| Objective1 The first part of this research is to investigate the potential molecular mechanisms by which 18 β-Glycyrrhetinic acid inhibits human liver cancer cells growth.2 The second part is to investigate the potential synergistic mechanism of 18β-Glycyrrhetinic acid and Glycyrrhizic acid combined with DDP in inhibiting human liver cancer cells growth.Methods1 The molecular mechanism of 18β-Glycyrrhetinic acid inhibits HCC cell lines growth1.1 In vitro1.1.1 The effect of 18 β-Glycyrrhetinic acid on HCC cells growth We detected the effect of18β-Glycyrrhetinic acid on growth of p53 wildtype HCC lines HepG2、QGY-7703 and p53 mutant HCC cell lines Huh-7、Sk-Hepl by MTS assays.We obtained the optimal time and optimal dose range of 18 β-Glycyrrhetinic acid by these experiments.Then,we treated two HCT116 cell lines,(p53 wild type and p53 knockout type)with the same conditions of 18 β-Glycyrrhetinic acid,to investigat the role of p53 in the inhibiting effects of 18 β-Glycyrrhetinic acid on cell growth.1.1.2 The effect of 18 β-Glycyrrhetinic acid on inducing apoptosis in HCC cellsWe pretreated HepG2 and QGY-7703 cells with increased doses of18 β-Glycyrrhetinic acid for 24H and 16H respectively,then,we dyed cells with Annexin V and PI to detect cell apoptosis by flow cytometry.Also,we detected the expression of PARP and cleaved-PARP which are closely associated with apoptosis by Western blotting in protein level.1.1.3 The effect of 18β-Glycyrrhetinic acid on DNA damageWe detected the foci numbers of γ-H2A.X,which is one marker of DNA double strand breakage,by immunofluorescence,to investigate whether 18 β-glycyrrhetinic acid can affect DNA damage in human hepatoma cells or not.We also detected the phosphorylation levels of γ-H2A.X and ATM by WB,to prove this in protein levels.1.1.4 The effect of 18 β-Glycyrrhetinic acid on p53 pro-apoptotic pathwaysIn HepG2 and QGY-7703 cells,we detected the effect of 18β-Glycyrrhetinic acid on p53 pro-apoptotic target genes PUMA,NOXA and MDM2 by Real-time PCR.Further on,we detected the effect of 18 β-glycyrrhetinic acid on phosphorylation level of p53 apoptosis-related phosphorylation site Ser15,to observe whether 18 β-glycyrrhetinic acid can activate p53 pro-apoptotic pathways.1.1.5 The effect of 18 β-Glycyrrhetinic acid on ROS productionWe pretreated HepG2 cells with 200μ M 18 β-Glycyrrhetinic acid for 2H、4H and 8H,pretreated QGY-7703 cells for 1H、3H and 5H,then we dyed cells with DCFH-DA to detect the intracellular ROS levels by flow cytometry.1.1.6 The effect of 18 β-Glycyrrhetinic acid on DNA damage after inhibiting ROS productionInorder to inhibit ROS producing by 18β-glycyrrhetinic acid,we pretreated HCC cells with NAC 1mM for 6 hours,and then incubated cells with 0.2mM Vitamin C,110 μ M β-mercaptoethanol and 18 β-glycyrrhetinic acid together,subsequently we detected cell proliferation rate with MTS assays and detected the expression of y-H2AX by western blotting to observe whether 18β-glycyrrhetinic acid induced DNA damage is due to ROS.1.2 Inhibition of GL on HCC xenograft in NOD SCID mice1.2.1 The effect of GL on inhibiting tumor growAfter established HCC xenograft through inoculating QGY-7703 cell suspensions subcutaneously in NODSCID mice,the mice were randomly divided into five groups and gave different drugs:(1)control group:NS,0.2ml;(2)GL lower dose group:45mg/kg GL;(3)GL higher dose group:135mg/kg GL;(4)DDP:2mg/kg;(5)Combination group:GL 45mg/kg + DDP 2mg/kg.All by intraperitoneal injection,gave DDP every other day,GL every day,for 14 days.Among the treatment,we weighed the mice every days and measured tumor volume every other day,using a caliper to measure the two mutually perpendicular diameters,long diameter A,minor axis B,then we calculate tumor relatively volume according to the formula V = AB2/2.Draw tumor volume growth curve and calculate the relative tumor proliferation rate(the relative tumor proliferation rate = T/C × 100%,T is the mean tumor volume of the treatment group,C is the mean tumor volume of the control group).24 hours after the whole treatment,mice were sacrificed with overdose sodium pentobarbital.Then we stripped the xenografts,weighed and calculated tumor inhibination rate by the following formula:tumor inhibination rate= 1-administered group/control group×100%.Compared the tumor weight and volumes with control group,and compared the combination group with the DDP group.1.2.2 The effect of GL on inducing HCC transplantation tumor apoptosisWe used the TUNEL staining kit to detect the apoptotic rate in xenograft which had been prepared to paraffin sections,by confocal laser scanning microscope.Compared the effects of the presence or absence of drugs in each group.2 The synergistic effect of 18 β-Glycyrrhetinic acid and GL combined with DDP2.1 In vitro2.1.1 The synergistic effect of 18 β-Glycyrrhetinic acid combined with DDP on inhibiting HCC cells proliferationWe used 100 μM、200 μM、300 μM 18 β-Glycyrrhetinic acid combined with 5g/L DDP to treat HepG2、QGY-7703 and Huh-7 cells for 24-48H respectivly,then detected the cell proliferation rates by MTS assay.2.1.2 The synergistic effect of 18 β-Glycyrrhetinic acid combined with DDP on p53 pro-apoptotic pathwayWe used qPCR to detect the synergistic effect of 18 β-Glycyrrhetinic acid combined with DDP in p53 pro-apoptotic target genes NOXA,PUMA and MDM2.2.2 The synergistic effect of Glycyrrhizic acid combined with DDP in xenograftThe methods are as described in 1.2.1,we observed the synergistic effect of Glycyrrhizic acid combined with DDP in mice.Results1.1 18 β-Glycyrrhetinic acid increases the production of ROS in HCC cells,induced DNA damage and cell apoptosis1.1.1 18 β-Glycyrrhetinic acid inhibits the growth of HCC cells in p53-independent wayMTS results showed that 200μM and 300μM of 18 β-Glycyrrhetinic acid could significantly inhibit the proliferation of the four HCC cell lines.In QGY-7703 cells,with 100μ M of 18 0-Glycyrrhetinic acid for 24 hours also showes a significant inhibitory effect.MTS results demonstrated that GA could effectively inhibit the proliferation of human hepatoma cells。In the four HCC cells in MTS experiments,HepG2,QGY-7703 are p53 wild type cells,Huh-7 and sk-Hepl are p53 mutant type cells.18 β-Glycyrrhetinic acid has significantly killing effect in both of these two types,which suggested that 18 3-Glycyrrhetinic acid may inhibit cell proliferation through p53-independent way.To further confirm whether 18β-Glycyrrhetinic acid inhibit liver cancer cells proliferation through p53-independent way or not,we treated a pair of human colon cancer cell lines HCT116,which have p53 wild type and p53 knock out type,with the same concentration gradient of 18 β-Glycyrrhetinic acid for the same time points,then we used MTS assays to detect the proliferation inhibition rates in these cells.The results showed that 200 μM and 300 μM of 18 β-Glycyrrhetinic acid could significantly kill these two cells,and with the same conditions the relatively proliferation inhibition rate didn’t have significant difference between these two cells.Above this,we can prove that 18β-Glycyrrhetinic acid inhibits HCC cell proliferation through p53-independent pathway.1.1.2 18 β-Glycyrrhetinic acid induces cell apoptosis in HCC cellsWe used 100 μM and 200 μM of 18 β-Glycyrrhetinic acid to treat HepG2 cells for 24 hours,and treat QGY-7703 cells for 16 hours,then we trypsinized these cells to single cell suspensions.After we dyed cell suspensions with Annexin V and PI,We detected the apoptotic rates by flow cytometry.The results showed that 18 β-Glycyrrhetinic acid can induce apoptosis in these two cells with a concentration-dependent way.We also detected the phosphorylation level of cleaved-PARP by Western blotting.After we treating QGY-7703 cells and HepG2 cells with different concentrations of 18 β-Glycyrrhetinic acid for 16 hours and 24 hours respectively,cleaved-PARP levels significantly increased,and the total PARP protein bands reducted compared to control group.Results of Annexin V-FITC/PI staining and Western blot showed that 18β-glycyrrhetinic acid could induce apoptosis in HCC cells HepG2 and QGY-7703.1.1.3 18β-Glycyrrhetinic acid induces DNA damage in HCC cellsImmunofluorescence test of γ-H.2AX showed that after treating HepG2 cells with 200μM 18 β-Glycyrrhetinic acid for 8 hours,the foci number in the nucleus of cells was more than that in control cells,while the number of nucleus without foci were significantly less than that in control group.Compared with control group,18 β-Glycyrrhetinic acid could significantly increase DNA damage in HepG2 cells.We also observed the effect of 18β-Glycyrrhetinic acid on foci in QGY-7703 cells,after treating with 200 μ M 18 β-Glycyrrhetinic acid for 6h,we found the number of nucleus with>5 foci significantly increased compared to control group.Results of WB showed that after treating HepG2 cells with 200 μM 18 β-Glycyrrhetinic acid for 4 hours,8 hours and 12 hours,phosphorylation levels of ATM(Ser1987)increased gradually with prolonged drug treatment.After treating QGY-7703 with 200 μM 18 β-Glycyrrhetinic acid for 1 hour,3 hours and 5 hours,we also observed that the phosphorylation levels of p-ATM(Ser1987)and γ-H2AX increased gradually with time compared to control groups.1.1.4 18 β-Glycyrrhetinic acid activates p53 pro-apoptotic pathwaysResults of qPCR showed that after treating HepG2 cells with 18 β-Glycyrrhetinic acid for 24 hours,expression of NOXA and PUMA significantly increased,while it had no influence on the expression of MDM2.The most significant effect was on NOXA,after treating HepG2 cells with 100 μ M 18 0-Glycyrrhetinic acid,it’s expression upregulated t0 14.35±1.086 times compared to control.After treating QGY-7703 cells with 18 β-Glycyrrhetinic acid for 16 hours,expressions of NOXA,PUMA and MDM2 all upregulated,and NOXA was the most significant one also.After treating with 200μM 18β-Glycyrrhetinic acid,expression of NOXA upregulated to 11.32±0.1528 times compared to control.Results of WB also showed that phosphorylation levels of p53(ser15)significantly increased in these two cells after 18β-Glycyrrhetinic acid treatment.1.1.5 18 β-Glycyrrhetinic acid increases ROS production in HCC cellsAfter treating HepG2 cells with 200 μM 18 β-Glycyrrhetinic acid for 2H,the fluorescence intensity of DCF began to significantly increase compared to control group,at 3H the fluorescence intensity was higher than 2H,and at 4H it began to decline,at 8H fluorescence intensity continued to decline.After treating QGY-7703 cells with 200 μM 18 β-Glycyrrhetinic acid for 1h,fluorescence intensity of DCF compared to the control group was significantly increased,but at 3H detection point,the fluorescence intensity began to decline compared to 1H,while the ROS positive cells didn’t have significant change.At 5H,the fluorescence intensity continued to reduce,but percentent of ROS positive staining cells were still higher than control group.1.1.6 DNA damage is alleviated after suppressing ROS generationAfter suppressing the ROS production with antioxidants,We found that the cell proliferation inhibitory of QGY-7703 cells and HepG2 cells all declined.After incubating QGY-7703 cells with anti-oxidants combining 18 β-Glycyrrhetinic acid together for 5 hours,the expression of γ-H2A.X significantly decreased.1.2 Glycyrrhizic acid has no significant effect on HCC xenograft in NODSCID1.2.1 Glycyrrhizic acid can’t inhibit the growth of HCC xenograftAs results of relative tumor volumes showed,either low concentration or high concentration of Glycyrrhizic acid could inhibit the proliferation of the HCC xenograft,both have no statistical difference in tumor weight when compared to control group.1.2.2 Glycyrrhizic acid can increase the apoptotic rate of tumor cells in HCC xenograftWe used Zeiss laser confocal microscope to photograph and count the positively cells stained with TUNEL in more than 1000 cells avoiding tumor necrosis areas,then calculated the apoptotic index.Compared to NS control group,the apoptotic rate all increased in two doses of Glycyrrhizic acid groups,cisplatin group and the combination group.The combination group incresed most obviously,and had significant difference with the cisplatin group and control group.2.1 18 β-Glycyrrhetinic acid and cisplatin produce a synergistic effect in inhibiting the proliferation of HCC cellsResults of MTS showed that after treating p53 wild-type cell lines HepG2,QGY-7703 and p53 mutant cell line Huh-7 with 100μM,200μM,300μM of 18β-Glycyrrhetinic acid respectively combining with 5g/L cisplatin,the inhibition rate of these 3 cells all significantly increased comparing to 18 β-Glycyrrhetinic acid and DDP alone,and the low concentration of 18β-Glycyrrhetinic acid could have a significant synergistic effect with DDP.In gene expression level,we found that in QGY-7703 cells treating with 18 β-Glycyrrhetinic acid combined DDP could significantly up-regulate the expression of p53 pro-apoptosis target genes PUMA,NOXA and MDM2,compared to monotherapy.This result showed that 18 β-Glycyrrhetinic acid and cisplatin had a synergistic effect in p53 pro-apoptotic pathway.2.2 Glycyrrhizic acid and DDP have a synergistic effect in inhibiting growth of HCC xenograft in miceThe terminal tumor volume of DDP group had a statistical difference when compared with control group.The combination group had a most obviously effect on inhibiting tumor proliferate,and had a statistic difference compared to DDP group.These results showed that Glycyrrhizic acid could enhance the effect of cisplatin anti HCC cells,and may inhibit the proliferation of DDP-resistant cells.Conclusions:1 Results in vitro suggested that the mechanism of 18 β-Glycyrrhetinic acid inhibiting the growth of HCC cell lines HepG2 and QGY-7703 may be:18 β-Glycyrrhetinic acid stimulates HCC cells to product more ROS,but the excess ROS can not be cleared,so the residual ROS attack the genomic DNA,which is resulting in DNA double strand breaks.Followed this,ATM is phosphorylated,which is one sensor of DNA damage in cell cycle checkpoint mechanism,and then ATM activates it’s downstream substrates,including p53,H2A.X etc.,ultimately lead to apoptosis of HCC cells.2 Results in vitro showed that 18 β-glycyrrhetinic acid inhibited HCC cells proliferation via a p53-independent pathway,and 18β-Glycyrrhetinic acid combined with cisplatin had a synergistic effect.In addition,in p53 wild-type cells,18β-Glycyrrhetinic acid could activate p53 pro-apoptotic pathways,and have synergistic effect in combination with cisplatin in p53 pro-apoptotic pathways.In mice bearing human hepatocellular carcinoma xenograft models,Glycyrrhizic acid combined cisplatin have the best inhibition effect comparing with DDP and Glycyrrhizic acid monotherapy.p53 is important in DDP anti-tumor effects,and p53 mutations also has been confirmed have a significant relationship in resistance mechanisms of cisplatin.The p53-independent way of 18 β-glycyrrhetinic acid in inhibiting HCC cell proliferations may caused this synergistic effect.But the more detail mechanism of their synergistic effect needs further research.3 18β-glycyrrhetinic acid is the efficacy ingredient hydrolysates of Glycyrrhizic acid in vivo,but 18 β-glycyrrhetinic acid is fat-soluble,which is difficult to administrate in vivo.Contrary,Glycyrrhizic acid has more flexible mode for administrating which has been used in clinical for several decades.Cisplatin is one of the first choices for liver cancer chemotherapy,but at the meantime,the platinum drugs have serious drug resistant problems,so,to find new and more effective alternatives to chemotherapy or to sensitize chemotherapy drugs is one of the currently research focuses.Combining with our in vitro data that glycyrrhetinic acid has synergistic killing efficiency with cisplatin on liver cancer cells,we used times of the clinical dose of glycyrrhizic acid in mice in order to get the anti-tumor concentration of 18β-glycyrrhetinic acid,and to explore whether the clinical used of glycyrrhizic acid combining with cisplatin can make a further effect in killing HCC cells in mice.But neither the low concentration nor the high concentration of Glycyrrhizic acid we used in mice xenograft has significant inhibitory effect,which may be related to the Glycyrrhizic acid we used can not metabolism efficient 18β-glycyrrhetinic acid to reach its anti-tumor concentration in NODSCID mice.In this regard,we need to directly use 18 β-glycyrrhetinic acid in vivo experiment to verify its in vivo anti-tumor effect.Low concentration of Glycyrrhizic acid combined with cisplatin has a significant better effect in inhibiting tumor growth than cisplatin monotherapy,and this is consist with which we found in vitro that low concentration of 18 β-glycyrrhetinic acid combining with DDP had significant synergic effect in inhibiting HCC cell proliferation and activating p53 pro-apoptotic pathway. |
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