Effects Of Ginkgo Biloba Extract On Proliferation And Apoptosis In B16 Cells Murine Melanoma

Objective: In recent years, as the continuous development the research of Chinese medicine, ginkgo biloba is paid attention to by the most scholars. Different degree pharmacology researchs are carried out to its leaves, flowers, fruits and testas, and the extracts of ginkgo biloba(ginkgobiloba extract, EGB) profound research. EGB is a standard ginkgo biloba extract, containing 24% flavonoids(contain: quercetin, naphthol and isorhamnetin) and 6% lactone(including 2. 8% ~ 3. 4% three lactones ginkgo and 2. 6% ~ 3. 2% new lactones ginkgo), which are the most important active substances in EGB, and the other components in EGB: procyanidins, glucose, rhamnose, organic acid, D- white sugar acid and tartaric acid. In recent years, many studies have proved that EGB had anti-tumor effect, and radiosensitizing effect, meanwhile,does not increase the acute radiation injury of the normal tissues, but EGB anti- melanoma research rarely reported, and the mechanism is still not clear. Malignant melanoma(MM) is a highly malignant tumor, occurs in the skin, accounting for 1% ~ 2% of all malignant tumor according to foreign statistics, accounting for about 1% in all the cancer death cases, and the incidence of MM is rising year by year. The early and extensive transfer, and the inefficiency to surgery, radiation and chemotherapy, and the bad side effect, make it become an intractable malignant tumour in the clinical treatment. Studies have found that EGB can inhibit the tumor proliferation, induce apoptosis, reduce tumor cell surface markers; can raise P53 m RNA gene expression, cut the Bcl- 2 m RNA gene expression and improve the activity of Caspase 3, inhibit tumour cells get into the split phase, promote cell apoptosis. As a result, the mechanism of EGB antitumor activity has become one of researchs hot spot, but the EGB effect in malignant melanoma B16 cells has not been reported. This experiment observes the influences of EGB on B16 cells murine melanoma proliferation, apoptosis and expression of Caspase 3, and analyzes the mechanism of EGB to the melanoma cells, to provide the experimental basis for the basic research and clinical application of EGB inducing tumor cell apoptosis.Medthods:1 Cells culture B16 cells Murine melanoma are cultured in RPMI- 1640 containing 10% fetal bovine serum(excluding double resistance) medium, placed on the cultivation of the 37℃ and 5% CO2, saturated humidity box for regular subculture.2 The experiment was divided into control group(EGB was 0mg/L) and experimental groups of different concentrations(20, 40, 80, 160 and 320mg/L) of ginkgo biloba extract(EGB). The proliferation of the B16 cells were tested by MTT method. Inhibition rates of the corresponding concentration and IC50 were calculated after the B16 cells groomed 48 h with different concentrations of EGB.3 The different concentration of EGB treatment B16 cells before and after 48 hours in cell morphology were observed and photographed by inverted microscope.4 the cell cycle of the B16 cells were analyzed with the PI staining method by FCM.5 the cell apoptosis of the B16 cells were analyzed with Annexin V/PI staining method by FCM.6 the expression of caspase-3 in the B16 cells were tested by Western blot method.7 Adopting SPSS13.0 software to analyse the statistics, and dividing them by P<0.05 is quite meaningful.Results:1 B16 cells were polygonal or rhombic, adherent growth, consistent in size, uniform distribution, proliferation, pyknotic phenomenon was not seen before the drugs action(Fig. 1). 2The result of MTT method indicated: EGB could resist the proliferation of the B16 cell. The OD figures: 0.615±0.026, 0.537±0.029, 0.414±0.016, 0.386±0.012, 0.347±0.037 from the EGB(20, 40, 80, 160 and 320mg/L) groups,and the OD figures of the control group: 0.839 ± 0.034. Each experimental group compared with the control group, the difference was statistically significant(P<0.05). Relative inhibition rate(%) :25.18, 34.67, 50.65, 53.99, 58.64(Table 1, Fig.3). IC50:87.65mg/L(Logit method of Statist-ics).3 The influence of EGB on B16 cells differentiation and cellular morphology. The control group(0 mg/L): The cell morphology were observed under inverted microscope after 48 hours, polygonal or rhombic, stick A wall visible growth and cascading phenomenon, almost the same size, uniform distribution, proliferation strongly, nuclear pyknosis phenomenon were rarely seen(Fig.2- A); cell morphology were visibled in concentration of 20 mg/L, 80 mg/L, 320 mg/L cultivating after 48 hours(Fig.2–B, Fig.2-C, Fig.2-D). The cell morphology of B16 cells that were trained in concentration of 80 mg/L, 320 mg/L after 48 hours had significantly changed: adherent cells density decreased, cells suspension increased. With the increase of the concentration, the change was more obvious.4 The result of PI staining method(FCM) showed that: the percentage of cells in G0/G1 phase had significantly increased after the B16 cells was groomed 48 h with different concentrations(20, 80, 320mg/L) of EGB:(58.767%±1.143%, 69.567%±1.089%, 75.700%±0.949%) compared with the control group(54.983%±1.087%)(P <0.05). While the percentage of cells in G2/M phase(8.533%±0.437%, 5.992%±0.270%, 4.740%±0.526%) and in S phase cells(32.700% ± 0.965%, 24.533% ± 1.046%, 19.617% ±1.055%)in experimental groups had decreased, compared with the control group in G2/M phase(9.712%±0.668%)(P <0.05)and S phase(35.467%±1.183%)(P <0.05).(Table 2, Fig.4-1, Fig.4-2).5 The result of Annexin V/PI staining method(FCM): the apoptosis rate significantly increased with the increasing concentration of EGB. In the experimental groups:the apotosis rates of the B16 cells: 11.7117±1.1929, 19.9800±1.9800, 25.4200±2.5777. In control groups, the rate of apotosis: 6.2467 ± 0.8676. The difference was statistically significant(P<0.05) Compared the control group with each experimental group.(Table 3,Fig.5, Fig.6).6 The result of Western blotting method showed that the expression of caspase-3 protein increased as the EGB concentration increased. In the experimental groups:the expression of caspase-3 protein in B16 cells: 0.40±0.03, 0.58±0.04, 0.69±0.05. the control group(EGB for 0mg/L): the relative expressions of caspase-3 protein in B16 cells 0.33±0.02. The difference between the experimental groups and the control group is meaningful to the statistics(P<0.05).(Table 4, Fig.7-A, Fig.7-B).Conclusions:1 EGB can significantly inhibit the proliferation and growth of B16 cells.2 EGB may induce cell cycle arrested in G0/G1 phase.3 EGB could make apoptosis rate increased of B16 cells.4 EGB may up-regulat the expression of caspase-3 protein in B16 cells.5 EGB inhibit the proliferation of B16 cells, arrest the cell cycle and induce apoptosis,It may be one of its that EGB can anti-tumor mechanisms. Therefore, it provides theoretical basis and experimental evidence for application of EGB as a single or adjuvant drug.