The Antitumor Screening And Mechanism In Vitro Of Furanodiene

1 Purpose Firstly, the purpose of this study is to observe the furandiene(FDE) in vitro inhibition of the 18 kinds of tumor cell lines,which is basised on the analysis of furandiene spectrum anti-tumor and evaluate its efficacy in vitro, and to further explore its role of sensitive tumor MGC-803 induced apoptosis of human gastric cancer cell lines; By the observation followedly of the tumor growth of human gastric cancer cell MGC-803 nu / nu mice transplanted and to evaluate its efficacy in vivo,the outcome will provide the foundation deeply for the pharmacological effect of the study of FDE.2 Materials and methods2.1 The antitumor screening of Furandiene To clarify effects of tumor cell proliferation in vitro of Furandiene, method using the MTT method to detected the inhibition of cell proliferation of various tumor cells in vitro with different concentrations of FDE. Drug concentration were designed as640,320,160,80,40,20,10 μg/ml, which acts on the test tumor cells(MGC-803,HT-29,DLD-1, NCI-H292,95- D, NCI-H460, NCI-H1650, A549, Hep G2, He La, HCT116, SK-OV-3, A375, HGC-27, HL-60, K562, Reh and THP-1), with fluorouracil as a positive control medicine, by using microplate reader could got its OD values and plotted with tumor cell growth curve and sorted data by using Origin8.0 software to determine the concentration of semi-efficient(IC50, μg/ml) and the maximuminhibition rate(Imax). While the preparation of bone marrow cells from normal rat can test the impact of normal cells after the using of FDE. Screening for the most sensitive cell line and studying for its further apoptotic mechanism.2.3 Studies on Inducing Apoptosis Effects and Mechanism of Furandiene for MGC-803 Gastric Cancer Cell Lines The screening experiments of anti tumor in vitro showed that the FDE have the effective growth inhibition on MGC-803 cells, p<0.01.To explore furtherly the mechanism, the methods of this present study including: ① MTT assay was used for screening tumor spectrum and detecting the viability of MGC-803 Cells with different concentrations and different timings;②Cell morphology was observed by light microscopy and changes of apoptosis of Hoechst 33242 staining were observed by fluorescence microscopy morphological; ③Annexin V-FITC/PI double staining which combined with flow cytometry were used to detect cell apoptotic;④Fluorescence probe DCFH-DA was employed to detect the changes of reactive oxygen species(ROS); ⑤ Rhodamine 123 staining was employed to detect the changes of mitochondrial membrane potential; ⑥PI staining and flow cytometry were used to detect cell cycle; ⑦Caspase-3, Caspase-8, Caspase-9 activity which combined with spectrophotometric detection kit were used to test the Caspase-3, Caspase-8,Caspase-9 activity.2.4 Evaluation of effects of the growth in vivo for MGC-803 cells of Furandiene Exponentially growing the sensitive MGC-803(5 × 107)cells in 200μl medium were seeded into 4-6 weeks-old nude mouse.Establishing the xenograft tumors model by the method of tumor block transplanting. Forty nude mice were randomly divided into 5 groups in the basis of the same tumor size. Vein injective administration was the way of drug delivery for the all groups. Fluorouracil was the positive control while FDE were experimental groups. The weight of animals were surveyed every other day while the diameter of the xenograft tumors in the same way at the same time.The xenograft tumors were surveyed in the end of the experiment and the animalswere executed to collect the tumors which were scalaged to collect the dota after then.The inhibitory effect was observed to occur in a dose-dependent manner to evaluate the anti-cancer activity of FDE.3 Results3.1 The antitumor screening of Furandiene The test results of FDE in the inhibition of proliferation in vitro which showed that the concentration of the test drug were setted by covering the drug effect window and repeated the test results same basically. The cytotoxicity of FDE was firstly assessed by MTT assay which showed that the experimental treatments had obvious effect on lung cancer, liver cancer, cervical cancer, colon cancer, stomach cancer, melanoma and individual human leukemia cells proliferation which were compared with the control group for 48 hours.The inhibitor concentration(IC50) were 16.58~55.30μg/ml,P<0.05.The results showed that the IC50 of MGC-803 cells were 16.58μg/ml.But leukemic cells Reh, THP-1 and SKOV-3 ovarian cancer cells were not sensitive which inhibitor concentration(IC50) value were 187.19 μg/ ml,239.5μg/ml, 206μg/ml.At the40μg/ml concentration FDE taking,there was not inhibitory effect on the bone marrow cells of normal rats.Positive drug fluorouracil were taked at a fixed exposure concentration(25ug/ml), which the range of cell growth inhibition rate were11.29%~91.46%.3.2 Discuss on Inducing Apoptosis Effects and Mechanism of Furandiene for MGC-803 Gastric Cancer Cell Lines①MTT results showed that FDE have better inhibition of growth of tumor cells with the drug for 24、48、72 hours on MGC-803 Gastric Cancer Cells. Furthermore,with the increasing concentration of drug and the extension of time,the time-dosage dependent relationship existed in the inhibition rate of tumor cells.②After treated with FDE, the growth of the cells was inhibited and the morphological changes in gastric cancer MGC-803 cells were found by the inverted microscope which behavedas chromatin margination, cell shrinkage and apoptotic cells.The Hoechst 33342 staining showed that morphological changes were observed in treated group.Chromatin margination,fragmentation,nuclei dissolution and white color were observed as well.③ The Annexin/PI double staining showed that no much apoptotic cells were observed in control group. After normal cells were treated with 80μg / ml FDE,the percentage of they has become less, apoptosis cells became more which averageapoptosis of rate was24.17±4.24%, and it showed significant difference.(P<0.05)④DCFH-DA results show that a significant generation of ROS was observed after 48 h of exposure to FDE. Compared to the control group,FDE at 80μg/ml promoted the increases in DCF fluorescence intensity by 1.35 folds respectively.⑤The Rh123 staining showed the Rh123 accumulation was in a great deal in control group which suggesting the high ΔΨm.While after treatment, the Rh123 accumulation was greatly decreased suggesting the significant decreased of ΔΨ m(p<0.05).⑥The PI staining showed that the distribution of gastric cancer MGC-803 cell cycle was significantly changed after(20,40,80μg/ml FDE) treatment.The PI positive cells in G0/G1 phase changed be 64.03%; PI positive cellsin G2/M phase changed be 6.59%;PI positive cells in S phase changed be 29.37% in experimental group.⑦Detection of Caspase-3﹠Caspase-8﹠Caspase-9 activity showed that the activity of Caspase-3﹠Caspase-8 ﹠ Caspase-9 were significantly increased after(20,40,80μg/ml FDE)treatment.After treated with 80μg/ml FDE for 48 h,the Caspase-3 ﹠ Caspase-8 ﹠Caspase-9 release from the cells were increased(1.06、0.82、1.38)times,(1.24、0.83、1.51)times and(1. 39、1.31、1.98)times as much ascontrol group(P <0.05).3.3 Evaluation of effects of the growth in vivo for MGC-803 cells of Furandiene The experiment results showed that the inhibition rate were 23.88%, 27.99% and33.00% respectively after(20,40,80mg/kg FDE) treatment according to the observation of xenograft tumors of MGC-803. The tumor volume and tumor weight of treatmental groups were inhibited while were compared with the control group. All of these results showed an correlation between the inhibition rate and the dosement of FDE but not obviously.4 Conclusions4.1 The cytotoxicity of Furandiene was availably assessed by MTT assay which showed that the experimental treatments had obvious effect on 18 human malignant cell lines proliferation.Furthermore, the inhibitory effect was observed to occur in a time-and dose-dependent manner as well.The result of its role in the study of tumor cells in vitro inhibition provide premise.4.2 Mitochondrial pathway may be an indicator of apoptosis and blocking cell cycle may be a key indicator of cellular viability after furandiene treatment.The motility ability and invasive ability of gastric cancer MGC-803 cells could be inhibited by furandiene. The detection of Caspase-3 ﹠ Caspase-8 ﹠ Caspase-9 activity may be related to its apoptosis.4.3 The growth of tumor xenografts could be slowed after furandiene treatment in a dose-dependent manner in vivo.Furandiene is experimental settings and low toxicity according to the appropriate dosing-schedules. All these results suggested that furandiene is a harmfulless compound while in a setting of the measuring range but not efficient significantly which was expected to develop into a species new anticancer drug which should need more studies.