RESUMO
Cold physical plasma, a partially ionized gas rich in reactive oxygen species (ROS), is receiving increasing interest as a novel anticancer agent via two modes. The first involves its application to cells and tissues directly, while the second uses physical plasma-derived ROS to oxidize liquids. Saline is a clinically accepted liquid, and here we explored the suitability of plasma-oxidized saline (POS) as anticancer agent technology in vitro and in vivo using the Ehrlich Ascites Carcinoma (EAC) model. EAC mainly grows as a suspension in the peritoneal cavity of mice, making this model ideally suited to test POS as a putative agent against peritoneal carcinomatosis frequently observed with colon, pancreas, and ovarium metastasis. Five POS injections led to a reduction of the tumor burden in vivo as well as in a decline of EAC cell growth and an arrest in metabolic activity ex vivo. The treatment was accompanied by a decreased antioxidant capacity of Ehrlich tumor cells and increased lipid oxidation in the ascites supernatants, while no other side effects were observed. Oxaliplatin and hydrogen peroxide were used as controls and mediated better and worse outcomes, respectively, with the former but not the latter inducing profound changes in the inflammatory milieu among 13 different cytokines investigated in ascites fluid. Modulation of inflammation in the POS group was modest but significant. These results promote POS as a promising candidate for targeting peritoneal carcinomatosis and malignant ascites and suggest EAC to be a suitable and convenient model for analyzing innovative POS approaches and combination therapies.
RESUMO
Malignant melanoma is the most dangerous form of skin cancer. Despite new therapies for melanoma treatment, effective therapy is mainly limited by excessive metastasis. Currently, the factors determining metastasis development are not elucidated, but oxidative stress was suggested to be involved. To this end, we analyzed oxidative stress parameters during the metastatic development using the syngeneic B16F10 melanoma model. An increase in blood plasma lipid peroxidation occurred at the earliest stage of the disease, with a progressive decrease in oxidative damage and an increase in antioxidant defense. Vice versa, increased lipid peroxidation and 3-nitrotyrosine, and decreased antioxidant parameters were observed in the metastatic nodules throughout the disease. This was concomitant with a progressive increase in vascular endothelial growth factor and proliferating cell nuclear antigen. We conclude that the oxidative stress in the bloodstream decreases during the metastatic process and that nitrosative stress increases during the proliferation and growth of metastatic nodules in the tumor microenvironment. These results will help to better understand the role of oxidative stress during melanoma metastasis.
Assuntos
Neoplasias Pulmonares/secundário , Melanoma/secundário , Metástase Neoplásica/patologia , Estresse Oxidativo/fisiologia , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Camundongos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
Cutaneous squamous cell carcinoma (SCC) is the most prevalent cancer worldwide, increasing the cost of healthcare services and with a high rate of morbidity. Its etiology is linked to chronic ultraviolet (UV) exposure that leads to malignant transformation of keratinocytes. Invasive growth and metastasis are severe consequences of this process. Therapy-resistant and highly aggressive SCC is frequently fatal, exemplifying the need for novel treatment strategies. Cold physical plasma is a partially ionized gas, expelling therapeutic doses of reactive oxygen and nitrogen species that were investigated for their anticancer capacity against SCC in vitro and SCC-like lesions in vivo. Using the kINPen argon plasma jet, a selective growth-reducing action of plasma treatment was identified in two SCC cell lines in 2D and 3D cultures. In vivo, plasma treatment limited the progression of UVB-induced SSC-like skin lesions and dermal degeneration without compromising lesional or non-lesional skin. In lesional tissue, this was associated with a decrease in cell proliferation and the antioxidant transcription factor Nrf2 following plasma treatment, while catalase expression was increased. Analysis of skin adjacent to the lesions and determination of global antioxidant parameters confirmed the local but not systemic action of the plasma anticancer therapy in vivo.
RESUMO
Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.
Assuntos
Fatores Imunológicos/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Gases em Plasma/uso terapêutico , Animais , Apoptose , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Camundongos , Gases em Plasma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Proteínas de Transporte de Cátions Orgânicos/genética , Gases em Plasma/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Técnicas de Cocultura , Terapia Combinada , Doxorrubicina/farmacologia , Epirubicina/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Melanoma Experimental , Camundongos , Proteínas de Transporte de Cátions Orgânicos/agonistas , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Oxaliplatina/farmacologia , Células THP-1 , Vorinostat/farmacologiaRESUMO
Despite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Oxidantes/toxicidade , Gases em Plasma/química , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , RNA Interferente Pequeno/metabolismoRESUMO
TGF-ß1 and oxidative stress are involved in cancer progression, but in melanoma, their role is still controversial. Our aim was to correlate plasma TGF-ß1 levels and systemic oxidative stress biomarkers in patients with melanoma, with or without disease metastasis, to understand their participation in melanoma progression. Thirty patients were recruited for melanoma surveillance, together with 30 healthy volunteers. Patients were divided into two groups: Non-metastasis, comprising patients with tumor removal and no metastatic episode for 3 years; and Metastasis, comprising patients with a metastatic episode. The plasmatic cytokines TGF-ß1, IL-1 ß, and TNF-α were analyzed by ELISA. For oxidative stress, the following assays were performed: malondialdehyde (MDA), advanced oxidation protein products (AOPP) levels, total radical-trapping antioxidant parameter (TRAP) and thiol in plasma, and lipid peroxidation, SOD and catalase activity and GSH in erythrocytes. Patients with a metastatic episode had less circulating TGF-ß1 and increased TRAP, thiol, AOPP and lipid peroxidation levels. MDA was increased in both melanoma groups, while catalase, GSH, and IL-1ß was decreased in Non-metastasis patients. Significant negative correlations were observed between TGF-ß1 levels and systemic MDA, and TGF-ß1 levels and systemic AOPP, while a positive correlation was observed between TGF-ß1 levels and erythrocyte GSH. Lower levels of TGF-ß1 were related to increased oxidative stress in Metastasis patients, reinforcing new evidence that in melanoma TGF-ß1 acts as a tumor suppressor, inhibiting tumor relapse. These findings provide new knowledge concerning this cancer pathophysiology, extending the possibilities of investigating new therapies based on this evidence.