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

Jeong Kim; Su-Mi Bae; Dae-Seog Lim; Sun-Young Kwak; Chang-Ki Lee; Yong-Seok Lee; IL-Ju Bae; Jin-Young Yoo; Young-Joo Lee; Chong-Kook Kim; Woong-Shick Ahn

doi:10.4143/crt.2005.37.5.307

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Original Article Tetraarsenic Oxide-mediated Apoptosis in a Cervical Cancer Cell Line, SiHa: Jeong Kim, M.D.¹, Su-Mi Bae, M.S.², Dae-Seog Lim, Ph.D.², Sun-Young Kwak, B.S.², Chang-Ki Lee, M.S.², Yong-Seok Lee, M.D.¹, IL-Ju Bae³, Jin-Young Yoo, Ph.D.⁴, Young-Joo Lee, Ph.D.⁵, Chong-Kook Kim, Ph.D.⁶, Woong-Shick Ahn, M.D., Ph.D.¹; Cancer Research and Treatment : Official Journal of Korean Cancer Association 2005;37(5):307-312.
DOI: https://doi.org/10.4143/crt.2005.37.5.307
Published online: October 31, 2005

¹Departments of Obstetrics and Gynecology, The Catholic University of Korea College of Medicine, Seoul, Korea.

²Cancer Research Center, Catholic Research Institutes of Medical Science, The Catholic University of Korea College of Medicine, Seoul, Korea.

³Laboratory of Chonjisan Institute, Seoul, Korea.

⁴Department of Pathology, The Catholic University of Korea College of Medicine, Seoul, Korea.

⁵Department of Bioscience and Biotechnology, Institute of Biotechnology, College of Life Science, Sejong University, Seoul, Korea.

⁶College of Pharmacy, Seoul National University, Seoul, Korea.

Correspondence: Woong-Shick Ahn, Department of Obstetrics and Gynecology, The Catholic University of Korea College of Medicine, 505, Banpo-dong, Seocho-gu, Seoul 137-040, Korea. (Tel) 82-2-590-2786, (Fax) 82-2-599-4120, ahnws@catholic.ac.kr

• Received: August 18, 2005 • Accepted: September 21, 2005

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
NOTES
REFERENCES

This article has been retracted. See "Retraction: Tetraarsenic Oxide-mediated Apoptosis in a Cervical Cancer Cell Line, SiHa" in Volume 39 on page 48.

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 1 µM, 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 dosedependent 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 0.5 µM. However, the apoptosis-related protein, Bax, was expressed to a significantly greater extent due to As₄O₆ than As₂O₃.
Conclusion
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.
Key words: Tetraarsenic oxide, Cervix neoplasms, Apoptosis

INTRODUCTION

Arsenical compounds are distributed as a natural toxicant, with no color, taste or smell. Diarsenic trioxide, As₂O₃, has been reported to induce almost complete remission of acute promyelocytic leukemia (APL) (1~3). Cytopathological studies have also shown that As₂O₃ induces apoptosis in APL cells. Recent reports have shown that As₂O₃ down-regulates the expression of the bcl-2 gene and induces the expression of the apoptosis-related proteins, caspases, as well as the degradation of promyelocytic leukemia (PML) and promyelocytic leukemia gene/retinoic acid receptor (PML/RAR) α proteins in APL cells (2,4). Similarly, arsenic trioxide has been shown to suppress the growth of tumor cells by cell cycle arrest, induction of cyclin-dependent kinase (CDK) inhibitors and apoptosis in a myeloma cell line, MC/CAR (5). Arsenic trioxide also causes cell death through apoptosis in a human leukemia cell line, NB4 (6), human papillomavirus (HPV) 16 infected cervical carcinoma cells (7) and human pancreatic cancer cells (8,9), which collective suggest that arsenic trioxide has anti-tumor activity in a variety of tumor cells.

Cervical cancer is mostly caused by infection within the high risk HPV group (10~12). After a high risk HPV infection, two viral oncogenic proteins, E6 and E7, play a critical role in the induction of cervical cancers by their interaction with p53 and retinoblastoma protein (pRb), respectively, for the inactivation of these cellular regulatory proteins (13~15). Presently, surgical, radiation and chemo- therapies have been used as approaches, but with limited success. However, the early detection of cervical cancer using the Pap smear has contributed to reducing the incidence of cervical cancers. However, relapsing cervical cancers have been problematic, adding importance to the development of anti-cervical cancer drugs.

Here, the ability of a new arsenical compound, As₄O₆ (tetraarsenic oxide), along with As₂O₃, were evaluated for their ability to suppress cell growth in human cervical carcinoma cells, SiHa cells. As₄O₆ was observed to be more effective at inhibiting SiHa cell growth than As₂O₃. Furthermore, As₄O₆ caused significantly greater increases in the sub-G1 cell population, DNA ladder formation and annexin V staining than As₂O₃. Furthermore, Western blotting analysis showed the expression of a cell proliferation marker was more significantly suppressed by As₄O₆ than As₂O₃. However, the expression of apoptosis-related protein was significantly increased by As₄O₆ than As₂O₃. Thus, these data suggest that a novel arsenic compound, As₄O₆, possesses more potent anti-proliferative effects on human cervical cancer cells, also with the induction of apoptosis, in vitro, compared to As₂O₃.

MATERIALS AND METHODS

1) Cell culture

HPV 16, immortalized a human cervical carcinoma cell lines, SiHa cells, containing wild type p53, were incubated at 37℃ in DMEM supplemented with 5% fetal bovine serum, 0.37% sodium bicarbonate, 30 µM HEPES and 100 µg/ml streptomycin/penicillin (cDMEM), in a CO₂ incubator.

2) Chemical reagents

The As₂O₃ was purchased from Sigma and the As₄O₆ (16~19) was provided from Chonjisan, Co., Seoul, Korea. The chemical structures of both As₂O₃ and As₄O₆ are shown in Fig. 1. These chemicals were diluted in phosphate-buffered saline (PBS), to a final concentration of 10^-3 M, and kept at 4℃. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma, which was dissolved in PBS to a final concentration of 5 mg/ml.

3) Cell growth inhibition assay

As a cell growth inhibition assay, the MTT assay was performed. First, SiHa cells (1×10³ cells/well) were divided into 96 well plates in 100 µl of cDMEM. After 24 h incubation, the cells were treated with different amounts of the two arsenic compounds. After 4 days incubation, 20 µl of MTT solution was added to each well of the plates, which were further incubated for 4 h at 37℃. The cell media were replaced with 100 µl of dimethyl sulfoxide (Sigma) per well. The plate was shaken for 10 sec and the optical density (OD) then measured at 570 nm, using an enzyme-linked immunosorbent assay-Reader (Spectromax 250, Molecular Devices). The growth inhibition rate (%) was calculated as follows: OD of non-treatment-OD of drug treatment/OD of non-treatment×100.

4) DNA fragmentation assay

SiHa cells were divided into 5×10⁵ cells per 100 mm dish plate. After 24 h incubation, different amounts of arsenic compounds were added to the cells, which were further incubated for 48 h at 37℃. The cells were then centrifuged, with lysis buffer [0.8% SDS, 0.1 M NaCl, 0.1 M EDTA, 50 µM Tris-HCl (pH8.0)] added to the subsequent cell pellets, followed by the addition of 20 µg/ml proteinase K (Sigma) and further incubated for 4 h at 56℃. DNA was extracted by phenol/chloroform treatment. Five mg of extracted DNA was analyzed on a 2% agarose gel containing ethidium bromide (0.1 µg/ml). The DNA ladder formation was visualized under UV light.

5) FACS analysis

Cells were washed twice with PBS and then resuspended in 1×binding buffer (10 µM Hepes/NaOH, pH 7.4, 140 µM NaCl, 2.5 µM CaCl₂). 5 µl of Annexin V-FITC and 10 ml of propidium iodide (BD, San Jose, CA) were added to 1×10⁵ cells per tube, followed by incubation at 22℃ for 15 min. 100 µl of 1×binding buffer was added to each tube, and the cells then analyzed by flow cytometry (BD). To analyze for the DNA contents, ethanol-fixed cells were incubated with RNase A (10 mg/ml) and propidium iodide (400 µg/ml), and then shaken for 1 h at 37℃ in the dark. The samples were read using flow cytometry (BD). The cell debris and fixation artifacts were gated out, and the G0/G1, S and G2/M populations were quantified using the CellQuest program.

6) Western blotting analysis

SiHa Cells were treated with 0.5 and 1 µM of As₂O₃ and As₄O₆ for 48 h. The cell lysates (approximately 30 µg of protein) was separated on 12% polyacrylamide SDS-gels and transferred to the nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membrane was then immersed in blocking buffer (5% skimmed milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h, at room temperature, and then incubated overnight with the primary antibodies (SantaCruz Biotechnology, Inc., Santa Cruz, CA), PCNA (1:200), CDK4 (1:200), p21 (1:200), Bax (1:200), Bcl-_XL/Bcl-_XS (1:500) and actin (1:5000), prepared in blocking buffer at 4℃. After incubation, the membrane was probed with the horseradish peroxidase-labeled anti-mouse IgG antibody (1:5000), prepared in PBS (containing of 0.05% Tween 20 and 5% skim milk powder), for 30 min at room temperature. The proteins bands in the membrane were detected using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK), and visualized using autoradiography with an X-ray film (Amersham).

RESULTS

1) As₄O₆ inhibited SiHa cell growth to a more significant level than As₂O₃

To investigate whether a new arsenical compound, As₄O₆, could suppress the growth of human cervical cancer cells, SiHa cells were treated with increasing amounts (0.5~5 µM) of As₄O₆ for 48 h. As a positive control, cells were also treated with As₂O₃. As shown in Fig. 2, the growth of SiHa cells was unaffected by their treatment with 0.5 µM As₂O₃. However, 1 µM As₂O₃ showed approximately 50% cell growth inhibition, while As₂O₃, ranging from 2 to 5 µM, displayed complete growth inhibition of the cancer cells. With As₄O₆, 0.5 µM displayed approximately 50% growth inhibition of the cancer cells, but within the range 1 to 5 µM showed complete growth inhibition of the cancer cells. This illustrates that As₄O₆ is more effective than As₂O₃ at inhibiting the growth of human cervical cancer cells in vitro.

2) As₄O₆ induced apoptosis more significant than As₂O₃ in SiHa cells

Next, the levels of apoptosis due to the addition of the two most sensitive doses, 0.5 and 1 µM, of the arsenical compounds (As₂O₃ and As₄O₆) were examined. As shown in Fig. 3A, As₄O₆ induced DNA ladder formation at 0.5 µM, indicative of apoptosis. However, few DNA ladders were formed on the addition of 0.5 µM As₂O₃. This suggests that As₄O₆ is more effective for the induction of apoptosis compared to As₂O₃. This pattern of apoptosis was confirmed by flow cytometry (Fig. 3B). In particular, As₄O₆ displayed 25% sub-G1 cell populations at 0.5 µM. However, few sub-G1 cell populations were observed on the addition of 0.5 µM As₂O₃. Similarly, 1 µM of each As₂O₃ and As₄O₆ showed 30 and 50% sub-G1 cell populations, respectively. Therefore, this data confirms that As₄O₆ is more potent at inducing apoptosis in SiHa cells.

3) As₄O₆ induced more early and late apoptotic cell populations in SiHa cells, as compared to As₂O₃

Next, the different apoptotic cell populations induced by these two drugs were counted. As shown in Fig. 4, the propidium iodide (+)/Annexin V (+) double positive cell populations (late apoptotic group) were 3.7, 3.7 and 4.8% at 0.0, 0.5 and 1 µM As₂O₃, respectively. The propidium iodide (-)/Annexin V (+) cell populations (early apoptotic group) were 1.4, 1.9 and 4.4% at 0.0, 0.5 and 1 µM As₂O₃, respectively. However, the propidium iodide (+)/Annexin V (+) double positive cell populations were 3.7, 8.0 and 11.5% at 0.0, 0.5 and 1 µM As₄O₆, respectively. Similarly, the propidium iodide (-)/Annexin V (+) cell populations were 1.4, 2.5 and 8.1% at 0.0, 0.5 and 1 µM As₄O₆, respectively. This shows that As₄O₆ induces greater increases in early and late apoptotic cells compared to As₂O₃.

4) Comparison of expression of cell growth- and apoptosis- related proteins by As₂O₃ and As₄O₆ in SiHa cells

To compare the anti-growth effects induced by As₂O₃ and As₄O₆ at the cell proliferation- and apoptosis-related protein levels, the cells were treated with the two arsenical compounds at 0.5 and 1 µM. As shown in Fig. 5, the expression of the cell proliferation marker, PCNA, was decreased by both arsenic compounds, whereas those of the apoptosis-related proteins, Bax and p21, were conversely increased. In particular, As₄O₆ was found to significantly inhibit the expressions of PCNA and Bcl-_XL compared to As₂O₃ at 0.5 µM and 1 µM, respectively. Similarly, the expressions of Bax and p21 were significantly increased by As₄O₆ compared to As₂O₃ at 1 µM and 0.5 µM, respectively. However, the expressions of CDK4, Bcl-XS and actin were unaffected by the two arsenic compounds. This illustrates that As₄O₆ can induce apoptosis, at least via the activation of the apoptosis-related protein, Bax, to a significantly greater level compared to As₂O₃.

DISCUSSION

The anti-tumor functions of the arsenic compound, As₂O₃, have been reported in leukemia both in vivo and in vitro (1~4). Here, As₂O₃ was also observed to induce apoptosis and cell growth inhibition in SiHa cell lines. This supports the previous findings where arsenic trioxide was found to induces anti-tumor effects via the induction of tumor cell apoptosis (1~5, 8, 9). In the case of promyeloleukemic cells, As₂O₃ has been found to down-regulate the expressions of bcl-2 and PML/RARα/PML proteins, which correlated with apoptosis (2,4). As₂O₃ also induces apoptosis in human pancreatic cancer cells, via changes in the cell cycle and caspase activation (8,9). Dai et al. also reported that the glutathione redox system was associated with apoptosis due to As₂O₃ (20). In one article, As₂O₃ was reported to inhibit cell growth and induce apoptosis in different cancers, including leukemia, myeloma, lymphoma and other solid tumors (21), suggesting apoptosis is a likely mechanism of arsenic trioxide in the suppression of tumor cell growth both in vivo and in vitro.

Herein, As₄O₆ was observed to be more effective at suppressing SiHa cells proliferation in vitro compared to As₂O₃. In particular, As₄O₆ showed significant anti-growth activity (approximately 50% cell growth inhibition) at a dose of 0.5 µM, but As₂O₃, at this concentration displayed few anti-growth effects. However, 1 µM As₂O₃ showed approximately 50% growth inhibition, which was consistent with our observation, where at a dose of 0.5 µM both DNA fragments and a sub-G1 cell population were significantly generated by As₄O₆ compared to As₂O₃. This is in line with the greater induction of early and late apoptotic cells observed in this study due to As₄O₆. These data support the notion that As₄O₆ could be a more potent drug against cervical cancer cell growth via apoptosis. The dose effects of arsenic trioxide on growth inhibition were consistent with many previous reports (5,7). The expression of the cell proliferation marker, PCNA (22), was also observed to be decreased by the two arsenic compounds. In particular, As₄O₆ significantly inhibited the expression of PCNA expression compared to As₂O₃. This is in line with our observation where As₄O₆ was shown to significantly inhibit the growth of cancer cells compared to As₂O₃. Furthermore, the expressions of the apoptosis-related proteins, Bax (23) and p21 (24), were increased by both arsenic compounds. In particular, the expression of Bax was significantly increased by As₄O₆ compared to As₂O₃. This correlates well with our observation that As₄O₆ significantly induced apoptosis in SiHa cells compared to As₂O₃, which is also compatible with the previous finding where As₂O₃ increased the expressions of the p21 and Bax proteins in human pancreatic cancer cells (9). However, the expressions of CDK4, Bcl-_XS and actin were unaffected by the two arsenic compounds. Thus, these data suggest that As₄O₆ mediates apoptosis, at least via the activation of the Bax pathway, in human cervical cancer cells, SiHa cells. Conversely, our thinking was that the Bcl_XL (25) and p21 proteins were more sensitive to changes in the concentrations of arsenic compounds.

CONCLUSIONS

The data presented in this study suggest that tetraarsenic oxide, As₄O₆, is more effective at suppressing the growth of SiHa cell growth compared to As₂O₃. In parallel with the inhibition of cell proliferation, As₄O₆ caused significant increases in the sub-G1 cell population, DNA ladder formation and annexin V staining compared to As₂O₃. Furthermore, the Western blotting analysis showed the expression of the apoptosis-related protein, Bax, was significantly increased by As₄O₆ compared to As₂O₃. Taken together, these findings suggest that a novel arsenic compound, As₄O₆, possesses more potent anti-growth effects toward human cervical cancer cells, with the induction of apoptosis in vitro, which might provide a new drug choice in the treatment of HPV-associated cervical cancer.

NOTES

This work was supported by a Korea Research Foundation Grant (KRF-2000-015-FP0047).

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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.

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).

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.

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.

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.

Figure & Data

REFERENCES

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

Cancer Res Treat. 2005;37(5):307-312. Published online October 31, 2005

DOI: https://doi.org/10.4143/crt.2005.37.5.307

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Figure

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

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

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).

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.

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.

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.