Selective Targeting of Tumorigenic Cancer Cell Lines by Microtubule Inhibitors

For anticancer drug therapy, it is critical to kill those cells with highest tumorigenic potential, even when they comprise a relatively small fraction of the overall tumor cell population. We have used the established NCI/DTP 60 cell line growth inhibition assay as a platform for exploring the relationship between chemical structure and growth inhibition in both tumorigenic and non-tumorigenic cancer cell lines. Using experimental measurements of “take rate” in ectopic implants as a proxy for tumorigenic potential, we identified eight chemical agents that appear to strongly and selectively inhibit the growth of the most tumorigenic cell lines. Biochemical assay data and structure-activity relationships indicate that these compounds act by inhibiting tubulin polymerization. Yet, their activity against tumorigenic cell lines is more selective than that of the other microtubule inhibitors in clinical use. Biochemical differences in the tubulin subunits that make up microtubules, or differences in the function of microtubules in mitotic spindle assembly or cell division may be associated with the selectivity of these compounds

Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines

Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions

A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration

The physiological determinants of drug-induced lysosomal stress resistance

<div><p>Many weakly basic, lipophilic drugs accumulate in lysosomes and exert complex, pleiotropic effects on organelle structure and function. Thus, modeling how perturbations of lysosomal physiology affect the maintenance of lysosomal ion homeostasis is necessary to elucidate the key factors which determine the toxicological effects of lysosomotropic agents, in a cell-type dependent manner. Accordingly, a physiologically-based mathematical modeling and simulation approach was used to explore the dynamic, multi-parameter phenomenon of lysosomal stress. With this approach, parameters that are either directly involved in lysosomal ion transportation or lysosomal morphology were transiently altered to investigate their downstream effects on lysosomal physiology reflected by the changes they induce in lysosomal pH, chloride, and membrane potential. In addition, combinations of parameters were simultaneously altered to assess which parameter was most critical for recovery of normal lysosomal physiology. Lastly, to explore the relationship between organelle morphology and induced stress, we investigated the effects of parameters controlling organelle geometry on the restoration of normal lysosomal physiology following a transient perturbation. Collectively, our results indicate a key, interdependent role of V-ATPase number and membrane proton permeability in lysosomal stress tolerance. This suggests that the cell-type dependent regulation of V-ATPase subunit expression and turnover, together with the proton permeability properties of the lysosomal membrane, is critical to understand the differential sensitivity or resistance of different cell types to the toxic effects of lysosomotropic drugs.</p></div

Clofazimine inclusions formed in macrophage-like cells <i>in vivo</i>.

<p>(<b>A</b>) Mice fed with clofazimine (above) showed reddish pigmentation visible in the ear, tail, and skin when compared to mice treated with vehicle only (below). (<b>B</b>) Weight gains in mice fed with and without clofazimine were comparable (N = 40, ▪, vehicle; ○, treated; *, P<0.01, end-point T-test). (<b>C</b>) Biochemical analysis of various organs revealed differences in the accumulation and retention of clofazimine after wash out (*, P<0.01, N = 5 per group, ANOVA). (<b>D</b>) Ruby red inclusions appeared in frozen sections of spleen, lung and liver, but not in kidneys of 8 wk supplemented diet. H, hepatocyte; V, blood vessel; M, microgranulomas. (<b>E</b>) Intracellular inclusions were extracted in perfusion-fixed liver upon ethanol-dehydration and staining with toluidine blue. Arrows indicate needle-like cavities remaining after extraction. (<b>F</b>) Histological sections revealed cellular changes in liver of mice fed with clofazimine. H&E staining, F4/80 macrophage specific marker, Masson's trichrome staining (MTS, collagen fibers), von Willebrand factor (vWF, endothelium) and alpha smooth muscle actin (αSMA). K, Kupffer cells. Scale bar  = 10 µm unless otherwise indicated.</p

The effect of a simultaneous inhibition of the transport of chloride and proton ions in spherical versus tubular lysosomes.

<p>(A) Dimensions of tubular and spherical lysosomes. (B) The simultaneous inhibitions of the cytoplasmic chloride and V-ATPase number per lysosome induced significant changes in lysosomal pH, Cl^-, and membrane potential. The effect was magnified in the tubular lysosome where the > 4 pH unit increment in lysosomal pH, > 150 mM reduction in lysosomal Cl^- accumulation, and > 250 mV increment in membrane potential were observed (as indicated by the black arrow signs).</p

The effect of a combination of lysosomal ion stressors in spherical versus tubular lysosomes.

<p>(A) Dimensions of tubular and spherical lysosomes. (B) The simultaneous inhibitions of V-ATPase and CLC7 numbers induced changes in both spherical and tubular lysosomes with minimal difference between the two lysosomal morphologies. The increment in V-ATPase inhibition induced the most significant change while only the complete depletion of CLC7 induced physiological perturbation which mainly arose from the significant change in membrane potential (> 250 mV, as indicated by the black arrow sign). (C) The simultaneous inhibition of V-ATPase and membrane proton permeabilization induced very similar and significant changes in the overall physiology of both spherical and tubular lysosomes.</p

Macrophages containing intracellular CLDIs were collected, plated and studied <i>in vitro</i>.

<p>(<b>A</b>) Bone marrow macrophage (BMM) and thioglycollate elicited peritoneal macrophages (PM) were obtained from mice fed with clofazimine, attached and spread on tissue culture plastic, and were stained with Hoechst 33342 to show nuclei. (<b>B</b>) Peritoneal macrophages with CLDIs migrated away from large clusters when plated on tissue culture dishes. (<b>C</b>) Illuminating peritoneal macrophages with blue (490 nm) light triggers clofazimine release (observed in TRITC channel) from CLDIs. (<b>D</b>) Once removed from cells, extracellular CLDIs grew in size and became irregular in morphology, unlike intracellular CLDIs. Red blood cells (d = 8 µm) in the background serve as size markers, for reference. (<b>E</b>) CLDIs inside bone marrow-derived cells in suspension, stained with Trypan Blue. Scale bars  = 10 µm unless otherwise indicated.</p

Deep-etch freeze-fracture electron microscopy of isolated CLDIs.

<p>(<b>A</b>) Pure isolated CLDIs stood out from surrounding ice and cytosolic debris based on their elongated polyhedral shape and internal layered structure. (<b>B</b>) Isolated CLDIs clearly lacked the outer double membrane covering. (<b>C</b>) Biochemically-isolated CLDI often showed outer layers of material that appeared to be peeling off from the structure.</p

CLDIs exhibited different chemical and physical properties from pure clofazimine crystals.

<p>Polarized light and epifluorescence microscopy (using the eGFP or Cy3 fluorescence channels) showed that pure clofazimine crystals (control, <b>A</b>) were unchanged by different treatments. These crystals appeared birefringent and fluoresced in the standard eGFP and Cy3 channels of the epifluorescence microscope. (<b>B</b>) Isolated CLDIs remained intact in isotonic solution of 10% sucrose in water, did not fluoresce in the eGFP channel but fluoresced in the Cy3 channel. (<b>C</b>) Isolated CLDIs burst and aggregated in distilled water, and became fluorescent in the eGFP channel. (<b>D</b>) After exposure to 1N NaOH, isolated CLDIs partially disintegrated in different parts. Arrows point to the tips of a CLDI that were fluorescent in the eGFP channel. (<b>E</b>) After 15 min at 100°C, CLDIs fragmented and changed to a pale orange color. (<b>F</b>) CLDIs appeared to remain partly intact when viewed after 30 min sonication and 1 hour trypsin treatment. Scale bars  = 10 µm. (<b>G</b>) Powder X-ray diffractogram for isolated CLDI and 8 wk treated mouse spleen homogenate showed a single peak at 2-theta  = 7.2°Control spleen homogenate from vehicle-only treated mouse did not show this peak. As a reference, pure, solid clofazimine crystals (monoclinic and triclinic) showed many peaks at higher angle indicative of a three dimensional, molecular lattice organization.</p