Targeting drug resistance in cancer cells by the anthelminthic drug, pyrvinium pamoate

Abstract

Chemotherapy is the first line of option for cancer treatment and drug resistance remains a major impediment to achieve positive therapeutic benefit often results in therapy failure and tumour relapse. Multiple mechanisms are at play in enforcing drug resistance in tumours, some which are inherently active in certain tumour types. Others are eflecled during the course of chemotherapy. Overcoming drug action and survival depends primarily on the ability of the cancer cell to adapt to the rapidly changing environment. This includes metabolic rewiring which enables the cancer cell to utilize available resources for synthesis of ATP and growth. The major pathways yielding ATP in any cell are glycolysis and milochondrial respiration. Tumour cells preferentially utilize glycolysis for growth even in the presence of adequate oxygen and this addiction to glycolysis is termed 'Warburg effect'. Although mitochondria of tumour cells were previously thought to be defective and the cause of 'Warburg effect', recent research has proof that tumour cell mitochondria are active and respire. Eventhough glycolysis is less efficient in terms of ATP synthesis compared to mitochondrial oxidative phosphorylation per glucose molecule used, it is preferentially used by tumours since it is ideally suited for hypoxic environment commonly found in tumours. Secondly, glycolytic intermediates are shuttled for the synthesis of macromolecules like nucleotides, fatty acid metabolism and amino acids essential for rapid growth and cell d ivis ion. G lyco lysis also synthesizes reducing equivalents like NADPH and NADH essential for sustaining biochemical reactions. Most anticancer drugs target the glycolytic pathway and a subset of cancer cells suivdve by rapidly shifting to oxidative phosphorylation. This ability to shuttle between pathways is termed metabolic plasticity, and is the prime mechanism that enables the cancer cells overcome drug effect. The second important mechanism to tide over drug efTect in cancer cell is the engagement of drug efflux pumps that actively exclude drugs from cancer cells thereby rendering them ineffective. Passive mechanisms include expression of chaperone proteins belonging to Heat Shock Protein (HSP) family that stabilize their substrates thwarting drug efficacy. Chemicals which are cost effective, possess good safety profile and having the capability for targeting multiple mechanisms will be the most potent agents to overcome drug resistance in tumours. Pyrvinium pamoate, an FDA approved anthelminthic agent is an ideal candidate that matches the description. This project explored if Pyrvinium pamoate (PP) as a single agent or along with regular chemotherapeutic drugs could be used to overcome cancer drug resistance. Cell viability assays with MCF-7 cells showed that IC50 of PP was 280nM whereas the IC50 values of5-Fluoro Uracii (5-FU) and Doxorubicin(Dox) were 2.77pM and 4.7 pM respectively. PP at 500nM was able to reduce the IC50 of Dox from 2.77pM to 0.59 pM, a five fold reduction of effective dose in combination. PP at SOOnM was able to reduce the IC50 of 5-FU from 4.7pM to 0.35 pM, a seven fold reduction of effective dose in combination. PP was equally effective in Dox resistant MCF-7 cells generated over a 21 day time frame. PP at 500nM reduced the IC50 of Dox in Dox resistant ceils from 5.33 pM to 1,35 pM a reduction of effective dose by more than tliree fold. PP rapidly induced mitochondrial reactive oxygen species generation evident from the diffused red fluorescence of Mito SOX dye in PP treated cells compared to control MCF-7 cells which showed little dye expression. PP also reduced mitochondrial respiration evident from the reduction of basal respiration rate from 700 pmol/s/mg to 350 pmol/s/mg of O2 consumption mea.sured in MCF-7 cells by respirometry experiment in OROBOROS. The ATP dependent respiration was reduced from 600 pmol/s/mg to 50 pmol/s/mg of O2 by treatment with PP. The maximal respiration was reduced from 1050 pmol/s/mg to 400 pmol/s/mg of O2. The respiratory reserve capacity of MCF-7 cells was reduced from 300 pmol/s/mg to 75 pmol/s/mg of O2. The significant reduction of mitochondrial respiration shows that cancer cells use mitochondrial respiration for survival and PP can effectively block the same. In the light of the results from this study we propose PP as a novel drug candidate to overcome drug resistance in cancer cells. PP targets metabolic plasticity of cancer cells by targeting mitochondrial respiration which the drug resistant cancer cells rely on predominantly. PP also is potent against Dox resistant MCF-7 cells. PP in combination with 5-FU and Dox is equally efficient in inducing ceil death in MCF-7 cells. PP induces rapid generation mitochondrial ROS causing oxidative damage from which cancer cells seldom recover. Kfniii