acute promyelocytic leukemia (APL).

PREVENTION RESEARCH □ ORIG INAL ARTICLE □

39

INTRODUCTION

Arsenical compounds are distributed as a natural toxicant with no color, no taste, and no smell. Arsenic trioxide, As

2

O

3

has been reported to induce almost complete remission from

acute promyelocytic leukemia (APL).

^1∼3)

Cytopathological stu- dies also showed that As

2

O

3

induces apoptosis in APL cells.

Recent reports have shown that As

2

O

3

down-regulates bcl-2 gene expression and induces the expression of apoptosis-related proteins, caspases as well as degradation of promyelocytic leukemia (PML) and promyelocytic leukemia gene/retinoic acid

Apoptosis-induced Cell Growth Inhibitory Effects of a Novel Compound, As 4 O 6 in a Cervical

Cancer Cell Line, SiHa in Vitro

Hong-Seok Chang¹, Su-Mi Bae², Sun-Young Kwak², Ae Ju Lee², Aery Lee²

Yong Seok Lee³, Il-Ju Bae⁴, Jin-Young Yoo⁵, Young-Joo Lee⁶, Chong-Kook Kim⁷ and Woong-Shick Ahn³

1Department of Therapeutic Radiology, ²Catholic Research Institutes of Medical Science,

3Department of Obstetrics and Gynecology, The Catholic University of Korea College of Medicine, Seoul 137-040, ⁴Laboratory of Chonjisan Institute, Seoul 150-727,

5Department of Pathology, The Catholic University of Korea College of Medicine, Seoul 137-040,

6Department of Bioscience and Biotechnology, Institute of Biotechnology, College of Life Science, Sejong University, Seoul 143-747, ⁷College of Pharmacy, Seoul National University, Seoul 151-742, Korea

As

2

O

3

has been reported to be effective for treating acute leukemia and induce apoptosis in many tumor cells. In this study, we evaluated the ability of a novel arsenical compound, As

4

O

6

, along with As

2

O

3

to induce cell growth inhibition as well as apoptosis in human cervical cancer cells, SiHa cells

in vitro. To examine the levels of apoptosis, SiHa cells were given two sensitive doses, 0.5 and 1μM

of arsenical compounds, and then, DNA fragmentation assay and FACS analysis were conducted. In addition, Western blotting assay was done for the identification of target molecules for apoptosis. Both As

2

O

3

and As

4

O

6

caused dose-dependent inhibition of SiHa cell proliferation. In particular, As

4

O

6

was more effective for suppressing SiHa cell growth, as compared to As

2

O

3

. In parallel with inhibition of cell proliferation, As

4

O

6

caused an increase of the sub-G1 cell population significantly more than As

2

O

3

, as determined by propidium iodide DNA staining. This was confirmed by DNA fragmentation assay and annexin V staining. Western blotting analysis also showed that the proliferating cell nuclear antigen (PCNA) expression was suppressed by As

4

O

6

significantly more than As

2

O

3

, and that Bcl-

XL

with se- quence homology to Bcl-2 was significantly suppressed by As

4

O

6

. However, apoptosis-related proteins, p21 and Bax, were expressed by As

4

O

6

significantly more than As

2

O

3

. Taken together, these findings suggest that a novel arsenic compound, As

4

O

6

possesses more potent anti-proliferative effects on human cervical cancer cells with induction of apoptosis at least through activation in p21 and Bax proteins

in vitro. (Cancer Prev Res 11, 39-45, 2006)

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: Cervical cancer, Arsenic trioxide, Arsenic hexoxide, Apoptosis

책임저자：안웅식 ꂕ 137-040, 서울시 서초구 반포동 505 가톨릭대학교 의과대학 산부인과학교실 Tel: 02-590-2786, Fax: 02-599-4120 E-mail: ahnws@catholic.ac.kr

접수일：2005년 12월 20일, 게재승인일：2006년 1월 18일

Correspondence to：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

E-mail: ahnws@catholic.ac.kr

receptor (PML/RAR) α proteins in APL cells.

^2,4)

Similarily, arsenic trioxide suppresses the growth of tumor cells by cell cycle arrest, induction of cyclin-dependent kinase (CDK) inhi- bitors 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⁾

a human papillomavirus (HPV) 16 infected cervical carcinoma cells,

⁷⁾

and a human pancreatic can- cer cells.

^8,9)

These collective studies suggest that arsenic trioxide has an anti-tumor activity in a variety of tumor cells.

Cervical cancer is caused mostly by infection with a high risk group of HPV.

^10∼12)

After high risk HPV infection, two viral oncogenic proteins, E6 and E7 play a critical role in inducing cervical cancers by interacting with p53 and retinoblastoma protein (pRb) for inactivation of these cellular regulatory pro- teins, respectively.

^13∼15)

Presently, surgical, radiation, chemo- therapies have been approached with limited success. However, an early detection of cervical cancer using the Pap smear has contributed to cut down the incidence of cervical cancers.

However, relapsing cervical cancers have been problematic, adding importance to developing anti-cervical cancer drugs.

Here we evaluated the ability of a new arsenical compound, As

4

O

6

along with As

2

O

3

to suppress cell growth in human cervical carcinoma cells, SiHa cells. We observe that As

4

O

6

is more effective for inhibiting the SiHa cell growth, as compared to As

2

O

3

. Furthermore, As

4

O

6

caused an increase of the sub-G1

cell population, DNA ladder formation and annexin V staining significantly more than As

2

O

3

. Furthermore, Western blotting analysis showed that expression of a cell proliferation marker and Bcl-

XL

is suppressed by As

4

O

6

significantly more than As

2

O

3

. However, expression of apoptosis-related proteins was increased by As

4

O

6

significantly more than As

2

O

3

. Thus, these data suggest that a novel arsenic compound, As

4

O

6

possesses more potent anti-proliferative effects on human cervical cancer cells with induction of apoptosis in vitro, as compared to As

2

O

3

.

MATERIALS AND METHODS

1. Cell culture

HPV 16 immortalized human cervical carcinoma cell lines, SiHa cells containing wild type P53 were incubated DMEM supplemented with 5% fetal bovine serum, 0.37% sodium bicarbonate, 30 mM HEPES, and 100μg/ml streptomycin/

penicillin (cDMEM) at 37

^o

C in the CO

2

incubator.

2. Chemical reagents

As

2

O

3

was purchased from Sigma Chemical Co. As

4

O

616∼19)

was provided from Chonjisan., Co., Seoul, Korea. The chemical structures of both As

2

O

3

and As

4

O

6

are inserted in Fig. 1.

These chemicals were diluted in phosphate-buffered saline (PBS) to a final concentration of 10

^-3

M and kept at 4

^o

C.

Fig. 1. Growth inhibition patterns of As2O3 and As4O6 in SiHa cells in vitro. Cells were treated with the indicated amount of arsenic compounds, As2O3 and As4O6 for 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 and average OD values and SD were recorded.

This was repeated two more times with similar results.

*Statistically significant at p＜0.05 using the paired Student's t test compared to no drug treatment (control, CTL). The chemical structures of As4O6 and As4O6 were inserted in each panel. The shaded bigger circles and empty smaller circles represent As and O, respectively.

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma and dissolved in PBS at a final concentration of 5 mg/ml.

3. Cell growth inhibition assay

For cell growth inhibition assay, MTT assay was performed.

First, SiHa cells (1×10

³

cells/well) were divided into 96 well plate in 100μl of cDMEM. After 24 h incubation, cells were treated with different amount of arsenic compounds. After 4 days incubation, each well of plates were added with 20μl of MTT solution for 4 h at 37

^o

C. The cell media were replaced with 100μl of dimethyl sulfoxide (Sigma) per well. The plate was shaken for 10 sec, and then optical density (OD) was measured at a wavelength of 570 nm using enzyme-linked immunosorbent assay-Reader (Spectromax 250, Molecular De- vices). 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 5×10

⁵

cells per 100 mm dish plate. After 24 h incubation, cells were added with different amounts of arsenic compounds for 48 h at 37

^o

C. The cells were then centrifuged and subsequent cell pellets were added with lysis buffer [0.8% SDS, 0.1 M NaCl, 0.1 M EDTA, 50 mM Tris-HCl (pH8.0)], followed by addition of 20μg/ml proteinase K (Sigma). This was incubated for 4 h at 56

^o

C. DNA was ex- tracted by phenol/chloroform treatment. Five μg of extracted DNA was analyzed on a 2% agarose gel containing ethidium bromide (0.1μg/ml). 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 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl

2

). One×10

⁵

cells per tube were added with 5μl of Annexin V-FITC and 10μl of propidium iodide (BD, San Jose, CA), followed by incubation at 22

^o

C for 15 min.

Each tube was added with 100μl of 1×binding buffer and then cells were analyzed by flow cytometer (BD). For DNA contents, ethanol-fixed cells were incubated with RNase A (10 mg/ml) and propidium iodide (400μg/ml) and shaken for 1 h at 37

^o

C in the dark. The samples were read using flow

cytometer (BD). Cell debris and fixation artifacts were gated out and 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 1μM of As

2

O

3

and As

4

O

6

for 48 h. The cell lysates (approximately 30μg of protein) was separated in 12% polyacrylamide SDS-gels and transferred to the nitrocellulose membrane (Schleicher &

Schuell, Dassel, Germany). This was then immersed in blocking buffer (5% skim milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h at room temperature and incubated with primary antibodies (SantaCruz Biotechnology, Inc., California, USA), PCNA (1：200), CDK4 (1：200), p21 (1：200), Bax (1：

200), Bcl-

XL

/Bcl-

XS

(1：500) and actin (1：5,000) in blocking buffer overnight at 4

^o

C . After the incubation, the membrane was probed with horseradish peroxidase-labeled anti-mouse IgG antibody (1：5,000) in PBS (containing of 0.05% Tween 20 and 5% skim milk powder) for 30 min at room temperature.

The proteins in the membrane were detected by enhanced chemiluminescence detection system (Amersham, Buckingham- shire, UK) and bands were visualized by autoradiography using 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, As

4

O

6

could suppress the growth of human cervical cancer cells, SiHa

cells were treated with an increasing amount (0.5∼5μM) of

As

4

O

6

for 48 h. As a positive control, cells were also treated

with As

2

O

3

. As shown in Fig. 1, the growth of SiHa cells was

unaffected by treating cells with 0.5μM of As

2

O

3

. However,

1μM of As

2

O

3

showed approximately 50% cell growth

inhibition while As

2

O

3

ranging from 2 to 5μM displayed

complete cancer cell growth inhibition. In the case of As

4

O

6

,

0.5μM of As

4

O

6

displayed approximately 50% cancer cell

growth inhibition. In contrast, As

4

O

6

ranging from 1 to 5μM

showed complete cancer cell growth inhibition. This illustrates

that As

4

O

6

is more effective than As

2

O

3

for inhibiting human

cervical cancer cell growth in vitro.

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

We were next interested in examining the levels of apoptosis achieved by addition of two most sensitive doses, 0.5 and 1μM

of arsenical compounds (As

2

O

3

and As

4

O

6

). As shown in Fig.

2A, As

4

O

6

induced a DNA ladder formation at 0.5μM, which is indicative of apoptosis. However, little DNA ladders were formed by addition of 0.5μM As

2

O

3

. This suggests that As

4

O

6

is more effective for induction of apoptosis, as compared to

Fig. 2. Induction of DNA ladder formation (A) and sub-G1 cell population (B) in SiHa cells by As2O3 and As4O6. Cells were treated with 0.5 and 1μM of As2O3 and 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 detection of sub-G1 population.

B

0 0.5 1

SubG1 cells (%) -

Concent ation (uM)r 0

10 20 30 40 50 60 70

As O_{4 6} As O_{2 3}

A

As O As O As O As O_{2 3 4 6 2 3 4 6} 0.5 Mµ 1 Mµ

Fig. 3. Induction of early and late apoptotic cells in SiHa cells by As2O3and As4O6. Cells were treated with 0.5 and 1μM of As2O3

and 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 cytometer.

PI

Annexin V 10⁰ 10¹ 10² 10³ 10⁴

10⁰ 10¹ 10² 10³ 10⁴

FL3H-

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 10³ 10⁴

FL3H-

Control

Control

17.2 3.7

1.4

17.2 3.7

1.4

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 10³ 10⁴

FL3H-

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 10³ 10⁴

FL3H-

As O 0.5 uM_{2 3'}

As O 0.5 uM_{4 6'}

6.1 3.7

1.9

9.0 8.0

2.5

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 10³ 10⁴

FL3H-

10⁰ 10¹ 10² 10³ 10⁴ 10⁰

10¹ 10² 10³ 10⁴

FL3H-

As O 1 uM_{2 3'}

As O_{4 6'} 1 uM

4.2 4.8

4.4

6.8 11.5

8.1

FL1-H

FL1-H FL1-H

FL1-H

FL1-H FL1-H

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

As

2

O

3

. This apoptosis pattern was confirmed by flow cytometry (Fig. 2B). In particular, As

4

O

6

displayed 25% sub-G1 cell populations at 0.5μM. However, little sub-G1 cell populations were observed by 0.5μM of As

2

O

3.

Similarly, 1μM of As

2

O

3

and As

4

O

6

showed 30 and 50% sub-G1 cell populations, respectively. Therefore, this data confirms that As

4

O

6

is a more potent arsenic compound to induce apoptosis in SiHa cells.

3. As4O6 induced more early and late apopto- tic cell populations in SiHa cells, as compa- red to As2O3

We next counted different apoptotic cell populations induced by these two drugs. As shown in Fig. 3, propidium iodide (+)/Annexin V (+) double positive cell populations (late apoptotic group) were 3.7, 3.7 and 4.8% at 0, 0.5 and 1μM of As

2

O

3

, respectively. Propidium iodide (-)/Annexin V (+) cell populations (early apoptotic group) were 1.4, 1.9 and 4.4%

at 0, 0.5 and 1μM of As

2

O

3

, respectively. However, pro- pidium iodide (+)/Annexin V (+) double positive cell popu- lations were 3.7, 8.0 and 11.5% at 0, 0.5 and 1μM of As

4

O

6

, respectively. In similar, propidium iodide (-)/Annexin V (+) cell populations were 1.4, 2.5 and 8.1% at 0, 0.5 and 1μM of As

4

O

6

, respectively. This shows that As

4

O

6

induced more increased early and late apoptotic cells, as compared to As

2

O

3

.

4. Comparison of expression of cell growth- and apoptosis-related proteins by As2O3

and As4O6 in SiHa cells

To compare anti-growth effects induced by As

2

O

3

and As

4

O

6

at the levels of cell proliferation- and apoptosis-related proteins, cells were treated with two arsenical compounds at 0.5 and 1μM. As shown in Fig. 4, expression of the cell proliferation marker, PCNA, and the anti-apoptotic protein, Bcl-

XL

, was decreased by these arsenic compounds, whereas expression of apoptosis- related proteins, Bax and p21 was instead increased.

In particular, As

4

O

6

inhibited PCNA and Bcl-

XL

expression significantly more than As

2

O

3

at 0.5 M and 1μM, respectively.

Similarly, Bax and p21 expression was increased by As

4

O

6

significantly more than As

2

O

3

. However, expression of CDK4, Bcl-

XS

and actin was unaffected by these two arsenic com- pounds. This illustrates that As

4

O

6

can induce apoptosis through activation of apoptosis-related proteins, Bax and p21 to a more significant level, as compared to As

2

O

3

.

DISCUSSION

Anti-tumor functions of an arsenic compound, As

2

O

3

have been reported in leukemia in vivo and in vitro.

^1∼4)

We also observed that As

2

O

3

induced apoptosis and cell growth inhibition in SiHa cell lines. This supports previous findings that arsenic trioxide induces anti-tumor effects through induc- tion of tumor cell apoptosis.

^1∼5,8,9)

In the case of promyeloleu- kemic cells, As

2

O

3

down-regulates the expression of bcl-2 and PML/RARα/PML proteins which are correlated with apopto- sis.

^2,4)

As

2

O

3

also induces apoptosis in human pancreatic cancer cells through changes in cell cycle and caspase activation.

^8,9)

Dai et al. also reported that glutathione redox system is associated with apoptosis by As

2

O

320)

. In one literature, As

2

O

3

can inhibit cell growth and induce apoptosis in different cancers including leukemia, myeloma, lymphoma, and other solid

Fig. 4. Western blots of cell proliferation marker and apopto- sis-related proteins in SiHa cells by As2O3 and As4O6. Cells were treated with 0.5 and 1μM of As2O3 and As4O6 for 48 h.

The cells were harvested and cell lysates were run on 12%

SDS-PAGE. Subsequent protein bands were transblotted into nitrocellulose membrane for immunoblot assay.

PCNA

CDK4

p21

Bax

BclXS

Actin 0 As O_{2 3} As O_{4 6} As O_{2 3} As O_{4 6}

0.5 uM 1 uM

tumors

²¹⁾

, suggesting that apoptosis is a likely mechanism of arsenic trioxide to suppress tumor cell growth in vivo and in

vitro.

In our observation, As

4

O

6

was more effective for suppressing SiHa cells proliferation in vitro, as compared to As

2

O

3

. In particular, As

4

O

6

showed a significant anti-growth activity (ap- proximately 50% cell growth inhibition) at a dose of 0.5

μ

M, at which As

2

O

3

displayed little anti-growth effects. Instead, 1 μM of As

2

O

3

showed approximately 50% growth inhibition.

This is consistent with our observation that at a dose of 0.5 μM both DNA fragments and sub-G1 cell population were generated by As

4

O

6

significantly more than As

2

O

3

. This is in line with more induction of early and late apoptotic cells by As

4

O

6

we observed. These data support the notion that As

4

O

6

could be a more potent drug against cervical cancer cell growth through apoptosis. The dose effects of arsenic trioxide on growth inhibition are consistent with many previous reports.

^5,7)

We also observed that the expression of cell proliferation marker, PCNA,

²²⁾

and the anti-apoptotic protein, Bcl-

XL23)

was decre- ased by these arsenic compounds. In particular, As

4

O

6

inhibited PCNA and Bcl-

XL

expression significantly more than As

2

O

3

. This is in line with our observation that As

4

O

6

inhibited cancer cell growth significantly more than As

2

O

3

. Furthermore, the expression of apoptosis-related proteins, Bax and p21

^22,24)

was increased by arsenic compounds. In particular, Bax and p21 expression was increased by As

4

O

6

significantly more than As

2

O

3

. This correlates well with our observation that As

4

O

6

induced apoptosis in SiHa cells significantly more than As

2

O

3

. This is also compatible with the previous finding that As

2

O

3

increased the expression of p21 and Bax proteins in human pancreatic cancer cells.

⁹⁾

However, expression of CDK4, Bcl-

XS

and actin was unaffected by these two arsenic compounds.

Thus, these data suggest that As

4

O

6

mediates apoptosis at least through activation of Bax and p21 pathways in human cervical cancer cells, SiHa cells.

In conclusion, the data presented here suggest that As

4

O

6

is more effective for suppressing SiHa cell growth, as compared to As

2

O

3

. In parallel with inhibition of cell proliferation, As

4

O

6

caused an increase of the sub-G1 cell population, DNA ladder formation and annexin V staining significantly more than As

2

O

3

. Furthermore, Western blotting analysis showed that expression of apoptosis-related proteins, p21 and Bax was in- creased by As

4

O

6

significantly more than As

2

O

3.

Taken toge- ther, these findings suggest that a novel arsenic compound,

As

4

O

6

possesses more potent anti-growth effects on human cervical cancer cells with induction of apoptosis in vitro, which might provide a new drug choice for treating HPV-associated cervical cancer.

ACKNOWLEDGEMENT

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

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