Antitumor Effect Of Ent-kaurene Diterpenoid Derivative In Wedelia Prostrata

Objective: In order to fine lead compounds with high activity and few side effect, toreconstruct the ent-kaurene diterpenoids from Wedelia prostrata, and detect theanti-growth activity in vitro and vivo, and investigate the antitumor effects and themechanism of the activity compound after reconstruction.Methods:1.To reconstruct the W1which is rich in the Wedelia prostrate. Add α-methylene cyclopentanone group to the W1and amide group on carboxyl in C-4.2.MTT and SRB assays were used to detect the viability of tumor cells treated with15-oxo-Ent-Kaur-16-en-19-butyrylamide (W1-6). Acridine orange stained SW480cells treated with W1-6at different concentrations. The photos were recorded withfluorescence microscope. Flow cytometry was used to test apoptotic ratio（AnnexinV-FITC/PI double staining assay）and cell cycle (PI staining assay). Western blottinganalysis was employed to analyze the expression of cleaved caspase-3and Bcl-2.3.Establishing the model of mice transplanted tumor U14to detect the antitumoractivity in vivo.Result:1. Structure of the synthesized with α-methylene cyclopentanone groupcompound is15-oxo-Ent-Kaur-16-en-19-butyrylamide (W1-6).2. Experimentalresults showed that W1-6could significantly inhibit the proliferation of tumor cellswith the highest IC50was0.88mg·L-1in a concentration–dependent manner.3. Thetumor inhibitory ratios of the mice treated with30mg·(kg·d)-1,15mg·(kg·d)-1and7.5mg·(kg·d)-1of W1-6were35.07%,10.23%and28.62%, respectively. The resultsuggested that W1-6had certain antitumor effect in vivo.4. SRB assay revealed thatW1-6had a strong antiproliferative effect on SW480cells. The IC50of the SW480cells treated with W1-6for24h,48h,72h, were0.86mg·L-1,0.40mg·L-1,0.19mg·L-1,respectively.5. Block cell cycle in S phase was observed in the cells treated with W1-6for24h. The percentages of SW480cells in S stage treated with0mg·L-1,0.25mg·L-1,0.50mg·L-1,1.00mg·L-1of W1-6for24h were27.92%,37.92%,47.65%,52.91%, respectively.6. Morphology changes of apoptosis were indicated byacridine orange staining. Compared to the untreated cells, SW480cell population intest groups decreased and there were morphological characteristics of apoptosisincluded cell shrinkage, nuclear condensation and formation of apoptotic bodies inSW480cells.7. The FCM analysis displayed that W1-6induced SW480cells earlyapoptosis. After treated with0mg·L-1,0.25mg·L-1,0.50mg·L-1,1.00mg·L-1of W1-6for24h, the percentages of early apoptotic cells were1.2%,6.9%,7.4%,9.3%, and thelate apoptotic ratios were8.5%,8.0%,10.1%,18.8%, respectively. Compared withcontrol group, the early apoptotic ratios were higher in test groups, and the differencewas significant (P<0.01). The difference of the late apoptotic ratios of test group (0.50mg·L-1) and the control group was significant (P<0.05).8. After treated with W1-6for24h, cleaved Caspase-3protein expressed was increased but Bcl-2decreased in aconcentration–dependent manner.Conclusion: W1-6was synthesized with α-methylene cyclopentanone group bystructure modification of W1.The experimental results revealed that W1-6hadantitumor activity in vitro and vivo. The mechanism may be to arrest cell cycle andinduced apoptosis by increased the cleaved Caspase-3protein and decreased the Bcl-2to activate the mitochondrial pathway and death receptor pathway.