In order to evade chemotherapeutic interventions, cancerous cells adopt many different strategies. One curious phenomenon is the concurrent development of resistances to multiple chemotherapeutic agents. At the heart of this phenomenon lies an ancient mechanism that was characterized by Victor Ling and his colleagues in Toronto (Figure 1)
After his postdoctoral training at Cambridge University with two-time Nobel laureate Frederick Sanger, Ling started his own lab at the Ontario Cancer Institute in 1971 (1). His goal was to study the relationship between genetics and cellular behavior at a time when the study of molecular genetics was just emerging; it had only been a few decades since it was confirmed that DNA was responsible for the inheritance of traits between generations (2). With only relatively crude techniques available, Ling sought to isolate cells that had abnormal cell division using a chemical called colchicine.
Colchicine is a small molecule drug originally extracted from the meadow saffron, a plant first documented by ancient Egyptians for the treatment of rheumatism and swelling (3) (Figure 2). While today it is commonly used for the treatment of gout, Ling utilized colchicine for its ability to bind tubulin, a key structural protein that is crucial for the division of cells (4). When colchicine binds tubulin, it loses its ability to polymerize into microtubule filaments and thus cannot undergo cell division, eventually killing the cell. Only mutant cells with abnormal cell division would be able to survive in the presence of colchicine, and Ling intended to isolate these mutants to better characterize the underlying mechanisms for their survival.
Ling and his group had successfully isolated cells that were resistant to colchicine, but they also observed something surprising in many of these cells: not only were some mutants resistant to colchicine, but they had also gained resistance to other structurally-unrelated molecules such as puromycin and daunorubicin, the former being an antibiotic and the latter a chemotherapeutic agent (5). This phenotype, termed multidrug resistance (MDR), was suspected to be facilitated by a transport protein as the drugs were retained in the cells if they were starved of their energy source, adenosine triphosphate (ATP). The implication that a single protein was responsible for this phenomenon bewildered Ling and his colleagues as it had been thought that there were unique transporter proteins responsible for pumping out different types of drugs from the cell.
“We found it difficult to envision how a single transporter could recognize so many different compounds!” - Victor Ling
The curiosities of MDR drove Ling, now collaborating with other scientists in Toronto, to identify the protein that caused it. This effort led to the identification of a protein that was termed P-glycoprotein (P-gp) (Figure 3), due to its association with colchicine permeability (1). Through the continued efforts of Ling and others (1), we now understand that P-gp belongs to an ancient family of energy-dependent transporters known as ATP-binding cassette (ABC) transporters. ABC transporters are important in all forms of life, from pumping out antibiotics in bacteria to moving cholesterol in mammals. P-gp exploits the hydrophobic nature of many drugs to “vacuum” and subsequently efflux them out of the cell, thus explaining the wide range of drug resistances it alone confers (6). It is thought that P-gp had evolved out of a necessity to protect vulnerable cells and tissues, such as the brain and reproductive organs, from toxic chemicals (7). Cancerous cells can exploit P-gp by increasing its abundance in the plasma membrane to pump out chemotherapeutics, which are often hydrophobic (8). In addition to cancer, P-gp also contributes to the growing threat of antibiotic resistance by pumping out antibiotics from bacterial cells (9).
Blocking P-gp could improve the pharmacological action of other chemotherapeutics, yet approved P-gp inhibitors remains elusive to this day. The failure to generate a clinically-viable P-gp inhibitor has been mainly attributed to the evolved poly-specificity of the substrate binding pocket of P-gp, such that small molecules designed to inhibit P-gp have often elicited off-target effects (10). One target of note is cytochrome P450, a major family of enzymes involved in metabolizing drugs in the body. Both cytochrome P450 and P-gp work to eliminate drugs from the body, and inhibitors of P-gp often interact with cytochrome P450 proteins as well, primarily due to the poly-specificity of each protein’s substrate binding pocket. By affecting two independent pathways of drug elimination, P-gp inhibitors can lead to unpredictable toxicities in clinical trials, as seen in clinical trials of tariquidar, a potent P-gp inhibitor. Furthermore, as a P-gp inhibitor would need to be administered in combination with another chemotherapeutic, it is crucial that the polypharmacological profile of any P-gp modulators be comprehensively understood to avoid adverse drug interactions. Accordingly, computational approaches, such as proteome-wide screening provided by Cyclica, can be used to predict both on- and off-target effects of putative P-gp inhibitors, as well as possible adverse interactions with other chemotherapeutics prior to clinical trials.
Victor Ling’s work in discovering and characterizing P-gp has improved our understanding of the MDR phenomenon, and overcoming the action of P-gp to improve the efficacy of chemotherapeutics remains an active field of research. Today, Ling continues to study MDR and ABC transporters as a professor at the University of British Columbia. He also currently serves as the president and scientific director of the Terry Fox Research Institute. Cyclica honours the contributions of Victor Ling to the field of pharmacology and cancer biology.
Stay tuned for our next blog outlining other great discoveries by Canadian scientists or check out our other blog posts for Canada150 found here.
1. Gottesman, M. M. & Ling, V. (2005) The molecular basis of multidrug resistance in cancer: The early years of P-glycoprotein research. FEBS Lett 580.
2. Hersey, A. D. & Chase, M. (1952) Independent functions of viral protein and nucleic acid in growth of bacteriophase. J Gen Physiol 36 (1), 39-56.
3. Graham, W. & Roberts, J. B. (1953) Intravenous colchicine in the treatment of gouty arthritis. Ann Rheum Dis 12 (1), 16-19.
4. Borisy, G. G. & Taylor, E. W. (1967) The mechanism of action of Colchicine. Colchicine binding to sear urchin eggs and the mitotic apparatus. J Cell Biol 34, 535-548.
5. Ling, V. & Thompson, L. H. (1974) Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J Cell Physiol 83, 103-116.
6. Shapiro, A. B. & Ling, V. (1998) Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein. Eur J Biochem 254, 181-188.
7. Bosch, I. & Croop, J. M. (1998) P-glycoprotein structure and evolutionary homologies. Cytotechnology 27, 1-30.
8. Cordon-Cardo, C. et al. (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38, 1277-1287.
9. Poole, K. (2005) Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 51, 13-17.
10. Callaghan, R., Luk, F., & Bebawy, M. (2014) Inhibition of the Multidrug Resistance P-Glycoprotein: Time for a Change of Strategy? Drug Metab Dispos 42, 623-631.
This blog was written by Tonny Huang, a graduate student at the Princess Margaret Cancer Center. Tonny has a deep interest in the applications of protein science for the betterment of human health. You can find him here on LinkedIn.