RESUMEN
Redox regulation of epidermal growth factor receptor (EGFR) signaling helps protect cells against oxidative stress. In this study, we investigated whether the cytotoxicity of an EGFR tyrosine kinase inhibitor, erlotinib (ERL), was mediated by induction of oxidative stress in human head and neck cancer (HNSCC) cells. ERL elicited cytotoxicity in vitro and in vivo while increasing a panel of oxidative stress parameters which were all reversible by the antioxidant N-acetyl cysteine. Knockdown of EGFR by using siRNA similarly increased these oxidative stress parameters. Overexpression of mitochondrial targeted catalase but not superoxide dismutase reversed ERL-induced cytotoxicity. Consistent with a general role for NADPH oxidase (NOX) enzymes in ERL-induced oxidative stress, ERL-induced cytotoxicity was reversed by diphenylene iodonium, a NOX complex inhibitor. ERL reduced the expression of NOX1, NOX2, and NOX5 but induced the expression of NOX4. Knockdown of NOX4 by using siRNA protected HNSCC cells from ERL-induced cytotoxicity and oxidative stress. Our findings support the concept that ERL-induced cytotoxicity is based on a specific mechanism of oxidative stress mediated by hydrogen peroxide production through NOX4 signaling.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Procesos de Crecimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Femenino , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Ratones Desnudos , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adenoid cystic cancers (ACC) in the head and neck are rare yet present a clinical dilemma. Although 5-y survivals are excellent, they have a propensity for late recurrences. Most of these cancers are initially treated with surgery followed by radiation. When recurrences happen, treatment options are limited both by the morbidity and low efficacy of re-irradiation and repeated surgical resection. Reported response rates to chemotherapy are low and targeted therapies may be one option. We, therefore, investigated signaling pathways that may be active in adenoid cystic cancers. Tissues from the last nine ACC patients resected at the University of Iowa were immunohistochemically stained with antibodies for EGFR, phosphorylated (P) Akt, and P-MAPK in order to molecularly characterize these tumors. An ACC cell line (ACC3) was also characterized by western blot. We found that seven of the nine tumor samples had strong expression of P-Akt and 5/9 had P-MAPK. None of them had EGFR expression. In the ACC3 cell line, similar data was found in that there was P-Akt and P-MAPK but no EGFR expression. We tested the HIV protease inhibitor nelfinavir (NFV) which has been shown to inhibit Akt signaling to see its effect on ACC3 cells. Both P-Akt and P-MAPK were inhibited with NFV in ACC3 cells and this resulted in growth inhibition and clonogenic death. In patients where re-irradiation or further surgery is not an option, a trial of NFV may be warranted.
Asunto(s)
Carcinoma Adenoide Quístico/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Transducción de Señal , Western Blotting , Carcinoma Adenoide Quístico/patología , Carcinoma Adenoide Quístico/terapia , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Inmunohistoquímica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nelfinavir/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de TiempoRESUMEN
PURPOSE: Human papilloma virus (HPV)-associated cancers of the head and neck (H&N) are increasing in frequency and are often treated with radiation. There are conflicting data in the literature regarding the radiation response in the presence of HPV infection, with some data suggesting they may be more sensitive to radiation. There are few studies looking at in vitro effects of HPV and further sensitization by inhibitors of specific signaling pathways. We are in the process of starting a clinical trial in H&N cancer patients using nelfinavir (NFV) (which inhibits Akt) and it would be important to know the effect of HPV on radiation response +/- NFV. METHODS AND MATERIALS: Two naturally infected HPV-16 cell lines (UPCI-SCC90 and UMSCC47) and the HPV-negative SQ20B H&N squamous carcinoma cells were used. Western blots with or without 10 uM NFV were done to evaluate signaling from the PI3K-Akt pathway. Clonogenic assays were done in the three cell lines with or without NFV. RESULTS: Both UPCI-SCC90 and UMSCC47 cells were sensitive to radiation as compared with SQ20B and the degree corresponded to Akt activation. The SQ20B cell line has an activating mutation in EGFR resulting in phosphorylation (P) of Akt; UMSCC47 has decreased P-phosphatase and TENsin (PTEN), resulting in increased P-Akt; UPCI-SCC90 had overexpression of P-PTEN and decreased P-Akt. NFV resulted in downregulation of Akt in all three cell lines, resulting in sensitization to radiation. CONCLUSIONS: HPV-infected H&N cancers are sensitive to radiation. The degree of sensitivity correlates to Akt activation and they can be further sensitized by NFV.
Asunto(s)
Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/radioterapia , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Tolerancia a Radiación , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Supervivencia Celular , Inhibidores Enzimáticos/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Papillomavirus Humano 16 , Humanos , Nelfinavir/uso terapéutico , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tolerancia a Radiación/efectos de los fármacosRESUMEN
BACKGROUND: Chronic alcoholics experience increased incidence and severity of infections, the mechanism of which is incompletely understood. Dendritic cells (DC) migrate from peripheral locations to lymph nodes (LN) to initiate adaptive immunity against infection. Little is known about how chronic alcohol exposure affects skin DC numbers or migration. METHODS: Mice received 20% EtOH in the drinking water for up to 35 weeks. Baseline Langerhans cell (LC) and dermal DC (dDC) numbers were enumerated by immunofluorescence (IF). LC repopulation after inflammation was determined following congenic bone marrow (BM) transplant and ultraviolet (UV) irradiation. Net LC loss from epidermis was determined by IF following TNF-alpha or CpG stimulation. LC and dDC migration into LN was assessed by flow cytometry following epicutaneous FITC administration. RESULTS: Chronic EtOH consumption caused a baseline reduction in LC but not dDC numbers. The deficit was not corrected following transplantation with non-EtOH-exposed BM and UV irradiation, supporting the hypothesis that the defect is intrinsic to the skin environment rather than LC precursors. Net loss of LC from epidermis following inflammation was greatly reduced in EtOH-fed mice versus controls. Ethanol consumption for at least 4 weeks led to delayed LC migration into LN, and consumption for at least 8 weeks led to delayed dDC migration into LN following epicutaneous FITC application. CONCLUSIONS: Chronic EtOH consumption causes decreased density of epidermal LC, which likely results in decreased epidermal immunosurveillance. It also results in altered migratory responsiveness and delayed LC and dDC migration into LN, which likely delays activation of adaptive immunity. Decreased LC density at baseline appears to be the result of an alteration in the skin environment rather than an intrinsic LC defect. These findings provide novel mechanisms to at least partially explain why chronic alcoholics are more susceptible to infections, especially those following skin penetration.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Etanol/administración & dosificación , Células de Langerhans/efectos de los fármacos , Piel/efectos de los fármacos , Consumo de Bebidas Alcohólicas/efectos adversos , Consumo de Bebidas Alcohólicas/inmunología , Animales , Movimiento Celular/inmunología , Femenino , Células de Langerhans/citología , Células de Langerhans/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Piel/citología , Piel/inmunologíaRESUMEN
As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.