Abstract
The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin.
Highlights
Dactinomycin is an important anticancer chromopeptide.[1]The drug is known to intercalate between DNA base pairs762 MOLECULAR PHARMACEUTICS VOL. 3, NO. 6, 762-772 and inhibit transcription.[2]
This toxic effect leads to cell death, primarily by apoptosis
Apoptosis is initiated by activating cellular targets, which permeabilize the outer mitochondrial membrane, causing release of soluble proteins from the mitochondrial intermembrane space
Summary
Dactinomycin is an important anticancer chromopeptide.[1]The drug is known to intercalate between DNA base pairs762 MOLECULAR PHARMACEUTICS VOL. 3, NO. 6, 762-772 and inhibit transcription.[2]. Dactinomycin is an important anticancer chromopeptide.[1]. The drug is known to intercalate between DNA base pairs. 6, 762-772 and inhibit transcription.[2] This toxic effect leads to cell death, primarily by apoptosis. The drug is commonly used in vitro to execute apoptosis. Apoptosis is initiated by activating cellular targets (e.g., the pro-apoptotic Bcl-2 family member Bid), which permeabilize the outer mitochondrial membrane, causing release of soluble proteins (e.g., cytochrome c and SMAC/Diablo) from the mitochondrial intermembrane space. In the presence of ATP (or dATP), released cytochrome c binds to Apaf-1
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