Acitretin induces apoptosis through CD95 signalling pathway in human cutaneous squamous cell carcinoma cell line SCL-1

Skin cancers are by far the most common human malignancies. Retinoids have shown promising preventive and therapeutic effects against a variety of human malignancies. The aim of this study was to investigate the apoptosis-inducing effect of acitretin on human skin squamous cell carcinoma (SCC) SCL-1 cells. We found that acitretin preferentially inhibited the growth of SCL-1 cells in a dose- and time-dependent manner, but not of non-malignant keratinocyte HaCaT cells. This inhibition appeared to be due to induction of apoptosis as revealed by enzyme-linked immunosorbent assay. AnnexinV/propidium iodide assay and morphological observation confirmed the pro-apoptotic effect of acitretin on SCL-1 cells. We further demonstrated that apoptosis was induced within 1–2 days and involved activation of caspases-8, -9, -3 and poly (ADP-ribose) polymerase (PARP). Caspase-8 inhibitor effectively suppressed acitretin-induced apoptosis whereas caspase-9 inhibitor did not. Acitretin increased the levels of CD95 (Fas), CD95-ligand and Fas-associated death domain. Neutralizing ZB4 anti-Fas antibody significantly inhibited the apoptosis in SCL-1 cells induced by acitretin. These results suggest that acitretin is able to induce apoptosis in skin cancer cells possibly via death receptor CD95 apoptosis pathway without affecting the viability of normal keratinocyte.

Some studies suggested that retinoid could be used as a chemopreventive and chemotherapeutic agent in various malignancies because of its anti-proliferating effect through induction of cell apoptosis [8][9][10]. Other hypotheses postulate that retinoids may affect growth factors, down-regulate proto-oncogenes and increase ceramides [11][12][13].
Apoptosis plays a critical role in carcinogenesis, its signalling pathways include death receptor (extrinsic) and mitochondrial (intrinsic) pathway [14]. The Fas (CD95)/Fas ligand (FasL; CD95L) system is an important extracellular pathway. The binding of FasL to the Fas receptor activates caspase-8 (an initiator caspase) via Fas-associated death domain protein (FADD), and the two types of cells have been defined. In type I cells, caspase-8 is sufficient to ensure execution of the final steps of apoptosis through direct activation of caspase-3 [15,16]. In type II cells, activation of caspase-8 leads to activation of mitochondria, which in turn results in cleavage of caspases-9, -3 [17,18]. On the other hand, in intrinsic pathway, caspase-9 (another initiator caspase) activates caspase-3, contributing to apoptotic cell death [19,20].
Although two major apoptotic pathways have been well defined in mammalian cells, the involvement of these two apoptotic pathways in acitretin-induced apoptosis has not been previously reported. Therefore, this study was to investigate the apoptosisinducing effect of acitretin on human SCC cell line SCL-1 and HaCaT cells.

AnnexinV/propidium iodide (PI) assay by flow cytometry
Apoptotic cells were quantified by annexinV-FITC and PI double staining using a staining kit from Roche Diagnostics. Briefly, SCL-1 cells and HaCaT cells were treated with either DMSO or retinoids dissolved in DMSO for the designated times, and then 1.0 ϫ 10 6 cells were washed with ice-cold phosphate buffer saline (PBS) and resuspended with 100 l binding buffer. Two microlitres FITC-conjugated annexinV and 2 l PI were added to the resuspended cells and incubated for 15 min. in the dark. The cells were then resuspended in 0.5 ml of binding buffer and analysed by flow cytometry.

Apoptotic morphological changes
Ultrastructural changes in cell apoptosis were determined by transmission electron microscopy. Briefly, treated and control cells in 75-cm 2

Detection of proteins by Western blotting
Cells were washed with PBS and then suspended in an extraction buffer (20 mM

Real-time PCR analysis
Real-time PCR was performed to assess Fas and FasL expression in SCL-1 cells. Briefly, total RNA was extracted from treated or untreated SCL-1 cells using Trizol reagent. Two micrograms of total RNA were reversely transcribed with TIANscript M-MLV transcriptase (Tiangen Biotechnology, Beijing, China) using random primers. The quantification of apoptosisrelated genes was carried out in triplicate for each gene. Primers for Fas receptor were sense, 5Ј-AAGTGACTGACATCAACTCC-3Ј; and antisense, 5Ј-CCACTTCTAAGCCATGTCC-3Ј, amplicon size was 270 bp. Primers for the FasL were sense, 5Ј-ACATGAGGAACTCTAAGTATCCC-3Ј; and antisense, 5Ј-CAAAATTGACCAGAGAGAGC-3Ј, amplicon size was 174 bp. Primers for ␤-actin (house keeper gene) were sense, 5Ј-TCATCACCATTG-GCAATGAGC-3Ј; and antisense, 5Ј-CAGCACTGTGTTGGCGTACAGGT-3Ј, amplicon size was 158 bp. The optimal PCR reaction was established using the Real Master Mix (SYBR Green) PCR Kit (Tiangen Biotechnology) according to the manufacturer's instructions. A relative quantification analysis (two standard curves method) was carried out with Rotor Gene 3000 software, version 6.0 (Corbett Research, Mortlake, Australia). The ratio of a target DNA sequence to a reference ␤-actin sequence in samples from untreated cells was served as a 'calibrator'. The results are expressed as a normalized ratio.

Assays for Fas
The expression of cell surface Fas was measured by immunofluorescence flow cytometry. After various treatments, a total of 1.0 ϫ 10 6 cells was collected by centrifugation, and washed twice with ice-cold PBS containing 1% bovine serum albumin. Cells were then incubated with 100 l of FITCconjugated anti-Fas antibody or control mouse IgG-FITC antibody for 40 min. at 4ЊC. After incubation in the dark, cells were washed twice and resuspended in ice-cold PBS. Immunofluorescence staining of cell surface Fas was analysed by flow cytometry. For Fas inhibitor assay, neutralizing ZB4 anti-Fas antibody was added at 1 g/ml 10 min. before treatment with acitretin for 24 hrs. Apoptosis was analysed by the ELISA assay as described above.

Inhibition of caspase activity
SCL-1 cells that were induced to undergo apoptosis were cultured in the presence or absence of caspase inhibitors that bind to the active site of their respective protease. The following caspase inhibitors were used: Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor). Inhibitors were dissolved in DMSO at a concentration of 10 M and diluted with normal culture medium to a final concentration of 10

Effects of acitretin on the cell proliferation of cell line SCL-1
We investigated the effects of acitretin on cell proliferation in SCL-1 and HaCaT at five different concentrations of acitretin for 3 days and 10 Ϫ5 M of acitretin for 1, 3, 5 days using a MTT assay (Fig. 1A and B). The results showed that acitretin inhibited cell growth of SCL-1 in a dose-and time-dependent manner. In contrast, acitretin exhibited a few inhibitory effects on the proliferation of HaCaT cells, indicating that acitretin showed less toxicity to nonmalignant keratinocytes.

Effects of acitretin on apoptosis in SCL-1 cells
The induction of apoptosis was determined by the Cell Death Detection ELISA, and the results showed acitretin induced apoptosis of SCL-1 cells in a dose-and time-dependent manner ( Fig. 2A  and B). However, the pro-apoptotic effect of acitretin on HaCaT was much less prominent than that on SCL-1. AnnexinV and PI double staining were performed to confirm the above findings in cultured SCL-1 cells and HaCaT cells incubated with either vehicle or acitretin for 1, 2 and 3 days. AnnexinV-positive apoptotic cells were found to increase time dependently in acitretin-treated SCL-1 cells, consistent with the results of ELISA (Fig. 2C and D

Effects of acitretin treatment on caspase activation
Since caspases play a pivotal role in the apoptosis signalling pathway [24,25], we examined the activation of caspases-8, -9, -3 by both active and inactive forms using Western blot analysis. An active fragment of caspase-8 occurred 12 hrs after acitretin treatment, this fragment increased time dependently, whereas the corresponding caspase-9 and caspase-3 active fragments occurred 24 hrs after acitretin treatment (Fig. 3A). To confirm the presence of active caspase-3 in acitretin-treated cells, the cleavage of PARP was determined. After treatment with acitretin for 24 hrs, the fulllength form of the PARP protein (116 kD), a substrate for caspase-3, was degraded into the cleaved form (85 kD; Fig. 3A). These results suggest that acitretin-induced apoptosis is associated with the activation of caspases-8, -9, -3 and caspase-8 may be the first caspase activated by acitretin in SCL-1 cells.
In order to confirm the role of caspases in acitretin-induced apoptosis, Cell Death Detection ELISA was performed with inhibitors of caspases-8 and -9. These caspase inhibitors, when used alone, did not exert any effect on the apoptosis of SCL-1 cells. However, combined treatment with inhibitor of caspase-8 effectively blocked acitretin-induced apoptosis in SCL-1 cells, whereas with inhibitor of caspase-9 failed to do so (Fig. 3B). These results indicate that acitretin-induced apoptosis is dependent on the activation of caspase-8.

Induction of cell surface Fas protein and mRNA expressions of Fas/FasL during acitretin-induced apoptosis
The fact that caspase-8 inhibitor was found to block acitretininduced apoptosis dramatically in SCL-1 cells encourage us to further investigate whether the cell surface Fas receptor is affected by acitretin with flow cytometric analysis using an anti-Fas monoclonal antibody. The results revealed that the level of the cell surface Fas receptor was significantly increased in acitretin-treated cells compared with the untreated controls (Fig. 4). Accordingly, the results with relative quantification analysis also demonstrated that both Fas and FasL mRNAs increased at 12 hrs after the addition of acitretin and continued to increase substantially in a timedependent manner (Fig. 5, Table 1).

Effects of acitretin on protein expressions in Fas apoptotic signalling pathway
To be sure whether acitretin affects the death receptor apoptotic pathway, we checked the status of Fas, FasL and FADD proteins. The results shown in Fig. 6

Fig. 1 Effects of acitretin on cell growth of SCL-1 cells. (A) SCL-1 cells and HaCaT cells were treated with acitretin at five different concentrations for 3 days. (B) SCL-1 cells and HaCaT cells were treated with 10 Ϫ5 M of acitretin for various times. Cell proliferation was assessed by MTT assay. Cell viability was estimated from the equation: % of cell viability ϭ 100 ϫ At/Ac, where At and Ac are the absorbencies in treated and control cultures, respectively. Data are shown as median of three different independent experiments (each with three cultures).
just after 12 hrs exposing to acitretin, and these changes were well matched to the appearance of the active caspase-8 fragment (Fig. 3A) Fig. 7, ZB4 alone did not exert any effect on the

Discussion
Acitretin, the second generation of retinoids, was reported to exhibit anti-tumour activity in actinic keratosis, verrucous carcinomas, SCC without significant side effects or toxicity [2,3,5,6]. However, the mechanism of its anti-carcinogenic effect is not fully understood. In this report, we demonstrate for the first time that acitretin-induced apoptosis in skin SCC cell line SCL-1 via CD95/CD95L apoptotic signalling pathway.
We found that SCL-1 cells were sensitive to the growth inhibitory effects of acitretin, which correlated with apoptosis induction. The apoptosis induction was further confirmed by annexinV/PI assay and apoptotic changes in morphology under electron microscope. Because caspases are central components in apoptotic signalling pathways [25,26,27], the involvement of caspases was examined. Among the caspases, caspases-3, -8 and -9 have been shown to play important roles in apoptosis [28], and caspase-3 is a key executioner involved in apoptosis. Caspase-3 activity is controlled by upstream regulators, such as caspase-9 or caspase-8, which modulates the mitochondria-and death receptor-dependent pathway, respectively [29]. Acitretin activated caspases-3, -8 and -9, it is plausible that both the mitochondrial and the death receptor pathways might be activated by acitretin. However, in our study, the inhibitor of caspase-9 had no significant effect on acitretin-induced apoptosis, indicating that intrinsic pathway did not play the major role in the apoptosis. Caspase-8 inhibitor effectively suppressed acitretin-induced apoptosis, implying that acitretin-induced apoptosis is dependent on the extrinsic pathway because caspase-8 is primarily activated during the death receptor apoptotic pathway. To determine whether the  death receptor pathway is really involved in acitretin-induced apoptosis, we evaluated the status of the cell surface Fas receptor, which is a representative member of the death receptor family, is affected by acitretin-treated cells. Interestingly, the expression of Fas on the plasma membrane was significantly increased 48 hrs after acitretin treatment. Corresponding to the appearance of the active caspase-8 fragment (Fig. 3A), similar changes were also detected by quantitating both mRNA and protein for Fas receptor using RT-PCR and Western blot (Figs 5 and 6, Table 1); these results indicate that the apoptotic pathway related to Fas seems to be involved in acitretin-induced apoptosis.
Fas death receptor can be activated to undergo oligomerization by binding its ligand FasL, then the activated Fas recruits the adapter molecule FADD by the death domain, and procaspase-8 into a complex resulting in activated caspase-8. We demonstrated that FasL levels were increased in SCL-1 cells during the treatment with acitretin by RT-PCR and Western blotting (Figs 5 and 6, Table 1). In addition, up-regulation of Fas, FasL and FADD during acitretin treatment corresponded to activation of caspase-8 (Figs 3A and 6).

These results suggest that signalling by the CD95/CD95L system may play an important role in the induction of apoptosis of SCL-1 cells by acitretin. The involvement of the Fas-mediated pathway in acitretin-induced apoptosis was corroborated by further investigating the effects of neutralizing ZB4 anti-Fas antibody on acitretininduced apoptosis by disturbing the Fas-FasL interaction. ZB4 was found to inhibit the apoptosis induced by acitretin in SCL-1 cells, suggesting that Fas receptor-ligand interaction was involved in acitretin-induced apoptosis pathway.
In CD95/CD95L apoptotic signalling, the mitochondrial activation-mediated pathway is required in type II cells [30,31]. Activation of caspase-8 induces the release of cytochrome c from the mitochondria [18]. Cytochrome c, with the help of apoptotic protease activating factor-1, activates caspase-9 and in turn activates caspase-3 [32], which is also transmitted in type I cells [33]. Because mitochondrial pathway is obligatory in mediating the Fas signalling in type II cells, any damage to mitochondrial pathway can only inhibit Fas-induced apoptosis in these cells, but not type I cells [34]. In this study acitretin substantially induced the activation of caspase-9; however, caspase-9 inhibitor failed to affect acitretin-induced apoptosis, whereas caspase-8 inhibitor or ZB4 effectively suppressed acitretin-induced apoptosis. From these findings, we conclude that activation of caspase-9 is triggered by Fas and acitretin enhances a type I CD95/Fas-mediated apoptotic signalling pathway in SCL-1 cells.
Skin cancers are by far the most common human malignancies [35], although the surgical removal is the major therapeutic modality, but it is practically limited in treating multiple lesions. Particularly in OTR patients with the high risk of transition from pre-malignant to malignant cutaneous lesions, the therapeutic options are limited to the local excision, cryotherapy, topical chemotherapeutic agents and radiotherapy [36]. The presence of multiple cancerous lesions makes it difficult to effectively treat cutaneous malignancies at the same time by dermatosurgery. Therefore, the preventative or suppressive modalities have been recommended in the population with high risk of SCC, and also with actinic keratosis, verrucous carcinomas. Oral retinoids, specifically acitretin, have been shown to have chemosuppressive effects against skin cancers in OTR [2]. Several uncontrolled trials, case reports and a few small controlled trials have supported the use of oral acitretin for chemoprevention of skin cancer in this population [7,37,38]. For instance, Bavinck et al. [39] [13]. In this study, acitretin remarkably inhibited cell proliferation of skin SCC cells by the