Single Cell Analysis of Doxorubicin into Cancer Cell : Distribution , Kinetics , and Quantification
Date Issued
2009
Date
2009
Author(s)
Liu, Chun
Abstract
Doxorubicin is a widely used anti-cancer drug that kills cancer cells by binding to DNA and topoisomerase to induce programmed cell death. Scientists usually evaluate the effectiveness of drugs to cells through the IC50 test which is denoted by the half-maxima inhibitory concentration. For example, in this thesis we used the alamarBlue dye to test our system. In healthy cells alamarBlue can be metabolized to its reduced form to emit fluorescence. However, in cells where cell growth is inhibited, an oxidized environment is maintained in which alamarBlue will remain non-fluorescent. Therefore when the fluorescence intensity of drug-treated cells is only half of the untreated cells, the concentration of the drug is the IC50. Although we can find out the effective concentration of drugs to the cells through the IC50 test, it does not tell us the details of drugs such as the distribution of drugs in the cell, the number of drug molecules in the cell, the different responses between cells and et cetera. Therefore, to study these problems we used confocal laser scanning microscope by monitoring the fluorescence of Dox. The advantage of confocal laser scanning microscopes lies in their optical sectioning effect, which can exclude out-of-focus light and provides better image quality than conventional fluorescent microscopes. On the other hand, we can do single cell observation and compare the difference of rate and quantity of drug uptake between cells that cannot be obtained from the population experiment. t first we expected that the number of Dox molecules in the cell should increase with time until it reaches saturation. However, the results didn’t meet the expectation . In our experiment, we observed that in some cells the amount of Dox being absorbed into the cells rises at first and then gradually slows down as it reaches saturation. However, in some cases the amount of Dox continues to rise shortly after saturation while in others the amount of Dox never reached saturation but instead continues to increase. rom this we observed the difference in the quantity and behavior of drug uptake between cells. Most of the concentration of Dox in the nuclei is higher than the concentration of Dox in the cell medium by about 100-fold; but some can even reach to 1000-fold or higher . We also used Dox itself as a nuclear marker to observe whether cells undergo nuclear condensation or DNA fragmentation. The advantage of using Dox itself as a nuclear marker is that we don’t have to add another nuclear marker which may cause cytotoxicity. After comparing cells subjected to the 4uM and 1uM Dox treatment we found that cells under the 4uM Dox treatment have higher death rates and higher Dox concentration in the nuclei than the cells under the 1 uM treatment. Therefore, from this we know that the higher the number of Dox molecules there are in the cells, the higher their death rates are and vice versa
Subjects
single cell
doxorubicin
distribution
quantitation
SDGs
Type
thesis
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