No abstract

Artesunate is a semisynthetic derivative from artemisinin, a natural product from the Chinese herb Artemisia annua L. It exerts antimalarial activity, and, additionally, artemisinin and its derivatives are active against cancer cells. The active moiety is an endoperoxide bridge. Its cleavage leads to the formation of reactive oxygen species and carbon-centered radicals. These highly reactive molecules target several proteins in Plasmodia, which is thought to result in killing of the microorganism. DNA damage induced by artemisinins has not yet been described. Here, we show that artesunate induces apoptosis and necrosis. It also induces DNA breakage in a dose-dependent manner as shown by single-cell gel electrophoresis. This genotoxic effect was confirmed by measuring the level of ;-H2AX, which is considered to be an indication of DNA double-strand breaks (DSB). Polymerase B-deficient cells were more sensitive than the wild-type to artesunate, indicating that the drug induces DNA damage that is repaired by base excision repair. irs1 and VC8 cells defective in homologous recombination (HR) due to inactivation of XRCC2 and BRCA2, respectively, were more sensitive to artesunate than the corresponding wild-type. This was also true for XR-V15B cells defective in nonhomologous endjoining (NHEJ) due to inactivation of Ku80. The data indicate that DSBs induced by artesunate are repaired by the HR and NHEJ pathways. They suggest that DNA damage induced by artesunate contributes to its therapeutic effect against cancer cells. [Cancer Res 2008;68(11):4347-51]

Since multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy, we report a microfluidic approach combined with the same-single-cell analysis to investigate the modulation of MDR, manifested as the inhibition of drug efflux. A microfluidic chip that was capable of selecting and retaining a single multidrug-resistant cancer cell was used to investigate drug efflux inhibition in leukemia cell lines. Three advantages of the microfluidic-based same-single-cell analysis (dubbed as SASCA) method have been revealed. First, it readily detects the modulation of drug efflux of anticancer compounds (e.g., daunorubicin) by MDR modulators (e.g., verapamil) among cellular variations. Second, SASCA is able to compare the different cellular abilities in response to drug efflux modulation based on the drug transport kinetics of single cells. Third, SASCA requires only a small number of cells, which may be beneficial for investigating drug resistance in minor cell subpopulations (e.g., cancer "stem" cells).

No abstract

No abstract

A microfluidic method to study the contraction of a single cardiac myocyte (heart muscle cell) has been developed. This method integrates various single-cell operations as well as on-chip dye loading, and quantitative analysis of intracellular calcium concentration, [Ca2+]i. After the channel enlargement by on-chip etching to accommodate large-sized cardiac myocytes, a single cell is selected and retained at a V-shaped cell retention structure within the microchip. Owing to the fragile property of the cardiac myocytes that could easily be damaged by centrifugation, the calcium-sensitive fluorescent dye was loaded in the cell by on-chip dye loading. This on-chip method minimized the damage to the cells from the use of a centrifuge in the conventional method and provided a way of cellular analysis of fragile cells. Subsequently, quantitative analysis of [Ca2+]i of a single cardiac myocyte by fluorescence measurement was achieved for the first time in a microfluidic chip, thanks to the intracellular calcium stimulant of ionomycin. The resting [Ca2+]i of the cardiomyocyte determined was consistent with the literature value. From the spontaneous contraction study, it was found that fluorescence intensity cannot represent the [Ca2+]i variation accurately, which implied the importance of the quantitative analysis of [Ca2+]i.

Due to the cellular heterogeneity in multidrug resistance (MDR) cell populations, positive drug effects on the modulation of MDR can be obscured in conventional methods, especially when only a small number of cells are available. To address cellular variations among different MDR cells, we report a new microfluidic approach to study drug effect on MDR modulation, by investigating drug accumulation of daunorubicin in MDR leukemia cells. We have demonstrated that the new approach of same-single-cell analysis by accumulation (denoted as SASCA-A) is not only superior to different-single-cell analysis, but also has key advantages over our previous approach of same-single-cell analysis. First, SASCA-A is much simpler as it does not require multiple cycles of drug uptake and drug efflux. Second, it is faster, only taking about one fourth of the time used in the previous approach. Third, it provides a more 'identical' and reliable control because it compares the time points just before MDR modulator tests. To help understand the dynamics of drug accumulation in MDR cells, we also developed a mathematical model to describe the kinetics of drug accumulation conducted in individual cells. The SASCA-A method will benefit drug resistance research in minor cell subpopulations (e.g., cancer "stem" cells) because this method requires only a small number of cells in identifying the MDR reversal effect.

A microfluidic microarray that is created in the double spiral format has produced a greater density of probes than in our previous report. Using this double-spiral format together with centrifugal pumping for liquid delivery, 384 x 384 hybridization assays have been performed on one circular disk at one time, at the intersections between the spiral channels and spiral probe lines. Each sample was introduced into each inlet reservoir leading to 4 spiral channels and was analyzed independently, and so the hybridization results were self-corrected among the 4 spiral channels. In this work, fast microarray hybridizations have been successfully achieved by using both complementary oligonucleotides as well as PCR products prepared from plant fungal pathogen cultures.