Inhibition of sonic hedgehog pathway by small molecule inhibitors: Targeting colon cancer stem cells
Date
2016-02-19
Authors
Omar, Aadilah
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Abstract
The existence of Cancer Stem Cells (CSC’s) has been proven extensively in recent years. CSC’s have been positively identified in, and isolated from colon cancer. They have been found to be resilient to therapy and are highly metastatic and it has been suggested that this could be attributed to the presence of CSC’s expressing cell surface CD133. An active Hedgehog-Gli pathway has been detected in human colon carcinoma cells as well as their stem cells. Sonic Hedgehog (Shh) inhibitors such as cyclopamine have been successful in the treatment of other cancer types.
CSC’s were isolated from HT29 and DLD1 cells using the CD133 surface protein and these cells were further characterized via their expression of key stem cell markers using fluorescence microscopy. These markers included c-Myc, KLF4 and alkaline phosphatase, all of which showed elevated expression levels in the CSC fraction. The growth pattern of CSC’s in comparison to non-CSC’s was analyzed using cell-impedance (xCELLigence) assays, with clear differences noted in their growth rates. Adhesion assays revealed that although CSC’s have a lower proliferation rate, their adhesive potential proved to be greater, possibly due to them being highly metastatic. The effects of the Shh inhibitors cyclopamine and SANT-2 on cellular adhesion of colon CSC’s were compared to the Shh pathway control molecule tomatidine, and a marked decrease in adhesion potential was noted. With regard to cell invasion, this was significantly decreased when CSC’s were treated with the small molecule inhibitors. In cell migration/scratch assays a similar trend was seen, wherein cell migration was retarded by Hh pathway inhibition. Using confocal microscopy, the cell surface expression of Shh and CD133 was observed following treatment with cyclopamine and while found to significantly reduce Shh expression, CD133 expression remained unaffected.
Considering these in vitro results small molecule inhibitors targeting the Shh pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSC’s. Further investigation into the genes involved using PCR arrays would provide more detailed information on the drug action and allow for the development of an enhanced therapeutic.
Description
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of Philosophy (Internal Medicine).
Johannesburg, 2015