Plasma Thermogram Parameters Differentiate Status and Overall Survival of Melanoma Patients.

Melanoma is the fifth most common cancer in the United States and the deadliest of all skin cancers. Even with recent advancements in treatment, there is still a 13% two-year recurrence rate, with approximately 30% of recurrences being distant metastases. Identifying patients at high risk for recurrence or advanced disease is critical for optimal clinical decision-making. Currently, there is substantial variability in the selection of screening tests and imaging, with most modalities characterized by relatively low accuracy. In the current study, we built upon a preliminary examination of differential scanning calorimetry (DSC) in the melanoma setting to examine its utility for diagnostic and prognostic assessment. Using regression analysis, we found that selected DSC profile (thermogram) parameters were useful for differentiation between melanoma patients and healthy controls, with more complex models distinguishing melanoma patients with no evidence of disease from patients with active disease. Thermogram features contributing to the third principal component (PC3) were useful for differentiation between controls and melanoma patients, and Cox proportional hazards regression analysis indicated that PC3 was useful for predicting the overall survival of active melanoma patients. With the further development and optimization of the classification method, DSC could complement current diagnostic strategies to improve screening, diagnosis, and prognosis of melanoma patients.

The Utility of Differential Scanning Calorimetry Curves of Blood Plasma for Diagnosis, Subtype Differentiation and Predicted Survival in Lung Cancer.

Early detection of lung cancer (LC) significantly increases the likelihood of successful treatment and improves LC survival rates. Currently, screening (mainly low-dose CT scans) is recommended for individuals at high risk. However, the recent increase in the number of LC cases unrelated to the well-known risk factors, and the high false-positive rate of low-dose CT, indicate a need to develop new, non-invasive methods for LC detection. Therefore, we evaluated the use of differential scanning calorimetry (DSC) for LC patients' diagnosis and predicted survival. Additionally, by applying mass spectrometry, we investigated whether changes in O- and N-glycosylation of plasma proteins could be an underlying mechanism responsible for observed differences in DSC curves of LC and control subjects. Our results indicate selected DSC curve features could be useful for differentiation of LC patients from controls with some capable of distinction between subtypes and stages of LC. DSC curve features also correlate with LC patients' overall/progression free survival. Moreover, the development of classification models combining patients' DSC curves with selected plasma protein glycosylation levels that changed in the presence of LC could improve the sensitivity and specificity of the detection of LC. With further optimization and development of the classification method, DSC could provide an accurate, non-invasive, radiation-free strategy for LC screening and diagnosis.

S1P Signaling in the Tumor Microenvironment.

Sphingosine-1-phosphate (S1P), together with other phosphosphingolipids, has been found to regulate complex cellular function in the tumor microenvironment (TME) where it acts as a signaling molecule that participates in cell-cell communication. S1P, through intracellular and extracellular signaling, was found to promote tumor growth, angiogenesis, chemoresistance, and metastasis; it also regulates anticancer immune response, modulates inflammation, and promotes angiogenesis. Interestingly, cancer cells are capable of releasing S1P and thus modifying the behavior of the TME components in a way that contributes to tumor growth and progression. Therefore, S1P is considered an important therapeutic target, and several anticancer therapies targeting S1P signaling are being developed and tested in clinics.

Increased methylation upstream of the MEG3 promotor is observed in acute myeloid leukemia patients with better overall survival.

BACKGROUND: The delta-like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3(MEG3) locus (DLK1-MEG3 locus) plays a critical role in the maintenance and differentiation of hematopoietic stem cells. Accumulating evidence implicates the imprinted genes from this locus, DLK1 and MEG3, in the development and progression of acute myeloid leukemia (AML). However, the contribution of this locus to the treatment response of patients and their survival is unknown. METHODS: DNA methylation of select CG dinucleotide-containing amplicons (CpG sites) within the DLK1-MEG3 locus and within differentially methylated regions of other imprinted loci was assessed in the mononuclear cells of 45 AML patients by combined bisulfite restriction analysis. Methylation results were compared with patient response to first-round induction therapy and overall survival. Multivariable analysis was employed to identify independent prognostic factors for patient overall survival in AML. RESULTS: Increased methylation at CpG sites within the MEG3 promotor region was observed in AML patients having longer overall survival. In addition, patients with shorter overall survival had increased expression of DLK1 and MEG3, and methylation at the MEG3-DMR CpG site inversely correlated with MEG3 expression. Multivariable analysis revealed that methylation at CG9, a non-imprinted CpG site within the MEG3 promotor region which contains a CCCTC-binding factor (CTCF)-binding DNA sequence, is an independent prognostic factor for the overall survival of AML patients. CONCLUSIONS: The results of our pilot study underscore the importance of the DLK1-MEG3 locus in AML development and progression. We identify CG9 methylation as an independent prognostic factor for AML patient survival, which suggests that distinct miRNA signatures from the DLK1-MEG3 locus could reflect varying degrees of cell stemness and present novel opportunities for personalized therapies in the future. These data provide a foundation for future studies into the role of higher-order chromatin structure at DLK1-MEG3 in AML.

[Corrigendum] Pituitary sex hormones enhance the pro­metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase­1.

After the publication of the article, the authors realize that they overlooked stating that the author Magda Kucia was the recipient of an OPUS grant (grant no. UMO­2016/21/B/NZ4/00201). Therefore, the Acknowledgements section of the paper should have read as follows (the added text is highlighted in bold): "This study was supported by NIH grants 2R01 DK074720 and R01HL112788, the Stella and Henry Endowment, and NCN Harmonia grant UMO­2014/14/M/NZ3/00475 to M.Z.R., and OPUS grant UMO­2016/21/B/NZ4/00201 to M.K." The authors regret their oversight in failing to include this information in the Acknowledgements section of their paper. [the original article was published in International Journal of Oncology 50: 317-328, 2017; DOI: 10.3892/ijo.2016.3787].

Analysis of the Paternally-Imprinted DLK1-MEG3 and IGF2-H19 Tandem Gene Loci in NT2 Embryonal Carcinoma Cells Identifies DLK1 as a Potential Therapeutic Target.

The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Estressores ocupacionais e estratégias de enfrentamento / Occupational stressors and coping strategies / Factores de estrés ocupacionales y estrategias de enfrentamiento / Stresseurs professionnels et les stratégies de coping

O presente estudo teve como objetivo conhecer a vivência dos estressores ocupacionais e estratégias de enfrentamento utilizadas na perspectiva dos professores. Participaram do estudo oito professoras provenientes de duas escolas públicas municipais de uma cidade da região metropolitana de Porto Alegre, RS. Como instrumento de coleta de dados foi utilizado o método do diário em sua modalidade qualitativa, estruturado em dois eixos temáticos estabelecidos a priori: estressores e estratégias de enfrentamento utilizadas. Os registros foram analisados por meio da técnica de análise de conteúdo categorial-temática. Como resultados, foram identificados cinco estressores de natureza psicossocial: relação professor-alunos; falta de apoio de familiares de alunos; sobrecarga de papel; conciliar trabalho-família e conciliar trabalho-vida pessoal. As estratégias de enfrentamento referidas foram as com foco na emoção e evitação.

The present study had as objective to know the experience of the occupational stressors and coping strategies used from the perspective of the teachers. Eight female teachers from two municipal public schools of a city in the metropolitan region of Porto Alegre, RS, participated in the study. As a data collection instrument, the journal method was used in its qualitative modality, structured in two thematic axes established a priori: stressors and coping strategies used. The records were analyzed using the categorical-thematic content analysis technique. As results, five stressors of a psychosocial nature were identified: teacher-student relationship; lack of support from student relatives; paper overload; reconciling work-family and reconciling work-life. The coping strategies referred to were those focused on emotion and avoidance.

Este estudio tuvo como objetivo conocer a la vivencia de los factores de estrés ocupacionales y estrategias de enfrentamiento utilizadas en la perspectiva de los profesores. Participaron del estudio ocho profesoras de escuelas públicas municipales de una ciudad de la región central de Porto Alegre, RS- Brasil. Como instrumento de colecta de datos fue utilizado el método del diario en su modalidad cualitativa, estructurado en dos ejes temáticos establecidos a priori: factores de estrés y estrategias de enfrentamiento utilizadas. Los registros fueron analizados por medio de la técnica de análisis de contenido categorial-temática. Como resultado, fueron identificados cinco factores de estrés de naturaleza psicosocial: relación profesor-alumno; falta de apoyo de familiares de alumnos; sobrecarga de papel; conciliar trabajo-familia y trabajo-vida personal. Las estrategias de enfrentamiento referidas fueron las con enfoque en la emoción y prevención.

La présente étude a eu l'objectif de connaître l'expérience des stresseurs professionnels et aussi des stratégies de coping utilisées sur le point de vue des enseignants. Huit enseignantes, de deux écoles publiques municipales d'une ville de la région métropolitaine de Porto Alegre, RS, ont participé à l'étude. Le journal intime - dans la modalité qualitative - a été utilisé comme outil pour collecter les donnés. Ils ont été structurés sur deux axes thématiques prévues à priori: des stresseurs et des stratégies de coping utilisées. Les enregistrements ont été analysés à l'aide de la technique d'analyse de contenu catégorielle thématique. On a pu identifier cinq stresseurs de nature psychosocial : de la relation enseignant-élèves; de l'absence de soutien par les familles des élèves; surcharge de rôles; difficulté de conciliation entre le travail et la famille et entre le travail et la vie personnelle. Les stratégies de coping mentionnées étaient celles axées sur l'émotion et l'évitement.

Novel evidence that pituitary sex hormones regulate migration, adhesion, and proliferation of embryonic stem cells and teratocarcinoma cells.

The pituitary sex hormones (SexHs): follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) regulate several functions crucial for reproduction, including oogenesis, spermatogenesis, and lactation. An important source of prolactin-like hormones, known as lactogens, is the placenta, and lactogens bind to the PRL receptor (PRLR) with high affinity and thereby mimic the actions of PRL. Recently, it has been demonstrated that pituitary SexHs were involved in metastatic lung cancer, certain sarcomas, and leukemia. In the present study we aimed to investigate whether FSH, LH, and PRL were able to stimulate stem cells involved in early development. To address this issue we employed a murine embryonic stem cell line (ES-D3) as well as two teratocarcinoma cell lines, P19 (murine) and NTera2 (human). We determined that all these cells expressed SexH receptors at the mRNA and protein levels and that stimulation of these receptors induced phosphorylation of p42/44 MAPK, p38 MAPK, and AKT. Moreover, ES-D3, P19, and NTera2 cells responded with increased migration and adhesion to physiological concentrations of pituitary SexHs. In view of these findings we proposed that maternal-derived pituitary SexHs regulate the biology of stem cells involved in early development.

Rinossinusite aguda / Acute rhinosinusitis

Objetivos: revisar a prevalência, a etiologia, o diagnóstico e o tratamento da rinossinusite aguda. A rinossinusite aguda é uma doença comum de prevalência mundial, caracterizada por uma inflamação da cavidade nasal e dos seios paranasais, podendo ter origem fúngica, viral ou bacteriana. Os sintomas são principalmente obstrução e secreção nasal, porém a sintomatologia pode ser ampla. Consequentemente, a correta identificação e manejo torna essa patologia um desafio na prática clínica. Metodologia: Foi realizado uma busca nas bases de dados Medline/ Pubmed e LILACS no mês de maio de 2018, incluindo artigos publicados em inglês ou português nos últimos 5 anos. Foram utilizados os seguintes descritores: "acute rhinosinusitis", "rhinitis" e "sinusitis". Os artigos foram selecionados com busca direta, considerando relevância do tema à proposta e fator de impacto. Resultados: De 228 publicações, 10 foram selecionadas, demonstrando que a rinossinusite aguda é uma doença cuja incidência ainda não está bem definida, mas é maior em indivíduos com idade entre 12 e 17 anos em relação à população geral, com prevalência de etiologia viral. Conclusão: A rinossinusite aguda é uma doença com alta incidência. O diagnóstico é predominantemente clínico, mas em casos restritos há exames de imagem, sendo o tratamento sempre sintomático.

Objectives: To review the prevalence, etiology, diagnosis and treatment of acute rhinosinusitis. Acute rhinosinusitis is a common global disease characterized by inflammation of the nasal cavity and paranasal sinuses, which may be of fungal, viral or bacterial origin. The symptoms are mainly obstruction and nasal secretion, but the symptomatology can be ample. Methodology: The search was performed in the Medline / Pubmed and LILACS databases in May 2018, including articles published in English or Portuguese in the last 5 years. The following descriptors were used: "acute rhinosinusitis", "rhinitis" and "sinusitis". The articles were selected with direct search, considering relevance of the theme to the proposal and impact factor. Results: Of 228 publications, 10 were selected, demonstrating that acute rhinosinusitis is a disease whose incidence is still not well defined, but is higher in individuals aged 12 to 17 years in relation to the general population, with a prevalence of viral etiology. Conclusion: Acute rhinosinusitis is a disease with a high incidence. The diagnosis is predominantly clinical, but in restricted cases there are imaging tests, and the treatment is always symptomatic.

Bloqueios do neuroeixo: uma revisão entre duas modalidades anestésicas / Central neuraxial blocks: a review between two anesthetic modalities

Objetivo: Levando em consideração a numerosa indicação do bloqueio raquidiano e da perianestesia na prática médica, o objetivo deste trabalho consiste em realizar uma revisão comparativa entre essas duas modalidades anestésicas. Métodos: Foi realizada busca nas bases de dados PubMed/MEDLINE, LILACS e Google acadêmico, e foram usadas as seguintes palavras-chave: Spinal Anesthesia e Epidural Anesthesia. Após isso, os artigos foram filtrados pelos autores. Resultados: na raquianestesia utiliza-se uma pequena dose de anestésico local para produzir uma profunda analgesia sensorial. Por outro lado, na perianestesia necessita-se de uma grande dose de anestésico local. Conclusão: essas duas anestesias neuroaxiais utilizadas na prática pré e pós-cirúrgica são seguras, porém necessitam de cuidados.

Objective: Considering the numerous indications of spinal block and perianesthesia in medical practice, the objective of this study is to perform a comparative review between these two anesthetic modalities. Methods: A search was performed at PubMed®, LILACS® and Google Academic® using the keywords "spinal anesthesia" and "epidural anesthesia". After this, the papers were filtered by the authors. Results: In spinal anesthesia, a small dose of local anesthetic is used to produce a deep sensory analgesia. On the other hand, perianesthesia requires a large dose of local anesthetic. Conclusion: these two neuroaxial blocks used in pre and post-surgical practice safe, although require care.

Novel pleiotropic effects of bioactive phospholipids in human lung cancer metastasis.

We previously proposed that one of the unwanted side effects of chemotherapy and radiotherapy is the increase in several peptide- and non-peptide based chemoattractants in damaged tissues, leading to induction of a prometastatic microenvironment for remaining cancer cells. Herein, we turned out our attention to a potential role of bioactive phospholipids (BphsLs), such as sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and lysophosphatidic acid (LPA) in lung cancer (LC) metastasis. We report that LC cells express several functional BphL receptors (for S1P, LPC, and LPA) as well as several enzymes involved in their metabolism and that BphsLs are potent chemokinetic and adhesion factors for these cells. We also demonstrate for the first time the novel role of C1P as a prometastatic factor in LC cells. In addition to their chemokinetic activities, BphsLs also sensitize or prime the chemotactic responsiveness of LC cells to known prometastatic factors such as hepatocyte growth factor/scatter factor (HGF/SF). Thus, for the first time we demonstrate a prometastatic effect that is based on the priming of a cell's responsiveness to chemotactic factors by chemokinetic factors. To our surprise, none of the bioactive lipids induced proliferation of LC cells or ameliorated toxic effects of vincristine treatment. Interestingly, BphsLs increase adhesion of LC cells to bone marrow-derived stromal cells and stimulate these cells to release ExNs, which additionally increase LC cell motility. In conclusion, our results show that BphsLs are important modulators of prometastatic environment. Therefore, their inhibitors could be considered as potential anti-metastatic drug candidates to be included as a part of post radio- and/or chemo- therapy treatment.

Do Cancer Cell Lines Have Fixed or Fluctuating Stem Cell Phenotypes? - Studies with the NTera2 Cell Line.

One of the important questions when studying established cancer cell lines is whether such cells contain a subpopulation of primitive cancer stem cells that maintains the expansion of the cell line. To address this issue, we performed studies on the established human embryonal carcinoma cell line NTera2 by evaluating the potential stemness of cells sorted according to their expression of the cell surface stem cell markers CD133 and SSEA4. By performing in vitro and in vivo assays, we observed different properties of cells expressing both, one, or neither of these antigens. While sorted SSEA4+ subpopulations exhibited the greatest propensity for migration toward normal serum and the highest seeding efficiency in the lungs of immunodeficient mice, CD133-SSEA4- cells displayed high seeding efficiency to the bone marrow after injection in vivo. It is worth noting that these properties did not depend on the size of the evaluated cells. To address the question of whether cancer stem cell phenotypes in cell lines are fixed or fluctuating, we sorted single cells according to their expression of CD133 and SSEA4 antigens and observed that cells which did not express these cancer stem cell markers gave rise to cells that express these markers after expansion in vitro. Therefore, our results support the idea that within established cancer cell lines, the phenotype of the cell subpopulation expressing cancer stem cell markers is not fixed but fluctuates during cell line expansion, and cells negative for these markers may acquire their expression.

Pituitary sex hormones enhance the pro­metastatic potential of human lung cancer cells by downregulating the intracellular expression of heme oxygenase­1.

We report that human lung cancer cell lines express functional receptors for pituitary sex hormones (SexHs) and respond to stimulation by follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL). Expression of these receptors has also been confirmed in patient lung cancer samples at the mRNA level. Stimulation of human lung cancer cell lines with FSH, LH, or PRL stimulated migration and chemotaxis, and some cell lines responded by enhanced proliferation. Moreover, priming of human lung cancer cells by exposing them to pituitary SexHs resulted in enhanced seeding efficiency of injected human lung cancer cells into bone marrow, liver, and lungs in an immunodeficient mouse model. The chemotaxis of lung cancer cell lines corresponded with the activity of heme oxygenase­1 (HO­1), as stimulation of these cells by FSH, LH, and PRL downregulated its expression in a p38 MAPK­dependent manner. Moreover, while downregulation of HO­1 by the small­molecule inhibitor tin protoporphyrin (SnPP) promoted migration, upregulation of HO­1 by the small­molecule activator cobalt protoporphyrin (CoPP) showed the opposite effect. Based on this finding, we propose that pituitary SexHs play a significant role in the pathogenesis of lung cancer, particularly when the blood level of FSH increases due to gonadal dysfunction with advanced age. Finally, we propose that upregulation of HO­1 expression by a small­molecule activator may be effective in controlling SexH­induced cell migration in lung cancer.

Involvement of BAFF and APRIL in Resistance to Apoptosis of Acute Myeloid Leukemia.

B-cell activation factor of the TNF family (BAFF), and a proliferation-inducing ligand (APRIL), two members of the tumour necrosis factor (TNF) superfamily, beyond playing a significant role in normal B-cell development, promote survival and proliferation of malignant B cells. Both ligands interact with 3 receptors: BAFF-R, specific to BAFF, and TACI and BCMA which are shared by both BAFF and APRIL. Here we wished to investigate the potential role of these proteins in resistance of acute myeloid leukaemia (AML) blasts to apoptosis. We found that the levels of both mRNA and proteins of APRIL, BAFF and their receptors were expressed in leukaemic cells of 24 newly diagnosed, untreated AML patients. We also demonstrated that patients who did not further respond to induction therapy (NR) presented with significantly higher baseline APRIL and BAFF expression on AML blasts as compared to these subjects who, after induction, achieved complete remission (CR) following induction therapy. Moreover, we observed striking differences in baseline levels of BCMA between CR and NR patients as we did not find detectable expression of this receptor in the latter group of patients. Interestingly, we found that AML blasts collected at baseline from NR patients cultured in presence of exogenous BAFF and APRIL were significantly more resistant to spontaneous or drug-induced apoptosis as compared with cells derived from CR patients. Altogether, our data confirm that BAFF and APRIL signaling play important role in AML pathogenesis and susceptibility to cytotoxic therapy while measuring of BCMA expression on AML cells can become a novel prognostic factor for chemotherapy response.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities.

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50-100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

Induction of a Tumor-Metastasis-Receptive Microenvironment as an Unwanted Side Effect After Radio/Chemotherapy and In Vitro and In Vivo Assays to Study this Phenomenon.

Besides surgical removal of tumor tissue, chemotherapy and radiotherapy are the most important and efficient treatment modalities employed to treat therapy-susceptible malignancies. The main aim of this treatment-to destroy tumor cells-is unfortunately usually associated with toxicity to nontumor cells and different degrees of tissue and organ damage. In damaged tissues several chemoattractants are upregulated and released that may attract tumor cells. Moreover, highly migratory radio/chemotherapy treatment may endow cells with several properties of cancer stem cells which survive and respond to these chemoattractants upregulated in collateral tissues. Based on this, one of the unwanted and underappreciated side effects of chemotherapy or radiotherapy is the creation of a metastasis-receptive microenvironment in bones as well as in other organs of the body. Herein we describe methods and assays that can be employed to study migratory properties of cancer cells in in vitro (chemotaxis) and in vivo (seeding efficiency assay) conditions in response to the induction of pro-metastatic microenvironments in various organs and tissues.

Vitamin D3 stimulates embryonic stem cells but inhibits migration and growth of ovarian cancer and teratocarcinoma cell lines.

BACKGROUND: Deficiency in Vitamin D3 (cholecalciferol) may predispose to some malignancies, including gonadal tumors and in experimental models vitamin D3 has been proven to inhibit the growth of cancer cells. To learn more about the potential role of vitamin D3 in cancerogenesis, we evaluated the expression and functionality of the vitamin D receptor (VDR) and its role in metastasis of ovarian cancer cells and of murine and human teratocarcinoma cell lines. METHODS: In our studies we employed murine embrynic stem cells (ESD3), murine (P19) and human (NTERA-2) teratocarcimona cells lines, human ovarian cancer cells (A2780) as well as purified murine and human purified very small embryonic like stem cells (VSELs). We evaluated expression of Vitamin D3 receptor (VDR) in these cells as well as effect of vitamin D3 exposure on cell proliferation and migration. RESULTS: We here provide also more evidence for the role of vitamin D3 in germline-derived malignancies, and this evidence supports the proposal that vitamin D3 treatment inhibits growth and metastatic potential of several germline-derived malignancies. We also found that the ESD3 murine immortalized embryonic stem cell line and normal, pluripotent, germline-marker-positive very small embryonic-like stem cells (VSELs) isolated from adult tissues are stimulated by vitamin D3, which suggests that vitamin D3 affects the earliest stages of embryogenesis. CONCLUSIONS: We found that however all normal and malignant germ-line derived cells express functional VDR, Vitamin D3 differently affects their proliferation and migration. We postulate that while Vitamin D3 as anticancer drug inhibits proliferation of malignant cells, it may protect normal stem cells that play an important role in development and tissue/organ regeneration.

Human rhabdomyosarcoma cells express functional pituitary and gonadal sex hormone receptors: Therapeutic implications.

Evidence has accumulated that sex hormones play an important role in several types of cancer. Because they are also involved in skeletal muscle development and regeneration, we were therefore interested in their potential involvement in the pathogenesis of human rhabdomyosarcoma (RMS), a skeletal muscle tumor. In the present study, we employed eight RMS cell lines (three fusion positive and five fusion negative RMS cell lines) and mRNA samples obtained from RMS patients. The expression of sex hormone receptors was evaluated by RT-PCR and their functionality by chemotaxis, adhesion and direct cell proliferation assays. We report here for the first time that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors are expressed in established human RMS cell lines as well as in primary tumor samples isolated from RMS patients. We also report that human RMS cell lines responded both to pituitary and gonadal sex hormone stimulation by enhanced proliferation, chemotaxis, cell adhesion and phosphorylation of MAPKp42/44 and AKT. In summary, our results indicate that sex hormones are involved in the pathogenesis and progression of RMS, and therefore, their therapeutic application should be avoided in patients that have been diagnosed with RMS.

Vitronectin in the ascites of human ovarian carcinoma acts as a potent chemoattractant for ovarian carcinoma: Implication for metastasis by cancer stem cells.

Vitronectin has been identified mainly as an adhesion protein that signals through uPAR and selected integrin receptors. In addition to its pro-adhesive properties, we identified recently vitronectin as a main chemoattractant present in diluted plasma/serum that directly stimulates migration of cancer cells. We also found that this pro-migratory activity of vitronectin can be quenched by fibrinogen. Based on this we hypothesized that this may explain preference of cancer cell to metastasize to fibrinogen-low microenvironments such as lymphatics or peritoneal cavity. Based on this, we decided to investigate a role of vitronectin in metastasis of ovarian cancer cells to peritoneal cavity. We tested migratory responsiveness of three human ovarian cancer cell lines to ascites isolated from ovarian cancer patients and characterize possible molecules involved in migration of ovarian cancer cells. The ascites samples were exposed to heat inactivation, proteinase K digested, dialyzed and charcoal stripped. We also performed cut-off filtration analysis and by employing ELISA assays to measure concentration of vitronectin in ascites fluid samples. Finally, we employed shRNA against uPAR and small molecular inhibitors of integrin receptors to assess their involvement in biological effects of vitronectin. From our studies, we found that the similarly to diluted plasma, vitronectin in absence of fibrinogen is a main chemotactic/chemokinetic protein present in ascites fluid. We also found that these pro-migratory properties of vitronectin can be quenched by addition of fibrinogen. Our studies also indicate that both uPAR and integrin receptors on ovarian cancer cells regulate migration of these cells to vitronectin gradient. In summary, we identified free soluble vitronectin as a potent direct chemoattractant for ovarian cancer cells and that its activity is suppressed after binding to fibrinogen. Since in ascites fluids vitronectin is present in free form because of a lack or low level of fibrinogen, this could explain preferences of ovarian cancer stem cells to metastasize within peritoneum. We propose that inhibitors which could sequester soluble vitronectin in similar fashion as fibrinogen, could be employed as a novel anti-metastatic drugs.