Study On Anti-tumor Activity And Mechanism Of Evodiamine

Objectives:To examine the anti-tumor activity and mechanism of evodiamine (Evo) in vivo and in vitro. Methods:1. Animal tumor inhibition experiments in vivoTumor inhibition experiment in Kunming mouse: Mice were injected with 10' S180 or HepA cells in 0.2 ml into the left axillary hypoderm .The next day, evodiamine(20 mg/kg, 10 mg/kg, 5 mg/kg) suspended in 0. 5% carboxymethyl cellulose (CMC) was taken orally once a day. Tumor weight was measured after 10 days and calculated the inhibition rate.2. Tumor cell inhibition experiments in vitroThe inhibition of evodiamine on 10 human tumor cell lines was determined by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Colony formation assay was used to examine the effect of evodiamine on the long-term viability of SPC-A1 cells.3. Cell cycle distribution and apoptosis assayThe morphological changes of SPC-A1 cells treated with evodiamine were observed by light microscope and transmission electronic microscope. The effects of evodiamine on the cell cycle distribution and apoptosis were determined using propidium iodide staining and through flow cytometry. Apoptotic SPC-A1 cells were determined using an ApoAlert Annexin V-FITC Apoptosis Kit. DNA fragmentation of SPC-A1 cells was assayed by agarose gel electrophoresis and APO-BRDU kit. The fluorescence intensity was determined using a spectrofluorometer to measure caspase -8,-3 activity in SPC-A1 cells. Changes in mitochondrial membrane potential (ΔΨm) in SPC-A1 cells after treatment with evodiamine were assayed using 3, 3,-dihexyloxacarbocyanine iodide [DiOC₆(3)] and through flow cytometry. The levels of PARP, Bcl-2, Bax associated with apoptosis and cyclin A, B, D, CDK 1, 2, 4, Rb associated with cell cycle of SPC-Al cells treated with evodiamine were measured by Western blots. Results:1. Tumor inhibition experiments in vivoEvodiamine had a remarkable inhibitory effect on the growth of S180 and HepA after administered to the tumor-bearing mice at the dose of 20 mg/kg, 10 mg/kg and 5 mg/mg. We got almost the same tumor inhibitory rate around 30%⁶0% after three independent experiment.2. Tumor cell inhibition experiments in vitroEvodiamine had different inhibitory intensity on 10 human tumor lines as follows: SPC-A1 > NCI - H446 > MCF - 7 > Hela > A375 - S2 > SMMC-7721 > K562 > HL -60 > Swillc > U251. Evo had stronger inhibitory effect on SPC-A1.NCI -H446 , MCF-7, Hela , A375-S2, SMMC—7721, K562 and HL—60 with their IC50 values less than 20 Mg/ml. The inhibitory effects of evodiamine on the proliferation of SPC-A1 cells were observed in a dose- and time -dependent manner. The ability of the inhibition to SPC-A1 cells growth were positively correlated with the concentration and the treatment time of evodiamine. The IC50 values at 24 hr, 48 hr and 72 hr were 9.680, 5.060> 2.619 Mg/ml, respectively. The long-term viability of SPOA1 cells, as measured by colony formation assay, was found to be significantly decreased after exposure of the cells to evodiamine with an IC50 value of 1.323 Mg/ml. 3.The influence of Evo on cell cycle distribution and apoptosisMorphological and ultrastructural changes of evodiamine-treated SPC-A1 cells were examined at various times using a light or an electron microscopy. In the evodiamine-treated cells, characteristics of apoptosis [namely, cell detachment, membrane blebbing, cell shrinkage with a condensed cytoplasm, and vesicle formation (abundant vacuoles with multivesicular bodies)] appeared. At 6 hr after the addition of evodiamine, the mitochondrial changes in the interruption and/or absence of the cristae and with loss of matrix density could be observed. Then the compaction and margination of nuclear chromatin into an amorphous and osmophilic mass was clearly detected.Analysis of SPC-A1 cell cycle was performed through flow cytometry. In the presence of different concentration of evodiamine, the cells were blocked at G2/M phase, and the effect was strengthened with the increase of the concentration. Likewise, in the presence of 0. 5 Mg/ml evodiamine, a net increa...