RESUMO
In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.
Assuntos
Receptor de Fator de Crescimento Neural , Neoplasias da Retina , Retinoblastoma , Canais de Cátion TRPM , Humanos , Linhagem Celular , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Proteínas de Membrana/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Receptor CB1 de Canabinoide/metabolismoRESUMO
BACKGROUND: Photodynamic therapy (PDT) is clinically approved to treat neoplastic skin diseases such as precursors of cutaneous squamous cell carcinoma (cSCC). In PDT, 5-aminolevulinic acid (5-ALA) drives the selective formation of the endogenous photosensitizer protoporphyrin IX (PpIX). Although 5-ALA PDT is clinically highly effective, resistance might occur due to decreased accumulation of PpIX in certain tumors. Such resistance may be caused by any fundamental step of PpIX accumulation: 5-ALA uptake, PpIX synthesis and PpIX efflux. METHODS: We investigated PpIX accumulation and photodynamically induced cell death in PDT refractory SCC-13, PDT susceptible A431, and normal human epidermal keratinocytes (NHEK). Expression of genes associated with cellular PpIX kinetics was investigated on mRNA and protein level. PpIX accumulation and cell death upon illumination were pharmacologically manipulated using drugs targeting 5-ALA uptake, PpIX synthesis or efflux. RESULTS: The experiments indicate that taurine transporter (SLC6A6) is the major pathway for 5-ALA uptake in cSCC cells, while being less important in NHEK. Downregulation of PpIX synthesis enzymes in SCC-13 was counteracted by methotrexate (MTX) treatment, which restored PpIX formation and cell death. PpIX efflux inhibitors targeting ABC transporters led to significantly increased PpIX accumulation in SCC-13, thereby fully overcoming resistance. CONCLUSIONS: The results indicate a conserved threshold for PpIX accumulation with respect to PDT-resistance. Cells showed increased viability after PDT at PpIX concentrations below 1.5 nM. Selective uptake of 5-ALA via taurine transporter SLC6A6 in cutaneous tumor cells is novel but unrelated to resistance. MTX can partially abrogate resistance by PpIX synthesis enzyme induction, while efflux mechanisms via ABC transporters seem the main driving force and promising drug targets.
Assuntos
Carcinoma de Células Escamosas , Fotoquimioterapia , Neoplasias Cutâneas , Transportadores de Cassetes de Ligação de ATP , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Protoporfirinas/metabolismo , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Retinal ischemia is a common pathomechanism in various eye diseases. Recently, evidence accumulated suggesting that the extracellular matrix (ECM) glycoprotein tenascin-C (Tnc) plays a key role in ischemic degeneration. However, the possible functional role of Tnc in retinal ischemia is not yet known. The aim of our study was to explore retinal function and rod-bipolar/photoreceptor cell degeneration in wild type (WT) and Tnc knock-out (KO) mice after ischemia/reperfusion (I/R) injury. Therefore, I/R was induced by increasing intraocular pressure in the right eye of wild type (WT I/R) and Tnc KO (KO I/R) mice. The left eye served as untreated control (WT CO and KO CO). Scotopic electroretinogram (ERG) recordings were performed to examine rod-bipolar and rod-photoreceptor cell function. Changes of Tnc, rod-bipolar cells, photoreceptors, retinal structure and apoptotic and synaptic alterations were analyzed by immunohistochemistry, Hematoxylin and Eosin staining, Western blot, and quantitative real time PCR. We found increased Tnc protein levels 3 days after ischemia, while Tnc immunoreactivity decreased after 7 days. Tnc mRNA expression was comparable in the ischemic retina. ERG measurements after 7 days showed lower a-/b-wave amplitudes in both ischemic groups. Nevertheless, the amplitudes in the KO I/R group were higher than in the WT I/R group. We observed retinal thinning in WT I/R mice after 3 and 7 days. Although compared to the KO CO group, retinal thinning was not observed in the KO I/R group until 7 days. The number of PKCα+ rod-bipolar cells, recoverin+ photoreceptor staining and Prkca and Rcvrn expression were comparable in all groups. However, reduced rhodopsin protein as well as Rho and Gnat1 mRNA expression levels of rod-photoreceptors were found in the WT I/R, but not in the KO I/R retina. Additionally, a lower number of activated caspase 3+ cells was observed in the KO I/R group. Finally, both ischemic groups displayed enhanced vesicular glutamate transporter 1 (vGlut1) levels. Collectively, KO mice showed diminished rod-photoreceptor degeneration and retinal dysfunction after I/R. Elevated vGlut1 levels after ischemia could be related to an impaired glutamatergic photoreceptor-bipolar cell signaling and excitotoxicity. Our study provides novel evidence that Tnc reinforces ischemic retinal degeneration, possibly by synaptic remodeling.