed,and observed by fluorescence microscope.Each group counted 300 cells at a time,and the apoptotic rate was calculated.4.Detection the effect of PTL on the HCT-8,HCT-8/VCR cell Bax,Bcl-2,protein expression by Western-blot.We collected HCT-8,HCT-8/VCR cell protein of the control group,PTL5?mol/L,10?mol/L,20?mol/L 24 h,,western blot was used to detect Bax,Bcl-2 protein expression.5.Detection the reversal effect on drug resistance of PTL in HCT-8/VCR cells by MTT(1)We take the logarithmic phase HCT-8,HCT-8/VCR cells,seeded in 96 well plates,added VCR of different concentrations,treated for 24 h.Then we assayed HCT-8,HCT-8/VCR cells half of the inhibition of cell concentration of VCR,and resistance index of HCT-8/VCR to the VCR.(2)We take the logarithmic phase HCT-8/VCR cells,seeded in 96 well plates,seted control group,PTL5?mol/L,VCR(0.25,0.5,1,2,4,6,10 ?g/ml)group,PTL5?mol/L + VCR group.After treating 24 h,we assayed PTL reversal effect on drug resistance of HCT-8/VCR by MTT.6.Assay PTL reversal effect on drug resistance of HCT-8/VCR by flow cytometry(Annexin V-FITC/PI staining)We digested and collected HCT-8/VCR cells of control group,PTL5?mol/L group,VCR2?g/ml group,PTL5?mol/L + VCR2?g/ml group treated for 24 h,then we conductedAnnexinV / FITC staining and flow cytometry,calculated the percentage of cell apoptosis.7.Detection the effect of PTL on the HCT-8/VCR cells P-gp,MRP,GST-? protein expression by Western-blotWe collected HCT-8/VCR cell protein of the control group,PTL5?mol/L,10?mol/L,20?mol/L treated for 24 h,western blot was used to detect P-gp,MRP,GST-? protein expression.? ? Emodin induces apoptosis in CACO-2 cells and regulates PI3K/AKT signaling pathway1?Detection of emodin on the inhibition of CACO-2 cell proliferation by MTT.CACO-2 cells in logarithmic growth phase were added 96-well plate,the experimental group was added with different concentrations of PTL,and the control group was added with the same amount of drug-free culture medium.After 24 hours of treatment,MTT method was used to detect the absorbance(OD)of each hole,and the inhibition rate of cell proliferation was calculated.2?The effect of emodin on apoptosis of CACO-2 cells was detected by flow cytometry.CACO-2 cells in logarithmic growth phase were digested and collected,and treated with emodin for 24 hours.The final concentrations were 0 mol/L,15 mol/L,30 mol/L and 60mol/L,respectively.The percentage of apoptosis was analyzed by Annexin V/FITC staining and flow cytometry.3?Detection of emodin on apoptosis of CACO-2 cells by DAPI stainingCACO-2 cells in logarithmic growth stage were inoculated into 6-well plates with 1*104 cells/ml and 2 ml cells per pore.Emodin concentration of 15 mol/L,30 mol/L and 60mol/L,and the control group were divided into four groups,each group had three multiple holes.After 24 hours of administration,the cover slides were removed,fixed,stained with DAPI and sealed with carbonic buffer glycerin.The excitation wavelength was 359 nm under fluorescence microscope.Apoptosis was observed.4?Effect of emodin on cell cycle of CACO-2 cells by flow cytometryCACO-2 cells in logarithmic growth phase were cultured in RPMI-1640 complete medium and inoculated into 6-well plate with 2 ml cells per hole for 12 hours.The culture medium was changed and the drug was added.The final concentration of emodin was 0 mol/L,15 mol/L,30 mol/L and 60 mol/L,respectively;the control group was added to the completemedium group,the blank control group was only added to the complete medium,after 24 hours in the incubator,the cells were digested with trypsin,the concentration was adjusted to1 x 106/ml,0.25 ml cells were taken,PBS was washed,1000 rpm,4 C was isolated.After 5minutes,the supernatant was discarded(repeated twice),the cells were suspended in 0.25 ml binding buffer,0.1 ml cell suspension was placed in 5 ml flow tube,PI 20 UL was added to the final concentration of 50 ug/ml,wrapped in tin foil,stained for 30 minutes with 4 degrees of light avoidance,and tested on the computer within 24 hours.Cell cycle was detected by flow cytometry and repeated 3 times.5?Detection of emodin on CACO-2 cell protein expression by Western-blotThe expression of Bax and Bcl-2 proteins in CACO-2 cells treated with 15 mol/L,30mol/L and 60 mol/L emodin for 24 hours was detected by Western blot.Result:1.The effect of PTL on the HCT-8,HCT-8/VCR cell proliferation inhibitionParthenolide has proliferation inhibition on colon cancer HCT-8,HCT-8/VCR cell line,which in a dose and time dependent.PTL12 h median inhibitory concentration IC50 was 20.21 ± 2.63?mol/L,21.52 ± 3.25?mol/L.PTL24 h median inhibitory concentration IC50 was 15.46 ± 1.22?mol/L,17.05 ± 2.78?mol/L.PTL48 h median inhibitory concentration IC50 was 11.44 ± 1.65?mol/L,10.85 ± 2.02?mol/L.2.The effect of PTL on the HCT-8,HCT-8/VCR cell apoptosisThe apoptosis rate of HCT-8 cells in control group was 4.23% ± 0.75%.Compared with the control group,after 5,10,20 ?mol/L treatment of PTL for 24 h,HCT-8 cell apoptosis was significantly increased(p <0.05),were 8.23% ± 1.45%,22.46% ± 2.75%,38.16% ± 3.25%respectively.The apoptosis rate of HCT-8/VCR cells in control group was 4.42%±1.16%.Compared with the control group,after 5,10,20 ?mol/L treatment of PTL for 24 h,HCT-8 cell apoptosis was significantly increased(p<0.05),were8.43%±0.95%,21.67%±3.53%,39.16%±3.51%respectively.3.Detection the effect of PTL on the HCT-8,HCT-8/VCR cells apoptosis by fluorescence stainingFluorescence staining results showed that,PTL induced HCT-8,HCT-8/VCR apoptosis in a dose-dependent manner.HCT-8 cells in the control group ratio of apoptotic cells was10.33% ± 2.05%,compared with the control group,after 5,10,20 ?mol/L of PTL treated for24 h,HCT-8 cell apoptosis was significantly increased(p <0.05),were 20.55% ± 3.55%,32.46% ± 3.76%,46.8% ± 4.28% respectively.HCT-8/VCR cells in the control group ratio of apoptotic cells was 9.42%±1.25%,compared with the control group,after 5,10,20 ?mol/L of PTL treated for 24 h,HCT-8 cell apoptosis was significantly increased(p<0.05),were 18.66%±2.15%,30.65%±2.87%,45.44%±3.81% respectively.4.Detection the effect of PTL on the HCT-8,HCT-8/VCR cell Bax,Bcl-2,protein expression by Western-blotHCT-8,HCT-8/VCR cells shows low expression of Bax in control group,compared with control group,Bax expression was significantly increased(p<0.05)in PTL 5?mol/L,10?mol/L,20?mol/L group.HCT-8,HCT-8/VCR cells shows high Bcl-2 expression in the control group,compared with control group,Bax expression in PTL 5?mol/L,10?mol/L,20?mol/L group was significantly decreased(p<0.05).These results suggest that,PTL promots of HCT-8,HCT-8/VCR cell expression of Bax,inhibits of Bcl-2 expression,induces HCT-8,HCT-8/VCR apoptosis in a concentration-dependent manner.5.Detection the reversal effect on drug resistance of PTL in HCT-8/VCR cells by MTT(1)VCR has proliferation inhibition effect on colon cancer HCT-8,HCT-8/VCR cell line(p<0.05)in concentration-dependent relationship(p <0.05),VCR half inhibitory concentration IC50 was 2.21 ± 0.24?g / ml,38.9 ± 3.04?g/ml,HCT-8/VCR on the VCR of the resistance index was 17.6 times.(2)VCR of 0.25,0.5,1,2,4 ?g / ml has no significant proliferation inhibition effect on HCT-8/VCR cells(p>0.05),VCR of 6,10 ?g / ml has slightly proliferation inhibition effect on on HCT-8/VCR cells(p<0.05),PTL5?mol/L combined with VCR(0.25,0.5,1,2,4,6,10 ?g/ ml)has no significant proliferation inhibition effect on HCT-8/VCR cells(p>0.05)can significantly increase the proliferation inhibition effect on HCT-8/VCR cells(p<0.05).Combined use VCR IC50 was 3.01 ± 0.45?g/ml,compared with VCR alone,IC50 was significantly lower(p<0.05),PTL can significantly reduce the resistance index of HCT-8/VCR to VCR(p<0.05).The results show,PTL can significantly increase the VCR inhibitory inhibition effect on HCT-8/VCR cells,reverse resistance of HCT-8/VCR cells.6.Assay PTL reversal effect on drug resistance of HCT-8/VCR by flow cytometry(Annexin V-FITC/PI staining)The results Annexin V-FITC/PI staining detecting apoptosis showed that the apoptosis rate 5.01% ± 0.47% in the control group,treated by2?g/ml VCR for 24 h,HCT-8/VCR apoptosis rate was 6.93% ± 1.45%,compared with the control group,there is no significant difference(p>0.05);treated by5?g/ml VCR for 24 h,HCT-8/VCR apoptosis rate was 10.02%± 0.86%,compared with the control group,apoptosis was significantly increased(p<0.05),treated by PTL5?mol/L + VCR2?g/ml for 24 h,HCT-8/VCR cells apoptosis was 34.7% ±2.33%,compared with VCR2?g/ml group and PTL5?mol/L group,the apoptosis rate increased significantly(p<0.05).The results show that,PTL can significantly increase the ability of VCR induced HCT-8/VCR apoptosis,reversed resistance HCT-8/VCR cells to VCR.7.Detection the effect of PTL on the HCT-8/VCR cells P-gp,MRP,GST-? protein expression by Western-blotWestern blot analysis showed that the expression of P-gp,MRP and GST-pi protein in HCT-8 control group was lower than that in HCT-8 control group.Compared with HCT-8control group,the expression of P-gp,MRP and GST-pi protein in HCT-8/VCR control group increased significantly(p < 0.05);compared with HCT-8/VCR control group,PTL5 mol/L,10mol/L and 20 mol/L treated HCT-8/V CR cells 24.After h,the expression of P-gp,MRP and GST-pi decreased significantly(p < 0.05),and the higher the concentration of PTL,the lower the expression of P-gp,MRP and GST-pi.8.With the gradual increase of emodin concentration,the survival rate of CACO-2 cells decreased gradually,and to 200 mol/L,the cell survival rate was almost 0.Emodin had a IC50 value of 30 mol/L for CACO-2 cells cultured in 24 h.9.With the increasing of emodin concentration,the apoptosis rate of CACO-2 cells increased from 1.85% in the control group to 42.66% in the 60 mol/L emodin group.Emodin induced apoptosis in CACO-2 cells in a dose dependent manner.10.After the intervention of 30 mol/L and 60 mol/L emodin,the nucleus was obviously condensed and the staining was deepened.Occasionally,the chromatin of nucleus gathered at the side of nuclear membrane in crescent shape.Especially after 60 mol/L emodin intervention,the apoptotic nucleus fragmented into round bodies of different sizes,which were surrounded by cell membrane and suspected to be apoptotic bodies.11.Most of the cells in the control group were in the G0/G1 phase,62.3%,33.7% in the S phase and 4.0% in the G2/M phase;after emodin intervention,the cell cycle stagnated significantly in the G2/M phase,and the number of cells in this phase increased significantly.Moreover,with the increase of emodin concentration,the cells that stagnate at G2/M phase also increase gradually.The cell cycle arrest of G2/M in 60-mol/L emodin group was most obvious.12.The expression level of Bcl-2 was opposite to that of Bax,while the expression level of Bcl-2,p-PI3 K and p-AKT were high in control group.The expression level of Bcl-2,p-PI3 K and p-AKT was gradually down-regulated with the increase of emodin concentration.There was no significant difference in the expression level of PI3 K and AKT between the two groups.Conclusion:1.Parthenolide can inhibit the cell proliferation and induce apoptosis of HCT-8,HCT-8/VCR,parthenolide can decreas expression of Bcl-2 to induce apoptosis though increase the expression of Bax.2.Parthenolide can significantly increase the proliferation inhibition and apoptosis induction ability of VCR on HCT-8/VCR cells.Pyrethrin can reverse the drug resistance of HCT-8/VCR cells.The mechanism is that PTL can decrease the expression of P-gp,MRP and GST-pi proteins in HCT-8/VCR cells and reverse the drug resistance of HCT-8/VCR cells.3.Emodin significantly inhibited the growth of CACO-2 colon cancer cells.Its anticancer effect is mainly due to the induction of apoptosis and cell cycle arrest.In addition,it can inhibit the PI3 / AKT signaling cascade in CACO-2 cells,suggesting the potential application of emodin in the treatment of colon cancer. |
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