0022-3565/08/3262-388–394$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics
JPET 326:388–394, 2008
Vol. 326, No. 2
134080/3358722
Printed in U.S.A.
Arsenic Trioxide, Arsenic Pentoxide, and Arsenic Iodide Inhibit
Human Keratinocyte Proliferation through the Induction
of Apoptosis
Wai-Pui Tse, Christopher H. K. Cheng, Chun-Tao Che, and Zhi-Xiu Lin
School of Chinese Medicine (W.-P.T., C.-T.C., Z.-X.L.) and Department of Biochemistry (C.H.K.C.),
The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
Arsenics are inorganic metalloids that are ubiquitously
distributed throughout the Earth’s crust. For centuries, some
of these inorganic compounds have been used to treat a
variety of ailments in many traditional medical systems. In
Chinese medicine, for example, arsenic-containing minerals
are primarily prescribed for the topical treatment of scabies,
carbuncles, herpes zoster, enduring ulcers, psoriasis, and
arthritis (Jiangsu New Medical College, 1986; Hua et al.,
2003). In our previous study, realgar, a mineral commonly
used in Chinese medicine for topical treatment of psoriasis
and the main chemical constituent of which is As2S2, was
found to be a potent antiproliferative agent on HaCaT cells
(Tse et al., 2006). This promising experimental finding stimulated us to further investigate whether other arsenic compounds also possess similar antiproliferative properties. The
This study was supported by a Direct Grant from The Chinese University of
Hong Kong (project 2030317).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.134080.
nuclear condensation and DNA fragmentation were observed
when the cells were exposed to arsenic compounds. Cell cycle
analysis with propidium iodide (PI) staining demonstrated the
appearance of sub-G1 peak and cell arrest at the G1 phase
in the presence of these compounds. Quantitative analysis by
annexin V-PI staining revealed that the arsenic-induced
apoptotic event was dose-dependent. Moreover, the arsenic
compounds were able to activate caspase-3 expression when
examined by Western blot analysis. Our experimental data
unambiguously demonstrated that induction of cellular apoptosis was mainly responsible for the observed antiproliferation
brought about by the arsenic compounds on HaCaT keratinocytes, suggesting that these arsenic compounds are putative
agents from which psoriasis-treating topical formulae could be
developed.
identification of active antiproliferative arsenics and the
elucidation of their action mechanism would lead to the development of topical agents for effective management of
psoriasis.
Affecting approximately 2% of the population worldwide,
psoriasis is a common chronic inflammatory skin disease
(Lebwohl, 2003; Nickoloff and Nestle, 2004). Histologically, a
typical psoriatic lesion features distinct epidermal acanthosis and parakeratosis resulted from hyperproliferation and
disturbed differentiation of keratinocytes (Camisa, 1998).
Among many essential alterations in the pathophysiology of
psoriasis, hyperproliferation and aberrant differentiation of
epidermal keratinocytes are two of the fundamental cellular
events in the onset, development, and maintenance of the
disease process. Compounds that inhibit keratinocyte proliferation and modulate keratinocyte differentiation are potentially useful in the treatment of psoriasis because a
balanced homeostatic control of keratinocyte growth and differentiation is crucial for recovery from psoriatic to normal
epidermis.
ABBREVIATIONS: PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling; PI, propidium iodide.
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ABSTRACT
Arsenic compounds have been traditionally used to treat a
variety of ailments, including skin diseases. Our previous study
identified the extract of realgar to possess potent antiproliferative action on HaCaT cells. The present study aimed at
evaluating whether several inorganic arsenics found in realgar
also possess similar antiproliferative properties. The results
showed that arsenic trioxide, arsenic pentoxide, and arsenic
iodide had significant antiproliferative action on HaCaT cells,
with IC50 values at 2.4, 16, and 6.8 ␮M, respectively. However, these compounds only modestly inhibited the growth of
Hs-68 cells, a normal human skin fibroblast cell line, with
IC50 values at 43.4, 223, and 89 ␮M, respectively, conferring
a favorable toxicity profile. In mechanistic studies, all three
compounds caused DNA fragmentation as demonstrated by
gel electrophoresis and the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling method. Morphologically,
Arsenic Compounds Induce Keratinocyte Apoptosis
Materials and Methods
Chemicals. Arsenic trioxide, arsenic pentoxide, and arsenic iodide were purchased from Sigma-Aldrich (St. Louis, MO). The compounds were dissolved in phosphate-buffered saline (PBS) to give 10
mM stock solutions that were then sterilized by filtration (0.2-␮m
pore-size filter; Corning Inc., Corning, NY) before use in cell culture
experiments. Other chemicals and reagents used were of analytical
grade.
General Cell Culture. HaCaT, an immortalized cell line of human epidermal keratinocytes (Boukamp et al., 1988), which has been
extensively used as an in vitro model for studies on the pathogenesis
of psoriasis and evaluation of antipsoriatic drugs (Garach-Jehoshua
et al., 1999; Farkas et al., 2001; Thielitz et al., 2004), was provided by
the China Centre for Type Culture Collection (Wuhan, China).
Hs-68, a human fibroblast cell line established from the foreskin of a
normal Caucasian newborn male, was purchased from the American
Type Culture Collection (Manassas, VA). Both cell lines were routinely maintained in Dulbecco’s modified eagle’s medium with 10%
fetal calf serum (Invitrogen, Carlsbad, CA), 10 ␮g/ml streptomycin,
and 10 U/ml penicillin, and they were incubated at 37°C in a 5% CO2,
95% air-humidified atmosphere. All cell culture experiments were
carried out when the culture was 60 to 90% confluent.
Proliferation Assay. The arsenic compounds together with HaCaT
cells were cultured in 96-well plates, with each well containing 2 ⫻ 104
cells in 200 ␮l of Dulbecco’s modified Eagle’s medium. By serial dilution,
the final concentrations of arsenic trioxide, arsenic pentoxide and arsenic iodide ranged from 100 to 0.4 ␮M, from 250 to 1 ␮M, and from 250
to 1 ␮M, respectively. The treated HaCaT cells were incubated for 12,
24, and 48 h, and the proliferation rates under the influence of these
inorganic compounds were determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay was
carried out as described previously (Tse et al., 2006). In brief, MTT was
added to the wells at a final concentration of 0.5 mg/ml, and the cells
were incubated at 37°C for 2 h. The medium was then completely
removed from the wells and replaced with 100 ␮l of dimethyl sulfoxide.
The absorbance of the dissolved formazan dye was recorded at 540 nm
using a microplate spectrophotometer (FLUOstar Optima; BMG
Labtech, Durham, NC). Likewise, Hs-68 cells were exposed to arsenic
trioxide, arsenic pentoxide, and arsenic iodide at concentrations from
100 to 0.4 ␮M by serial dilution. MTT assay was also used to determine
the proliferation of Hs-68 cells after 48-h incubation. The IC50 values
were determined using a GraphPad Prism 3.0 computer program
(GraphPad Software Inc., San Diego, CA).
Fluorescent Staining of HaCaT Cells for Morphological
Evaluation. Approximately 7.5 ⫻ 105 HaCaT cells per well were
seeded in six-well plates. The cells were treated with 12 ␮M arsenic
trioxide, 40 ␮M arsenic pentoxide, and 24 ␮M arsenic iodide for 48 h,
and then they were washed with PBS and fixed in 4% paraformaldehyde for 30 min. Subsequently, they were stained with 20 ␮g/ml
Hoechst 33342 (Invitrogen) for 15 min at room temperature in the
dark. Morphological changes of the arsenic compound-treated cells
were evaluated using an inverted fluorescent microscope (Olympus,
Tokyo, Japan) according to the method described previously (Abrams
et al., 1993).
DNA Fragmentation Assay. One million HaCaT cells were
seeded on 100-mm plates and exposed to 48 ␮M arsenic trioxide, 120
␮M arsenic pentoxide, and 72 ␮M arsenic iodide for 48 h. After
harvest, cells were lysed in 200 ␮l of DNA lysis buffer at 37°C for 15
min. The supernatant was sequentially incubated with 0.4 ␮g/ml
RNase and then with 1.5 ␮g/ml proteinase K at 56°C for 1.5 h. The
DNA of the cells was then precipitated with sodium acetate and
centrifuged at 20,000 ⫻ g for 30 min. Finally, 30 ␮l of Tris-EDTA
buffer was added to the sample, and the sample was incubated at
37°C for 30 min. To analyze the fragmented DNA, 10 ␮l of the
extracted cellular DNA was separated on a 1.5% agarose gel by
electrophoresis, and DNA ladders in the gels were visualized under
UV light after staining with ethidium bromide.
Terminal Deoxynucleotidyl Transferase Biotin-dUTP NickEnd Labeling Assay. To further analyze the DNA fragmentation,
terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assay in which the DNA strand breaks could be detected by enzymatic labeling of the free 3⬘-OH termini with modified
nucleotides was used according to methods described previously
(Gavrieli et al., 1992; Portera-Cailliau et al., 1994; Sgonc et al.,
1994). In brief, 7.5 ⫻ 105 HaCaT cells per well were seeded on a
six-well plate and exposed to 18 ␮M arsenic trioxide, 65 ␮M arsenic
pentoxide, and 42 ␮M arsenic iodide at 37°C for 48 h. Cells were then
fixed in 2% paraformaldehyde for 1 h and permeabilized with 0.1%
Triton X-100 at 4°C for 2 min. The cells were then incubated at 37°C
in the dark for 1 h with 50 ␮l of TUNEL reaction mixture of the In
Situ Cell Death Detection kit (Roche Applied Science, Philadelphia,
PA). Finally, cells were resuspended in 0.5 ml of PBS, and then they
were analyzed by FACSort flow cytometry (BD Biosciences, Franklin
Lakes, NJ).
Cell Cycle Analysis with PI Staining. Approximately 7.5 ⫻ 105
HaCaT cells per well seeded on six-well plates were exposed to
arsenic trioxide at 6, 12, 24, and 36 ␮M; arsenic pentoxide at 40, 60,
80, and 100 ␮M; and arsenic iodide at 24, 36, 48, and 60 ␮M,
respectively, and they were incubated for 48 h. After washed by PBS,
cells were fixed in 70% ethanol at 4°C overnight. The cells were then
resuspended in 43 ␮g/ml PI solution with 1 mg/ml RNase and incubated in the dark at 37°C for 30 min. They were then subject to DNA
content analysis using a FACSort flow cytometer (BD Biosciences),
in which the CellQuest program was used to analyze the results.
Different phases of the cell cycle were assessed by collecting the
signal at channel FL2-A. The percentage of the cell population at a
particular phase was estimated by ModFit LT for Mac version 3.0
software (Verity Software House, Topsham, ME) according to the
methods described previously (Nicoletti et al., 1991; Tounekti et al.,
1995).
Quantitative Analysis of Apoptotic Cells by Annexin V-Green
Fluorescent Protein Staining. In our experiments, 7.5 ⫻ 105 HaCaT
cells per well were seeded on the six-well plate, and they were incubated
with arsenic trioxide at 3, 12, 24, and 36 ␮M; arsenic pentoxide at 40,
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Given the intrinsic hyperproliferative nature of epidermal
cells in psoriatic lesions, it has been postulated that acanthosis of psoriasis is a direct result from diminished apoptotic
cell death of keratinocytes; and indeed, resistance of epidermal keratinocytes to apoptosis has been found in psoriatic
lesions (Wrone-Smith et al., 1997). Apoptosis enables the
elimination of dysfunctional cells without evoking an inflammatory response. Because of this unique function, apoptosis
plays a crucial role in maintaining homeostasis in continually renewing tissues such as skin (Bianchi et al., 1994; Reed,
1998), and it counterbalances proliferation to maintain epidermal thickness and contributes to normal stratum corneum formation. On the contrary, defects in epidermal
apoptosis will result in hyperproliferation of keratinocytes,
the underlying pathogenesis of psoriasis (Kawashima et al.,
2004). Indeed, the apoptotic index of the basal cell layer in
psoriatic epidermis (0.035%) is significantly lower than that
of healthy skin (0.12%) (Laporte et al., 2000). Agents that
induce keratinocyte apoptosis could therefore be useful in the
treatment of psoriasis.
Our present study focuses on the hyperproliferation and
apoptotic dysfunction of epidermal keratinocytes in psoriasis.
This article reports the growth inhibitory action of three
arsenic compounds, namely, arsenic trioxide (As2O3), arsenic
pentoxide (As2O5), and arsenic iodide (AsI3), on a cultured
HaCaT human keratinocyte model and the elucidation of the
mechanism for the observed cellular growth inhibition.
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Results
␮M, respectively. It is clear that the arsenic compounds exhibited differential cytotoxic profiles on the HaCaT and
Hs-68 cells, and they only showed mild cytotoxic action toward the normal human Hs-68 fibroblasts.
Alteration of Cellular Morphology. After exposure to
12 ␮M arsenic trioxide, 40 ␮M arsenic pentoxide, and 24 ␮M
arsenic iodide for 48 h, a greater number of HaCaT cells
showed detachment from the culture plate compared with
the medium control (Fig. 1). The Hoechst 33342-stained HaCaT
keratinocytes seemed to be shrunken, and they displayed
fewer intercellular connections and exhibited typical apoptotic morphology characterized by chromatin condensation and
DNA fragmentation.
Detection of DNA Fragmentation. Detection of DNA
laddering on electrophoresis was used to confirm the morphological finding regarding the apoptotic action of the arsenic compounds. DNA laddering was evident when HaCaT
cells were exposed to 48 ␮M arsenic trioxide, 120 ␮M arsenic
pentoxide, and 72 ␮M arsenic iodide for 48 h (data not
shown). The appearance of DNA laddering is indicative of
cellular DNA fragmentation. This was confirmed by the
TUNEL assay, which constitutes another method to detect
the fragmented DNA by identifying the apoptotic cells in situ
using terminal deoxynucleotidyl transferase to transfer biotin-dUTP to the strand breaks of cleaved DNA. The DNA
strand breaks can be detected by enzymatic labeling of the
free 3⬘-OH terminal with modified nucleotides. Compared
with the control (Fig. 2a), 18 ␮M arsenic trioxide (Fig. 2b), 65
␮M arsenic pentoxide (Fig. 2c), and 42 ␮M arsenic iodide
(Fig. 2d) were all capable of inducing the appearance of
apoptotic peaks, indicative of the occurrence of apoptosis in
the HaCaT cells.
Action of Arsenic Compounds on Cell Cycle Progression. The flow cytometric measurement of PI-stained DNA is
shown in Fig. 3. Arsenic trioxide at 24 ␮M, arsenic pentoxide
Action of Arsenic Compounds on HaCaT and Hs68
Cell Proliferation. The antiproliferative action of arsenic
trioxide, arsenic pentoxide, and arsenic iodide on the cultured HaCaT keratinocytes and Hs-68 cells as determined by
MTT assay is shown in Table 1. The arsenic compounds
exerted potent antiproliferative action on HaCaT keratinocytes in a dose- and time-dependent manner. The IC50 values
of arsenic trioxide were 9.0, 6.9, and 5.1 ␮M; those for arsenic
pentoxide were 35.5, 25.0, and 18.6 ␮M; and those for arsenic
iodide were 19.2, 18.0, and 7.3 ␮M when the cells were
incubated for 12, 24, and 48 h, respectively. These results
demonstrated the significant growth inhibitory effect of the
arsenic compounds on HaCaT keratinocytes. The IC50 values
of arsenic trioxide, arsenic pentoxide, and arsenic iodide on
Hs-68 cells after 48-h incubation were 43.4, 223.0, and 89.0
TABLE 1
Comparison of the IC50 values of As2O3, As2O5, and AsI3 on cultured
HaCaT and Hs-68 cells
IC50
Cell Type
Incubation Time
As2O3
Hs-68
12
24
48
48
AsI3
␮M
h
HaCaT
As2O5
9.0
6.9
5.1
43.4
35.5
25.0
18.6
223.0
19.2
18.0
7.3
89.0
Fig. 1. Action of arsenic compounds on HaCaT cell morphology as examined by fluorescent microscopy. a, control HaCaT cells stained with
Hoechst. b, HaCaT cells treated with 12 ␮M arsenic trioxide. c, HaCaT
cells treated with 40 ␮M arsenic pentoxide. d, HaCaT cells treated with
24 ␮M arsenic iodide. Morphological examinations were carried out at
48 h of incubation. Note that the Hoechst-stained HaCaT keratinocytes
seemed to be shrunken, and they showed apoptotic morphology characterized by chromatin condensation.
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60, 80, and 100 ␮M; and arsenic iodide at 24, 36, 48, and 60 ␮M,
respectively, for 48 h. Trypsinized cells were pooled and stained concomitantly with annexin V and PI. The annexin V used was a chimeric
recombinant protein produced by fusing green fluorescent protein to the
N terminus of annexin V (Ernst et al., 1998). The stained cells were
subsequently analyzed by flow cytometry (BD Biosciences). The signals
were detected on the FL1 and FL3 channels, and quadrant markers
were set on dotplots of unstained and stained cells.
Western Blot Analysis of Caspase-3. A million cells seeded on
each 100-mm plate were exposed to arsenic trioxide at 6, 12, 24, and
32 ␮M, arsenic pentoxide at 40, 60, 80, and 100 ␮M, and arsenic
iodide at 24, 36, 48, and 60 ␮M, respectively, for 48 h. The cells
removed from the culture plates by scraping were lysed with lysis
buffer for 3 h, and the resultant lysates were boiled for 10 min. The
supernatant was collected and stored at ⫺20°C. The protein concentrations were measured with the bicinchoninic acid protein assay kit
(Sigma-Aldrich). Equal amounts of protein were resolved by SDSpolyacrylamide gel electrophoresis on a 15% gel. Separated proteins
were then electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), which was then blocked with 10%
nonfat milk. Afterward, the membrane was sequentially probed with
the primary anti-caspase-3 antibody (Calbiochem, San Diego, CA)
and then the secondary peroxidase-conjugated goat anti-rabbit IgG
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The
immunoreactive bands were visualized with an enhanced chemiluminescence Western blotting detection kit (Amersham Life Sciences,
Sydney, Australia) on light-sensitive films (AGFA, Mortsel, Belgium). Rainbow molecular weight markers were used as size markers for the determination of protein size.
Statistical Analysis. Data were expressed as mean ⫾ S.E.M.
Statistical comparisons between arsenic compounds treatment and
control were carried out using one-way analysis of variance, followed
by post hoc Dunnett’s test using the nontreatment as the control
group on SPSS for Windows version 14.0 (SPSS Inc., Chicago, IL).
Differences were considered significant at p ⬍ 0.05, and they were
denoted as ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; and ⴱⴱⴱ, p ⬍ 0.001.
Arsenic Compounds Induce Keratinocyte Apoptosis
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Fig. 2. TUNEL analysis of arsenic compounds-mediated apoptosis in
HaCaT cell. a, cells cultured in the absence of arsenic compounds. b to d,
cells cultured in the presence of 18 ␮M arsenic trioxide, 65 ␮M arsenic
pentoxide, and 42 ␮M arsenic iodide, respectively. Cells were incubated
for 48 h. Green, control for autofluorescence of cells in the presence of
label or enzyme solution. Black, medium control incubated with label
solution only. Red area indicates cells incubated with TUNEL reaction
mixture with both label and enzyme solution.
at 80 ␮M, and arsenic iodide at 60 ␮M were able to induce the
appearance of the sub-G1 phase, which is an indicator of the
occurrence of cellular apoptosis. After 48 h of treatment with
arsenic trioxide and when the concentration was increased
from 6 to 36 ␮M, the amount of cells in sub-G1 phase was
elevated from 0.64 to 33.7% (Fig. 4a). Likewise, the sub-G1
population gradually increased from 2.9 to 28.0% when the
arsenic pentoxide concentration was increased from 40 to 100
␮M (Fig. 4b). As for arsenic iodide, an increase of concentration from 24 to 60 ␮M corresponded to an augmentation of
sub-G1 population from 2.4 to 16.5% (Fig. 4c). Taken together, our experimental results clearly demonstrated that
the arsenic compounds tested were able to induce apoptosis
in HaCaT cells.
Quantitative Analysis of Apoptotic Cells by Annexin
V-PI Staining. The discrimination between apoptotic and
necrotic cells could be achieved by quantitatively estimating
the relative amount of the annexin V and PI-stained cells in
the population. The majority of cells were intact when exposed to lower concentration of the arsenic compounds. However, when the concentration of arsenic trioxide increased
from 3 to 36 ␮M, the percentage of the apoptotic cells was
significantly elevated from 5.5 to 63.0%; and accordingly, the
percentage of viable cells was decreased from 86.1 to 16.8%
Discussion
The use of arsenic minerals, notably realgar, in Chinese
medicine can be dated back to approximately 2000 years ago
when its application was documented in the Divine Husbandman’s Classic of Materia Medica, the first specialist book on
Chinese herbal medicines. In Chinese medicine practice, the
arsenic-containing minerals are primarily formulated into
various topical applications for dermatological conditions
such as eczema, ulcers, carbuncles, and fungi or parasitic
infestations (Lei et al., 1995). In the early 1900s, arsenic
compounds were developed for use among the first generation of chemotherapeutic agents in cancer treatment. However, the past 100 years have seen a precipitous decline in the
use of arsenics for cancer treatment; much of this was due to
the advent of other cytotoxic chemotherapeutic agents, and
in conjunction with concerns over their toxicity and carcinogenicity. In 1990s, Chinese researchers reported the dramatic clinical response with the use of arsenic trioxide in the
treatment of acute promyelocytic leukemia (Sun et al., 1991;
Zhang et al., 1996). The accumulated clinical evidence
showed that a stable solution of arsenic trioxide given by
intravenous infusion was remarkably safe and effective both
in patients with newly diagnosed and refractory acute promyelocytic leukemia (Waxman and Anderson, 2001). The
discovery resulted in renewed interests in this traditional
remedy. As a result of several decades of intensive research,
today, arsenic trioxide has been approved by the Food and
Drug Administration as the first-line chemotherapeutic
agent for the treatment of relapsed and refractory acute
promyelocytic leukemia. More recently, this inorganic salt is
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after 48 h of arsenic trioxide treatment (Fig. 5a). Likewise,
the percentage of apoptotic cells was markedly increased
from 16.9 to 68.5%, and the viable cells were significantly
decreased from 64.2 to 14.0% as the concentration of arsenic
pentoxide increased from 40 to 100 ␮M (Fig. 5b). Likewise,
when the arsenic iodide concentration increased from 24 to
60 ␮M, the apoptotic cells also were elevated from 11.7 to
61.1%, and correspondingly, the viable cells were decreased
from 75.3 to 17.0% (Fig. 5c). Because apoptotic cells in vitro
will eventually undergo “secondary necrosis,” the percentage
of necrotic cells was thus increased from 7.7 to 21.9%, from
17.7 to 19.2%, and from 12.3 to 20.6% for arsenic trioxide,
arsenic pentoxide, and arsenic iodide, respectively, as the
concentrations increased. These results unambiguously demonstrated that induction of cellular apoptosis is mainly responsible for the arsenic compound-mediated HaCaT keratinocyte growth inhibition and that the apoptotic action of
these arsenic compounds is dose-dependent.
Western Blot Analysis. Caspase-3 is the apoptosis-promoting enzyme responsible for cleaving cellular substrates
leading to the characteristic cell morphology alterations. The
results of caspase-3 activation by different concentrations of
arsenic compounds are shown in Fig. 6. The arsenic compounds were able to significantly increase the activity of
caspase-3 (19 and 17 kDa) and to decrease the procaspase-3
(32 kDa) in a dose-dependent manner. These results demonstrated that the underlying mechanism of the arsenic compound-induced apoptosis in HaCaT cells involves the cleavage of procaspase-3 into the activated form of caspase-3.
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being explored for other forms of hematological cancers, including multiple myeloma (Kalmadi and Hussein, 2006).
In an attempt to explore the potential use of arsenic-containing Chinese medicine for psoriasis treatment, we have shown in
our previous study that the extract of realgar consistently possess potent inhibitory action on the proliferation of cultured
HaCaT cells (Tse et al., 2006). In the present study, we evaluated the antiproliferative activity of three arsenic chemicals,
namely, arsenic trioxide, arsenic pentoxide, and arsenic iodide.
Our experimental results demonstrated that these arsenic compounds possess potent inhibitory action on the growth of HaCaT
keratinocytes, with arsenic trioxide being the most potent and
arsenic pentoxide the least potent. It is also worth noting that
all three arsenic compounds showed only modest inhibitory
effect on the growth of normal human fibroblast Hs-68 cells,
exhibiting discernible differential cytotoxic profiles between the
fast-growing HaCaT cells and normal human fibroblasts. This
favorable toxicity profile of the arsenic compounds is important
because it enables formulating topical applications of arsenic
compounds that could exert significant therapeutic effect without evoking harmful side effects on normal skin cells. Our data
also showed that arsenic trioxide and arsenic iodide, as trivalent salts, possessed higher inhibitory action but also higher
toxicity than the pentavalent salt arsenic pentoxide. These observations are congruent with other findings that the inorganic
trivalent salts of arsenic are generally more toxic than the
pentavalent salts (Lederer and Fensterheim, 1983).
The elucidation of the underlying cellular and biochemical
mechanisms for the observed growth inhibitory action is necessary for the bioactive arsenic compounds to be developed as
an effective therapy for psoriasis treatment. Because cellular
apoptosis and/or necrosis could be responsible for growth
inhibition of cultured cells, experiments were designed to
elucidate, at morphological, molecular, and biochemical levels, whether induction of cellular apoptosis is responsible for
the arsenic compounds-mediated growth inhibition on human keratinocytes. It is well recognized that hyperproliferation of epidermal keratinocytes seen in psoriasis is the result
of the aberrant expression of many regulatory molecules
associated with proliferation, and defects in apoptosis are
believed to play an important role in the pathogenesis of
psoriasis (Boehm, 2006). Arsenic compounds that are able to
inhibit keratinocyte proliferation and induce keratinocyte
apoptosis would conceivably possess good potential for being
developed into effective agents for treating psoriasis.
Several assays were used to detect arsenic-induced apoptosis, because no single assay is capable of unambiguously
confirming the occurrence of apoptosis. In our experiments,
arsenic compound-treated HaCaT cells were found to have
hypercondensed nuclei when stained with the Hoechst stain
followed by observation under the microscope. DNA cleavage
is a biochemical hallmark of apoptosis, and assays that measure prelytic DNA fragmentation are especially useful for the
determination of apoptotic cell death (Compton, 1992). In the
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Fig. 3. Flow cytometric analysis of cell
cycle distribution of HaCaT cells with
PI staining. a, medium control. b to d,
cells treated with 24 ␮M arsenic trioxide, 80 ␮M arsenic pentoxide, and 60
␮M arsenic iodide for 48 h, respectively. Note the appearance of sub-G1
phase upon the treatment with arsenic salts.
Arsenic Compounds Induce Keratinocyte Apoptosis
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Fig. 5. Distribution of viable, apoptotic, and necrotic HaCaT keratinocytes in the presence of arsenic compounds as measured by annexin V-PI
staining. a to c, bar chart presentation of the distribution of viable,
apoptotic, and necrotic cell populations after treatment with arsenic
trioxide, arsenic pentoxide, and arsenic iodide, respectively, for 48 h.
Fig. 4. Dose-dependent effect of arsenic compounds on the induction of
sub-G1 phase on HaCaT cells. a to c, treatment with arsenic trioxide,
arsenic pentoxide, and arsenic iodide, respectively. The incubated time
was 48 h.
current investigation, arsenic compounds were able to induce
DNA fragmentation as illustrated by gel electrophoresis. Using the TUNEL method, we further confirmed that DNA
strand breaks were induced in the HaCaT cells by arsenic
compounds. Cell cycle progression analysis by flow cytometry
revealed that arsenic compounds significantly increased the
population of HaCaT cells in the sub-G1 phase (apoptotic
peak) while reducing the number of cells in the G2/M and S
phases. This finding suggests that the arsenic compounds are
able to induce cell cycle arrest at the G1 phase, thereby
causing apoptosis in the HaCaT cells.
Early in apoptosis, phosphatidylserine is translocated from
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the inner to the outer surface of the plasma membrane.
Phosphatidylserine exposure therefore represents a useful
target for evaluating apoptosis (Fadok et al., 1992; Martin et
al., 1995; Vermes et al., 1995). Quantitative analysis of
apoptotic cells by concomitant annexin V-PI staining also
demonstrated that the arsenic compounds were capable of
inducing apoptosis on the HaCaT keratinocytes in a concentration-dependent manner. The physical destruction of the
apoptotic cells is mediated by a class of enzymes called cysteine proteases, or caspases, which are responsible for the
cleavage of specific protein substrates at an amino acid position immediately after an aspartic acid residue. Caspase-3
is the major active caspase in apoptotic cells, and its activation is the point of no return for the execution of apoptosis
(Hoshi et al., 1998; Kirsch et al., 1999). In our study, the
activation of caspase-3 was detected when the HaCaT keratinocytes were exposed to the arsenic compounds, indicating
unequivocally the occurrence of cellular apoptosis.
Taking our experimental results together, we conclude that
the arsenic compounds are capable of inducing programmed cell
death in cultured HaCaT keratinocytes. The apoptotic actions
observed in the present study provide an explanation to the
underlying mechanism of the potent antiproliferative property
exhibited by arsenic compounds on HaCaT cells. The successful
identification of arsenic compounds as potent antiproliferative
and apoptogenic agents not only places the traditional use of
arsenic-containing minerals for psoriasis on a scientific footing
but also renders them promising candidates for further development into topical therapeutic formulae for psoriasis treatment. Further in vivo experiments to evaluate the antipsoriatic
potential of several topical formulations containing arsenic
compounds on psoriasis-relevant animal models are currently
ongoing in our laboratory.
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