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

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

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

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

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

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