Human Tumor Cell Proliferation Evaluated Using Manganese-Enhanced MRI

Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn(2+) [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro.Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl(2) for one hour and then thoroughly washed. MEMRI R(1) values (longitudinal relaxation rates), which have a positive linear relationship with Mn(2+) concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn(2+)-induced increases in R(1) compared to cells not exposed to Mn(2+). C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R(1) values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R(1) for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet.These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI

Uptake Rate of Cationic Mitochondrial Inhibitor MKT-077 Determines Cellular Oxygen Consumption Change in Carcinoma Cells

<div><h3>Objective</h3><p>Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake.</p> <h3>Methods</h3><p>R3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells.</p> <h3>Results</h3><p>Cells took up MKT-077 with a time constant of ∼1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of ∼30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45–73% (p = 0.003).</p> <h3>Conclusions</h3><p>MKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity <em>in vivo</em>.</p> </div

Manganeseenhanced MRI of human choroidal melanoma xenografts. Invest Ophthalmol Vis Sci

PURPOSE. To test the hypothesis that the structure and function of an experimental human choroidal melanoma xenograft and neighboring non-tumor-bearing retina can be simultaneously assessed by using manganese-enhanced MRI (MEMRI). METHODS. Spheroids grown from the human choroidal melanoma cell line C918 were implanted in the superior suprachoroidal space of 11 WAG/Nij-rnu nude rats. Two weeks later, MRI data were collected 4 hours after intraperitoneal injection of saline or MnCl 2 , an MRI contrast agent that can act as a biomarker of cellular demand for ions, such as calcium. The following parameters were measured: (1) tumor signal intensity, (2) inner and outer retinal signal intensity in non-tumorbearing inferior retina, and (3) whole and inner retinal thickness of inferior retina. Separate MEMRI experiments were performed on spheroids in vitro after MnCl 2 exposure and washing. RESULTS. In vitro, spheroids exposed to MnCl 2 retained sufficient Mn 2ϩ to demonstrate contrast enhancement during MEMRI. In vivo, injection of MnCl 2 resulted in a 30% increase in tumor signal intensity compared with tumors in rats injected with saline (P Ͻ 0.05). In inferior retina of tumor-bearing eyes, outer retinal signal intensity increased by 17% relative to a similar region in control eyes (P Ͻ 0.05), but there was no change in the inferior inner retinal intensity. Total retinal thickness of the inferior retina in the tumor-bearing eyes increased by 8%, compared with that in the non-tumor-bearing eyes (P Ͻ 0.05). CONCLUSIONS. The present identification of regions of enhanced Mn 2ϩ uptake in choroidal melanoma and a somewhat unexpected edema and increased outer retinal ion demand in neighboring non-tumor-bearing retina highlights MEMRI as a potentially powerful method for noninvasively monitoring tumor progression and treatment response and efficacy. (Invest Ophthalmol Vis Sci. 2007;48:963-967

HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in <i>Pseudomonas aeruginosa</i> Keratitis

<div><p>Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with <i>Pseudomonas (P.) aeruginosa</i>. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in <i>P. aeruginosa</i>-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.</p></div

Theoretical relative decrease in oxygen consumption of R3230Ac cells as a function of MKT-077 uptake.

<p>R3230Ac cells were treated with 2, 4, or 6 µg/ml MKT-077 at t = 0 minutes. Percent decrease in consumption, %Δq, was calculated from Equation 26 using the mean fitted parameters (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037471#pone-0037471-t003" target="_blank">Table 3</a>). Points indicate values after 30 minutes (•) or 2 hours (▴) of MKT-077 exposure.</p

Effect of extracellular MKT-077 concentration on cellular uptake.

<p>A) Data from a typical uptake experiment. R3230Ac cells were treated with 4 µg/ml MKT-077 at time 0. Solid line represents total cellular MKT-077 uptake, dashed line represents intracellularly bound or organelle-sequestered drug, and dotted line indicates free drug in the cytoplasm as determined by the model. B) MKT-077 uptake by R3230Ac cells as a function of drug concentration and time. Values are the mean ± SEM. p<0.0001 using a two-way ANOVA. Curves are fits of the mean values to Equation 1. 2 µg/ml: β = 11.1, k = 0.458 min⁻¹ (n = 11); 4 µg/ml: β = 18.0, k = 0.502 min⁻¹ (n = 10); 6 µg/ml: β = 18.0, k = 0.632 min⁻¹ (n = 7). C) Initial drug uptake rate as a function of treatment concentration. Line is calculated from Equation 5 with a slope of 1.44×10⁻⁴ ml/(10⁵ cells min). D) Steady-state MKT-077 uptake as a function of treatment concentration. Line is calculated from Equation 2 with a slope of 0.00845 ml/10⁵ cells. In panels C and D, the points are staggered along the abscissa for clarity, and the horizontal bars represent the mean values. One-way ANOVA with Bonferroni's Multiple Comparison Tests: * p<0.001 vs. 2 µg/ml value, ^# p<0.001 vs. 4 µg/ml value, ^̂ p<0.05 vs. 2 µg/ml value.</p

Fitted and calculated parameters and goodness-of-fit values for the model fits of MKT-077 uptake.

<p><i>Values are means</i> ± <i>SD. Values in italics are the means of the 95% confidence intervals. One-way ANOVA with Bonferroni's Multiple Comparison Tests:</i></p>a<p> <i>p<0.001 vs. 2 µg/ml value,</i></p>b<p> <i>p<0.001 vs. 4 µg/ml value,</i></p>c<p> <i>p<0.05 vs. 2 µg/ml value.</i></p

Effect of MKT-077 on pO₂ and oxygen consumption in MDA-MB-231 cells.

<p>A) Change in pO₂ measured in MDA-MB231 cell suspension before and after addition of different concentrations of MKT-077 (0, 2, or 4 µg/ml) to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the “relative rate” model (Equations 14 and 20). B) Percent decrease in oxygen consumption of MDA-MB231 cells, %Δq, as a function of steady-state MKT-077 uptake. The solid curve is the predicted relationship calculated from Equation 25 using the medians of the fitted parameters: q₁ = 2.91×10⁻⁶ ml O₂/(10⁵ cells min), α_r = 6.04×10⁻⁴ ml O₂ (10⁵ cells)^0.5]/(ng MKT-077)^0.5, and κ = −0.101 min⁻¹. (▪): 2 µg/ml MKT-077; (▴): 4 µg/ml MKT-077; (•): 6 µg/ml MKT-077.</p

Fits of a typical pO₂ data set to the three oxygen consumption models.

<p>A) Change in pO₂ measured in R3230Ac cell suspension before and after addition of 6 µg/ml MKT-077 to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the following models: dq/dC_C = −α₀ (“constant”, dotted line); dq/dC_C = −α₁C_C (“uptake”, dashed line); dq/dC_b = −α₂ (dC_C/dt) (“rate”, solid line) [see Equations 14–17]. The inset shows the same data from −20 to 15 minutes. B) Corresponding changes in oxygen consumption, q, predicted by the three models (Equations 10–13).</p

Examples of the inhibition of oxygen consumption by MKT-077 and resultant model fits.

<p>A) Change in pO₂ measured in R3230Ac cell suspension before and after addition of different concentrations of MKT-077 (0, 2, or 6 µg/ml) to air-saturated medium at t = 0 minutes. Points are measured pO₂ data. Curves are fits of data to the “rate” model (Equations 14 and 17). B) Changes in oxygen consumption, q-q₁, predicted by the “rate” model (Equations 10 and 13).</p