RESUMO
Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.
Assuntos
Antivirais/farmacologia , Heparina/farmacologia , Cloreto de Magnésio/farmacologia , Aciclovir/farmacologia , Adenovírus Humanos/efeitos dos fármacos , Adenovírus Humanos/fisiologia , Animais , Antivirais/química , Células CHO , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Fibroblastos , Heparina/química , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Humanos , Cloreto de Magnésio/química , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Cultura Primária de Células , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Relação Estrutura-Atividade , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
PURPOSE: COVID-19 infection has manifested as a major threat to both patients and healthcare providers around the world. Radiation oncology institutions (ROI) deliver a major component of cancer treatment, with protocols that might span over several weeks, with the result of increasing susceptibility to COVID-19 infection and presenting with a more severe clinical course when compared with the general population. The aim of this manuscript is to investigate the impact of ROI protocols and performance on daily practice in the high-risk cancer patients during this pandemic. METHODS: We addressed the incidence of positive COVID-19 cases in both patients and health care workers (HCW), in addition to the protective measures adopted in ROIs in Germany, Austria and Switzerland using a specific questionnaire. RESULTS: The results of the questionnaire showed that a noteworthy number of ROIs were able to complete treatment in SARS-CoV2 positive cancer patients, with only a short interruption. The ROIs reported a significant decrease in patient volume that was not impacted by the circumambient disease incidence, the type of ROI or the occurrence of positive cases. Of the ROIs 16.5% also reported infected HCWs. About half of the ROIs (50.5%) adopted a screening program for patients whereas only 23.3% also screened their HCWs. The range of protective measures included the creation of working groups, instituting home office work and protection with face masks. Regarding the therapeutic options offered, curative procedures were performed with either unchanged or moderately decreased schedules, whereas palliative or benign radiotherapy procedures were more often shortened. Most ROIs postponed or cancelled radiation treatment for benign indications (88.1%). The occurrence of SARS-CoV2 infections did not affect the treatment options for curative procedures. Non-university-based ROIs seemed to be more willing to change their treatment options for curative and palliative cases than university-based ROIs. CONCLUSION: Most ROIs reported a deep impact of SARS-CoV2 infections on their work routine. Modification and prioritization of treatment regimens and the application of protective measures preserved a well-functioning radiation oncology service and patient care.
Assuntos
COVID-19/prevenção & controle , Infecção Hospitalar/prevenção & controle , Controle de Infecções/métodos , Neoplasias/radioterapia , Pandemias , Recursos Humanos em Hospital/estatística & dados numéricos , SARS-CoV-2/isolamento & purificação , Agendamento de Consultas , Áustria/epidemiologia , COVID-19/diagnóstico , COVID-19/epidemiologia , Teste para COVID-19/estatística & dados numéricos , Institutos de Câncer/estatística & dados numéricos , Comorbidade , Infecção Hospitalar/epidemiologia , Estudos Transversais , Alemanha/epidemiologia , Hospitais Comunitários , Hospitais Universitários/estatística & dados numéricos , Humanos , Incidência , Controle de Infecções/organização & administração , Máscaras/estatística & dados numéricos , Máscaras/provisão & distribuição , Neoplasias/epidemiologia , Cuidados Paliativos/estatística & dados numéricos , Utilização de Procedimentos e Técnicas , Risco , Inquéritos e Questionários , Suíça/epidemiologia , Telemedicina/estatística & dados numéricos , Teletrabalho/estatística & dados numéricosRESUMO
BACKGROUND: Several physiological (fertilization, placentation, wound healing) and pathophysiological processes (infection with enveloped viruses, cancer) depend on cell fusion. In cancer it was postulated that the fusion of cancer cells with normal cells such as macrophages or stem cells may not only give rise to hybrid cells exhibiting novel properties, such as an increased metastatic capacity and drug resistance, but possibly also cancer stem/ initiating cell properties. Hence, hybrid clone cells (M13HS, M13MDA435 and M13MDA231) that were derived from spontaneous fusion events of human M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg, MDA-MB-435-Hyg and MDA-MB-231-Hyg cancer cells were investigated regarding potential in vitro cancer stem/ initiating cell properties. METHODS: CD44/CD24 expression pattern and ALDH1 activity of parental cells and hybrid clones was determined by flow cytometry. A colony formation and mammosphere formation assay was applied to determine the cells' capability to form colonies and mammospheres. Sox9, Slug and Snail expression levels were determined by Western blot analysis. RESULTS: Flow cytometry revealed that all hybrid clone cells were CD44+/CD24-/low, but differed markedly among each other regarding ALDH1 activity. Likewise, each hybrid clone possessed a unique colony formation and mammosphere capacity as well as unique Snail, Slug and Sox9 expression patterns. Nonetheless, comparison of hybrid clones revealed that M13HS hybrids exhibited more in vitro cancer stem/ initiating cell properties than M13MDA231 and M13MDA435 hybrids, such as more ALDH1 positive cells or an increased capacity to form colonies and mammospheres. CONCLUSION: The fate whether cancer stem/ initiating cells may originate from cell fusion events likely depends on the specific characteristics of the parental cells.
Assuntos
Neoplasias da Mama/patologia , Células Epiteliais/patologia , Células Híbridas/patologia , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Fusão Celular , Movimento Celular , Células Epiteliais/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Células Híbridas/metabolismo , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células Tumorais CultivadasRESUMO
Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , NF-kappa B/metabolismo , Faringite/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Movimento Celular , Proliferação de Células , Celulose/química , Biologia Computacional , Simulação por Computador , Regulação para Baixo , Granuloma/metabolismo , Proteínas Hemolisinas/sangue , Sistema Imunitário , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ácido N-Acetilneuramínico/metabolismo , Fagocitose , Fenolsulfonaftaleína/química , Extratos Vegetais/uso terapêutico , Ratos , Saliva/metabolismo , Transdução de Sinais , Baço/metabolismo , Temperatura , Xerostomia/terapiaRESUMO
BACKGROUND: In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α). METHODS: The gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody. RESULTS: The data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. CONCLUSIONS: The matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline-based antibiotic minocycline might exhibit anti-fusogenic properties because it inhibits a cell fusion-related mechanism.
Assuntos
Fusão Celular , Células Epiteliais/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Mama/citologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Claudina-1/antagonistas & inibidores , Claudina-1/genética , Claudina-1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Integrases/genética , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. METHODS: Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. RESULTS: M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS hybrid clones exhibited a mesenchymal phenotype and, with the exception of one hybrid clone, responded to EGF with an increased migratory activity. CONCLUSION: Fusion of human breast epithelial cells and human breast cancer cells can give rise to hybrid clone cells that possess certain CS/IC properties, suggesting that cell fusion might be a mechanism underlying how tumor cells exhibiting a CS/IC phenotype could originate.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Evolução Clonal , Células Epiteliais/metabolismo , Células Híbridas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Família Aldeído Desidrogenase 1 , Biomarcadores , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Evolução Clonal/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Células Híbridas/patologia , Isoenzimas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Retinal Desidrogenase/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Esferoides Celulares , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The biological phenomenon of cell fusion plays an important role in several physiological processes, like fertilization, placentation, or wound healing/tissue regeneration, as well as pathophysiological processes, such as cancer. Despite this fact, considerably less is still known about the factors and conditions that will induce the merging of two plasma membranes. Inflammation and proliferation has been suggested as a positive trigger for cell fusion, but it remains unclear, which of the factor(s) of the inflamed microenvironment are being involved. To clarify this we developed a reliable assay to quantify the in vitro fusion frequency of cells using a fluorescence double reporter vector (pFDR) containing a LoxP-flanked HcRed/DsRed expression cassette followed by an EGFP expression cassette. Because cell fusion has been implicated in cancer progression four human breast cancer cell lines were stably transfected with a pFDR vector and were co-cultured with the stably Cre-expressing human breast epithelial cell line. Cell fusion is associated with a Cre-mediated recombination resulting in induction of EGFP expression in hybrid cells, which can be quantified by flow cytometry. By testing a panel of different cytokines, chemokines, growth factors and other compounds, including exosomes, under normoxic and hypoxic conditions our data indicate that the proinflammatory cytokine TNF-α together with hypoxia is a strong inducer of cell fusion in human MDA-MB-435 and MDA-MB-231 breast cancer cells.
Assuntos
Neoplasias da Mama/fisiopatologia , Fusão Celular/métodos , Glândulas Mamárias Humanas/fisiologia , Western Blotting , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Integrases , Microscopia Eletrônica de Varredura , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-ß, TNF-α, IL-1ß and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.
Assuntos
Neoplasias da Mama/metabolismo , Células Epiteliais/metabolismo , Células Híbridas/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genéticaRESUMO
After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.
Assuntos
Fibroblastos/metabolismo , Macrófagos/metabolismo , Melanoma/metabolismo , Fenótipo , Células Estromais/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Fusão Celular , Células Cultivadas , Fibroblastos/patologia , Humanos , Células Híbridas , Macrófagos/patologia , Melanoma/patologia , Monócitos/metabolismo , Monócitos/patologia , Células Estromais/patologiaRESUMO
Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell's phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAF(V600E) melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAF(V600E) protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAF(V600E) with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAF(V600E) mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAF(V600E) mutation or protein in the peritumoral stroma of BRAF(WT) melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome.
Assuntos
Melanoma/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/genética , Células Estromais/metabolismo , Fusão Celular , Humanos , Melanoma/patologia , Células Estromais/patologiaRESUMO
Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRß. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.
Assuntos
Receptores ErbB/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Anticorpos Neutralizantes , Comunicação Autócrina , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Humanos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that "accidental" tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells.
Assuntos
Evolução Clonal , Células Híbridas/fisiologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Fusão Celular , Instabilidade Genômica , Humanos , Neoplasias/genética , RegeneraçãoRESUMO
We have shown previously that norepinephrine induces migratory activity of tumour cells from breast, colon and prostate tissue via activation of beta-2 adrenergic receptors. Consequently, this effect can be inhibited pharmacologically by clinically established beta-blockers. Tumour cell migration is a prerequisite for metastasis formation, and accordingly we and others have shown that breast cancer patients, which take beta-blockers due to hypertension, have reduced metastasis formation and increased survival probability as compared to patients without hypertension or using other anti-hypertensive medication. Unlike the aforementioned tumour cells, pancreatic cancer cells show a reduced migratory activity upon norepinephrine treatment. By means of our three-dimensional, collagen-based cell migration assay, we have investigated the signal transduction pathways involved in this phenomenon. We have found that this conflicting effect of norepinephrine on pancreatic cancer cells is due to an imbalanced activation of the two pathways that usually mediate a pro-migratory effect of norepinephrine in other tumour cell types. Firstly, the inhibitory effect results from activation of a pathway which causes a strong increase of the secondary cell signalling molecule, cAMP. In addition, activation of phospholipase C gamma and the downstream protein kinase C alpha were shown to be already activated in pancreatic cancer cells and cannot be further activated by norepinephrine. We hypothesize that this constitutive activation of the phospholipase C gamma pathway is due to a cross-talk with receptor tyrosine kinase signalling, and this might also deliver an explanation for the unusual high spontaneous migratory activity of pancreatic cancer cells.
Assuntos
Carcinoma/metabolismo , Movimento Celular/efeitos dos fármacos , Norepinefrina/farmacologia , Neoplasias Pancreáticas/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Fosfolipase C gama/metabolismo , Proteína Quinase C-alfa/metabolismo , Transdução de SinaisRESUMO
HER2 signalling by heterodimerisation with EGFR and HER3 in breast cancer is associated with worst outcome of the afflicted patients, which is attributed not only to the aggressiveness of such tumours but also to therapy resistance. Thus, in the present study we investigated the role of EGFR, HER2 and HER3 lateral signalling in cell migration by applying the MDA-MB-468-HER2 (MDA-HER2) breast cancer cell line, representing a valid model system. Knockdown of HER3 expression by siRNA resulted in decreased phosphorylated AKT (pAKT) levels, abrogated epidermal growth factor (EGF)-mediated PLC-γ1 activation and a diminished EGF-induced migratory activity, depicting the interplay of EGF receptor (EGFR)/HER2/PLC-γ1 and HER2/HER3/PI3K signalling in mediating the migration of EGFR/HER2/HER3-expressing breast cancer cells. Since therapy failure usually arises from metastatic cells, we further investigated whether HER3 signalling was active in established breast cancer disseminated tumour cell (DTC) lines as well as in primary DTCs derived from breast cancer patients. EGF treatment of DTC lines resulted solely in increased pAKT S473 levels, whereas in MDA-HER2 cells both pAKT S473 and pAKT T308 levels were increased upon EGF stimulation. Moreover, despite active HER3 molecules, as indicated by pTyr1222 staining, about 90% of analysed breast cancer patient DTCs exhibited very low or even no detectable pAKT S473 levels, suggesting that these cells might have fallen into dormancy. In summary, our data indicate the important role in EGFR, HER2 and HER3 lateral signalling in breast cancer cell migration. Moreover, our data further show that primary tumour cells and DTCs can vary in their HER activation status, which is important to know in the context of cancer therapy.
Assuntos
Neoplasias Ósseas/enzimologia , Neoplasias da Mama/enzimologia , Movimento Celular , Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfolipase C gama/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor ErbB-3/genética , TransfecçãoRESUMO
S100A8 and S100A9 belong to the damage-associated molecular pattern molecules. They are upregulated in a number of inflammatory skin disorders. Owing to their abundance in myeloid cells, the main function of S100A8/A9 has been attributed to their role in inflammatory cells. However, it is becoming increasingly clear that they also exert important roles in epithelial cells. In this review, we discuss the context-dependent function of S100A8/A9 in epithelial cells and their impact on wound healing, psoriasis and other skin diseases.
Assuntos
Calgranulina A/fisiologia , Calgranulina B/fisiologia , Células Epiteliais/fisiologia , Fenômenos Fisiológicos da Pele , Humanos , Psoríase/fisiopatologia , Dermatopatias/fisiopatologia , Cicatrização/fisiologiaRESUMO
BACKGROUND: The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-ß/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. RESULTS: Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. CONCLUSIONS: Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.
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BACKGROUND: Tumor tissue resembles chronically inflamed tissue. Since chronic inflammatory conditions are a strong stimulus for bone marrow-derived cells (BMDCs) it can be assumed that recruitment of BMDCs into cancer tissue should be a common phenomenon. Several data have outlined that BMDC can influence tumor growth and metastasis, e.g., by inducing a paracrine acting feedback loop in tumor cells. Likewise, cell fusion and horizontal gene transfer are further mechanisms how BMDCs can trigger tumor progression. RESULTS: Hygromycin resistant murine 67NR-Hyg mammary carcinoma cells were co-cultivated with puromycin resistant murine BMDCs from Tg(GFPU)5Nagy/J mice. Isolation of hygromycin/puromycin resistant mBMDC/67NR-Hyg cell clones was performed by a dual drug selection procedure. PCR analysis revealed an overlap of parental markers in mBMDC/67NR-Hyg cell clones, suggesting that dual resistant cells originated by cell fusion. By contrast, both STR and SNP data analysis indicated that only parental 67NR-Hyg alleles were found in mBMDC/67NR-Hyg cell clones favoring horizontal gene transfer as the mode of origin. RealTime-PCR-array analysis showed a marked up-regulation of Abcb1a and Abcb1b ABC multidrug transporters in mBMDC/67NR-Hyg clones, which was verified by Western Blot analysis. Moreover, the markedly increased Abcb1a/Abcb1b expression was correlated to an efficient Rhodamine 123 efflux, which was completely inhibited by verapamil, a well-known Abcb1a/Abcb1b inhibitor. Likewise, mBMDCs/67NR-Hyg clones revealed a marked resistance towards chemotherapeutic drugs including 17-DMAG, doxorubicin, etoposide and paclitaxel. In accordance to Rhodamine 123 efflux data, chemotherapeutic drug resistance of mBMDC/67NR-Hyg cells was impaired by verapamil mediated blockage of Abc1a/Abcb1b multidrug transporter function. CONCLUSION: Co-cultivation of mBMDCs and mouse 67NR-Hyg mammary carcinoma cells gave rise to highly drug resistant cells. Even though it remains unknown whether mBMDC/67NR-Hyg clones originated by cell fusion or horizontal gene transfer, our data indicate that the exchange of genetic information between two cellular entities is crucial for the origin of highly drug resistant cancer (hybrid) cells, which might be capable to survive chemotherapy.
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BACKGROUND: Some breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines. METHODS: Migration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested. RESULTS: Hypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration. CONCLUSIONS: Hypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients.
Assuntos
Comunicação Autócrina/fisiologia , Neoplasias da Mama/patologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Citocinas/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neutrófilos/efeitos dos fármacosRESUMO
Although cell fusion is an omnipresent process in life, to date considerably less is still known about the mechanisms and the molecules being involved in this biological phenomenon in higher organisms. Cell Fusion in Health and Disease Volume 2 is covering the dark side of cell fusion: namely its role in pathophysiological processes. International leading experts will present up-to-date overviews about cell fusion mediated horizontal gene transfer in bacteria and viruses, class III viral membrane fusion proteins, trophoblast fusion in trisomy 21, and the role of microvesicles in malignancies. Particular attention is paid on cell fusion in cancer and how this biological phenomenon may initiate the origin of (recurrence) cancer stem cells as well as drive the progression of multiple myeloma, colon cancer, breast cancer, and malignant melanoma. Thus, Cell Fusion in Health and Disease Volume 2 represents a state-of-the-art work for researchers, physicians or professionals being interested in reflecting the dark side of cell fusion.
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Fusão Celular , Publicações Periódicas como Assunto , Animais , HumanosRESUMO
Although cell fusion is an omnipresent process in life, to date considerably less is still known about the mechanisms and the molecules being involved in this biological phenomenon in higher organisms. In Cell Fusion in Health and Disease Volume 1 international leading experts will present up-to-date overviews about the current knowledge about cell fusion-mediating molecules in C. elegans and mammalian cells. Further topics of the book will focus on cell fusion in physiological processes including fertilization, placentation, skeletal muscle development, and tissue repair and will sum up the use of artificial cell fusion for cellular reprogramming and cancer vaccine development. Thus, Cell Fusion in Health and Disease Volume 1 represents a state-of-the-art work for researchers, physicians or professionals being interested in the biological phenomenon of cell fusion in physiological processes and beyond.