Mechanisms Of Heparanase Regulation By Endoplasmic Reticulum Stress And Effects On The Invasion And Migration Of Breast Caner Cells

Aims:1. To investigate the effects of endoplasmic reticulum stress (ER stress) on invasion and migration of breast cancer cells.2. To investigate ER stress activates the expression and activity of heparanase (Hpa).3. To explore whether the heparanase inhibitors OGT2115and Low molecular weight heparin (LMWH) can regulation of invasion and migration caused by ER stress of breast cancer cells.4. Detect changes of metastasis-related protein and explore the mechanism of ER stress induce Hpa.Methods:1. We first examined cell viability by ADM of breast cancer cell lines, MDA-MB-231and MDA-MB-435. Cells have been treated with different concentrations of ADM for24,48and72h, then cell viability was detected with MTT assay. The concentration on invasion and migration experiments were according to less impact on cell viability.2. Chemotherapeutic reagents ADM and TM are treatment with breast cancer cells after24h, western blot detect ER stress classical protein of GRP-78, CHOP.3. Taking MDA-MB-231and MDA-MB-435as tool cells and investigate the effects on the invasion and migration by ADM and TM. The metastatic activity was observed by wound healing assay. ER stress inducers are treatment with breast cancer cells for24h,36h, Western blot and ELISA assay to detect the expression and activity of Hpa.4. Use the heparanase inhibitor OGT2115/LMWH inhibit Hpa then using Transwell assay detect invasion and migration caused by ER stress. To detect Hpa and Hpa-related protein such as MMP-9, Nf-κB, epithelial-mesenchymal transition (EMT) proteins. Results:1. ADM and TM can increased the invasion and migration of breast cancer cells MTT assay showed that cell proliferation can be inhibited by ADM. However, a low concentration of ADM (0.2μmol·L-1) and TM (0.75μmol·L-1) did not have a very effective to cell death, but increased cell invasion and migration to some extent (P<0.05)2. TM and ADM induced ER stress and Hpa in breast cancer cellsADM is a chemotherapeutic reagent which can induce ER stress. Using ADM to examined whether the increase in the invasion and migration of breast cancer cells is due to ER stress. To monitor, we assayed for the expression of GRP78after treatment with ADM/TM in breast cancer cells by western blot analysis. GRP78is an indicator of ER stress, ER stress are kept in an inactive state through binding to the ER chaperone GRP78, results show that cells exposed to TM expressed a higher level of GRP78. Hpa has a major role in the tumor metastasis, and to determine whether ER stress induces Hpa activation in breast cancer cells, we used western blot analysis and the ELISA assay to detect expression and activity of Hpa. Results show a change in bands from50kD to65kD, indicating the activation of Hpa. In addition, the increased signal in the ELISA assay also reflects changes of Hpa, results told us ER stress inducer can active Hpa in breast cancer cells, it increased at16h and24h, then causing a series of after-effects of the cell invasion and migration.3. Hpa inhibitor OGT2115decreased the invasion and migration induced by ER stressAs Hpa promotes rumor cell invasion and migration, we then examined whether ER stress can enhance cell invasion and migration is through induction of Hpa. We used OGT2115to prove inhibit Hpa can regulate invasion and migration by ER stress. OGT2115which can exhibits anti-angiogenic properties in vitro by directly suppressing Hpa activity. MTT assay results show that OGT2115can be not decrease the anti-proliferative effect of ADM and ELISA results confirmed that OGT2115can suppress Hpa induced by ER stress. Then we tested whether OGT2115can alter the effects of TM/ADM on cell invasion and migration. OGT2115can suppress the invasion and migration of breast cancer cells but it is not particularly obvious. But compared with the control group, the number and rate of migrated cells was obviously reduced when exposed to TM+OGT2115. OGT2115significantly enhanced the anti-invasion and anti-migration activity of ADM.4. LMWH decreased the invasion and migration induced by ER stressIn order to validate the results above, we also chose another heparanase inhibitor in the following studies. LMWH as an exogenous supplement of heparins is susceptible to cleavage by Hpa in vitro, and this cleavage significantly neutralizes the anticoagulant properties of these polysaccharides. LMWH exhibited a moderate anti-tumor activity and it can decrease heparanase induced by ADM or TM. However, it significantly reduced cell invasion and migration when used in combination with TM/ADM.5. Detect changes of metastasis-related protein under ER stress.By the above experiments, Hpa is an important role in breast cancer cell invasion and metastasis under ER stress. At the same time Hpa will cause other invasion and metastasis-associated protein change, such as MMP-9. TM0.75μmol·L-1treat with breast cancer cells MDA-MB-231and MDA-MB-435after24h, western blot assay found that IκB level down, maybe induce EMT. EMT protein E-cadherin-pan, b-catenin, vimentin changed. Using OGT2115and LMWH can regulation of these proteins, they may participate in the process of ER stress induced Hpa.Conclusion1. Chemotherapeutic reagents ADM and TM induce ER stress can increase breast cancer cells MDA-MB-231, MDA-MB-435invasion and migration, this is mainly due to activation of Hpa.2. Inhibit Hpa can reduce breast cancer cells MDA-MB-231, MDA-MB-435invasion and migration caused by ER stress.3. EMT maybe appear in ER stress induce Hpa. Their common participation in the breast cancer cell invasion and migration caused by ER stress.