Antitumor Activities Of Ethyl Acetate Extracts From Selaginella Doederleinii Hieron In Vitro And In Vivo And Its Possible Mechanism

Selaginella doederleinii Hieron belongs to the family Selaginellaceae(Pteridophyta) and is one of the species of plants for medicinal use.It has unique advantages in resources due to its wide distribution in China and its easy propagation. Selaginella doederleinii Hieron has the sterilization, detoxication, detumescence and anti-cancer functions,it also has been used as folk medicine for cancers.Now, the relevant researches of Selaginella doederleinii Hieron are still not systematic and deep yet, so we deeply studied the anti-tumor activity,functional mechanism and material basis of the ethyl acetate extracts of Selaginella doederleinii Hieron based on the preliminary experiment which has suggested that the acetic ether portion showed better anti-tumor activity than other portions.Objective: To study the antitumor activities of ethyl acetate extracts from Selaginella doederleinii Hieron(SD) in vitro and i n v i v o a n d i t s p o s s i b l e m o l e c u l a r m e c h a n i s m Method: HPLC method was developed for chemical analysis of theethyl acetate extracts of Selaginella doederleinii. Experiment on the ethyl acetate extractions of Selaginella doederleinii Hieron and cell lines DU145, Hep G2, Hela, MCF-7, A549, PC12, Vero, the anti-tumor affects in vitro was determined by MTT assay.Inverted microscope was used to observe the morphological changes of cells after they were incubated with the ethyl acetate extractions of Selaginella doederleinii Hieron. Experiment on the ethyl acetate extractions of Selaginella doederleinii Hieron and cancer cell line Hep G2, Annexin VFITC/PI staining assay was used to investigate the apoptotic rate of tumor cells.RT-PCR assay was applied to measure the m RNA expression of 5-lox, cox-2, flap, 12-lox, bcl-2, bax, caspase-3, survivin genes after Hep G2 cells treat with the ethyl acetate extract from Selaginella doederleinii Hieron. The anti-tumor activity of Selaginella doederleinii Hieron extracts was conducted in vivo using H-22 xenograft tumor mice. We adopted molecular docking approach to study the interactions of the six biflavonoids and cox-2,5-lox,12-lox,15-lox.The docking process was accomplished by Auto Dock(version 4.2) and the docking results were quantified by Auto Dock 4.2 scoring functionsResults:(1) Six biflavonoids was identified from Selaginella doederleinii Hieron ethyl acetate extracts through HPLC analysis.(2) Selaginella doederleinii Hieron ethyl acetate extracts caused the tumor cells death in a dose-dependent manner, and the level of cytotoxicity of SD extracts against Vero and PC12 cells were much lower than cancer cells(3) After treatment with Selaginella doederleinii Hieron ethyl acetateextracts, we can see the tested tumor cells lost their surface morphology significantly and become small and round, made fewer cellular contacts, reduced in size and number obviously through inverted microscope. These cell morphological changes were in a dose- and time-dependent manner.For PC12, the cell toxicity of Selaginella doederleinii Hieron ethyl acetate extracts was much lower than the positive drug.(4) The Annexin V-FITC/PI staining assay indicated that the cell apoptosis rate increase along with the increase of Selaginella doederleinii Hieron ethyl acetate extracts concentrations and Selaginella doederleinii Hieron ethyl acetate extracts induced the tumor cells apoptosis in a dose and time-dependent manner.(5)RT-PCR assay suggested Selaginella doederleinii Hieron ethyl acetate extracts suppressed the m RNA expression of cox-2, 5-lox, flap, 12-lox gene, reduced bcl-2, survivin and increased bax, caspase-3 m RNA expression with the action time of the drug extending.(6) Animal experiments in vivo revealed that Selaginella doederleinii extracts significantly decreased the tumor weights of H-22 tumor-bearing mice and the inhibitory rates were 23.8%, 39.8%, and 57.7% at the dosages of 4, 8 and 16 g/kg/day, respectively. Compare to positive drug, Selaginella doederleinii extracts caused less side effects.(7) The molecular docking assay revealed that amentoflavone, 3’,3’’’-binaringenin,2’’and 3’’-dihydro-3’,3’’’-biapigenin all exhibited strong interactions with cox-2, 5-lox, 12-lox respectively.Conclusion: The results showed that Selaginella doederleiniiHieron ethyl acetate extracts had considerable anti-tumor activity in vitro and could induce Hep G2 cells apoptosis. Meanwhile, the animal experiments revealed that Selaginella doederleinii extracts had good anti-tumor activity in vivo. The mechanisms of cell apoptosis induced by Selaginella doederleinii ethyl acetate extracts may be associated with decreasing the gene expression of cox-2, 5-lox, flap, and12-lox m RNA, decreasing the ratio of bcl-2 and bax m RNA level, activating caspase-3, suppressing survivin m RNA expression. These results offering evidence of possible mechanisms of Selaginella doederleinii Hieron suppress cell proliferation and promote cells apoptosis and provide the molecular theoretical basis of clinical application of Selaginella doederleinii Hieron for cancer therapy.