Profiling of Human Normal and Cancer Cell Lines using Phenotype Microarray Analysis
Authors:Shelley Alvarado, Shane Kennedy, Josh Mytych, NITIKA PARMAR, Nichole Wetton
Mentor:Nitika Parmar, Associate Professor of Biology, California State University, Channel Islands
The overall aim of this research project was to determine whether the insulin/mTOR pathway is being targeted in women’s cancer cell lines when treated with anti-cancer agents. In cancer cells, dysregulation of this pathway leading to increased mTOR activity and tumor growth has been well documented. Five different cancer cell lines- ovarian (OV90, TOV112D), uterine (MES-SA, KLE), breast (SKBR) and non-cancerous kidney (HEK293) were studied using the BIOLOG Phenotype Microarray anti-cancer panels. Preliminary experimentation involved treatment of cells seeded on panels consisting of 96 different anti-cancer agents for 24-36h, followed by examination of growth using a standard MTT assay. Growth profile results showed that six reagents produced the greatest growth inhibition - berberine chloride, azathioprine, celastrol, gossypol, miltefosine, and etoposide. All cell lines were then treated with 5µM of these six reagents to determine whether the growth inhibition was due to targeting of the insulin/mTOR pathway. Controls included untreated cells or cells treated with solvent alone. We utilized an mTOR specific ELISA based biochemical assay for this purpose. The ELISA results indicated that the mTOR pathway is being targeted by celastrol in TOV112D, SKBR, MES-SA cells; by etoposide in TOV112D, SKBR cells; by berberine chloride in TOV112D cells; by azathioprine in HEK293cells; by miltefosine in MES-SA, HEK293 cells and by gossypol in SKBR cells. We are currently conducting a dose response profile of all six reagents by treating the cells with doses varying from 0.05-5M. Data gathered for celastrol and etoposide so far indicates that growth inhibition occurs 0.5M and above in cancer cells but not in HEK293 cells. To confirm these results we will be monitoring the phosphorylation of another protein in the mTOR pathway, phospho-S6, by immunoblotting. Our collective findings indicate that growth inhibition observed in some cell lines is due to the mTOR signaling being targeted.