The Effect Of Nerve Growth Factor To The Breast Cancer Cell MDA-MB-231 And The Screen Of Its Inhibitory Factors

This experiment investigated the expressions of nerve growth factor (NGF) and its receptor TrkA in human breast cancer cell MDA-MB-231; the signaling pathway of NGF. We also have selected the breast cancer cell proliferation inhibitory peptide aptamers from Ras binding peptide aptamers and studied the mechanism. This study is expecting to find the potential therapeutic target for breast cancer. Results as follows:1. Immunofluorescence and enzyme linked immunosorbent assay to detect autocrine of NGF in MDA-MB-231 cells. Results show that MDA-MB-231 cells expressed and autocrined NGF.2. MTT assay was used to investigate the effect of NGF antagonist Ro 08-2750 on cell viability; 5-bromodeoxyuridine (BrdU) used to detect cell proliferation; flow cytometry employed to analyse cell apoptosis as well as changes in cell cycle; TUNEL used to detect cell apoptosis. Ro 08-2750 inhibited the proliferation in a dose-dependant manner, and NGF counteracted this effect; Ro 08-2750 inhibited cell proliferation rate from 25% to 10%; Ro 08-2750 made S-phase cells increase but G2/M-phase cells decrease and induced cells to enter apoptosis program; apoptosis rate counted to 9%.3. Immunofluorescence and Western blot used to detect expression of high affinity receptor of NGF in MDA-MB-231; MTT used to show the effect of k252a to cell viability; PCR and Western blot employed to study the signaling pathway. MDA-MB-231 expressed NGF receptor TrkA, and k252a inhibited cell viability; Ro 08-2750 stimulated the long-time transcription of p53 and short-time transcription of caspase-3 and p21, and enhanced the transcription of Bax; Ro 08-2750 weakened the phosphorylation of MAPK and Akt proteins but enhanced the expression of p53 and Bax.4. 6 specific Ras binding peptide aptamers were transfected to MDA-MB-231 cells and MTT was used to detect the viability of MDA-MB-231 cells after transfection; PCR and Western blot was used to investigate the mechanism of inhibition. pPER 41R and pPER 105R inhibited cell viability effectively; pPER 41R and pPER 105R stimulated the transcription of p53, Bax and caspase-3; pPER 41R weakened the transcription of Aktl significantly, and pPER 105R had a weaker effect; pPER 41R and pPER 105R inhibited the phosphorylation of MAPK and Akt proteins drastically; pPER 41R and pPER 105R enhanced the expression of p53 and Bax.This study shows that Proliferation of breast cancer cell MDA-MB-231 was dependent on NGF. NGF promoted the survival of MDA-MB-231 cells. NGF-specific antagonist Ro 08-2750 could inhibit the proliferation of MDA-MB-231 cells, increase S-phase cells, decrease G2/M-phase cells and induce cell apoptosis. In MDA-MB-231 cells, NGF was probably to up-regulate p-ERKl/2 and p-Akt and down-regulate p53 and Bax. pPER 41R and pPER 105R could effectively inhibit the proliferation and survival of MDA-MB-231 cells. This result may be caused by the up-regulation of p53 and Bax proteins and down-regulation of p-ERK1/2 and p-Akt. It is suggested that pPER 41R and pPER 105R had special inhibition for Ras and had a potential value in treatment of breast cancer.