Antitumor Activity Of Curcumol Derivative CD-2e On HepG2 Cell Line And Preparation Of CD-2e-loaded PEG-PCL Micelles

Liver cancer is one of the most common cancers in China,and what we should notice is that its death rate is the second highest.And as clinical study showed anticarcinogen can kill normal cells,result in serious side effects.Therefore,Chinese researchers paid more attention to the traditional Chinese medicine,and tried to find some new compounds which have significant efficacy with less side effects.As one of the major antitumor active components of zedoary,curcumol was used in the treatment of cervical cancer as early as 1970s.However,its anti-cancer function was limited because of its poor solubility in water.Curcumol derivative CD-2e was one of the derivatives of curcumol.It was a new compound entity obtained by modifying the hydroxyl of curcumol.We studied the antitumor activity of curcumol derivative CD-2e.Its IC₅₀0 values of hepatoma HepG2cell line,chronic myeloid leukemia K562 cell line,colonic adenocarcinoma Caco2cell line,human gastric cancer MKN45 cell line were 96.39±0.71?M?257.08±1.69?M?215.65±1.22?M?158.33±0.81?M.Compared with curcumol the IC₅₀ value of each tumor cell was improved,and anti-proliferative effect on HepG2was best.Therefore,HepG2 cell line was selected as the follow-up study object of the anti-tumor effect of curcumol derivative CD-2e.Clone formation inhibition assay was one of the most effective method for determination of cell proliferative activity.The result of this test showed that the number of cell colony at concentrations of 50?M and 100?M under curcumol derivative CD-2e are 80%and 50%of curcumol group.In this way,we demonstrated that the anti-tumor ablity of curcumol derivative CD-2e was significantly stronger than curcumol in same concentration.Subsequently,Annexin v-FITC and PI double labeling were used to determine the effect of curcumol derivative CD-2e on apoptosis assay by flow cytometry.The results showed that curcumol derivative CD-2e could induce HepG2 cells apoptosis.From the results of AO-EB staining we saw the changes in cell morphology after the effect of curcumol derivative CD-2e and curcumol.With the extension of acting time chromosome shrinkage the dye penetrated into the dead cell,stained the chromatin of the dead cell into orange,the phenomena of cell breakage were observed 24 hours later.Through detecting the impact of curcumol derivative CD-2e on cell cycle of HepG2 cell line,we known that curcumol derivative CD-2e increase the proportion of cells in S phase and inhibit the proliferation of HepG2 cells in the interdivision phase.In addition,we studied the effect of curcumol derivative CD-2e on HepG2 cell line on protein level,we found curcumol derivative CD-2e have significantly inhibitory effect on Total-JNK and P-JNK1 expression,and no significant inhibitory effect on JNK2and P-JNK2.Finally,for the purpose that making curcumol derivative CD-2e have better solubility and bioavailability,we designed curcumol derivative CD-2e-loaded PEG-PCL micelles to prolong its circulation time.The process were as follows:firstly,molecular dynamics simulation study was conducted on the micellar formation process of curcumol derivative CD-2e-loaded PEG-PCL micelles.We speculate the feasibility of preparation method.Then,through single factor exploration and orthogonal design the best formula was determined.The average nanoparticles particle size was 190nm,the Zeta potentials of nanopartial was appropriate which ensure the nanopartials were stable,and the encapsulation efficiency was 90%.Micelles in vitro stability was observed under 4?and 25?for 14 days,the small variations in size,PDI and Zeta show that the stability of curcumol derivative CD-2e-loaded PEG-PCL micelles was great.In vitro release at pH6.5 and pH7.4release media,the cumulative release of micelles was higher at pH6.5 which is 80%.Because rhodamine–loaded PEG-PCL micelles could be found in HepG2 cells,we could draw a conclusion that it can be uptaken by HepG2 cells,and it was helpful for the curcumol derivative CD-2e-loaded PEG-PCL micelles localizing in HepG2 cells and released in weak acid environment.And drug concentration-time curve proved that curcumol derivative CD-2e-loaded PEG-PCL micelles could change the pharmacokinetics of curcumol derivative CD-2e,prolong the circulation time in vivo and make a slow release.Through the above experimental studies,the following conclusions could be obtained:Curcumol derivative CD-2e could induce apoptosis of HepG2 cells by inhibiting the expression of Total-JNK and P-JNK1 and it could block HepG2 cells in S phase.Curcumol derivative CD-2e-loaded PEG-PCL micelles can improve its solubility,prolong circulation time and increase its bioavailability.