Cancer Res Treat.  2005 Oct;37(5):307-312.

Tetraarsenic Oxide-mediated Apoptosis in a Cervical Cancer Cell Line, SiHa

• Kim J
• Bae SM
• Lim DS
• Kwak SY
• Lee CK
• Lee YS
• Bae IJ
• Yoo JY
• Lee YJ
• Kim CK
• Ahn WS

Abstract

PURPOSE
Diarsenic oxide, As2O3, has been reported to be effective in treating acute leukemia, and induce apoptosis in many tumor cells. In this study, the ability of a novel arsenical compound, As4O6 (tetraarsenic oxide), along with As2O3, for its ability to induce cell growth inhibition, as well as apoptosis, in human cervical cancer cells, SiHa cells, were evaluated in vitro. MATERIALS AND METHODS: To examine the levels of apoptosis, SiHa cells were given two sensitive doses, 0.5 and 1micrometer, of arsenical compounds, and a DNA fragmentation assay and FACS analysis were then conducted. In addition, a Western blotting assay was performed to identify target molecules for apoptosis. RESULTS: Both As2O3 and As4O6 induced dose-dependent inhibition of SiHa cell proliferation. In particular, As4O6 was more effective at suppressing SiHa cell growth than As2O3. In parallel with the inhibition of cell proliferation, As4O6 caused a significantly greater increase in the sub-G1 cell population than As2O3, as determined by propidium iodide DNA staining. This was confirmed by a DNA fragmentation assay and annexin V staining. The Western blotting analysis also showed that the expression of proliferating cell nuclear antigen (PCNA) was suppressed to a significantly greater extent by As4O6 than As2O3 at a dose of 0.5micrometer. However, the apoptosis-related protein, Bax, was expressed to a significantly greater extent due to As4O6 than As2O3. CONCLUSIONS: Taken together, these findings suggest that a novel arsenic compound, As4O6, possesses more potent anti-proliferative effects on human cervical cancer cells, with the induction of apoptosis also, at least via the activation of Bax protein in vitro.

Keyword

Tetraarsenic oxide; Cervix neoplasms; Apoptosis

MeSH Terms

Annexin A5
Apoptosis*
Arsenic
bcl-2-Associated X Protein
Blotting, Western
Cell Line*
Cell Proliferation
DNA
DNA Fragmentation
Humans
Leukemia
Proliferating Cell Nuclear Antigen
Propidium
Uterine Cervical Neoplasms*
Annexin A5
Arsenic
DNA
Proliferating Cell Nuclear Antigen
Propidium
bcl-2-Associated X Protein

Figure

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  Fig. 1 The chemical structures of As2O3 and As4O6. The shaded bigger circles and empty smaller circles represent As and O, respectively.

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  Fig. 2 Growth inhibition patterns of As2O3 and As4O6 in SiHa cells in vitro. Cells were treated with the indicated amount of the two arsenic compounds, As2O3 and As4O6, over 4 day incubation periods. Cell growth suppression was measured as described in "Methods and Materials." OD was measured at 570 nm. The assay was performed in triplicate, with the average OD values and SD recorded, which was repeated twice more, with similar results. *Statistically significant at p<0.05, using the paired Student's t-test, compared to no drug treatment (control, CTL).

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  Fig. 3 Induction of DNA ladder formation (A) and sub-G1 cell population (B) in SiHa cells due to treatment with As2O3 and As4O6. Cells were treated with 0.5 and 1 µM of As2O3 or As4O6 for 48 h. (A) DNA was analyzed on a 2% agarose gel and photographed under UV light (Con: Control, SM: Size marker). (B) Cells were stained with propidium iodide, and analyzed using flow cytometer, for the detection of the sub-G1 population.

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  Fig. 4 The induction of early and late apoptotic cells in SiHa cells due to treatment with As2O3 and As4O6. Cells were treated with 0.5 and 1 µM of As2O3 or As4O6 for 48 h. Cells were stained with both annexin V-FITC and propidium iodide, and then analyzed for different apoptotic cell populations using flow cytometry.

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  Fig. 5 Western blots of the cell proliferation marker and apoptosis-related proteins in SiHa cells due to treatment with As2O3 and As4O6. Cells were treated with 0.5 and 1 µM of As2O3 or As4O6 for 48 h. The cells were harvested and the cell lysates run on 12% SDS-PAGE. Subsequent protein bands were transblotted onto a nitrocellulose membrane for an immunoblot assay.

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