The Effect Of Shikimic Acid From Illicium Verum On Cell Proliferation And Apoptosis In Hepatoma Carcinoma Cell Line

Objective: To study the effects of shikimic acid from Illicium verum on theproliferation and apoptosis of hepatoma carcinoma cell line HepG-2. To observethe influences on the tumor cell’s proliferation, apoptosis, cell cycle and theexpression of protein Bax and Bcl-2. To explore shikimic acid’s mechanism ofantitumor preliminarly and provide the references for the development ofclinical new antitumor drugs.Methods：1. Shikimic acid was extracted fromIllicium verum by microwave-assisted extraction. Then the extractive wasfurther separated and detected.2. The growth inhibition rate of hepatomacarcinoma cell line HepG-2was determined with MTT methods by differentconcentration of shikimic acid which effected the tumor cells for24,48,72hour.3. The liver tumor cell morphology which was impacted by differentconcentration of shikimic acid was observed in the inverted microscope.4.Apoptotic cell were detected by immunofluorescence techniques withHoechst33342staining after different concentrations of shikimic acid affecting48h.5. The liver tumor cell cycle was detected by FCM after differentconcentrations of shikimic acid affecting48h.6. The expression of protein Baxand Bcl-2were analyzed by immunohistochemistry chemical method.Results： 1. Shikimic acid was extracted from Illicium verum by microwave-assistedextraction and then the extraction efficiency was measured out37.45mg/g afterthe extractive being separated.2. Growth inhibition of hepatoma carcinoma cellline HepG-2treated with shikimic acid was worked out time dependence anddose dependence.3. It could be found that the negative control group cells withspindle shape, uniform cytoplasm, big nucleus and full form arranged in a groupeach other by inverted microscope. However, among the group of cells treatedwith shikimic acid for48hour, it could be observed that with the increase ofdrug concentration, the tumor cells with irregular profile arranged in a mess, thecell membrane not only shrinked,but became round and shed, moreover, the cellvolume became smaller and the number of cells were less.4. It was observedthat with the increase of concentration a part of cytomembrane shrinked andsome nucleus became corrugated, which were more and more obvious. As theconcentration of shikimic acid reached1mg/ml, a number of incomplete cellnucleus and cell debris were watched treated with hoechst33342staining byfluorescent inverted microscope.5. The results on FCM showed that the cellcycle progression of hepatoma carcinoma cell line HepG-2treated with ofshikimic acid for48hour delayed at G1phase.With the drug concentrationincreased, the percentage of cells at G1phase increased, however, thepercentage of cells at S phase reduced.And variation of each group was distinctfrom the control group (p<0.05).6. Immunohistochemical experiment revealed,the expression of protein Bax was intensive, which presented that the number ofpositive cells rasied and the dyeing deepen (p<0.05). However, the expression ofprotein Bcl-2decreased, which presented that the number of positive cellsreduced, the cytoplasm was shallow and the nucleus was nearlycolorless(p<0.05).Conclusions: Shikimic acid can markedly control the growth of hepatoma carcinoma cell and make the cell cycle stay G1phase. In addition,shikimic acid can induce tumor cells into apoptosis as well, which may beconnected with the high expression of protein Bax and the low expression ofprotein Bcl-2. Therefore, shikimic acid can be used as a new type of drugs whichcan resist liver cancer, and will become a new chemotherapy method.