Purifacation And Its Mechanisms On Inhibition Of Cell Proliferation In Vitro Of Antitumour Active-protein Fraction From Camel Whey In Xinjiang

Xinjiang is a major distribution area of the Chinese Bactrian camel, accounting for52percent of the distribution of country. In the traditional medicine of ethnic minorities in Xinjiang, there is a record of camel milk as an adjunctive treatment to inhibit tumor cell growth, but the ingredients of its anti-tumor activity and its mechanism is still unclear.The milk from Camelus bactrianus in Xinjiang was used as the investigative using ammonium sulfate fractional precipitation to preliminary separate camel whey, and screened its tumor-suppressor components and sensitive tumor cell lines.To explore the xinjiang bactrian camel milk suppressor component that inhibit tumor cell proliferation in vitro and its mechanism, we adopt MTT to screen the whey protein components with anticancer activity. Eca-109cell morphological changes were studied by inverted microscope, scanning electron microscopy (sem) and transmission electron microscopy and fluorescence microscopy. Flow cytometry was used to detect apoptosis rate, the change of mitochondrial membrane potential and caspase3,8,9apoptosis protein expression in the Eca-109cells after24h treatment with camel whey tumor-suppressor component. Results show that the ammonium sulfate fractionation components of camel whey have a certain inhibitory effect on cancer cell, but35%-50%ammonium sulfate fractionation of camel whey component (TR35) suppressed proliferation of Eca-109cells significantly, and had a certain amount of time-dose dependent manner. TR35had tumor suppressor activity on11strains of malignant tumor cells including Eca-109cells and the time-dose dependent (P<0.01). TR35components can induce the apoptosis of Eca-109cells, the apoptotic body, mitochondrial membrane potential depolarization, and cell cycle arrest in S phase. Under the light microscope and electron microscope showed typical apoptosis characteristics. Annexin-V/PI double dye detection appeared early apoptosis cells and Caspase3,9protein up-regulated expression. Conclusion:xinjiang bactrian camel whey protein components TR35inhibits proliferation of esophageal Eca-109cell, and could induce apoptosis of Eca-109by raising expression level of caspase-3, the induced way may be associated with mitochondrial pathway.In order to explore internal mechanism of TR35which inhibit Eca-109cell proliferatio and induce its apoptosis, RNA-Seq and two-dimensional electrophoresis combined with mass spectrometry technology were used to study the transcriptome and proteomics research on Eca-109cell treated with4mg/mL TR35for24hours. The differentially expressed genes between control and treatment were analyzed based on RNA-Seq data. There were259differentially expressed genes up-regulated and145down-regulated(|log2Ratio|(4mgA/conA)≥1and FDR (False Discovery Rate)≤0.001). GO functional enrichment analysis and KEGG pathway analysis of these genes, illustrated that this reaction involve in multiple life processes, cellular processes and the regulation of stress reaction. This showed that the influence of TR35on the the Eca-109cells is the result of the various synergy. To further analyzed and discussed the function of34up-regulated genes and31down-regulated genes. Results show that most cancer related genes were related on the apoptosis pathway and this was coincided with early experimental results. Analysis of approximately2,000protein spots on each two-dimensional electrophoresis gel revealed33differentially expressed proteins. Among them,32differentially expressed protein spots were identified by mass spectrometry. We analyzed the function of12proteins which expression was changed in both experiments. The results revealed the anticancer mechanism of esophageal cancer cells by adding TR35in protein levels, and laid the foundation of explore its internal mechanism in the future.