Research On Antitumor Effect Of HTMF Isolated From Laggera Pterodonta In Vitro

Objective:This study was to observe the cytotoxicity on the normal cells and antiproliferative effects on the tumor cells in vitro of 3,5-hydroxy -6,7,3',4'-tetramethoxyflavone (HTMF) isolated from Laggera pterodonta. Moreover, the mechanism of inducting the tumor cell apoptosis was explored. Our results will provide the experiental basis for the exploitation of antitumor new drugs.Methods:1 Cytotoxicity of HTMF on the normal cells and the antiproliferative effects of HTMF on the tumor cellsCells were cultured by the common cell culture method. The cytotoxicity on the normal cells (EVC304 and Vero) and inhibitory rate on the tumor cells (A549, HepG2, HEp-2 and CNE) were determined. The normal cell's CC50 (50% Cytotoxicity Concentration) and tumor cell's IC50 (50% Inhibitory Concentration) were accounted. Furthermore, the cells (HEp-2 and CNE) inhibited significantly by HTMF were chosed and quantity-effect and time-effect relationships of the inhibitory effect were analysed by MTT assay. The inhibitory rate of A driamycin (ADM) in clinical used commonly antineoplastic agent on HEp-2 and CNE cells and cytotoxicity of DMSO on HEp-2, CNE, EVC304 and Vero cells were also determined by MTT assay.2 Effects of HTMF to changes of the cell and nuclear morphological characteristics, and cell apoptosis rate on the tumor cellsAfter the tumor cells were treated with different concentrations of HTMF, the changes of the cell morphological characteristics were observed under the inverted microscope and take a photograph. The changes of cell nuclear morphological characteristics were observed under the fluorescence microscope through the Hoechst 33258 staining and take a picture. The cell apoptosis rate on HEp-2 and CNE cells was displayed by flow cytometry (FCM). 3 Effects of HTMF to the expression of Caspase-3 and Caspase-9The expressions of Caspase-3 and Caspase-9 were detected by Western-blotting with different concentrations or for different times with the same concertration treatments of HTMF.4 Effects of HTMF to mitochondrial membrane potential (ΔΨm)The mitochondrial membrane potential was analyzed by FCM with Rho 123 staining and by laser confocal microscope with JC-1 (a kind of fluorescent probe was used generally to detect mitochondrial membrane potential, and the changes of the mitochondrial membrane potential were observed by the changes of the flourescence color) staining.Results:1 MTT assay results showed that the IC50 values were 61.49,74.61,27.81 and 28.31μg/mL at 48 h treatments of HTMF on A549, HepG2, HEp-2 and CNE cells respectively, while CC50 values on EVC304 and Vero were 56.49 and 87.64μg/mL respectively. The inhibitory rate on HEp-2 and CNE cells increased with the increasing of concentration and time prolongation respectively, and showed up dose and time dependent manners (P< 0.05, P< 0.01). The cytotoxicity results exhibited DMSO with different volume percentagein in different groups had no cytotoxicity to HEp-2 and CNE cells (P> 0.05). The inhibitory effects of ADM were better than that of HTMF.2 The changes of the cell morphological characteristics showed that the cells became round, the volume was shrinked, and the adherence capability was decreased. The cell population accounting and cell density were decreased significantly compaired with the control group with the increasing of concentration of HTMF after 48 h on HEp-2 and CNE cells. The cells showed karyopycnosis, nuclear volume shrinking, and maldistribution by Hoechst 33258 staining. A part of cell nuclears showed up to break into pieces, and to form some apoptotic body with thick white-blue fluorescent in Hoechst 33258 staining cells. The apoptosis rate is significantly increased with the increasing of concentration of HTMF on HEp-2 and CNE cells (P< 0.05, P < 0.01). 3 Western-blotting results showed that Caspase-3 and Caspase-9 were activated in dose dependent manners on HEp-2 and CNE cells with various concentrations of HTMF (P<0.01). After treated with 40μg/mL of HTMF, Caspase-3 and Caspase-9 were activated in time dependent manners on HEp-2 and CNE cells with time prolongation (P< 0.01).4 Laser confocal microscope with JC-1 fluorescence staining showed that the flourescence color changed from red to green with the increasing of concentration and time prolongation, and at the same time mitochondria numbers became more and more less and maldistribution. FCM with Rho123 staining results showed that the number of the mean fluorecence index of the mitochondria decreased with the increasing of concentration and time prolongation, respectively (P< 0.05, P< 0.01).Conclusion:1 HTMF had different inhibitory effects on A549, HepG2, HEp-2 and CNE cells. Furthermore, HTMF had an significantly antiproliferative effects on HEp-2 and CNE cells in dose and time dependent manners, while exhibited low cytotoxicity to the two normal cells, Vero and EVC304. This showed that HTMF had the selectivity, high performance and low cytotoxicity on antitumor effect.2 HTMF inhibited significantly the proliferation of HEp-2 and CNE cells by inducting the cell apoptosis. Furthermore, the cell apoptosis rate showed the dose dependent manner.3 Caspase-3 and Caspase-9 were activated obviously in dose and time dependent manners in the pathway of the cell apoptosis induction by HTMF on HEp-2 and CNE cells.4 HTMF decreased in mitochondrial membrane potential in dose and time dependent manners, activated caspase cascade reaction, and inducted the apoptosis of HEp-2 and CNE cells by the mitochondrial passway.