Study On The Anti-hepatocarcinogenic Effect And Related Mechanisms Of Valeric Acid Encapsulated By Liposome Nanoparticle

HCC is the 5 th most common cancer worldwide,and also the top three cause of death.In USA and Asian-Pacific region,its morbidity was showing a upwards trend in recent years.HCC possesses the characteristics of concealed,rapid progress,poor prognosis and high mortality;Patients with advanced HCC lose the of surgical opportunity of resection and liver transplantation,have limited therapeutic approaches.Even though some novel drugs like immunotherapy agents,target therapy drugs are under development and has achieved some progressions,the effective drug for HCC treatment is still in need.Chinese herb has the characteristics of multiple targets,multiple functions,low risk of developing drug-resistance and safe to use.Therefore,the application of functional monosomy of herb has drawn many attention worldwide.Besides,the application of LNP in HCC has became much more mature than ever,Thus the combination of these two together has more potential for HCC treatment.Purpose and Significance1 In vitroTo study the proliferation inhibition effect of VA,the main component of valerian,in various tumor cells and some normal cells,and to compare the delivery efficiency of selected liposome nanoparticles encapsulated with VA on various cells,and evaluate the safety of selected nanoparticles,based on the predicting functions of VA,further related researches will be conducted,and combined with the results from analysis of data selected from multiple public databases,the relevant anti-tumor pathways and targets will be located,and the anti-tumor mechanisms of VA will be initially explored.2 In vivoEstablish stable hepatocellular carcinoma cell lines containing the luciferase gene,and subsequently establish bioluminescence liver cancer xenograft nude mouse model for studying how the LNP encapsulated VA complexes effects tumor-bearing mouse.The effectiveness and safety of the drug were preliminarily studied by dynamically observing the systemic tumor imaging,general conditions and survival rate of the transplanted mice.Research Methods1 In vitroMTS experiments were carried out to study the proliferation inhibition effect of VA and LV on various cancer cell lines and some corresponding normal cells,the corresponding inhibition rate and IC50 have also be calculated.Screened for the most effective tumor type and then used wound healing assay,transwell chamber invasion assay,and colony formation assay to evaluate the comprehensively effect of VA on those cell lines.The chemical 3D structure imaging website tool(http://www.chemspider.com)and the Chemical Functional Prediction tool(http://www.swisstargetprediction.ch)have been used for predicting the biological functional targets of VA,and then used the HD AC Activity Colorimetric kit to confirm the result of the predictions;Based on Oncomine(?) dataset and Kaplan-Meier public database data analysis,The negative correlation between the expression of E2F1/3 gene related to HD AC and the onset/prognosis of clinical HCC was evaluated.Then the effect of VA on these genes was explored by PCR.Then the functions of HD AC,E2F1,3 were used to find downstream targets of relative pathways,and then located its pro-apoptotic effect as the research focus.With caspACETM assay system,the effect of VA on CASPS3 activity or even apoptosis of hepatoma cells have been evaluated.Furthermore,the effect of VA on E2F1 and E2F3 in SNU-449 cells was confirmed by Whole gene expression array,and new treatment-related genes were also been explored.The GENT public database was used to help us to understand the expression of newly discovered genes in various organs of the body and provided theoretical data for further research.2 In vivoBy using well-stabilized hepatocellular carcinoma cell lines,SNU-449 and Hep3B,which contains the luciferase gene,and certain proportion of Matrigel,the bioluminescence tumor-bearing mouse models have been established.To our best understanding,the method developed by author for creating percutaneous liver cancer model,has not been reported yet.mouse models were then randomly divided into a treatment groups(LV,tail vein injection)and the control groups were started to be treated on the 15th day,and the tumor images were recorded weekly by using the IVIS fluorescence imaging system.Tumor images and corresponding fluorescence values,body weight and general conditions,and plot survival curves were also been recorded.Results1 In vitro①Different concentrations of VA possesses inhibition effect on all tested cell lines.The inhibition rate increased with increasing concentration,dose-effect relationship has been displayed.Especially for liver cancer cells(human hepatoma cell line Hep3B,human hepatoma cell line SNU-449,human hepatoblastoma cell line HepG2)and human non-Hodgkin’s lymphoma cell line Farage,VA has caused more stronger inhibitory effect on them,and have a weaker inhibitory effect on normal liver cells.② LNP in different concentrations did not significantly inhibit the cells,and the inhibition rate of LV on Hep3b,SNU-449 and TH1E-3 cells was higher than that of free VA(P<0.05).The inhibition rate of LV on HepG2 was lower than that of free VA(P<0.05).③ Compared with the control group,VA significantly inhibited the migration ability,colony cloning ability and invasion ability of Hep3b,SNU-449 and HepG2(P<0.05);Compared with the control group,VA significantly inhibited the 3D formation ability of Hep3b and SNU-449 cells.④Predictive tools showed that VA and HD AC have higher potential of interaction,by compared with the control groups,VA showed significant inhibition effect on the activity of HD AC in Hep3b,SNU-449 and HepG2 cells(P<0.05).⑤Compared with the control group,VA can improve the activity of CASP3 in HepG2,Hep3b and SNU-449 cells.⑥Whole gene expression array indicated the expression FABP,SLCO1B3,SLC22A9,E2F3,Bcl-XL,Bak,had been changed significantly.⑦IPA experiments showed multiple pathways of HDACs,E2F1,3,Bcl-XL,Bak,Bcl-2,Bax,caspase,etc.(P<0.05).⑧RT-PCR showed that the expression changes of E2F1,3,Bcl-XL,Bak,Bcl-2,Bax,FABP1,SLCO1B3 and SLC22A9 genes in SNU-449 cells were consistent with the results of whole gene expression analysis.Similar changes in expression of those gene can been seen in the results in Hep3B cells.⑨The KMP survival curve showed that the expression of E2F1/3 was negatively correlated with the prognosis of patients with clinical HCC(P<0.001 and P=0.003).PCR showed that VA inhibited the expression of E2F1,3 in Hep3B and HepG2 cells(P<0.05).⑩U133A analysis showed that FABP1,SLC22A9 and SLCO1B3 genes were highly expressed in normal liver and liver cancer cells.The KMP survival curve showed that the expression of E2F1/3 was negatively correlated with the prognosis of HCC patients(P<0.001 and P=0.003,respectively).PCR showed that VA inhibited the expression of E2F1 and 3 in Hep3B and HepG2 cells(P<0.05),meanwhile,the expression of E2F3 in SNU-449 was decreased,and the result was consistent with the result from Whole gene expression array.⑥ Compared with the control groups,VA significantly increased the activity(SA)of CASPS3 in Hep3b,SNU-449 and HepG2 cells(P<0.001).⑦Whole gene expression array showed that VA significantly down-regulated the expression of FABP1,SLC22A9 and SLCO1B3 in SNU-449 cells,and U133A analysis showed that these three genes were highly expressed in normal liver and liver cancer cells.2 In vivoIn both cell lines,the tumor signal of the control groups increased rapidly.Compared with each treatment groups,the tumor fluorescence value showed significant difference from the 21st day(P<0.05).In the two groups of cell lines,the LV groups have shown slow tumor growth,and the tumor could even shrink as time went by.In the SNU-449 treatment group,the fluorescence signal of the tumor gradually decreased after one week of treatment,while the signal of the tumor in the control group showed a steady upward trend.In the LV group,the signal of one single mice continued to be strong,but The signal was stable between 7th and 21st day.In the Hep3B LV group,the tumor signal showed a downward trend from the first week,but there was a slight increase in the signal on the 21st day,and there was no significant difference within the group,while the NC group model increased significantly and steadily.Besides,the NC group of SNU-449 liver cancer mice died on the 27th,36th,49th,and 50th day respectively from the first treatment,while the LV group found 1 death in 39 days,and the rest survived more than 60d.As for the Hep3B liver cancer mice,in the NC group,one has died for severe ascites on the 24th day,other two died in 37th days,and the remaining one died on the 46th day.Compared with the intervention group,one died on the 42nd day,the other one died on the 54th day,and the other two survived more than 60 days.Conclusion and Significance1 VA has inhibitory effects on a variety of tumors,especially on hepatocellular carcinoma cells and Farage cells,and has a weak inhibitory effect on normal liver cells.VA has multiple anti-cancer effect and can inhibit the growth of 3D formation,has potential for HCC treatment;2 Each concentration of LNP did not significantly inhibit the growth of tested cells,showing a very high safety of LNP.The inhibition rates of LV on SNU-449,Hep3B,THLE-3 cells were higher than that of free VA respectively,while the inhibition rates of LV group in other type of cells were lower or unchanged compared with VA groups,showing that the LNP has excellent targeted delivery effeciency on hepatocytes and hepatocellular carcinoma cells;expand the original application of LNP.3 VA can specifically act on HD AC,induce cell apoptosis through inhibiting the activity of HD AC,therefore improve the activity of CASP3 then inhibit the growth of cancer cell.It has the potential of becoming an HD AC inhibitor.4 E2F1,3 have the negative correlation with HCC patients,are important therapeutic targets and indexs for predicting prognosis of HCC patients.VA may significantly reduce the expression of E2F1,3 though the pathwaty of HDAC/E2F1/E2F3/CASP3 and modulate the expression of Bcl-XL/Bak though the pathwaty of HDAC/Bcl-XL/Bak/CASP3 therefore inducing cell apoptosis.5 Add TCIM theory of "liver meridian" for V.officinalis based research.6 VA can exert anti-cancenr effect by modulating the expression of FABP1、SLCO1B3.7 LV suppressed the growth of liver cancer in mouse,showed its efficacy and safty.To our best knowledge,our study first revealed the anticancer effect potential of VA and related mechanisms.Futhermore,we combined the herbal monomer and modern technics together to provide safe and effective delievery approach for future use of VA in clinical while we proved the potetianl of VA to be an HADC inhibitor.Our study also combined the theory of Chinese medicine and modern technics,deeply revealed the content of nature quality of VA,enhanced and expanded the understading of the therapeutic functions and potential of VA,more importantly,this study might provide a novel and better therapeutic agent for HCC patients.