NEMO-binding domain peptide inhibits proliferation of human melanoma cells

Cancer Letters 274 (2009) 331–336

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NEMO-binding domain peptide inhibits proliferation of human melanoma cells Angela Ianaro a,*, Mariaroberta Tersigni a, Giuseppe Belardo b, Silvana Di Martino c, Maria Napolitano d, Giuseppe Palmieri e, MariaCristina Sini e, Anna De Maio f, MariaNeve Ombra g, Giuseppina Gentilcore c, Mariaelena Capone c, MariaLibera Ascierto c, Rocco Alfredo Satriano h, Benedetta Farina f, MariaRosaria Faraone-Mennella f, Paolo Antonio Ascierto c,1, Armando Ialenti a,1 a

Department of Experimental Pharmacology, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy Department of Biology, University of Rome Tor Vergata, Rome, Italy Medical Oncology and Innovative Therapy National Tumor Institute, Fondazione Pascale, Naples, Italy d Clinical Immunology National Tumor Institute, Fondazione Pascale, Naples, Italy e Unit of Cancer Genetics, Institute of Biomolecular Chemistry, C.N.R., Sassari, Italy f Department of Biochemical and Biochemistry, Federico II University of Naples, Italy g Institute of Food Sciences, C.N.R., Avellino, Italy h Clinical Dermatology, Second University of Naples, Italy b c

a r t i c l e

i n f o

Article history: Received 21 July 2008 Received in revised form 23 September 2008 Accepted 30 September 2008

Keywords: Apoptosis Melanoma NBD peptide NF-jB

a b s t r a c t Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-jB (NF-jB) pathway. In vitro studies have shown that NF-jB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-b NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFa-induced NF-jB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-jB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-jB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-jB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process. Ó 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has in* Corresponding author. Tel.: +39 081678663; fax: +39 081678403. E-mail address: [email protected] (A. Ianaro). 1 These Authors contributed equally. 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.09.038

creased in the last decade. Important predisposing factors are considered sun exposure and genetic susceptibility [1]. Due to the complex nature of the disease, metastatic melanomas have proven to be typically resistant to radiation, immunotherapy and biochemotherapy (combining conventional chemotherapies like dacarbazine and IL-2 or IFN-a), suggesting that some melanomas maintain an anti-apoptotic phenotype, thus achieving both advanced

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survival capacity and resistance to chemotherapeutic agents [2]. Currently, there is a critical need for the development of therapeutic agents that could target aberrant survival pathways in metastatic melanomas and improve the treatment of this disease [3]. Recent advances in the understanding of the underlying biology in progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-jB (NF-jB) pathway [1]. NF-jB is a critical regulator of the immediate early pathogen response, playing an important role in inflammation, cell proliferation and survival, as well as in the regulation of virus replication. The mammalian NF-jB family contains five proteins, RelA/p65, NFjB1 (p50), NF-jB2 (p52), c-Rel and RelB, which can form a variety of homodimers and heterodimers to differently control gene expression [4]. A wide range of stimuli, including cytokines, viral and bacterial products, activate NF-jB, mostly through IjB kinase (IKK)-dependent phosphorylation and subsequent degradation of specific inhibitors, the IjBs, that retain NF-jB in the cytoplasm. Upon activation, NF-jB dimers enter the nucleus, where they modulate transcription of many genes encoding cytokines, growth factors, cell adhesion molecules, and antiapoptotic proteins [5]. The IKK complex is composed of three subunits: the catalytic subunits IKK-a and IKK-b, and the regulatory subunit IKK-c (also known as NEMO). It has been previously reported that a very small region in the COOH terminus of IKK-a and IKK-b was essential for stable interaction with NEMO and for the assembly of the heteromeric IKK–NEMO complex. This region was termed the NEMO-binding domain (NBD) [6]. It has also been found that a short cell-permeable peptide spanning the IKK-b NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFa-induced NF-jB activation in vivo. Moreover it has been demonstrated that administration of the NBD peptide effectively ameliorates inflammatory responses in animal models of inflammation without overt signs of toxicity [7]. Recently, NF-jB activation has been connected with multiple aspects of oncogenesis, including the control of apoptosis, cell migration, cell cycle progression, and cell differentiation. Numerous studies have indicated that NF-jB activation also suppresses cell death pathways by switching on genes that dampen proapoptotic signals [8]. In vitro studies have shown that NF-jB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. This observation correlates with data from clinical specimens as well, where Rel A expression is significantly elevated in human nevi and melanomas relative to normal skin. This elevation in expression also correlated with increased phosphorylation and nuclear translocation of Rel A [1]. In this perspective, inhibition of NF-jB is expected to be an ideal therapeutic target in those tumours where NF-jB appears to play a unique survival role. In the present study we investigated the effect of the NBD peptide on NF-jB activity and survival of A375 hu-

man melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-jB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-jB DNA-binding activity. We also show that the inhibition of A375 cell growth is due to NBD peptide large induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process. Moreover, this event could be directly correlated to the inhibition of NF-jB-DNA binding activity. In fact it has been widely demonstrated that unmodified PARP-1 negatively down regulates formation of NF-jB-DNA complex via its physical association with the transcription factor [9]. 2. Materials and methods 2.1. Reagents and cell culture Wild-type and mutant NBD peptides were purchased from Genosphere Biotech (Paris, France). The human melanoma cell line A375 and normal human epidermal melanocytes (NHEM) were purchased from PromoCell (Heidelberg, D-69126, Germany). A375 were grown in complete Dulbecco’s modified Eagle’s medium (DMEM). NHEM were grown in Melanocyte growth medium 2 (PromoCell). Cells were grown at 37 °C in a humidified incubator under 5% CO2. To determine cell viability A375 cells were plated at 1  105 in 6-well plates and in 2 mL DMEM complete medium containing different concentrations of the NBD peptide. After incubation for 24 h, cells were collected and an aliquot of cell suspension was mixed with equal volume of trypan blue 0.4%. Viability was determined at the designed intervals by vital-dye exclusion assay. 2.2. Electrophoretic mobility shift assay (EMSA) Aliquots of whole-cell extracts (12 lg protein/sample) were incubated 32P-labeled jB DNA probe in binding buffer for 30 min as described [8]. DNA-protein complexes were analyzed by non-denaturing 4% polyacrylamide gel electrophoresis. Quantitative evaluation of NF-jB-jB DNA complex formation was determined by densitometric analysis performed with a GS-700 imaging densitometer (Bio-Rad, Italy) and a data computer program (Molecular Analyst; IBM). 2.3. Flow cytometry In order to asses the cells viability, 1  106 A375 cells were stained with 20 lL 7-AAD (BD Biosciences, San Diego, CA) and incubated at 4 °C protected from light for approximately 20 min or until analysis on the flow cytometer. The active form of caspase-3 was measured by FACScalibur cytometer (BD Biosciences, San Diego, CA) using the PE-conjugated anti-human-active caspase3 monoclonal antibody (mAb) (BD Biosciences, San Diego, CA).

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2.4. PARP electophoretic analyses and immunoblotting

3.3. Inhibiting NF-jB/p65 nuclear translocation leads to A375 cells apoptosis

PARP-1 electrophoretic analyses and immunoblotting were performed as previously described [10].

The loss of plasma membrane integrity can be evidenced by cell permeability to vital fluorescent DNA dyes such as 7-AAD. As shown in Fig. 2A, the percentage of early apoptotic cells, defined by low 7-AAD staining, is 28.8%, 34.0%, 49.0% and 62.1% for the cells treated with NBD peptide at the concentration of 6.25, 12.5, 25 and 50 lM, respectively (untreated cells 9.3%). To characterize the mechanism underlying the inhibition of proliferation, A375 melanoma cells were exposed to the NBD peptide (6.25, 12.5, 25, and 50 lM) for 3 h and were then analyzed by flow cytometry using the monoclonal antibody (mAb) PE-conjugated anti-human-active caspase-3. Our results clearly showed a concentration-dependent activation of caspase-3 (a downstream caspase) that resulted in about 20.3%, 25.0%, 30.9% and 40.2% activation of caspase-3 (Fig. 2B). Activation of caspases by NBD peptide was further confirmed by the cleavage of the well-known caspase-3 substrate, poly(ADPribose) polymerase (PARP) (Fig. 3). Western blotting with anti-PARP antibodies revealed almost comparable expression of PARP (113 kDa) in both treated and untreated cells (Fig. 3A). However densitometric analysis of immunopositive bands corresponding to both native (113 kDa) and apoptotic (89 kDa) PARP, indicated that PARP (89 kDa)/PARP (113 kDa) ratio increased from 3 to 6 h in treated cells (Fig. 3B).

2.5. Statistical analysis Values are expressed as the mean ± SEM of n experiments run in triplicate for in vitro experiments. Comparisons were calculated by one-way analysis of variance and Bonferroni-corrected p value for multiple comparisons. The level of statistically significant difference was defined as p < 0.05. 3. Results 3.1. The NBD peptide suppresses the proliferation of human melanoma cells in vitro To investigate whether treatment with the NBD peptide would lead to cell death, cell survival was measured by trypan blue dye exclusion on A375 cell line, whether NHEM were used as control. Cells were plated at the concentration of 1  105 cells/well and were cultured in medium with NBD peptide at 0, 6.25, 12.5 and 25 lM concentrations. After 24 h of culture, the cell viability was determined by hemocytometer counting. The NBD peptide induced a concentration-dependent inhibition of melanoma cells viability (Fig. 1A) while the NBD-mut peptide had no effect on cell viability at every concentration tested (data not shown).

4. Discussion Metastatic melanoma is an aggressive skin cancer that is notoriously resistant to current cancer therapies. It has been recently shown that inhibition of constitutive NF-jB activity blocks the oncogenic potential of neoplastic cells by different ways: by sensitizing tumour cells to chemotherapeutic drug-induced apoptosis, by decreasing the highly proliferative rate which characterizes transformed cells, by inhibiting tissue invasiveness and metastatic potential of highly malignant cells [11]. IKK is a key mediator of the NF-jB signalling pathway [12]. For example, in melanoma cell lines, IKK activity is 3- to 14-fold higher and NF-jB activity is 3.4- to 6.8-fold higher than in normal human epidermal melanocytes [13]. Moreover, it has been recently demonstrated that selective inhibition of NF-jB through down-regulation of IKK activity induces apoptosis of human melanoma cells [2], suggesting that the most effective and selective ap-

3.2. Inhibition of human melanoma cells proliferation by NBD peptide correlates with suppression of NF-jB activation A375 cells were treated with 12.5 lM NBD peptide in order to investigate its effect on NF-jB activity. Whole-cell extracts were analyzed by EMSA at different time points after treatment and the levels of NF-jB DNA-binding activity were quantified. A375 cell line was found to display a constitutively high NF-jB DNA binding activity which was dramatically reduced by the NBD peptide in a time-dependent manner (Fig. 1B). In fact, NBD peptide treatment (12.5 lM) for 3 and 6 h caused a significative (p < 0.001) inhibition of NF-jB DNA-binding activity by 80% and 70%, respectively, in A375 cell line. Treatment with NBD-mut did not have any influence on the NF-jB DNA-binding activity (data not shown). The major NF-jB band in A375 cells consisted of the p50 and p65 subunits, as determined by supershift analysis (data not shown).

n.s.

A375 (105)

0

1.8 ± 0.20

1.60 ± 0.14

6.25

1.8 ± 0.10

1.20 ± 0.10

12.5

1.7 ± 0.09

0.54 ± 0.04

25.0

1.6 ± 0.12

0.32 ± 0.03

3h

6h

100

(arbitrary units)

NF-κB

Normal Human Epidermal Melanocytes (105)

NF-κBbinding activity

C

NBD Peptide (μM)

80 60 40 20 0 0

3

6 h

Fig. 1. Treatment with wt-NBD peptide inhibits growth of A375 melanoma cells in a concentration-dependent manner (A). Constitutive NF-jB activity was examined in nuclear extracts from of A375 melanoma cells in electrophoretic mobility shift assay. wt-NBD peptide (12.5 lM) treatment down regulates NF-jB activity in a time-dependent manner (B).

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Fig. 2. A375 melanoma cells were stained with 7-AAD (A) and with the monoclonal antibody (mAb) PE-conjugated anti-human-active caspase-3 (B), then were analyzed by flow cytometry. Treatment with wt-NBD peptide-induced apoptosis of A375 melanoma cells in a concentration-dependent manner (A–D, 6.25–50 lM; E, untreated; F–I, 6.25–50 lM).

proach for inhibition of NF-jB activation might be offered by inhibitors of IKK activity. The NBD peptide is a short cell-permeable peptide spanning the IKK-b NBD fused with a sequence derived from the Drosophila antennapedia homeodomain that mediates membrane translocation. Due to its structural characteristics, effectively blocks the interaction of IKK-b with NEMO and inhibits activation of NF-jB both in vitro and in vivo [7,14–16]. In the present study we investigated the effect of the NBD peptide on NF-jB activity and survival in A375 human melanoma cells. In melanoma, constitutive activation of NF-jB confers tumour survival capacity and escape from apoptosis. We

hypothesized that the NBD peptide-induced suppression of NF-jB activation would lead to inhibition of melanoma tumour cells growth. To study this possibility, the cell viability was assessed by haemocytometer counting. As hypothesized, NBD peptide treatment resulted in a concentration-dependent inhibition of melanoma cell proliferation. This effect was associated with direct inhibition of constitutive NF-jB DNA-binding activity. To discriminate if NBD peptide-induced inhibition of cell growth was due to a process of necrosis or apoptosis we performed FACS analysis for both 7-AAD and caspase-3. Flow cytometry results indicated a concentration-dependent increase in the percentage of apoptotic melanoma cells by specific inhibition of NF-jB with NBD peptide. A significant increase of

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Density INT/mm2

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 1

2

3

Fig. 3. A375 melanoma cells were untreated (lane 1) or treated with wt-NBD peptide (12.5 lM) for 3 (lane 2) and 6 h (lane 3). Whole cell lysates were immunoblotted with anti-PARP antibodies (A). Densitometric analysis of immunopositive bands corresponding to both native (113 kDa) and apoptotic (89 kDa) PARP, indicated that PARP (89 kDa)/PARP (113 kDa) ratio increased from 3 to 6 h in treated cells (B).

apoptosis occurred with all concentration of NBD peptide used. In contrast, no effect was observed when the mutant control peptide was added to cells. Our results show that NBD peptide-induced inhibition of human melanoma cells proliferation was due to induction of apoptosis were further strengthened by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process. Moreover, this event could be directly correlated to the inhibition of NF-jB-DNA binding activity. In fact it has been widely demonstrated that unmodified PARP-1 negatively down regulates formation of NF-jB-DNA complex via its physical association with the transcriptional factor [9]. NF-jB inhibition is considered a very promising weapon to fight cancer. The finding that NF-jB is a key player in cancer has prompted researchers to focus their efforts in looking for drugs able to suppress NF-jB activity in malignant cells. NF-jB activation could be blocked at different levels targeting the various components of its signalling cascade, such as the IKK complex, the IjBa inhibitory protein, the p65 subunit of the transcriptionally active heterodimer, the proteasome [17]. Therefore, an increasing number of compounds able to block NF-jB by inhibiting one or more of the molecules involved in the pathway activating it, have been tested and have shown to suppress the growth of those cancer cells whose tumourigenicity depends on NF-jB activity [18]. Many of these drugs have given promising results in preclinical models for NF-jB-dependent solid tumours (breast, lung, colon, bladder, ovary, pancreas and prostate cancers), but their clinical efficacy have shown to be poorly appreciable.

Actually, the only pharmacological inhibitors of NF-jB activation approved for clinical use are represented by proteasome inhibitors for treatment of some hematological malignancies, such as multiple myeloma, or adult T-cell leukaemia, for whose pathogenesis it has been clearly demonstrated the key role of NF-jB [19,20]. One of the most problematic aspects of a cancer therapy based on inhibition of NF-jB activity is represented by the difficulty to find compounds which block the oncogenic activity of NF-jB without interfering with its physiological roles in immunity, inflammation and cellular homeostasis. Unfortunately, most of the drugs analyzed so far also affect other cellular signalling pathways involved in the regulation of apoptosis and proliferation other than of inflammatory and immunological response, thereby determining a number of highly toxic side effects. To this end, all the efforts are now concentrated on the ability to identify novel NFjB targets specifically activated in tumours, but not in normal cells. Thus, the inhibition of these targets should block the oncogenic potential of NF-jB in cancer cells without affect its role in normal tissues. In this light, the results of our paper showing, for the first time, that treatment of melanoma cells with the specific IKK inhibitor, NBD peptide, resulted in effective blockage of hyper-proliferation of melanoma cells in vitro through induction of apoptosis, assume a relevant significance. Our data argue for the powerful role of NF-jB in the future of melanoma therapy targets. The importance of NF-jB in melanoma tumour progression is evident in many recent studies utilizing various inhibitors of NF-jB for the treatment of melanoma. The use of NF-jB inhibitors has resulted in significant anti-tumour effects in mela-

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noma tumour xenograft models and some have led to ongoing clinical trials [1]. As we are eagerly awaiting the results of these studies, it is important to note that NF-jB targeting for treatment of cancers such as melanoma is a rather new field and in need of optimization in both drug design as well as drug delivery in patients. We believe that one of the most important aspects of our study is that it provides further support for the hypothesis that selective inhibition of NF-jB activation can be an effective strategy for challenging melanoma. 5. Conflict of interest None declared. Acknowledgements Work was supported by Italian Ministry of Health ‘‘Programma Ricerca Finalizzata”. A special thank to Roberto Santelia for providing us with the peptide. References [1] K.I. Amiri, A. Richmond, Role of nuclear factor-jB in melanoma, Cancer and Metastasis Reviews 24 (2005) 301–313. [2] J. Yang, K.I. Amiri, J.R. Burke, J.A. Schmid, A. Richmond, BMS-345541 targets inhibitor of kappaB kinase and induces apoptosis in melanoma: involvement of nuclear factor kappaB and mitochondria pathways, Clinical Cancer Research 12 (2006) 950– 960. [3] S.E. McNulty, R. DelRosario, D. Cen, F.L. Meyskens Jr., S. Yang, Comparative expression of NF-jB proteins in melanocytes of normal skin vs. benign intradermal naevus and human metastatic melanoma biopsies, Pigment Cell Research 17 (2004) 173–180. [4] J. Yang, W.H. Pan, G.A. Clawson, A. Richmond, Systemic targeting inhibitor of kappab kinase inhibits melanoma tumor growth, Cancer Research 67 (2007) 3127–3134. [5] Y. Yamamoto, R.B. Gaynor, IjB kinases: key regulators of the NF-jB pathway, Trends in Biochemical Sciences 29 (2004) 72–79. [6] M.J. May, R.B. Marienfeld, S. Ghosh, Characterization of the Ikappa Bkinase NEMO binding domain, The Journal of Biological Chemistry 277 (2002) 45992–46000.

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