Accession Number : ADA599355

Title :   Proteomic Analysis to Identify Functional Molecules in Drug Resistance Caused by E-Cadherin Knockdown in 3D-Cultured Colorectal Cancer Models

Descriptive Note : Annual rept. 1 Sep 2012-31 Aug 2013

Corporate Author : NOTRE DAME UNIV IN

Personal Author(s) : Yue, Xiaoshan ; Hummon, Amanda B

Full Text :

Report Date : Sep 2013

Pagination or Media Count : 40

Abstract : During the first year of this research, we put our effort mainly into establishing and optimizing the inducible CDH1 knock down model with 3D culturing system, and evaluating its characteristics and performance. We successfully generated two inducible CDH1-knock-down cell lines (HT29-Green and HT29-Blue) from the parental colorectal cancer cell line HT29, which can express shRNAs targeting to CDH1 under the treatment of doxycyclin. These two cell lines together with one negative control cell line (HT29-Vector) which contains non-specific targeting shRNA are used to establish a model for EMT study in 3D-culture system. We optimized the conditions for initiating 3D structure formation, and found the best conditions for inducing CDH1 knock-down in 3D structures. The optimal conditions are 3000 cells/well seeding density, culturing for less than 10 days, inducing with 2 g/ml of doxycyclin for 3 days. The CDH1 down regulation was confirmed with RT-PCR analysis, and the cell morphological changes were confirmed with SEM and confocal microscopy observation. The increase of cell migration when down regulating CDH1 was confirmed with Boyden Chamber assay. We also optimized the SILAC methods and developed a new method for phosphoproteomic studies, which was published in Journal of Proteome Research. In conclusion, during the first year, we accomplished most of the work we proposed in our original proposal, and finished the first part of the second year s tasks. The successfully established 3D EMT model will be used for further downstream pathway analysis, and is expected to provide more details on how CDH1 functions during metastatic process.


Subject Categories : Anatomy and Physiology
      Medicine and Medical Research

Distribution Statement : APPROVED FOR PUBLIC RELEASE