Identification of anticancer OATP2B1 substrates by an in vitro triple-fluorescence-based cytotoxicity screen.

Membrane transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The cellular accumulation of many drugs is the result of the net function of efflux and influx transporters. Efflux transporters such as P-glycoprotein/ABCB1 have been shown to confer multidrug resistance in cancer. Although expression of uptake transporters has been confirmed in cancer cells, their role in chemotherapy response has not been systematically investigated. In the present study we have adapted a fluorescence-based cytotoxic assay to characterize the influence of key drug-transporters on the toxicity of approved anticancer drugs. Co-cultures of fluorescently labeled parental and transporter-expressing cells (expressing ABCB1, ABCG2 or OATP2B1) were screened against 101 FDA-approved anticancer drugs, using a novel, automated, triple fluorescence-based cytotoxicity assay. By measuring the survival of parental and transporter-expressing cells in co-cultures, we identify those FDA-approved anticancer drugs, whose toxicity is influenced by ABCB1, ABCG2 or OATP2B1. In addition to confirming known substrates of ABCB1 and ABCG2, the fluorescence-based cytotoxicity assays identified anticancer agents whose toxicity was increased in OATP2B1 expressing cells. Interaction of these compounds with OATP2B1 was verified in dedicated transport assays using cell-impermeant fluorescent substrates. Understanding drug-transporter interactions is needed to increase the efficacy of chemotherapeutic agents. Our results highlight the potential of the fluorescence-based HT screening system for identifying transporter substrates, opening the way for the design of therapeutic approaches based on the inhibition or even the exploitation of transporters in cancer cells.

Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

Structure-Activity Relationships of 8-Hydroxyquinoline-Derived Mannich Bases with Tertiary Amines Targeting Multidrug-Resistant Cancer.

A recently proposed strategy to overcome multidrug resistance (MDR) in cancer is to target the collateral sensitivity of otherwise resistant cells. We designed a library of 120 compounds to explore the chemical space around previously identified 8-hydroxyquinoline-derived Mannich bases with robust MDR-selective toxicity. We included compounds to study the effect of halogen and alkoxymethyl substitutions in R5 in combination with different Mannich bases in R7, a shift of the Mannich base from R7 to R5, as well as the introduction of an aromatic moiety. Cytotoxicity tests performed on a panel of parental and MDR cells highlight a strong influence of experimentally determined pKa values of the donor atom moieties, indicating that protonation and metal chelation are important factors modulating the MDR-selective anticancer activity of the studied compounds. Our results identify structural requirements increasing MDR-selective anticancer activity, providing guidelines for the development of more effective anticancer chelators targeting MDR cancer.