Abstract
With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, 2¢,7¢-dichlorofluorescin diacetate (DCFH-DA) and Fluo 3-AM, we simultaneously observed arsenic trioxide (As2O3)-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration [Ca2+]i within human skin squamous carcinoma cells (Colo-16 cells). Our results indicated that As2O3 induced [Ca2+]i elevation and ROS production within Colo-16 cells, and both [Ca2+]i elevation and ROS production were involved in the apoptosis of Colo-16 cells. These results suggested that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multi-parameter observation of As2O3-induced apoptosis at the single cell level. Key words: Two-photon laser scanning microscopy, confocal laser scanning microscopy, human skin squamous carcinoma cells (Colo-16 cells), arsenic trioxide, apoptosis.
Highlights
Arsenic is poisonous and chronic arsenic exposure from industrial or natural sources can cause serious toxicity, arsenic has been used therapeutically for more than 2,400 years (Klaassen, 1996); in traditional Chinese medicine, arsenic trioxide (As2O3) is used to treat syphilis, rheumatosis, and psoriasis (Shen et al, 1997)
Our results indicated that As2O3 induced concentration-dependent apoptosis of Colo-16 cells and both As2O3 -induced [Ca2+]i elevation and reactive oxygen species (ROS) production involved in the apoptosis of Colo-16 cells, suggesting that two-photon and confocal laser scanning microscopy might throw new insight into the anti-skin-cancer mechanism of As2O3
As2O3 (1 to 12 μM), cell viability were analyzed by MTT and the viable cell number was compared with the untreated control
Summary
Arsenic is poisonous and chronic arsenic exposure from industrial or natural sources can cause serious toxicity, arsenic has been used therapeutically for more than 2,400 years (Klaassen, 1996); in traditional Chinese medicine, arsenic trioxide (As2O3) is used to treat syphilis, rheumatosis, and psoriasis (Shen et al, 1997). Fluorescence microscopy is an essential tool of modern biology and has emerged as a novel and noninvasive method for visualization of living cells and biological tissue (Gustafsson, 1999). The effective sensitivity of fluorescence microscopy measurements is often limited by out-of-focus flare. This limitation is greatly reduced in a confocal laser scanning microscopy, where the out-offocus background is rejected by a confocal pinhole to produce thin (< 1 μm) and unblerred optical sections from within thick samples (White et al, 1987). The two-photon laser scanning microscopy is a new alternative to confocal microscopy and is a promising technique to observe biological specimens because of its inherent advantage such as three-dimensional resolution without a
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