The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation
Introduction
Retinoblastoma is the most common primary malignant intraocular tumor in infants and children. In the United States, it affects 12 per million children aged 0–4 years, representing 6.1% of all childhood cancers under the age of 5 years (Broaddus et al., 2009). Slightly more than half of the patients have the sporadic or non-inherited form of the disease, which results from the spontaneous inactivation of the retinoblastoma gene (RB1). Despite progress in the treatment of retinoblastoma, significant problems remain unsolved and metastatic disease is all too often fatal (Rodriguez-Galindo et al., 2003). Although several treatment modalities are available for retinoblastoma, including local control of small to intermediate size tumors with laser and/or cryotherapy sometimes in combination with radiation and/or chemotherapy, or enucleation with or without systemic chemotherapy to control metastatic disease, each of them has major drawbacks, especially in pediatric patients. For example, conventional external beam radiation, which is used to control large tumors, has many complications, including an increased incidence of secondary malignancies, such as osteosarcoma. This complication occurs more frequently in patients with the hereditary-form of retinoblastoma. The 30-year cumulative incidence of second malignancies is >35% for patients who received external beam therapy vs 6% for those patients without radiation (Roarty et al., 1988). Intra-atrerial chemotherapy is a more recent treatment option for retinoblastoma, however variables that affect blood flow can greatly affect drug delivery and therapy success (Marr et al., 2012) and can be complicated by retinal and choroidal vasculopathy in up to 10%–20% of patients (Bianciotto et al., 2012, Muen et al., 2012). Direct intravitreal injection of melphalan has also been tested as an effective modality in controlling active vitreous seeds, however there is concern for tumor dissemination (Ghassemi and Shields, 2012, Munier et al., 2012). Systemic chemotherapy used for treatment for intraocular retinoblastoma with subsequent consolidation with photocoagulation, cryotherapy, or radiotherapy has a recurrence rate of 24% by 5 years (Shields et al., 2002). This increases to 50% for patients with vitreous seeds (Shields et al., 2003). The recent reports (Sussman et al., 2003, Schefler et al., 2007, Shields, 2009, Shields et al., 2009) revealed success for local control approaching 90–100% for group A–C, but in less than 50% for group D (new international classification). In addition, significant morbidity with the chemotherapy has been described previously (Benz et al., 2000). One of the drugs used for chemotherapy (etoposide) is thought to be associated with increased incidence of acute myeloblastic leukemia although the actual cases implicated so far have been low with ∼20 cases reported (Nishimura et al., 2001). For these reasons, there is a pressing need for alternative treatment modalities for retinoblastoma with better safety and efficacy profiles.
Verteporfin (VP) belongs to the porphyrin family, which contains aromatic heterocyclic cyclic molecules composed of four modified pyrrole units which are interconnected at their carbon atoms via methine bridges (Liu-Chittenden et al., 2012). Photodynamic therapy (PDT) using verteporfin is a clinically approved, minimally invasive therapeutic procedure that involves administration of a photosensitizing agent followed by irradiation at a wavelength of 693 nm corresponding to an absorbance band of the sensitizer (Agostinis et al., 2011). In wet age related macular degeneration (AMD), liposomal VP (trade name Visudyne) accumulates in abnormal blood vessels, where it is activated by nonthermal laser at 693 nm generating reactive oxygen radicals. This results in local damage to the endothelium as well as blockage, and potential elimination of the vessels. It has been clinically used worldwide resulting in vision preservation in many patients (Miller et al., 1999). VP has also been tested but not yet approved as a light-based therapeutic modality for several human cancers (Pogue et al., 2003, Harbour, 2004, Isola et al., 2006, Celli et al., 2011; Tripursky et al., 2011, Tamella et al., 2011). In human cancers PDT with visudyne may act not only at the cancer vasculature but also directly on the cancer cells and act as an inducer of apoptosis or autophagy (Kessel et al., 2006).
Yes Associated Protein (YAP) is or has been suggested to be a major candidate oncogene in the human chromosome 11q22 amplicon, and mutations or abnormal expression of Hippo-pathway components are associated with human tumorigenesis (Fernandez-L et al., 2012, Liu et al., 2012, Mo et al., 2012). As a transcriptional coactivator, YAP has been reported to bind to several DNA-binding transcription factors, and among the reported YAP binding partners, the TEAD transcription factors are the best characterized (Vassilev, 2001). Recent studies indicate that VP may disrupt YAP-TEAD complex and inhibit growth of hepatocellular carcinoma without light activation (Liu-Chittenden et al., 2012).
Since YAP-TEAD pathway is usually not active in normal tissues, drugs disrupting this interaction have the potential for increased cancer specificity with minimal healthy tissue toxicity making them valuable novel potential therapeutic option. For this reason we investigated the effects of non-light activated -VP on human retinoblastoma cells.
Section snippets
Reagents
Verteporfin (Visudyne) was obtained from Novartis (Novartis, Basel, Switzerland).
Metformin, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ribonuclease-A were purchased from Sigma Aldrich (St.Louis, MO, USA). Propidium iodide was purchased from Invitrogen (Carlsbad, CA, USA). The following primary antibodies were purchased from Cell Signaling technology (Danvers, MA, USA) and used diluted 1:1000 unless stated otherwise: c-myc, Axl, phospho-S6 ribosomal protein
Verteporfin inhibits cell growth and viability, and increases doubling time of human retinoblastoma cell lines Y79 and WERI
In order to determine whether VP without light activation affects human retinoblastoma cell growth and proliferation, we analyzed its effect on the human retinoblastoma cell line Y79 and WERI. Cell lines were treated with two different concentrations of VP (2 μg/ml and 10 μg/ml) or vehicle control (PBS). All cells were protected from light at any time. Five days of VP treatment resulted in a decrease in cell colony sizes and a remarkable increase of single dysmorphic cells in suspension. Cell
Discussion
Verteporfin has been widely and safely used as a photosensitizer in PDT for neovascular macular degeneration as well as treatment of several human tumors, after it is activated by laser light to eliminate abnormal blood vessels (Miller et al., 1999). In the present study, we demonstrate that verteporfin has a potential to induce growth inhibition, apoptosis, and G0/G1-phase cell cycle arrest in human retinoblastoma cells in vitro without any light activation by interfering with the YAP-TEAD
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