Studies On Antitumor Activity Of Anthriscus Lactone Derivatives

Objective To design and synthesize 4’structural transformation of derivatives of deoxypodophyllotoxin to achieve synergistic effect of toxicity attenuated and potency augmentation; investigate to synthesize a series of derivatives of deoxypodophyllotoxin on a variety of human cancer cells in vitro growth inhibitory activity; investigate the influence of derivatives on SNU-387、HT-29 cell apoptosis; investigate the influence of derivatives on a variety of human cancer cells Bax、Bcl-2 gene expression.Methods Removal the 4’-methyl group of deoxypodophyllotoxin to get 4’-demethyl-4-deoxypodophyllotoxin containing reactive hydroxyl groups, and then with cinnamic acid, hydrocinnamic acid, p-nitrobenzoic acid and other ligand for esterification to get the related derivatives.MTT assay to observing the concentration gradient (0、1.5、3、6、 12.5、25、50、100ug/mL) of the four derivatives in vitro of inhibiting HepG2、SNU-387、 HT-29、MG-63、MCF-7 cell proliferation, cisplatin 50ug/mL as a positive group. Using Annexin V-FITC/PI apoptosis detection kit, the concentration of the four derivatives were 1、40、80ug/mL as a dosage group,10、40μg/mL of cisplatin as a positive group,0.1% DMSO as a control group, drug groups on SNU-387 and HT-29 cells affected 48h, apoptosis was detected by flow cytometry.The four derivatives was 50ug/mL as a dosage group,50ug /mL of cisplatin as a positive group,0.1% DMSO as a control group, by ELISA kit to detect the effect on MCF-7、HepG2、HT-29、MG-63 tumor cells after 48h of Bax, Bcl-2 gene expression.Results (1) 5 derivatives of deoxypodophyllotoxin was designed and synthesized successfully.They are 4’-demethyl-4’-O-cyclobutane carbonyl group-deoxypodophyllotoxin (cyclobutanecarboxylic acid derivatives),4’-demethyl-4’-O-(3-phenyl-2-allylacyl)-deoxypodophyllotoxin (cinnamic acid derivatives),4’-demethyl-4’-O-(3-phenyl-propionyl)-deoxypodophyllotoxin (hydrocinnamic acid derivatives),4’-demethyl-4’-O-4-nitrobenzoic acyl-deoxypodophyllotoxin (p-nitrobenzoic acid derivatives),4’-demethyl-4’-O-4 methyl benzoyl-deoxypodophyllotoxin (p-methylbenzoic acid derivatives),by H-NMR to confirm its structure.(2) Cyclobutanecarboxylic acid derivatives, cinnamic acid derivatives, hydrocinnamic acid derivatives, p-nitrobenzoic acid derivatives of selected tumor cells were shown to inhibit the proliferation activity, the best cytostatic effect of each derivative’s IC50 values are as follows:cyclobutanecarboxylic acid derivative on SNU-387 cells was 4.346μg/mL, on MCF-7 cells was 7.201μg/mL;hydrocinnamic acid derivative on HT-29 cells was 6.218ug/ mL;p-nitrobenzoic acid derivative on HepG2 cells was 9.894μg/mL; on MG-63 cells was 8.982μg/mL.In concentration of 100μg/mL, each group had the highest inhibition rate reach or close to 80%, showing good antitumor activity, and showed a dose-dependent manner. But cinnamic acid derivatives, hydrocinnamic acid derivatives on MG-63 cells, hydrocinnamic acid derivatives on MCF-7 cells proliferation inhibition less effective.(3) Detection of apoptosis by flow cytometry results showed that the dosage group and control group were shifted to the right compared to the scatter of two cells, suggesting the occurrence of apoptosis. Late apoptotic for SNU-387 cells, Annexin V+/PI+region expressed more cells with increasing concentration derivative showing a rising trend, positively correlated with the concentration; cyclobutanecarboxylic acid derivative on HT-29 cells, the early apoptotic, late apoptotic cells were significantly increased, while the other three derivatives although caused apoptosis, but the degree of apoptosis rate related to the concentration of derivatives is not high.(4) ELISA kit test results showed that cinnamic acid derivatives, hydrocinnamic acid derivatives, p-nitrobenzoic acid derivative, cyclobutanecarboxylic acid derivatives acting on MCF-7、HepG2、HT-29、MG-63 cells compared to each dosing group, the ratio of Bcl-2 and Bax decreased compared with the control group. The statistical t-test test showed, in addition to cinnamic acid derivatives on MCF-7、MG-63 cells as well as the p-nitrobenzoic acid derivatives on MCF-7 cells, the ratio of the rest groups compared with the control group has a significant difference(*P<0.05).Hydrocinnamic acid derivatives on MG-63 cells, p-nitrobenzoic acid derivatives, cyclobutanecarboxylic acid derivatives on HepG2 cells, Bcl-2/Bax ratio compared with positive group has a significant difference (#P<0.05).Conclusion Deoxypodophyllotoxin as the precursor for 4’structural transformation is feasible route to obtain new derivatives; cyclobutanecarboxylic acid derivatives, cinnamic acid derivatives, hydrocinnamic acid derivatives, p-nitrobenzoic acid derivative has inhibition on proliferation of tumor cells; derivatives enables SNU-387、HT-29 cells scatter moved to the apoptosis region, suggest that can induce apoptosis; by reducing the expression of Bcl-2 gene, promoting the expression of Bax gene to induce apoptosis of tumor cells.