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

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

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

Relative change in oxygen consumption as a function of extracellular and intracellular MKT-077 concentration.

<p>A) Percent decrease in oxygen consumption of R3230Ac cells, %Δq, as a function of [MKT-077] in medium. The points are staggered along the abscissa for clarity, and the horizontal bars represent the mean values. The %Δq values were significantly dependent on dose (ANOVA, p = 0.003). The dashed line is a linear regression: %Δq = 6.99C_M0+32.2, r = 0.706, p = 0.0005, n = 20. The solid curve is the predicted relationship calculated from Equation 24 using the mean parameters. *p<0.01 compared to the 2 µg/ml group using one-way ANOVA with Bonferroni's Multiple Comparison Test. B) Percent decrease in oxygen consumption of R3230Ac cells, %Δq, as a function of steady-state MKT-077 uptake. The dashed line is a linear regression: %Δq = 0.664C_C,∞+41.7, r = 0.621, p = 0.004, n = 20. The solid curve is the predicted relationship calculated from Equation 25 using the mean parameters. (▪): 2 µg/ml MKT-077, n = 7; (▴): 4 µg/ml MKT-077, n = 7; (•): 6 µg/ml MKT-077, n = 6.</p

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

Comparison of the fits of all 20 data sets to the three different models of oxygen consumption change.

<p><i>Values are means</i> ± <i>SD. Repeated Measures One-Way ANOVA: Goodness-of-fit values (r² and RMS error) - p<0.0001. Bonferroni's Multiple Comparison Tests:</i></p>a<p> <i>p<0.001 vs. “uptake” model value,</i></p>b<p> <i>p<0.01 vs. “uptake” model value. Paired t-tests:</i></p>c<p> <i>p<0.02 vs. “constant” model value,</i></p>d<p><i>p<0.002 compared to q₁ values determined from linear fit of pre-injection pO₂ data (t≤0) {q₁ = </i>4.37±1.02×10^−<i>6</i><i> [ml O₂/(10⁵ cells min)], mean ± SD, n = 20}.</i></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