Dual Targeting of Hypoxic and Acidic Tumor Environments with a Cobalt(III) Chaperone Complex

The rational design of prodrugs for selective accumulation and activation in tumor microenvironments is one of the most promising strategies for minimizing the toxicity of anticancer drugs. Manipulation of the charge of the prodrug represents a potential mechanism to selectively deliver the prodrug to the acidic tumor microenvironment. Here we present delivery of a fluorescent coumarin using a cobalt­(III) chaperone to target hypoxic regions, and charged ligands for pH selectivity. Protonation or deprotonation of the complexes over a physiologically relevant pH range resulted in pH dependent accumulation of the fluorophore in colon cancer cells. Furthermore, in a spheroid solid tumor model, the anionic complexes exhibited preferential release of the fluorophore in the acidic/hypoxic region. By fine-tuning the physicochemical properties of the cobalt–chaperone moiety, we have demonstrated selective drug release in the acidic and hypoxic tumor microenvironment

Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics

<div><p>The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and <i>in vivo</i> in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is <i>in vivo</i> or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.</p></div

N- and C-terminal tagged Tpm3.1 constructs have similar mobile fractions but dissimilar recovery rates.

<p>(A,B) Representative images of FRAP assay in MEFs transfected with either N- or C-Tpm3.1. FRAP zones (white arrows) were bleached and cells imaged at 1 fps for 2 min. (inset A,B). Enlarged images of FRAP zones over time (s). (C,D) FRAP curves of N- or C-Tpm3.1 transfected MEFs. (E) Half-times of N- and C-Tpm3.1 recovery (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s003" target="_blank">S1 Table</a>). Data obtained from 6 experiments, 3–15 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

Tpm3.1 maintains constant and rapid cycling on stress fibers in the presence of jasplakinolide.

<p>(A) Representative image and FRAP sequence of MEFs transfected with C-Tpm3.1. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves of C-Tpm3.1 in control and drug-treated conditions. (C) Mobile fraction of control and drug-treated condition (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s007" target="_blank">S5 Table</a>). (D,E) Curve fits for C-Tpm3.1 in control (D) and drug-treated condition (E). Data obtained from 3 separate experiments, 3–8 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

The majority of actin in stress fibers is stable.

<p>(A) Representative image and FRAP sequence of MEFs transfected with GFP-beta-actin. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves for GFP-actin in control and jasplakinolide treated condition. (C) FRAP curves for Lifeact-RFP in control and jasplakinolide treated condition. (D) Mobile fractions of control and drug treated GFP-actin and Lifeact-RFP (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s005" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s006" target="_blank">S4</a> Tables). (E) Curve fits for GFP-Actin control. (F) Curve fit for GFP-Actin treated with jasplakinolide. Data obtained from 3 separate experiments, 2–8 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 5 μm.</p

Intracellular intravital imaging of the kinetics of N- and C-terminal tagged Tpm3.1 constructs transfected into rat salivary gland acinar cells.

<p>(A) Confocal image of an acinus from rat submandibular salivary gland section stained with anti-Tpm3.1 (2G10) antibody. Tpm3.1 is enriched on the apical plasma membranes that form the canaliculi of acinar cells (white arrow). (B) Confocal image of a C-Tpm3.1 transfected cell (arrow) in a single acinus of a rat salivary gland <i>in situ</i>. Extracellular space outside the acinus was stained with 10kDa dextran Alexa 647 conjugate. (C) Illustration of the transfected acinar cell in (B), arrow shows apical membrane/canaliculi, arrowhead shows basolateral membrane. (D,E) Intravital microscopy and FRAP analysis of N- and C-Tpm3.1 constructs in live transfected rats. Numbers indicate time in sec. White arrows indicate FRAP zones on the canaliculi of rat acinar cells. (F) FRAP curves for N- and C-Tpm3.1. (G) Mobile fraction of N- and C-Tpm3.1. (H) Half-times for N- and C-Tpm3.1. (I, J) Curve fits for N- and C-Tpm3.1. 11–16 cells assayed from at least 3 animals per construct (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s004" target="_blank">S2 Table</a>). Error bars are +/- <i>SEM</i>. Scale bars = 5 μm.</p

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

<div><p>The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.</p></div

Nanomechanical properties of mouse adipose tissue from Tpm3.1 transgenic mice.

<p>Elastic modulus values of epididymal fat isolated from the Tpm3.1-overexpressing TG and control (WT) mice. A total of 6 mice per group were tested, 10–50 indentation points per area (area was 30 μm²) was conducted and 5–10 areas per epididymal fat pad (both left and right) were employed. Data is visualised as a box plot showing the median with the interquartile range and all the data points. Data points represent the mean of all indentations conducted per animal. A nonparametric, Mann-Whitney test shows no statistical differences between WT and TG samples.</p

Tpm3.1 knockdown leads to a decrease in the cell’s elastic modulus.

<p>(A) Representative westerns of 10 μg of total cellular protein isolated from the Tpm3.1- overexpressing cells untreated (untreat) or exposed to lipofectamine (Lipofec), scramble siRNA (scRNA), Tpm2.1 siRNA or human Tpm3.1 siRNA probed with the γ/9d (mouse and human Tpm3.1) and GAPDH as loading control. (B) Quantification of the total levels of Tpm3.1 levels, <i>n</i> = 3. (C) The elastic modulus for each of the treated Tpm3.1-overexpressing cells was determined. All the data points are presented as box and whisker plots/scatter dots with horizontal line (inside box) indicating median and outliers. ≥ 25 cells for each treatment from <i>n</i> = 3 independent experiments. **<i>P</i><0.01, compared to control.</p

Model proposing the potential impact of distinct actin filament populations on cell stiffness.

<p>We propose that Tpm can define different actin filament populations by dictating the recruitment of different actin binding proteins. Shown are crosslinked filaments associating with αactinin, tension bearing filaments with myosinII, bundles with fascin and branched actin filaments with Arp2/3 and Capping protein. The distinct organisation and functional properties of these Tpm-containing cortical actin filaments impact on cell stiffness.</p