Document Type

Dissertation - Open Access

Award Date

2023

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Douglas Raynie

Keywords

Anticancer, Drug Delivery, Efflux Assay, MRP1 Substrate, Multidrug Resistance, Therapeutic Deep Eutectic Solvent

Abstract

Multidrug resistance (MDR) is the major burden behind chemotherapeutic treatment failure. It is the principal mechanism by which cancer cells evade chemotherapeutic treatment. As a result, aggressive cancer cells survive and continue uncontrolled cell division. Multidrug resistance affects survival rate of almost all types of cancer patients and death toll rises at an alarming rate. There are seven different mechanisms for evolving MDR. The most common mechanism in efflux activity of overexpressed ABC transporters. MRP1 is a prominent ABC transporter that pumps out a wide variety of anticancer drugs from the cells and thereby reduces intracellular drug concentrations and develops chemoresistance. Currently there are several protocols available to assess interaction between MRP1 and probable substrate anticancer drugs. However, these protocols have several limitations in common, such as expansive instrument set up, trained personnel, complex image analysis, separation of drugs from cell lysate, preparation of membrane vesicle, and results are based on accumulation of secondary fluorescence or radiolabeled substrates. To the best of our knowledge, there are no known protocols that can directly detect interaction between MPP1 and chemotherapeutic agents and categorize as MRP1 substrates. To solve this issue, in first project we developed an easy to follow and efficient novel mammalian cell-based efflux assaying using HPLC-UV technique to detect whether MRP1 considers anticancer drug as substrate and directly pumps the drug of the cancer cells. We chose MDCK-II parental and MDCK-II MRP1 overexpressed cells to establish the assay. To evaluate the efficacy of novel protocol, the result was compared with a known MRP1 substrate anticancer drug vincristine (positive control). To conduct the assay, we chose total seven MRP1 modulator anticancer drugs identified previously in our laboratory. We exclusively focused on extracellular media for pumped-out drugs from both cell lines. Initially, incubation (1 hour) and efflux time points (2 hours) were optimized. In the final step, parental and MRP1 overexpressed cells were incubated with drugs for 1 hour. After that, cells were washed and replaced with fresh transport buffer and waited for 2 hours to collect pumped out drugs from the cells. Then the solution was injected into HPLC for determining concentration. Our research idea is that if the drug works as MRP1 substrate, it will be pumped out directly by MRP1 and extracellular concentration will significantly increase compared to parental cells (no MRP1 overexpression). Our idea worked perfectly and we identified some anticancer drugs namely, alisertib, mesalamine and celecoxib that are highly susceptible to MRP1 mediated drug resistance. Next, we validated our novel protocol using popular MTT assay. We used same cell lines for MTT. From MTT we noticed that for these substrate drugs cell viability was high in MRP1 overexpressed cells compared to parental cells. It confirmed the outcome of the novel efflux assay. The novel protocol will pioneer direct and rapid detection of new MRP1 substrates with accuracy. It could also be applied to other prominent ABC transporters to identify specific substrates. The novel assay will also promote development of ABC transporter specific inhibitors to inhibit activity of transporters and restore the pharmacological potential of chemotherapeutic agents.

Library of Congress Subject Headings

Antineoplastic agents.
Multidrug resistance.
Drug delivery systems.
Eutectics.
Solvents.

Number of Pages

193

Publisher

South Dakota State University

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Rights Statement

In Copyright