RESUMO
Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer type with no targeted therapy and hence limited treatment options. Modulated electrohyperthermia (mEHT) is a novel complementary therapy where a 13.56 MHz radiofrequency current targets cancer cells selectively, inducing tumor damage by thermal and electromagnetic effects. We observed severe vascular damage in mEHT-treated tumors and investigated the potential synergism between mEHT and inhibition of tumor vasculature recovery in our TNBC mouse model. 4T1/4T07 isografts were orthotopically inoculated and treated three to five times with mEHT. mEHT induced vascular damage 4-12 h after treatment, leading to tissue hypoxia detected at 24 h. Hypoxia in treated tumors induced an angiogenic recovery 24 h after the last treatment. Administration of the cardiac glycoside digoxin with the potential hypoxia-inducible factor 1-α (HIF1-α) and angiogenesis inhibitory effects could synergistically augment mEHT-mediated tumor damage and reduce tissue hypoxia signaling and consequent vascular recovery in mEHT-treated TNBC tumors. Conclusively, repeated mEHT induced vascular damage and hypoxic stress in TNBC that promoted vascular recovery. Inhibiting this hypoxic stress signaling enhanced the effectiveness of mEHT and may potentially enhance other forms of cancer treatment.
RESUMO
Triple-negative breast cancer (TNBC) is a leading cause of cancer mortality and lacks modern therapy options. Modulated electro-hyperthermia (mEHT) is an adjuvant therapy with demonstrated clinical efficacy for the treatment of various cancer types. In this study, we report that mEHT monotherapy stimulated interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) expression, and consequently cyclooxygenase 2 (COX-2), which may favor a cancer-promoting tumor microenvironment. Thus, we combined mEHT with nonsteroid anti-inflammatory drugs (NSAIDs): a nonselective aspirin, or the selective COX-2 inhibitor SC236, in vivo. We demonstrate that NSAIDs synergistically increased the effect of mEHT in the 4T1 TNBC model. Moreover, the strongest tumor destruction ratio was observed in the combination SC236 + mEHT groups. Tumor damage was accompanied by a significant increase in cleaved caspase-3, suggesting that apoptosis played an important role. IL-1ß and COX-2 expression were significantly reduced by the combination therapies. In addition, a custom-made nanostring panel demonstrated significant upregulation of genes participating in the formation of the extracellular matrix. Similarly, in the B16F10 melanoma model, mEHT and aspirin synergistically reduced the number of melanoma nodules in the lungs. In conclusion, mEHT combined with a selective COX-2 inhibitor may offer a new therapeutic option in TNBC.
Assuntos
Benzenossulfonamidas , Hipertermia Induzida , Melanoma , Pirazóis , Neoplasias de Mama Triplo Negativas , Humanos , Melanoma/tratamento farmacológico , Ciclo-Oxigenase 2 , Neoplasias de Mama Triplo Negativas/terapia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Aspirina/farmacologia , Aspirina/uso terapêutico , Microambiente TumoralRESUMO
Our aim was to detect the effect of modulated electro-hyperthermia (mEHT) on cell viability and to examine if hyperthermia can augment the cell killing effect of various chemotherapeutic agents. B16F10 melanoma cells were treated for 30, 60, 90 and 120 minutes with mEHT using LabEHY100 (OncothermTM). Cell viability was measured using MTT assay and apoptosis by annexin V/7-AAD staining using flow cytometry 24 hours post-treatment. For analyzing gene expression with qPCR cells were harvested after 60 minutes treatment. In combined protocols, cells were treated with paclitaxel (40 nM), dacarbazine (40 µM) or nutlin-3a (10 µM) after mEHT. mEHT induced nuclear translocation of p53 which in turn regulates pro- and anti-apoptotic gene expression accounting for decreased cell viability. In combination with chemotherapy, mEHT augmented the cell killing effect of dacarbazine or nutlin-3a but not that of paclitaxel determined 48 hours post-treatment. The sensitizing effect on chemotherapeutics demonstrate the efficiency of mEHT as an adjuvant modality in cancer treatment.
Assuntos
Hipertermia Induzida , Melanoma , Apoptose , Linhagem Celular Tumoral , Humanos , Hipertermia , Melanoma/tratamento farmacológicoRESUMO
The poor outcome of pancreas ductal adenocarcinomas (PDAC) is frequently linked to therapy resistance. Modulated electro-hyperthermia (mEHT) generated by 13.56 MHz capacitive radiofrequency can induce direct tumor damage and promote chemo- and radiotherapy. Here, we tested the effect of mEHT either alone or in combination with radiotherapy using an in vivo model of Panc1, a KRAS and TP53 mutant, radioresistant PDAC cell line. A single mEHT shot of 60 min induced ~50% loss of viable cells and morphological signs of apoptosis including chromatin condensation, nuclear shrinkage and apoptotic bodies. Most mEHT treatment related effects exceeded those of radiotherapy, and these were further amplified after combining the two modalities. Treatment related apoptosis was confirmed by a significantly elevated number of annexin V single-positive and cleaved/activated caspase-3 positive tumor cells, as well as sub-G1-phase tumor cell fractions. mEHT and mEHT+radioterapy caused the moderate accumulation of γH2AX positive nuclear foci, indicating DNA double-strand breaks and upregulation of the cyclin dependent kinase inhibitor p21waf1 besides the downregulation of Akt signaling. A clonogenic assay revealed that both mono- and combined treatments affected the tumor progenitor/stem cell populations too. In conclusion, mEHT treatment can contribute to tumor growth inhibition and apoptosis induction and resolve radioresistance of Panc1 PDAC cells.
Assuntos
Carcinoma Ductal Pancreático/terapia , Hipertermia Induzida/métodos , Neoplasias Pancreáticas/terapia , Apoptose , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Terapia Combinada , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , Terapia por RadiofrequênciaRESUMO
Modulated electro-hyperthermia (mEHT) is a non-invasive treatment modality of cancer where electric field generated by 13.56 MHz radiofrequency can selectively accumulate in malignant tumors compared to adjacent normal tissues. This effect is based on the metabolic shift in cancer cells which upregulates glycolysis even under oxygenated conditions (Warburg effect), resulting in elevated lactate and ion concentration. The concomitant increased permittivity can induce dielectric polarization and rotational friction of dipole molecules resulting in elevated core temperature, which can be controlled at 42 °C with the treating instrument. Complementary application of loco-regional mEHT can improve the efficiency of chemo-, radio- and recently molecular targeted therapies based on increasing local perfusion and xenobiotic concentration, resolving tumor hypoxia and improved immune surveillance supported by high-fever range hyperthermia. We earlier showed that mEHT has its own tumor inhibiting/destructing effect, however, its mechanism had not been clarified. In this project we have investigated the molecular mechanism of action of mEHT treatment using in vitro and in vivo models of colorectal adenocarcinoma.
Assuntos
Adenocarcinoma , Neoplasias Colorretais , Hipertermia Induzida , Apoptose , HumanosRESUMO
Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21waf1 and p27kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication.
Assuntos
Hipertermia Induzida , Melanoma/fisiopatologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Proteína HMGB1 , Proteínas de Choque Térmico HSP70 , Macrófagos/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Transplante de Neoplasias , Estresse Fisiológico , Proteína Supressora de Tumor p53/fisiologiaRESUMO
OBJECTIVE: Modulated electro-hyperthermia (mEHT), a noninvasive complementary treatment of human chemo- and radiotherapy, can generate selective ~42°C heat in cancer due to elevated glycolysis (Warburg-effect) and electric conductivity in malignant tissues. Here we tested the molecular background of mEHT and its combination with doxorubicin chemotherapy using an in vitro model. METHODS: C26 mouse colorectal adenocarcinoma cultures were mEHT treated at 42°C for 2 × 60 minutes (with 120 minutes interruption) either alone or in combination with 1 µmol/L doxorubicin (mEHT + Dox). Cell stress response, apoptosis, and cell cycle regulation related markers were detected using qPCR and immunocytochemistry supported with resazurin cell viability assay, cell death analysis using flow-cytometry and clonogenic assay. RESULT: Cell-stress by mEHT alone was indicated by the significant upregulation and release of hsp70 and calreticulin proteins 3 hours posttreatment. Between 3 and 9 hours after treatment significantly reduced anti-apoptotic XIAP, BCL-2, and BCL-XL and elevated pro-apoptotic BAX and PUMA, as well as the cyclin dependent kinase inhibitor p21waf1 mRNA levels were detected. After 24 hours, major elevation and nuclear translocation of phospho-p53(Ser15) protein levels and reduced phospho-Akt(Ser473) levels were accompanied by a significant caspase-3-mediated programmed cell death response. While mEHT dominantly induced apoptosis, Dox administration primarily led to tumor cell necrosis, and both significantly reduced the number of tumor progenitor colonies 10 days post-treatment. Furthermore, mEHT promoted the uptake of Dox by tumor cells and the combined treatment additively reduced tumor cell viability and augmented cell death near to synergy. CONCLUSION: In C26 colorectal adenocarcinoma mEHT-induced irreversible cell stress can activate both caspase-dependent apoptosis and p21waf1 mediated growth arrest pathways, likely to be driven by the upregulated nuclear p53 protein. Elevated phospho-p53(Ser15) might contribute to p53 escape from mdm2 control, which was further supported by reduced phospho-Akt(Ser473) protein levels. In combinations, mEHT could promote the uptake and significantly potentiate the cytotoxic effect of doxorubicin.
Assuntos
Neoplasias Colorretais/metabolismo , Doxorrubicina/farmacologia , Hipertermia Induzida/métodos , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Terapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and ß-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.
Assuntos
Colo/efeitos da radiação , Sequestradores de Radicais Livres/uso terapêutico , Mucosa Intestinal/efeitos da radiação , Lesões por Radiação/prevenção & controle , Radiação Ionizante , Protetores contra Radiação/uso terapêutico , Junções Íntimas/efeitos da radiação , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Citoesqueleto de Actina/metabolismo , Animais , Colo/efeitos dos fármacos , Colo/metabolismo , Suplementos Nutricionais , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/farmacologia , Absorção Intestinal , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Inulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Lesões por Radiação/tratamento farmacológico , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/farmacologia , Compostos de Sulfidrila/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismoRESUMO
Lysophosphatidic acid (LPA) is a growth factor-like mediator and a ligand for multiple GPCR. The LPA2 GPCR mediates antiapoptotic and mucosal barrier-protective effects in the gut. We synthesized sulfamoyl benzoic acid (SBA) analogues that are the first specific agonists of LPA2, some with subnanomolar activity. We developed an experimental SAR that is supported and rationalized by computational docking analysis of the SBA compounds into the LPA2 ligand-binding pocket.
Assuntos
Benzoatos/química , Receptores de Ácidos Lisofosfatídicos/agonistas , Sítios de Ligação , Técnicas de Química Sintética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Relação Estrutura-AtividadeRESUMO
The effects of acute exposure to low- and high-dose radiation on the quantitative and functional parameters of the immune system were analyzed. C57BL/6 mice were irradiated with different doses of γ radiation (0.01, 0.05, 0.1, 0.5 and 2 Gy) and splenocytes were isolated at various times. Alterations in the distribution and surviving fraction of splenocyte subsets such as CD4(+) and CD8(+) T lymphocytes, regulatory T cells (Treg), natural killer (NK) cells, dendritic cells (DCs) and B lymphocytes were analyzed by flow cytometry. Apoptosis frequency was quantified by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method 4 h after irradiation. Cytokine expression was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Low doses decreased apoptosis in the splenocyte subpopulations studied most prominently in NK cells and DCs. Exposure to 2 Gy increased apoptosis in all splenocyte subpopulations; B cells were the most sensitive and NK cells and DCs the least sensitive. The lowest cell numbers were measured 3 days after irradiation, with minor changes by day 7. CD8(+) and B cells were rather resistant to low doses but were very sensitive to 2 Gy, while NK cells, DCs and Treg cells were much more resistant to high doses. Expression of the T-helper 1 (Th1)- and helper 2 (Th2)-type cytokines decreased after low doses and increased after high doses. Interleukin 6 (IL-6) reacted at early times and IL-10 at later times. IL-5 levels were consistently elevated. These data highlight the differences in the responses of different splenocyte subpopulations to low- and high-dose radiation.