Title

Analysis of cytotoxicity of anticancer drugs

Date of Award

2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Keywords

Cytotoxicity, Anticancer drugs, Apoptosis, Cellular respiration, Doxorubicin

Subject Categories

Chemistry | Life Sciences | Pharmacology | Pharmacology, Toxicology and Environmental Health | Physical Sciences and Mathematics

Abstract

Apoptosis, induced in cancer cells by anticancer agents such as doxorubicin, dactinomycin, and the platinum drugs, is characterized by impaired respiration, decreased ATP, and activated caspases. Respiration rates of Jurkat or HL-60 cells treated with drugs are measured using a Pd (II) phosphor to monitor [O 2 ]. Cellular ATP is determined using the luciferin-luciferase bioluminescence system, and intracellular caspase activation measured by allowing caspases to cleave Ac-DEVD-AFC to the fluorescent AFC, which is detected on HPLC. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2 to 4 h of incubation with 20 μM doxorubicin, paralleling the decline in respiration rate. In the presence of dactinomycin, k decreased gradually with time. The decrease was more pronounced at higher concentrations of the drug. Cellular ATP remained constant for 5 hr in untreated cells, but decreased gradually in the presence of 20 μM dactinomycin (to one tenth the value at 5 hr for untreated cells). The drug-induced inhibition of respiration and decrease in ATP level were both blocked by the pancaspase inhibitor zVAD-fmk. Caspase activity was first noted after about 2 h of incubation with doxorubicin or dactinomycin, the production of AFC being linear with time afterwards. Caspase activation by doxorubicin was delayed in HL-60/MX2 cells, reflecting the critical role of topoisomerase-II in doxorubicin cytotoxicity. For both drugs, caspase activity increased rapidly between ∼2 and ∼6 hours, went through a maximum, and decreased after ∼8 hours ("caspase storm"). Cisplatin treatment induced noticeable caspase activity only after ∼14 h of incubation, and the fluorescent intensity of AFC became linear with time at ∼16 h. Exposure of the cells to all of the drugs studied led to impaired cellular respiration and decreased cellular ATP, concomitant with caspase activation. Thus, the mitochondria are rapidly targeted by active caspases. Comparing the ways in which respiration, ATP level, and caspase activity vary with time points up differences between the mechanisms of actions of doxorubicin, dactinomycin, and the platinum drugs. These results should also be useful in choosing the most effective drug for a particular clinical application.

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