Inhibitive Effect Of Camellia On The Growth Of CNE-2 Cells In Vitro And The Gene Expression Of VEGF-C \VEGFR-3

Objective:Camellia chrysantha (Hu) Tuyama is a kind of high medicinal value of natural herbs. After determed by authority of the department of modern medicine and related scientific research institutes, Camellia is nontoxic tea, leaf contains many amino acids, polyphenols, saponins, flavonoids and other beneficial ingredients which the body need [1-2], it also rich in an important human health effects of trace elements. After several decades of research, researchers have been convinced that Camellia has a special effect in reducing blood lipids, blood sugar, prevent cancer, cancer, and so. The effect and mechanism of tumor suppression has been preliminarily discussed, it was been studied that Camellia were used in rats in vivo antitumor and has significant effect. However, studies on nasopharyngeal carcinoma cells rarely relevant reports at home and abroad. The experiment was made on the purpose of laying a theoretical foundation for further study on finding low toxic and high efficiency natural anti-tumor drugs by observing the Camellia inhibited the CNE-2 cells proliferation and induced its apoptosis and a preliminary study on mechanisms of impaction of expression of VEGF-C \VEGFR-3 protein.Methods :1. MTT assay was used to determine to different concentrations (25μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 400μg / ml) of ethanol extract of Camellia at different times (24h, 48h, 72h) under the action of the CNE-2 cell proliferation and inhibition in vitro.2. Inverted phase contrast microscope was used to observe the CNE-2 cells morphological changes under impaction of different concentrations (50μg / ml, 100μg / ml, 200μg / ml) of ethanol extract of Camellia.3. Fluorescence microscope was used to observe apoptotic cells by using hochest 33342 staining which appeared from Camellia after impact by ethanol extract (50μg / ml, 100μg / ml, 200μg / ml) of CNE-2 cells .4. Flow cytometry was used to analysis different concentrations (50μg / ml, 100μg / ml, 200μg / ml) of the alcohol extract of Camellia impact on CNE-2 cell cycle and apoptosis.5. Protein blotting techniques (Western Blot) was used to detect the expression of caspase-3 protein impacted by various concentrations (50μg / ml, 100μg / ml, 200μg / ml) of alcohol extract of Camellia.6. Enzyme-linked immunosorbent assay (ELISA) was used to detect the change of vascular endothelial growth factor (VEGF-C \VEGFR-3) protein in the supernatant of CNE-2 cells. after impacting of various concentrations (50μg / ml, 100μg / ml, 200μg / ml) effect of ethanol extract of CamelliaResults:1. Camellia alcohol extract has effect on inhibiting the proliferation of the CNE-2 cells growth, which presented a character of time and dose dependent, with the time going and concentration rising, the inhibitory effect increasing gradually. Compared the affection of ethanol soluble extract of Camellia concentration greater than 50μg/ml to the negative control group, the difference was significant in statistically (p <0.01).2. Under observation by the inverted phase contrast microscope,as drug concentration increased, the CNE-2 cells grow slowly after 48h impacted by the ethanol extract of Camellia, cell shrinkage gradually and became smaller and more round, shedding, and finally see a large number of contracted , broken and exfoliated cells suspended in culture medium.3. Under the fluorescence microscope, different concentration of ethanol extract of Camellia impacted on CNE-2 cells was observed after 48h.It could be observed that cells revealed a dense concentration of the strong blue fluorescence staining, nuclear condensation, nuclear cleavage, nuclear chromatin aggregation apoptosis cell morphology.As the concentration increased,the apoptosis cells increased.4. After impacted by ethanol extract of Camellia 48h, with the concentration increased, the proportion of cells in G1 phase increased in CNE-2 cells, while the G2, S phase cells have different levels of reduction, cells were arrested in G1 phase. At the same time, as the concentration increased, the apoptosis rate also increased. Compared to the negative control group, the differences of apoptosis rate had statistically significant (p <0.05).5. Western Blot: With the increase of drug concentration, activation of the Caspase-3 protein bands gradually deepened, expression of Caspase-3 protein increased. Analysis of gel image processing system with integrated optical density measured with the GAPDH band and the negative control group integrated optical density were statistically significant differences (p <0.05).6. ELISA: As the drug concentration increased, VEGF-C \VEGFR-3 protein level in CNE-2 cell culture supernatant decreased.Conclusions:1. Ethanol extract of Camellia can inhibit the proliferation of CNE-2 cells in vitro, and presents the dose and time effects. 2. The mechanism of induction of apoptosis may be induced G1 arrest and the promot activation of Caspase-3.3. ELISA test confirmed that VEGF-C\VEGFR-3 expression in human nasopharyngeal cancer may play an important role in controlling metastasis.