Cancer Res Treat.  2006 Feb;38(1):54-60.

Arsenic Trioxide Induces A poptosis in Human Colorectal Adenocarcinoma HT-29 Cells Through ROS

• Cha Y
• Park DW
• Lee CH
• Baek SH
• Kim SY
• Kim JR
• Kim JH

Abstract

PURPOSE
Treatment with arsenic trioxide (As2O3) results in a wide range of cellular effects that includes induction of apoptosis, inhibition of cell growth, promotion or inhibition of cellular differentiation, and inhibition of angiogenesis through a variety of mechanisms. The mechanisms of As2O3-induced cell death have been mainly studied in hematological cancers, and those mechanisms in solid cancers have yet to be clearly defined. In this study, the mechanisms by which As2O3 induces apoptosis in human colorectal adenocarcinoma HT-29 cells were investigated.
MATERIALS AND METHODS
To examine the levels of apoptosis, HT-29 cells were treated with As2O3 and then we measured the percentage of Annexin V binding cells, the amount of ROS production and the mitochondrial membrane potential. Western blot analysis was performe to identify the activated caspases after As2O3 exposure, and we compared the possible target molecules of apoptosis. As2O3 treatment induced the loss of the mitochondrial membrane potential and an increase of ROS, as well as activation of caspase-3, -7, -9 and -10.
RESULTS
As2O3 induced apoptosis via the production of reactive oxygen species and the loss of the mitochondrial membrane potential. As2O3 induced the activation of caspase-3, -7, -9 and -10. Furthermore, As2O3 treatment downregulates the Mcl-1 and Bcl-2 expressions, and the release of cytochrome c and an apoptosis-inducing factor (AIF). Pretreating the HT-29 cells with N-acetyl-L-cysteine, which is a thiol-containing antioxidant, inhibited the As2O3- Induced Apoptosis and Caspase Activation.
CONCLUSION
Taken together, these results suggest that the generation of reactive oxygen species (ROS) by As2O3 might play an important role in the regulation of As2O3-induced apoptosis. This cytotoxicity is mediated through a mitochondria-dependent apoptotic signal pathway in HT-29 cells.

Keyword

Apoptosis; Arsenic trioxide; Mitochondria; Reactive oxygen species

MeSH Terms

Acetylcysteine
Adenocarcinoma*
Annexin A5
Apoptosis
Apoptosis Inducing Factor
Arsenic*
Blotting, Western
Caspase 3
Caspases
Cell Death
Cytochromes c
HT29 Cells*
Humans*
Membrane Potential, Mitochondrial
Mitochondria
Reactive Oxygen Species
Signal Transduction
Acetylcysteine
Annexin A5
Apoptosis Inducing Factor
Arsenic
Caspase 3
Caspases
Cytochromes c
Reactive Oxygen Species

Figure

• 

  Fig. 1 Effects of As2O3 on cell survival in human colorectal adenocarcinoma HT-29 cells. The cells were treated with increasing concentrations of As2O3. Cell survival was measured by the MTT assay. The values are the mean±SD of three independent experiments.

• 

  Fig. 2 Induction of apoptosis by As2O3-mediated cytotoxicity. The cells were treated with increasing concentrations of As2O3 for 48 h. The cells were incubated with Annexin V (A), H2DCF-DA (B), or JC-1 (C), and then they were analyzed by flow cytometry. The cell lysates were further analyzed by Western blotting with using anti-caspase and anti-PARP antibodies (D). The GAPDH level was used to demonstrate equal loading of proteins on each lane.

• 

  Fig. 3 Effects of As2O3 on the levels of Mcl-1, Bcl-2, cytochrome c and AIF. The cells were treated with increasing concentrations of As2O3 for 48 h. The cell lysates were analyzed by Western blotting using anti-Mcl-1, anti-Bcl-2, anti-cytochrome c and anti-AIF antibodies. The GAPDH level was used to demonstrate equal loading of proteins on each lane.

• 

  Fig. 4 The effects of NAC on the As2O3-mediated cytotoxicity. The cells were treated with As2O3 in the presence or absence of NAC. After 48 h, cell survival was determined by the MTT assay. The values are the mean±SD of three independent experiments.

• 

  Fig. 5 The effects of NAC on As2O3-mediated apoptosis. The cells were treated with 5 mM NAC for 30 min and then they were treated with 20 µM As2O3 for an additional 48 h. The cells were incubated with Annexin V (A), H2DCF-DA (B), or JC-1 (C) and next analyzed by flow cytometry. The cell lysates were analyzed by Western blotting with anti-caspase and anti-PARP antibodies (D). The GAPDH level was used to demonstrate equal loading of proteins on each lane.

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