Curcumin Induces Apoptosis in Tumor Necrosis Factor-Alpha-Treated Hacat Cells

International Immunopharmacology 13 (2012) 170–174

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International Immunopharmacology

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Curcumin induces apoptosis in tumor necrosis factor-alpha-treated HaCaT cells

Jun Sun, Jinzhao Han, Yi Zhao, Quangang Zhu, Jinhong Hu ⁎

Department of Pharmacy, Changhai Hospital, the Second Military Medical University, Shanghai 200433, China article info abstract

Article history: Psoriasis is a benign, chronic skin disease characterized by keratinocyte hyperproliferation and abnormal Received 5 February 2012 differentiation. Curcumin, a selective phosphorylase kinase inhibitor, is a natural phytochemical present in Received in revised form 15 March 2012 turmeric. Curcumin has been confirmed to have anti-inflammatory properties as well as the ability to inhibit Accepted 27 March 2012 proliferation and decrease the expression of pro-inflammatory cytokines in psoriatic keratinocytes. However, Available online 10 April 2012 the pro-apoptotic effect of curcumin in keratinocytes remains unclear. In the present study, we investigated the effect of curcumin on apoptosis induction in TNF-α-treated HaCaT cells. These results show that curcumin Keywords: fi α Curcumin exhibited a signi cant pro-apoptotic effect on HaCaT cells only in the presence of TNF- and/or TRAIL. The Apoptosis pro-apoptotic effect of curcumin resulted from the increased expression of TRAIL-R1/R2 and the decreased Keratinocyte expression of anti-apoptotic proteins. Our results indicate that both curcumin and TNF-α up-regulated the

TNF alpha expression of TRAIL-R1/R2. In addition, the expression of anti-apoptotic proteins (IAP1, IAP2, Bcl-XL) was up- TRAIL receptor regulated by TNF-α but suppressed by curcumin in HaCaT cells. Because these proteins are regulated by NF- κB, we examined the role of curcumin in NF-κB activation. As expected, curcumin inhibited TNF-α-induced activation of NF-κB, including NF-κB-P65. Curcumin also inhibited the TNF-α-induced production of IL-6/IL- 8 in HaCaT cells. These results imply that curcumin-induced apoptosis of HaCaT cells only occurs when TNF-α or/and TRAIL are present. Therefore, we believe that curcumin is able to reverse the anti-apoptotic function of TNF-α in HaCaT cells and thus expect curcumin to be successful in the treatment of psoriasis. © 2012 Elsevier B.V. All rights reserved.

1. Introduction curcumin in the treatment of active psoriatic plaques, Pol [12] found that curcumin notably inhibited keratinocyte proliferation, and Miquel Tumor necrosis factor-alpha (TNF-α) is an important cytokine [13] reported that curcumin decreases pro-inflammatory cytokine that mediates immune response, inflammation and apoptosis [1]. expression in keratinocytes. However, the effect of curcumin on TNF- Only a low level of TNF-α is present in the upper layer of the healthy α-treated keratinocyte apoptosis remains unclear. epidermis. However, abnormal production of TNF-α is associated This study was designed to investigate the effect of curcumin on with a wide spectrum of diseases, including psoriasis [2,3]. Anti-TNF apoptosis in TNF-α-treated human keratinocyte cell line (HaCaT). agents developed by rheumatologists have presented impressive effects in the treatment of severe plaque psoriasis and psoriatic arthritis and have been proven to be effective therapeutic strategies 2. Materials and methods by blocking the TNF-α pathway in psoriasis [4,5]. TNF-α promotes apoptosis through binding to TNF-receptor1. However, psoriatic 2.1. Chemicals lesions are usually hyper-proliferative despite increased TNF-α. This paradox is partially explained by NF-κB activation, which inhibits The chemicals used in this study include TNF-related apoptosis- TNF-α-induced apoptosis [6]. inducing ligand (TRAIL) and recombinant human TNF-α (PeproTech, Curcumin is a component of turmeric and has been used as a London, UK); curcumin and propidium iodide (PI) (Sigma, St. Louis, medical remedy in Southeast Asia for centuries. This compound has MO, USA); Human Annexin V Apoptosis Detection Kit (Bender anti-carcinogenic, anti-inflammatory and antioxidant properties [7–9], MedSystems, San Diego, USA); antibodies against human TRAIL and evidence has shown that its anti-inflammatory role arises as a receptor (TRAIL-R)- TRAIL-R1 to R4) (Alexis, Plymouth Meeting, result of inhibiting NF-κB activation [10]. As a selective phosphorylase USA); purified mouse IgG1, biotin anti-mouse IgG1 and streptavidin- kinase inhibitor, several previous studies have shown curcumin's phycoerythrin (Biolegend, San Diego, CA); antibodies against NF-κB- potential utility in the treatment of psoriasis. Heng [11] topically used p65, IAP1, IAP2, XIAP, Bcl-XL, c-FLIP, survivin and TRAIL (Cell Signal Technology, Boston, USA); and human IL-6, IL-8 ELSA Testing Kit (R&D, Minneapolis, USA). Curcumin was dissolved in 50% propylene − ⁎ Corresponding author. Tel.: +86 2181873746; fax: +86 2181873747. glycol PBS solution, stored at 20 °C and further diluted in medium E-mail address: [email protected] (J. Hu). prior to each experiment.

2.2. Cell culture 2.4. Apoptosis assays

HaCaT cell lines were maintained and passaged in RPMI-1640 Using the Annexin V Apoptosis Detection Kit combined with flow medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) cytometry, the effect of curcumin on the induction of apoptosis in and penicillin (100 U/mL)/ streptomycin (100 μg/mL) in our labora- HaCaT cells was observed. After incubation with curcumin, 2×105 tory. Cultures were maintained in a humidified incubator at 37 °C HaCaT cells were harvested, washed with PBS and resuspended in with 5% CO2. 200 μL binding buffer. After the addition of 5 μL Annexin V conjugate and a 10 min incubation, the samples were resuspended in 200 μL binding buffer and 5 μL propidium iodide (PI) and analyzed using 2.3. Flow cytometric analysis of TRAIL receptors MACSQuant® Analyzer (Miltenyi Biotec, Germany). Annexin V-positive cells were designated as apoptotic cells. The data were analyzed using To examine the surface staining of TRAIL receptors in HaCaT cells, FlowJo software. flow cytometric analysis was performed as described previously [14]. Briefly, 2×105 HaCaT cells were trypsinized, washed with PBS, and 2.5. Western blot analysis incubated with monoclonal antibodies against TRAIL-R1 to R4 or isotype-purified mouse IgG1 at 4 °C for 20 min and then with secondary To determine the level of protein expression in the cytoplasm and biotinylated anti-mouse IgG1 antibody and phycoerythrin-labeled nucleus, cell lysates were prepared as described elsewhere [15].A streptavidin under the same conditions. The working concentration of total of 20–40 μg of protein was subjected to SDS-PAGE, separated by the antibodies in the experiments was 10 μg/mL. A total of 2×104 cells electrophoresis and transferred to a polyvinylidene difluoride (PVDF) were analyzed using MACSQuant® Analyzer (Miltenyi Biotec, Germany). membrane. The membrane was incubated with primary antibody

Fig. 1. Effects of curcumin on TRAIL and its receptors expression. (A) Curcumin enhanced TRAIL-R1/R2 expression. HaCaT cells were seeded in 12-well plates, incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 16 h. The cell TRAIL receptors were detected by flow cytometry as described in Materials and methods section. (B) TRAIL expression. HaCaT cells were incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 16 h. Whole-cell extracts were prepared and analyzed by Western blotting using an anti-TRAIL antibody. The results shown are representative of 3 independent experiments. Cur, curcumin. 172 J. Sun et al. / International Immunopharmacology 13 (2012) 170–174 followed by incubation with the species-appropriate secondary anti- TNF- α-induced NF-κB activation, nuclear extracts from TNF-α body coupled to horseradish peroxidase as described with minor stimulated cell were incubated with a primary antibody against the modifications [16]. Bands were visualized using an ECL detection kit. p65 (RelA) subunit of NF-κB. These results suggest that the NF-κB complex inhibited by curcumin contains the p65 subunit (Fig. 3 B). 2.6. Electrophoretic mobility shift assay (EMSA) 3.4. Curcumin inhibits the expression of TNF-α-induced NF-κB- EMSA was performed using nuclear extracts of HaCaT cells as dependent anti-apoptotic proteins described previously [17]. Because apoptosis is regulated by anti-apoptotic proteins, such as 2.7. ELISA assay IAP1, IAP2, XIAP, BcL-XL, c-FLIP and survivin [18], we examined whether curcumin modulated the expression of anti-apoptotic proteins in HaCaT IL-6 and IL-8 secretion levels from HaCaT cell cultures were cells following TNF- α treatment (Fig. 3C). The results of western blot measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA). analysis show that Bcl-X , IAP1 and IAP2 expression were enhanced by The supernatants were centrifuged to remove cell debris and stored at L TNF-α but inhibited by curcumin. In this experiment, the expression of -80 °C before being assayed. XIAP, survivin and c-FLIP was undetected. 2.8. Statistics 3.5. Curcumin inhibits TNF-α-induced IL-6 and IL-8 production in HaCaT All experiments were performed in triplicate with a minimum of cells three replicates. The means and standard deviations were calculated. The significance of the differences among the treatments was determined Following curcumin or TNF-α treatment, the HaCaT cell media using either an analysis of variance (ANOVA) or the Kruskal–Wallis test. was harvested for ELISA. These results show that curcumin markedly A P-value of 0.05 was considered statistically significant. decreased the TNF-α-induced production of IL-6 and IL-8 in cultured HaCaT cells (Fig. 3 D and E). 3. Results

The goal of this study was to evaluate the effect of curcumin on apoptosis induction and the inhibition of NF-κB in HaCaT cells. Because TNF-α is known to play an important role in the pathogenesis of psoriasis, cells were treated with TNF-α to examine the effect of curcumin on HaCaT cells.

3.1. Curcumin enhances the expression of TRAIL receptors in HaCaT cells

To study the effect of curcumin and TNF-α on the expression of TRAIL and TRAIL receptors, HaCaT cells were exposed to a combination treatment of 7.37 μg/mL curcumin and 20 ng/mL TNF-α for 24 h; the cells were then harvested for western blot and ﬂow cytometric analyses. As shown in Fig. 1 A, TRAIL-R1/R2 expression increased markedly after TNF-α and curcumin treatment. A similar increase in the expression of TRAIL-R3/R4 was also observed. TRAIL expression increased after treatment with both TNF-α and curcumin but remained unchanged upon single-agent treatment with either compound (Fig. 1 B).

3.2. Curcumin induces apoptosis in HaCaT cells

The effect of curcumin and TNF-α on apoptosis was examined using a flow cytometric Annexin V assay. After exposure to 100, 200 and 400 ng/mL of TRAIL, the percentage of apoptotic cells fluctuated between 1.00% and 1.79%, showing insignificant differences among the experimental groups. (Fig. 2 A). In another experiment, we examined the effect of curcumin on HaCaT apoptosis as modulated by TNF-α and TRAIL (Fig. 2 B). Compared with the control groups, curcumin treatment did not lead to a significant increase in the percentage of apoptotic cells, but the combination treatment of TNF-α and TRAIL led to an increase in the percentage of apoptotic cells to 16.12%–20.83%. These results indicate that curcumin plays a pro- apoptotic role in combination with TNF-α or TRAIL in HaCaT cells. Fig. 2. Effects of curcumin on HaCaT cell apoptosis. (A) Apoptosis of HaCaT cells α κ induced by TRAIL. HaCaT cells were seeded in triplicate in 12-well plates. Cells were 3.3. Curcumin inhibits TNF- -induced NF- B activation treated with TRAIL at three concentrations (100, 200 and 400 ng/mL) and cultured for 16 h. Cell apoptosis was detected by flow cytometry as described in Materials and To investigate the effect of curcumin on TNF-α-induced NF-κB methods section. (B) Apoptosis of HaCaT cells induced by curcumin, TNF-α and TRAIL. activation, HaCaT cells were pretreated with 7.37 μg/mL curcumin for HaCaT cells were seeded in triplicate in 12-well plates. Cells were incubated with 7.37 μg/mL curcumin for 4 h, and then continued with 20 ng/mL TNF-α or 100 ng/mL 24 h and then stimulated with 20 ng/mL TNF-α for 30 min. As indicated TRAIL for 16 h. Apoptosis detection was the same as above. The results shown are α κ by EMSA, TNF- promoted NF- B activation in HaCaT cells, while representative of 3 independent experiments. *Pb0.05 vs. the blank group. Cur, curcumin inhibited this activation. (Fig. 3 A). To further examine the curcumin. J. Sun et al. / International Immunopharmacology 13 (2012) 170–174 173

4. Discussion with TNF-α. Moreover, curcumin also induces apoptosis when TRAIL is present in HaCaT cells. In another experiment, TRAIL failed to show Psoriasis is a chronic skin disease characterized by keratinocyte any pro-apoptotic effects on HaCaT cells. We believe that TNF- α hyperproliferation and anti-apoptotic features [19]. Over-expression treatment is also responsible for this result, which leads us to the of TNF-α is a key element in pathogenesis of psoriasis vulgaris and hypothesis that curcumin could reverse the anti-apoptotic features of psoriatic arthritis. Previous studies in vitro [20] show that serum or psoriatic lesions. synovial ﬂuid concentrations of IL-1, IL-6, IL-8 and TNF-α were TRAIL and its receptors (TRAIL-R1/R2) are well-known key regula- increased in psoriasis patients. Blocking the TNF-α pathway has tors of cellular apoptosis. But there still had evidence to support that become an important option in the treatment of psoriasis [4,5]. TRAIL sensitivity in keratinocytes is primarily regulated at the A recent clinical study [21] showed that TNF-α, TRAIL-R1/R2 and intracellular level rather than at the receptor level [22]. TRAIL-R1/R2 TRAIL expression in psoriatic lesions was up-regulated compared expression can be up-regulated by P53 [23,24] and NF-κBP65[25],and with the healthy epidermis; however, the keratinocytes of psoriatic TRAIL-R2 can also be regulated by SP1 [26,27].NF-κB P65 activity lesions show opposite anti-apoptotic features. TNF-α may contribute promoted by TNF-α may be responsible for the up-regulation of TRAIL- to this pathogenesis because, as shown herein, it enhanced the R1/R2 expression. Although TRAIL-R1/R2 and anti-apoptotic protein expression of anti-apoptotic proteins. expression are NF-κB dependent, we observed differential responses In this study, curcumin shows a pro-apoptotic effect on HaCaT modulated by curcumin. Therefore, we predict that curcumin and TNF- cells only when combined with TNF-α treatment. Concurrently, we α up-regulate TRAIL-R1/R2 in different ways. More detailed studies are found that TRAIL-R1/R2 expression was signiﬁcantly up-regulated by needed to support this hypothesis. curcumin and that the increase in TRAIL expression was also Additionally, our results show that curcumin inhibited TNF-α- modulated by the combination treatment of TNF-α and curcumin. induced NF-κB activation and the production of IL-6/8 in HaCaT cells,

In addition, TNF-α-induced IAP1, IAP2 and Bcl-XL increases in which is consistent with our prior conclusion [28]. Extensive evidence expression were inhibited by curcumin. Our results clearly demon- supporting the anti-inﬂammatory properties of curcumin in different strate that curcumin exhibits pro-apoptotic effects in combination cell types has previously been shown. We believe that curcumin can

Fig. 3. Effects of curcumin on NF-κB, anti-apoptotic proteins and IL-6/8. (A) Curcumin inhibits NF-κB activation. HaCaT cells were incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 30 min. The nuclear extracts were assayed for NF-κB activation by EMSA. (B) Curcumin inhibits nuclear translocation of P65. HaCaT cells were incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 1 h. The nuclear extracts were prepared and analyzed by Western blotting using anti-P65 antibody. 6 (C) Curcumin modulates the expression of anti-apoptotic gene products including IAP1, IAP2 and Bcl-XL. HaCaT cells (2×10 /mL) were incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 16 h. Whole-cell extracts were prepared, and 30 μg whole-cell lysate was analyzed by Western blotting using antibodies against IAP1, IAP2, Bcl- xl, XIAP, cFLIP, and survivin as indicated. (D) Curcumin inhibits HaCaT cells to produce IL-8. HaCaT cells (1×106/mL) were incubated with 7.37 μg/mL curcumin for 4 h, and then with 20 ng/mL TNF-α for 24 h. The medium was harvested for IL-8 ELISA assay. (E) Curcumin inhibits HaCaT cells to produce IL-6. Cell pretreatment was the same as above. The medium was harvested for IL-6 ELISA assay. The results shown are representative of 3 or 5 independent experiments. *Pb0.05 vs. the TNF group; **Pb0.05 vs. the blank group. Cur, curcumin. 174 J. Sun et al. / International Immunopharmacology 13 (2012) 170–174 reduce keratinocyte-linked inflammation by inhibiting NF-κB activa- [14] Leverkus M, Sprick MR, Wachter T, Mengling T, Baumann B, Serfling E, et al. Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by tion. In view of report [29] on permeation study of curcumin with removing the resistance-mediating block of effector caspase maturation. 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