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CELLULAR AND MOLECULAR
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
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; 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 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.
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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Materials and Methods |
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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 µg/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 x 104 cells in 200 µl 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 µM, from 250 to 1 µM, and from 250 to 1 µM, 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-2-yl)-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 µl 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 µM 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 x 105 HaCaT cells per well were seeded in six-well plates. The cells were treated with 12 µM arsenic trioxide, 40 µM arsenic pentoxide, and 24 µM 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 µg/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 µM arsenic trioxide, 120 µM arsenic pentoxide, and 72 µM arsenic iodide for 48 h. After harvest, cells were lysed in 200 µl of DNA lysis buffer at 37°C for 15 min. The supernatant was sequentially incubated with 0.4 µg/ml RNase and then with 1.5 µg/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 x g for 30 min. Finally, 30 µl 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 µl 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 Nick-End 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 x 105 HaCaT cells per well were seeded on a six-well plate and exposed to 18 µM arsenic trioxide, 65 µM arsenic pentoxide, and 42 µM 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 µl 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 x 105 HaCaT cells per well seeded on six-well plates were exposed to arsenic trioxide at 6, 12, 24, and 36 µM; arsenic pentoxide at 40, 60, 80, and 100 µM; and arsenic iodide at 24, 36, 48, and 60 µM, 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 µg/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 x 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 µM; arsenic pentoxide at 40, 60, 80, and 100 µM; and arsenic iodide at 24, 36, 48, and 60 µM, 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 µM, arsenic pentoxide at 40, 60, 80, and 100 µM, and arsenic iodide at 24, 36, 48, and 60 µM, 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 SDS-polyacrylamide 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 *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
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Results |
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Alteration of Cellular Morphology. After exposure to 12 µM arsenic trioxide, 40 µM arsenic pentoxide, and 24 µM 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.
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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 µM, 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% 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 µM (Fig. 5b). Likewise, when the arsenic iodide concentration increased from 24 to 60 µM, 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.
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Discussion |
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). 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 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 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 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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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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.
Address correspondence to: Dr. Zhi-Xiu Lin, School of Chinese Medicine, Faculty of Science, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. E-mail: linzx{at}cuhk.edu.hk
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