Tuesday, February 23, 2010

Detection of Drug-Induced Lysosomal Cytoxicity

The identification of cytotoxic effects is a critical element in the pre-clinical small molecule drug discovery process. In-vitro cell-based assays are typically used early in the process following screening which requires the ability to automate workflows to handle the number of compounds tested. Cytotoxic effects of drug molecules are often first observed as perturbations of normal cellular organelle functionality. For example, Lysosome perturbation as a result of ion trapping of amine containing compounds has been demonstrated to cause the formation of autophagosomes and autophagic cytopathology. Cationic amphiphilic compounds (i.e. small molecule drug compounds) can be absorbed by cells by simple diffusion and accumulate inside the acidic cellular organelles, a process referred to as lysosomotropism. While many drugs require the presence of a cationic moiety for bioactivity, their accumulation into subcellular organelles can lead to many undesirable effects [1].

Image of lysosomal staining by Lyso-ID® Red dye
Figure 1. Image of lysosomal staining by Lyso-ID® Red dye. Increase in lysosome volume and number resulting from 200 µM verapamil.
Many drugs cause an accumulation of phospholipids and lysosomes in the cytoplasm. For example amioderone induces an abnormal accumulation of phospholipids that appear as vacuoles with multilamellar inclusions often referred to as autophagosomes [2]. Other organic amines cause vacuolar-ATPase driven ion trapping, which has been associated with vacuolar and autophagic cytopathology.
Here we describe the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the Lyso-ID® Red Detection kit, part of the CELLestial® Live Cell Analysis platform from Enzo Life Sciences.
Lyso-ID® Red dye is a fluorescent reagent that accumulates in lysosomes. An increase in signal is indicative of an increase in the number or size of cellular lysosomes and lysosome-derived vacuoles. In addition to the lysosomal specific dye, the assay also uses Hoechst 33342 nuclear stain. A decrease of 30% or greater of the Hoechst signal is indicative of generalized cytotoxicity.

H-mesothelioma (H-Meso) cells were seeded at 20,000 cells per well and allowed to attach overnight. The following morning the cells were treated with increasing doses of chloroquine, verapamil or thiostrepton. After an 18-hour exposure to the drugs the cells were washed and Lyso-ID® Red and Hoechst 33342 dyes were added using the EL406 Washer Dispenser. After 30 minute incubation, excess dye was removed by washing 3 times with wash solution, followed by a final addition of 80 µL of wash solution. The fluorescence was then determined using a Synergy Mx reader. Lyso-ID® Red dye (red fluorescence) was measured using an excitation of 540 nm and an emission of 680 nm, while Hoechst dye (blue fluorescence) was determined with an excitation of 340 nm and an emission of 480 (Figure 2).


Schematic of the Automated Lyso-ID® Red Process Carried out by the EL406 Washer Dispenser and Synergy™ Mx Reader.
Figure 2. Schematic of the Automated Lyso-ID® Red Process Carried out by the EL406 Washer Dispenser and Synergy™ Mx Reader.
Initial experiments focused on the response of human mesothelioma cells to agents known to cause drug-induced phospholipidosis. When H-meso cells were treated with various concentrations of verapamil a dose dependent increase in Lyso-ID® Red signal is observed (Figure 3). The presence of 100 µM verapamil resulted in a 2-fold increase in fluorescence. This indicates that this drug causes an increase in the number of cellular lysosomes and/or an increase in lysosomal volume in H-Meso cells. Staining with Hoechst 33342, a monitor for cytotoxic cell loss, did not change over the tested concentrations.

Figure 3. Dose Dependent Drug Induced Increase in Lysosomal Content as Measured by Increase in Lyso-ID® Red Fluorescence

Figure 3. Dose Dependent Drug Induced Increase in Lysosomal Content as Measured by Increase in Lyso-ID® Red Fluorescence
Thiostrepton is an antibiotic related to Siomycin A, first described by Donivick et. al. in 1955 [3]. This thiazole compound, while typically used in topical antibiotic mixtures due to its poor water solubility, has been found to target breast cancer cells through inhibition of forkhead box M1 expression [4]. Because tumor cells are selectively targeted as compared to normal cells and the chemical structure of thiostrepton contains several amino groups we sought to examine whether or not this compound is differentially partitioned into lysosomes depending on cellular transformation.

Response change in Lyso-ID® Red dye Fluorescence caused by thiostrepton and verapamil in two mesothelial cell lines

Figure 4. Response change in Lyso-ID® Red dye Fluorescence caused by thiostrepton and verapamil in two mesothelial cell lines
The effect of thiostrepton on lysosomal content was tested with a H-meso malignant mesothelioma cell line, as well as control LP9 mesothelial cells. The verapamil dose response curves were run in parallel as a positive control. As seen in figure 4, verapamil elicited an approximately 2-fold increase in Lyso-ID® staining in both cell lines at higher drug concentrations with no increase in Hoechst signal. This indicates that these cell lines are capable of responding to agents known to cause phospholipidosis, resulting in increased lysosome content. When the effect of thiostrepton was examined, only the non-transformed cell line LP9 was found to respond with increased lysosome content as a result of exposure to the compound. Lyso-ID® Red dye fluorescence increased almost 3-fold with the addition of 5 µM thiostrepton in LP9 cells, while the H-Meso cells displayed no increase as a result to exposure to thiostrepton (Figure 4).

The Lyso-ID® Red kit in conjunction with the EL406 Combination Washer Dispenser provides an optimized workflow to investigate and screen compounds for phospholipidosis. The Lyso-ID® Red fluorescent assay kits provide a much easier way to screen for this potential problem than the gold standard method of electron microscopy. The ELx406 Washer Dispenser provides the liquid handling capabilities to automate many of the tedious fluid handling steps required of this assay. The EL406 instrument has the ability to quickly and reliably add and remove fluid from 96 or 384-well microplates containing live cells, replacing numerous manual pipetting tasks. In addition the device can dispense accurate amounts of Lyso-ID® Red reagent as needed.


References
1.Ikeda , K., M. Hirayama, Y. Hirota, E. Asa, J. Seki and Y. Tanaka (2008) Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes, BBRC, 377:268-274.
2.Lewis,J.H., F. Mullick, , K.G. Ishak, R.C. Ranard, B. Ragsdale, R.M. Perse, E.J. Rusnock, A. Wolke, S.B. Benjamin, L.B. Seeff, H.J. Zimmerman. (1990) Histopatholic Analysis of Suspected Amiodarone hepatoxicity. (Hum. Path. 21:59-67.
3.Donovick R, Pagano JF, Stout HA, Weinstein MJ (1955). Thiostrepton, a New Antibiotic I. In. vitro Studies Antibiotics Annual 3: 554–9.
4.Kwok, J. M-M., S.S. Myatt, C.M. Marson, R.C. Coombies, D. Constantinidou, and E. W-F. Lam (2008) Thiostrepton Selectively Targets Breast Cancer Cells through Inhibition of Forkhead box M1 Expression. Mol. Cancer Ther. 7(7):2022-2032


By, BioTek Instruments

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