Korean J Physiol Pharmacol.  2016 Jul;20(4):367-378. 10.4196/kjpp.2016.20.4.367.

Minimal systems analysis of mitochondria-dependent apoptosis induced by cisplatin

• Hong JY
• Hara K
• Kim JW
• Sato EF
• Shim EB
• Cho KH

• KMID: 2371054
• DOI: http://doi.org/10.4196/kjpp.2016.20.4.367

Abstract

Recently, it was reported that the role of mitochondria-reactive oxygen species (ROS) generating pathway in cisplatin-induced apoptosis is remarkable. Since a variety of molecules are involved in the pathway, a comprehensive approach to delineate the biological interactions of the molecules is required. However, quantitative modeling of the mitochondria-ROS generating pathway based on experiment and systemic analysis using the model have not been attempted so far. Thus, we conducted experiments to measure the concentration changes of critical molecules associated with mitochondrial apoptosis in both human mesothelioma H2052 and their ρ0 cells lacking mitochondrial DNA (mtDNA). Based on the experiments, a novel mathematical model that can represent the essential dynamics of the mitochondrial apoptotic pathway induced by cisplatin was developed. The kinetic parameter values of the mathematical model were estimated from the experimental data. Then, we have investigated the dynamical properties of this model and predicted the apoptosis levels for various concentrations of cisplatin beyond the range of experiments. From parametric perturbation analysis, we further found that apoptosis will reach its saturation level beyond a certain critical cisplatin concentration.

Keyword

Apoptosis; Cisplatin; Mitochondria-dependent pathway; Simulation; Systems approach

MeSH Terms

Apoptosis*
Cisplatin*
DNA, Mitochondrial
Humans
Mesothelioma
Models, Theoretical
Oxygen
Systems Analysis*
Cisplatin
DNA, Mitochondrial
Oxygen

Figure

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  Fig. 1 Cellular responses to cisplatin-induced DNA damage.MtDNA damage increases ROS generation, decreases mitochondrial membrane potential, and releases signals for cell death, including cytochrome c (Cyt c), second mitochondria activator of caspase (Smac) and apoptosis-inducing factor (AIF). Once released into the cytosol, cytochrome c triggers procaspase-9 activation and induces cell death. Solid arrows denote chemical reactions or upregulation; those terminated by a bar denote inhibition or down-regulation.

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  Fig. 2 Sensitivity analysis of rate constants for the model.(A) Sensitivity of each rate constant for apoptosis. (B) Sensitivity of each rate constant for each initial condition.

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  Fig. 3 Effect of cisplatin on cytochrome c localization and caspase activation.Cytosolic cytochrome c and activated caspases were analyzed by SDS-PAGE and Western blotting after incubation with (A) 50 µM and (B) 12.5 µM cisplatin. The levels of cytosolic cytochrome c, activated caspase-9, -3, and β-actin were quantified by densitometric analysis.

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  Fig. 4 Model predictions and experimental validation.(A) Apoptosis levels with increasing concentrations of cisplatin for 24 hr. (B) Cellular generation of ROS for 24 hr in the presence of 50 µM cisplatin.

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  Fig. 5 Comparisons between simulated and experimental obser vations in 50 µM cisplatin.H2052 cells were cultured for 24 hr in the presence of 50 µM cisplatin, and the mitochondria-dependent apoptotic mechanisms were explored. (A) Mitochondrial generation of ROS was analyzed using MitoSOX and flow cytometry. (B) Mitochondrial membrane potential (Δψm) was assessed using JC-1 and flow cytometry. Cytosolic cytochrome c (C), and activated caspase-9 (D) and -3 (E) were quantified by densitometric analysis. (F) Apoptosis was analyzed using the trypan blue exclusion test.

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  Fig. 6 Comparisons between simulated and experimental ob servations at 12.5 µM cisplatin con centration.

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  Fig. 7 Model predictions and experimental validation.(A) Apoptosis levels with increasing concentrations of cisplatin for 24 hr. (B) Interaction between apoptosis and mitochondrial ROS. mitROS, ROS at baseline value; mitROS × 3, ROS increased by three fold; mitROS × 0.5, ROS decreased by two fold; mitROS × 0.1, ROS decreased by ten fold; mitROS=0. (C) Interaction between apoptosis and the amount of cytochrome c in the mitochondria. CytCmit × 2, amount of cytochrome c in the mitochondria increased by two fold; CytCmit=0. (D) Interaction between apoptosis and the amount of cytochrome c in the mitochondria or cytosol.

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