Research Of Hsp90α Secretion And Its Function Mechanism In Breast Cancer Cell

BackgroundHeat shock protein 90α(HSP90α) has been mostly know as an abundant intracellular chaperone with more than 100 target proteins often involved in control of cell metabolism, survival, growth and differentiation. Hsp90αhas been a target for anti-cancer drugs. People all know that Hsp90αis one of the most abundant proteins in cells. However, exactly how much Hsp90αis present in a cell has never been shown experimentally. Furthermore, recent studies have discovered a surprising need for cancer cell to constitutively secrete Hsp90αfor invasion and metastasis. However, the mechanism of Hsp90αsecretion and secreted Hsp90αpromotes cancer cell motility remains unclear. In this case, we carried out these experiments to answer the above questions.Objectives1. To investigate the relative amount of Hsp90αin cells. Try to answer this question experimentally. 2. To figure out the link between the Hypoxia-inducible-factor (Hif-1α/Hif-1β) and Hsp90αsecretion.3. To show that secreted Hsp90αpromote cell motility via the cell surface receptor LRP1.Methods1. Prepare the human recombinant Hsp90αsample and measure its amount in advanced. Use cell lysate and human recombinant Hsp90αfor western blot analysis. Exposure both of them on one film. Measure the density of Hsp90αbands, calculate the amount of Hsp90αin cell lysate via comparing the their density.2. Grow the breast cancer cell line (MDA-MB-231), use siRNA down regulate Hif-1αor Hif-1βexpression, to see if this can have an effect on Hsp90αsecretion.3. Grow the breast cancer cell line (MDA-MB-231), use siRNA down regulate Hif-1α, Hif-1βand LRP1expression, then do cell migration and cell invasion experiments, to check secreted Hsp90αpromote cell motility and its functional mechanism.4. Animal experiment: Use SCID mice as model, each mouse is injected 1×106 cancer cell. Transfect luciferase reporter gene into breast cancer cell lines (MDA-MB-231 and MDA-MB-468). Experimental group mice are injected MDA-MB-231-Luc cell while control group mice are injected LRP1down-regulated MDA-MB-231-Luc cell or MDA-MB-468-Luc cell. Use an in vivo imaging system to compare their tumor formation ability.Results1. The relative amount of Hsp90αin cells: HDF: 2.33%±0.16; HKC: 2.35%±0.26; HEMN-LP: 2.25%±0.62; HBL-100: 3.47%±0.36; SCC-12: 3.79%±1.31; MDA-MB-231: 3.66%±0.21; A431: 4.53%±0.70; M24: 6.77%±1.56.2. Down-regulate either Hif-1αor Hif-1βexpression in MDA-MB-231 cell line can block Hsp90αsecretion and lower cell motility.3. Down-regulate LRP1 expression in MDA-MB-231 cell line can decline its invasiveness.Conclusions1. The Hsp90αaccounts for 2-3% of the total cellular proteins in normal cells and up to 3-7% in several cancer cell lines.2. MDA-MB-231 cell line expresses HIF-1α→HIF-1αgoes into nuclear to form dimerization with HIF-1β→HSP90αsecretion→secreted HSP90αbinds to cell surface receptor LRP1→promote cell motility.