A Unique Multiplex HTS Assay Enables the Discovery of a Novel Pro-Differentiate anti Colorectal Cancer Compounds

Alexander Plotnikov, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel (alexander.plotnikov@weizmann.ac.il)

Measurement of Alkaline Phosphatase (ALP) is a widely used procedure in clinical and basic research. At the basic research, ALP quantification is frequently used in the study of cancer physiology and for evaluation of pluripotency in stem cells. Current methods for examination of ALP expression and per cell normalization have certain disadvantages such as: relatively high cost, multiplicity of reagents and washing steps, low-precision methods of detection, and the necessity of dedicated instruments.  We discovered a simple and inexpensive luminescence-based method that allows multiplexed measurement and normalization of intracellular ALP levels in one sample well. The method comprises two commercially available reagents enabling quantification of ALP levels and cell number by two sequential luminescence readouts. Using this method we were able to detect and analyze reprogramming of pluripotent stem cells. We apply our multiplex assay in High Throughput Screening (HTS) campaigns for discovery of pro-differentiate anti colon cancer compounds. 

ALP is a ubiquitous marker of intestinal epithelial cell differentiation. While differentiated intestinal epithelial cells express relatively high level of the enzyme, ALP expression in poor differentiated colon cancer cells is known to be very low, almost negligible. We used this principle to screen compounds for their ability to induce ALP expression and differentiation of colon cancer cells (HT-29).

5760 compounds (from different chemical libraries) were screened. Potential hits were tested using normal colon cells (CCD-841) to ensure selective differentiate effect on cancer cells. Compounds were further validated in a dose dependent manner for their effect on pro-differentiating markers expression (E-Cad and ZO1). A methyl transferase inhibitor was identified as a compound that enhances differentiation and significantly delay proliferation of HT-29 cells without any effect on normal cells.