Study On Anti-growth Activity And Its Mechanism In Human Lung Cells By Limonoids

Limonoids are a group of modified high oxygenated triterpenes derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton, which mainly exists in the plant of Rutaceae and Meliaceae families. All limonoids taste bitter and showed the anti tumor, antiviral, wide spectrum bacteriostasis, anti-SARS, analgesia, calm hypnosis, anti-malarial parasite and other biological medicine actions. Xylocarpus granatum grows in tropical coastal areas of south China, which was used to treat red dysentery as folk medicine in Hainan. Seeds were used as a tonic, and used to treat diarrhea, cholera and fever caused by malaria in Southeast Asian. It was also be used as insect antifeedant.This work examined eight limonoid compounds that purified from Xylocarpus granatum seed. Bioassay showed that limonoids suppress the human lung tumor cell multiplication for the first time and exploid the mechanism of suppressing the tumor cell multiplied using apoptosis related protein p53 and Bax. This study will provide information for the new drug development with fewer side effects and more potential antitumor activity. Part one The inhibitory effects of eight Limonin compounds on human lung tumor cell proliferation activityObjective: To observe the inhibitory effects of eight Limonoids-Xylocarpin H?1?, Xylogranatin C?2?, Xylomexicanin A?3?, xylogranatin D?4?, 7-Deacetyl-7-oxogedunin?5?, Xyloccensin K?6?, xylocarponoid A?7? and Odoratone?8? on human lung tumor cell proliferation activity; and to screen out the compounds with significantly inhibitory proliferative activity on tumor cells.Methods: The proliferation of Human lung cancer cells was measured by monotetrazolium?MTT? colorimetric assay. Human lung cancer cells treated with solvent control?final concentration: 0.1%DMSO?, positive control?Cisplatin? and eight Limonin compounds?final concentrations: 100 ?mol/L and 100 ?mol/L initial concentration, dilution of 8 concentration gradien? were inoculated in 96-pore plate?1×104 cells in each pore?. There were three multiple pores in each group, and they were cultured in incubators of 37?, 5% CO2 and saturated humidity for 48 hours. 5 mg/m L MTT was added at the time of four hours before ending. At the end of the experiment, the OD values of each pore was tested and Origin 7.0 was adopted to make the statistical analysis. The result was presented with mean±standard deviation? ?± s?. The concentration effect curve of experimental compounds on the tumor cells was fit by Hill mathematical model to calculate the IC₅₀ of medicine.Results:?1? 100 ?mol/L cisplatin inhibited the proliferation of human nonsmall-cell lung cancer cells?A549?RERF-LC-kj and QG-56? strongly. The inhibition rate of cell proliferation was 28.98%, 44.66% and 39.54%. Compounds 2, 4, 5, 7, 8?100 ?mol/L final concentrations? showed the proliferation inhibition of A549, RERF-LC-kj and QG-56 cell lines as the same or better than the effect of cisplatin;?2? 100 ?mol/L cisplatin and eight kinds of limonoid compounds 1⁸ acted on human small-cell lung cancer cells?QG-90 and PC-6?, compounds 2, 4, 5, 7, 8 display inhibited the proliferation of QG-90 and PC-6 cells stronger than cisplatin;?3? Through logarithm regression equation of proliferative rate of tumor cells on the concentration of Xylogranatin C, IC50 values of Xylogranatin C on A549, RERF-LC-kj, QG-56, QG-90 and PC-6 cells were 4.79 ?mol/L, 12.07 ?mol/L, 23.22 ?mol/L, 4.40 ?mol/L and 4.57 ?mol/L respectively.Conclusions: Xylogranatin C significantly inhibited the proliferation of human lung cancer cells in a dose-dependence manner. 9,10-Seco- mexicanolide-type limonoids with a smaller substitute such as hydroxyl group at C-30 showed more potential antitumor activities on lung cell. anticancer drugs can induce different types of sensitive tumor cells apoptosis. Numerous monomeric chemicals extracted from herbs played an important role in anti-tumor by inducing cell apoptosis. The first part of our experiment had demonstrated that limonin compounds Xylogranatin C significantly inhibited the proliferation of human lung cancer cells. In this part, to identify the antitumor mechanism of limonin compounds Xylogranatin C, we explored whether limonin compounds Xylogranatin C induce human lung cancer cells?A549? apoptosis by FCM and observed the expressions of p53, Bax, caspase-3 protein by Western blot.Part two Effect of limonin compounds Xylogranatin C on apoptosis of human lung cancer cells and related mechanismsanticancer drugs can induce different types of sensitive tumor cells apoptosis. Numerous monomeric chemicals extracted from herbs played an important role in anti-tumor by inducing cell apoptosis. The first part of our experiment had demonstrated that limonin compounds Xylogranatin C significantly inhibited the proliferation of human lung cancer cells. In this part, to identify the antitumor mechanism of limonin compounds Xylogranatin C, we explored whether limonin compounds Xylogranatin C induce human lung cancer cells?A549? apoptosis by FCM and observed the expressions of p53, Bax, caspase-3 protein by Western blot. Objective: At previous, a lot of experiments have shown that manyMethods: We examined cell apoptosis by FCM. Cells were incubated 24 h by Xylogranatin C at final concentrations of 10 ?mol/L, and collected cells. They were washed by PBS three times respectively, digested by trypsin, fixed by 70% ethanol, added with 10% chicken erythrocytes as internal reference standard and stained simultaneously with the samples. While added with propidium iodide DNA fluorescent staining?Propidium Iodide, PI: 50 mg/L,triton-x100 1.0%?. The samples were stained away from light for 30 minutes in 4 ?refrigerator, filtrated by 500 copper mesh, and made into qualified single cell suspension. And regulated cell concentration of 5×105⁵ ×10⁶/L. Epics-XL?FCM produced by American Beckman Coulter was used to detect DNA.?2? We examined the p53, Bax and caspase-3 protein by Western blot. According to a 2×10⁶ cells/dishes, A549 cells in the logarithmic growth phase were inoculated. Cells were incubated by cisplatin and Xylogranatin C at 10 ?mol/L final concentrations for 48 h, and collected cells. They were washed by PBS respectively. Resolved proteins were then transferred to NC membranes which probed with appropriate dilution of primary antibodies [anti-53 polyclonal antibody?1:1000?, anti-Bax polyclonal antibody?1:1000?, anti-caspase-3 polyclonal antibody?1:800? and ?-actin?1:1000?] and secondary antibodies?1:20000?. Chemiluminescent photographic detected the expressions of proteins. Image analysis software was used to make semi-quantitative analysis. Data were presented as mean±standard deviation?? ±s? and analyzed by one way ANOVA and least difference test. A level of P<0.05 was considered statistically significant.Results:?1? Effects of Xylogranatin C on apoptosis of A549 cells: After be incubated 24 h by Xylogranatin C at final concentrations of 10 ?mol/L, A549 cells' early apoptosis rates were?31.7±2.6?%, compared with blank?5.4±0.4?%, have statistically significant?P<0.05?. Xylogranatin C increased apoptosis rate of A549 cells.?2? Xylogranatin C could increase the expression of p53, Bax and caspase-3 of A549 cells.?3? The reversal effects of caspase-inhibitor on Xylogranatin C's inhibition on proliferation of A549 cells: After treated on A549 cells with 1, 10 and 100 ?mol/L cisplatin and Xylogranatin C, the cell survival rates were 83.66%, 56.27%, 28.98% and 59.87%, 24.31%, 2.76%. After treated on A549 cells with 20 ?mol/L caspaseinhibitor and cisplatin or Xylogranatin C, the cell survival rates were 97.77%, 74.90%, 56.34% and 82.78%, 46.27%, 10.98%?Conclusions: Limonin compounds Xylogranatin C could significantly induce the apotosis of human lung cancer cells?A549 cells?; Xylogranatin C could increase the expressions of p53, Bax and caspase-3 of A549 cells, which was probably one of the mechanisms of A549 cell apotosis induced by limonin compounds Xylogranatin C. And caspase-inhibitor has the reversal effects of Xylogranatin C's inhibition on proliferation of A549 cells.