Cancer, multi-drug resistance, and P-gp

The leading cause of treatment failure in cancer patients is multi-drug resistance. One type of multidrug resistance is the result of a tumor cell excreting the drugs that would destroy it; these drugs are rejected via specific transport proteins. P-glycoprotein (P-gp) is a membrane protein in normal human cells, but it is also expressed in tumor cells and contributes to multidrug resistance.¹ The goal one of my projects during my time in undergraduate research (at the University of Denver) was to purify P-gp and test its functionality. A fluorescent dye, rhodamine 123, has been identified as a P-gp substrate. P-gp has a relatively unspecific binding capacity, so dyes structurally similar to rhodamine 123 may also be P-gp substrates. My ultimate goal for this project—working alongside another undergraduate student—was to more thoroughly understand the mechanism by which P-gp rejects toxins in order to better design chemotherapy drugs in the future.

In terms of methods, we used multiple procedures to purify P-gp and test its functionality, including:

a. Yeast transformation: the MDR1 gene which expresses the P-gp was transformed into the yeast cells.

b. Western blot: Once the yeast cells were grown, they were lysed and homogenized in order to isolate P-gp from the yeast membranes. A western blot was performed to determine that P-gp was present.

c. Purification: P-gp was purified by exposing the cells to: centrifugation, homogenization, and Ni-NTA resin. The elution from the resin was concentrated using a filter with a 150 kDa cutoff for better detection on a western blot. A western blot was performed to ensure that P-gp was still intact after the purification.

d. Fluorimetry: The intensity of two rhodamine dyes, tetramethyl rhodamine methyl ester (TMRM) and rhodamine B (RhB), were each measured in ethanol and phosphate buffered saline (PBS). The goal was to determine which dye would provide the greatest difference in intensity in ethanol and PBS.

At the conclusion of our project, P-gp was not intact after the purification process, so our next task was to determine what caused P-gp to be lost. Once we successfully purified enough P-gp in subsequent projects, our goal became to place P-gp into liposomes and use rhodamine to track the efflux mechanism of P-gp. To learn more about PGP, please refer to a great review article in Nature. Here’s the citation: Gottesman, M. M. et al. (2002).Multidrug Resistance in Cancer: Role of ATP-Dependent Transporters. Nature Reviews: Cancer. 2, 48-58. http://www.nature.com/nrc/journal/v2/n1/abs/nrc706.html

Sources:

Figler, R. A. et al. (2000).Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein. Archives of Biochemistry and Biophysics. 376, 34-36.