The Survival Inhibition Effect Of Nitidine Chloride On Pituitary Adenoma GH3 Cells

Background:Pituitary adenoma is a common kind of intracranial benign tumor, its incid ence is about 10% from all the intracranial tumors, and the quantity of Pituitar y adenoma patients were increasing in recent years. Though surgical treatment is the most common treatment for pituitary adenoma for now, unfortunately, th ere are still some kinds of tumors that cannot be wholly cut by surgical treat ment, such as Invasive pituitary adenoma and Refractory pituitary adenoma. No w, there are less types of drugs for the treatment of pituitary adenoma, the ad verse reactions are more and the postoperative recurrence rate of Pituitary aden oma is high, so searching for new efficient, safe and less-adverse reactions che mical drugs for pituitary adenoma treatment becoming urgent and a hot spot in research.Nitidine Chloride (NC) is a kind of natural alkaloid extracted from the root of Zanthoxylum Nitidum, early intensive studies showed that Nitidine Chloride plays a potent part in anti-tumor activities inside and outside of body by inhibiting proliferation and inducing apoptosis to many human solid cancer cells, such as Hepatic carcinoma, breast cancer and renal cancer. However, there are less research in the effects and mechanisms of NC on pituitary adenoma GH3 cells.Objective:By investigating the effect and analyzing the specific mechanism of NC o n the growth, cycle and apoptosis in pituitary adenoma GH3 cells, researching the effect of NC on the cellular pathway of pituitary adenoma GH3 cells, then discuss the possibility of NC to be used as a drug for pituitary adenoma’s tre atment.Materials and methods:1.Cell grouping:group the GH3 cells into 5 groups according to the different drug concentration of NC as 0μmol, 1μmol,2μmol,4μmol, and 8μmol, the group of 0μmol is used as blank control group.2. Cell viability assay: detecting GH3 cell viability of 5 groups of NC treated cells in different time by the method of CCK-8 assay.3. Apoptosis assay by flow cytometry:Cells treated with NC for 24h, dyed by the apoptosis kit of Annexin V/PI, detected the apoptosis of all groups of GH3 cells by flow cytometry.4.Cell cycle analysis by flow cytometry:Cells treated with NC for 12h, dyed by the way of PI and analyzed the cell cycle by flow cytometry.5.Detected the gene mRNA expression by RT-PCR methods:extracted the total RNA by the way of TRIzol from all the cells treated with different concentration of NC, evaluated the mRNA expression level changes to the apoptosis related genes of Bax, Bc1-2 and cell cycle related genes of Cyclin B1, CDK1, p21, p27 by the method of Real-time RT-PCR.6.Detected the protein levels by Western bloting:Cells treated with different concentration of NC were lysed in Cell lysis buffer of Western and IP, protein concentration determined by BCA kit. The whole cell lysates were fractionated by SDS-PAGE or Tricine-SDS-PAGE, the protein levels of the Bax, Bcl-2, Cyclin B1, CDK1 protein and AKT, ERK signaling pathway protein were determined by Western bloting methods.7.Statistical analysis:Statistical analysis was conducted using the software of SPSS 17.0, the obtained data were expressed by x±s, the difference among the groups inspected by t-test. P< 0.05 was considered statistical significant.Resμlts:1.The GH3 Cell proliferation effected by NC:NC had significant inhibitory effects on the GH3 Cell proliferation, and it showed positive correlation with dosage and time.2.The GH3 Cell apoptosis effected by NC:NC significantly increased the GH3 Cell apoptosis, and it showed positive correlation with dosage; the mRNA expression level of treated GH3 Cells by NC increased significantly, the mRNA expression level of Bcl-2 reduced significantly, the specific value of Bax/Bcl-2 increased markedly.3.The GH3 Cell cycle effected by NC:NC induced and accumulation of the GH3 Cells in the G2/M; NC significantly inhibited Cyclin B1 and CDK1 expression, whereas increased p21 and p27 expression in GH3 Cells.4.The GH3 Cellular pathway activation effected by NC:NC decreased the phosphorylation of AKT and ERK in GH3 Cells, held-up the activation of AKT and ERK pathway.Conclusion:1.NC can hold-up the proliferation and promotes apoptosis of Pituitary adenoma GH3 Cells.2.NC effects the proliferation and apoptosis of GH3 Cells by influencing cell cycle and hoding-up AKT and ERK signaling pathways’ activations.3.NC can be used as potential drug for the therapy of Pituitary adenoma, further research may excogitate effective solution for the therapy of Pituitary adenoma.