Three dimensional reconstruction of skin with melanoma: A model for study of invasion in vitro.

Melanoma is a type of tumor skin with high metastatic potential. Reconstructed human skin, development for pre-clinic assay, are make using primary human cells, but with same limitations. The aim this study was to characterize a cell culture model, with structure similar to human skin containing melanoma cells entirely from cell lines. Reconstructed skin with melanoma were development using human fibroblasts (MRC5), human epidermal keratinocytes (HaCat), and human melanoma (SK-MEL-28) embedded in collagen type I. The structure was characterized by hematoxylin-eosin stained, as well as points of melanoma cell invasion, which was associated with activity of MMPs (MMP-2 and MMP-9) by zymographic method. Then, the gene expression of the target molecular mechanisms involved in melanoma progression were evaluated. Here, the model development showed a region epidermis organized and separated from the dermis, with fibroblast cells confined and melanoma cells form delimited area invasion. MMP-2 and MMP-9 were identified during of cell culture and gene expression of BRAF, NRAS, and Vimentin was confirmed. The proposed model provides one more opportunity to study in vitro tumor biology of melanoma and also to allows the study of new drugs with more reliable results then whats we would find in vivo.

Regulation of cellular and molecular markers of epithelial-mesenchymal transition by Brazilin in breast cancer cells.

Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.

Machine learning-driven prediction of brain metastasis in lung adenocarcinoma using miRNA profile and target gene pathway analysis of an mRNA dataset.

BACKGROUND: Brain metastasis (BM) is common in lung adenocarcinoma (LUAD) and has a poor prognosis, necessitating predictive biomarkers. MicroRNAs (MiRNAs) promote cancer cell growth, infiltration, and metastasis. However, the relationship between the miRNA expression profiles and BM occurrence in patients with LUAD remains unclear. METHODS: We conducted an analysis to identify miRNAs in tissue samples that exhibited different expression levels between patients with and without BM. Using a machine learning approach, we confirmed whether the miRNA profile could be a predictive tool for BM. We performed pathway analysis of miRNA target genes using a matched mRNA dataset. RESULTS: We selected 25 miRNAs that consistently exhibited differential expression between the two groups of 32 samples. The 25-miRNA profile demonstrated a strong predictive potential for BM in both Group 1 and Group 2 and the entire dataset (area under the curve [AUC] = 0.918, accuracy = 0.875 in Group 1; AUC = 0.867, accuracy = 0.781 in Group 2; and AUC = 0.908, accuracy = 0.875 in the entire group). Patients predicted to have BM, based on the 25-miRNA profile, had lower survival rates. Target gene analysis of miRNAs suggested that BM could be induced through the ErbB signaling pathway, proteoglycans in cancer, and the focal adhesion pathway. Furthermore, patients predicted to have BM based on the 25-miRNA profile exhibited higher expression of the epithelial-mesenchymal transition signature, TWIST, and vimentin than those not predicted to have BM. Specifically, there was a correlation between EGFR mRNA levels and BM. CONCLUSIONS: This 25-miRNA profile may serve as a biomarker for predicting BM in patients with LUAD.

Vimentin takes a hike - Emerging roles of extracellular vimentin in cancer and wound healing.

Vimentin is a cytoskeletal protein important for many cellular processes, including proliferation, migration, invasion, stress resistance, signaling, and many more. The vimentin-deficient mouse has revealed many of these functions as it has numerous severe phenotypes, many of which are found only following a suitable challenge or stress. While these functions are usually related to vimentin as a major intracellular protein, vimentin is also emerging as an extracellular protein, exposed at the cell surface in an oligomeric form or secreted to the extracellular environment in soluble and vesicle-bound forms. Thus, this review explores the roles of the extracellular pool of vimentin (eVIM), identified in both normal and pathological states. It focuses specifically on the recent advances regarding the role of eVIM in wound healing and cancer. Finally, it discusses new technologies and future perspectives for the clinical application of eVIM.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-ß.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-ß. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-ß.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Gonadotropin Releasing Hormone (Gnrh) Triggers Neurogenesis in the Hypothalamus of Adult Zebrafish.

Recently, it has been shown in adult mammals that the hypothalamus can generate new cells in response to metabolic changes, and tanycytes, putative descendants of radial glia, can give rise to neurons. Previously we have shown in vitro that neurospheres generated from the hypothalamus of adult zebrafish show increased neurogenesis in response to exogenously applied hormones. To determine whether adult zebrafish have a hormone-responsive tanycyte-like population in the hypothalamus, we characterized proliferative domains within this region. Here we show that the parvocellular nucleus of the preoptic region (POA) labels with neurogenic/tanycyte markers vimentin, GFAP/Zrf1, and Sox2, but these cells are generally non-proliferative. In contrast, Sox2+ proliferative cells in the ventral POA did not express vimentin and GFAP/Zrf1. A subset of the Sox2+ cells co-localized with Fezf2:GFP, a transcription factor important for neuroendocrine cell specification. Exogenous treatments of GnRH and testosterone were assayed in vivo. While the testosterone-treated animals showed no significant changes in proliferation, the GnRH-treated animals showed significant increases in the number of BrdU-labeled cells and Sox2+ cells. Thus, cells in the proliferative domains of the zebrafish POA do not express radial glia (tanycyte) markers vimentin and GFAP/Zrf1, and yet, are responsive to exogenously applied GnRH treatment.

L1 cell adhesion molecule (L1CAM) in stage IB cervical cancer: distinct expression in squamous cell carcinomas and adenocarcinomas.

AIMS: L1 cell adhesion molecule (L1CAM) has been shown to be correlated with tumour progression, attributed to its possible association with epithelial-mesenchymal transition (EMT), characterised by the expression of vimentin and loss of e-cadherin. Herein, we investigate the associations between L1CAM and clinicopathological parameters, as well as the expression of vimentin and e-cadherin, in carcinomas restricted to the cervix. METHODS: The study was retrospective observational and included 45 squamous cell carcinomas (63.4%) and 26 adenocarcinomas (36.6%) submitted to primary surgical treatment. Patient age, FIGO (International Federation of Gynecology and Obstetrics) stage, tumour size and follow-up were obtained from the medical records. All the slides were revised to evaluate histological differentiation, lymphovascular space invasion, depth of infiltration, disease-free cervical wall thickness, pattern of invasion front, Silva pattern (for adenocarcinomas) and the percentage of tumour-infiltrating lymphocytes. Tissue microarrays were constructed for immunohistochemical staining for L1CAM, e-cadherin and vimentin. RESULTS: Adenocarcinomas were associated with lower disease-free and overall survival. L1CAM and vimentin expressions were more frequent among adenocarcinomas, although loss of e-cadherin expression was more common among squamous carcinomas. L1CAM expression was associated with larger tumours, vimentin expression and lower disease-free survival. No association was observed between the expression of either L1CAM or vimentin and loss of e-cadherin. High levels of tumour-infiltrating lymphocytes were more frequent in squamous cell carcinoma, high-grade tumours, destructive pattern at front of invasion and loss of e-cadherin expression. CONCLUSIONS: Our results confirm the prognostic role of L1CAM in cervical carcinomas, but suggest a role for mechanisms other than EMT.

Sertoli Cell Alterations in Peripubertal Varicocelized Rats: Evidence of Primary Damage on Spermatogenesis.

Idiopathic varicocele is closely associated with male infertility or subfertility. Sertoli cell is a very important regulator of spermatogenesis. We investigated the morphofunctional alterations in the Sertoli cell and its possible involvement in the establishment of testicular primary lesion in experimental left-sided varicocele, induced from peripuberty. Twenty-five male peripubertal rats (44 days postpartum [dpp]) were distributed into two groups: control (C) and varicocele (V). Experimental left varicocele was induced in rats through the partial ligature of the left renal vein. Euthanasia was performed at 100 dpp. Testicular histopathology and testosterone plasmatic level were evaluated. Transferrin and vimentin proteins were, respectively, used as immunomarkers of Sertoli cell function and structure. Significant reductions in vimentin and transferrin expressions were noticed in androgen-dependent stages (VII and VIII) of the seminiferous epithelium cycle in V rats; testosterone plasmatic level was also reduced. Bilateral testicular histopathological alterations were found in V rats, mainly massive germ cell desquamation. The histological damage and changes in protein expressions occurred bilaterally. The relevant impairment of the functional and structural characteristics of the Sertoli cell, together with the typical massive germ cell desquamation, indicates that Sertoli cell changes can primarily contribute to the significant testicular dysfunction associated with varicocele.

Cardiac Inflammation after Ischemia-Reperfusion of the Kidney: Role of the Sympathetic Nervous System and the Renin-Angiotensin System.

BACKGROUND/AIMS: To investigate the role of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in renal ischemia/reperfusion-induced (I/R) cardiac inflammatoryprofile. METHODS: Left kidney ischemia was induced in male C57BL/6 mice for 60 min, followed by reperfusion for 12 days, and treatment with or without atenolol, losartan, or enalapril. The expression of vimentin in kidney and atrial natriuretic factor (ANF) in the heart has been investigated by RT-PCR. In cardiac tissue, levels of ß1-adrenoreceptors, adenylyl cyclase, cyclic AMP-dependent protein kinase (PKA), noradrenaline, adrenaline (components of SNS), type 1 angiotensin II receptors (AT1R), angiotensinogen/Ang II and renin (components of RAS) have been measured by Western blotting and HPLC analysis. A panel of cytokines - tumour necrosis factor (TNF-α), interleukin IL-6, and interferon gamma (IFN-γ) - was selected as cardiac inflammatory markers. RESULTS: Renal vimentin mRNA levels increased by >10 times in I/R mice, indicative of kidney injury. ANF, a marker of cardiac lesion, increased after renal I/R, the values being restored to the level of Sham group after atenolol or enalapril treatment. The cardiac inflammatory profile was confirmed by the marked increase in the levels of mRNAs of TNF-α, IL-6, and IFN-γ. Atenolol and losartan reversed the upregulation of TNF-α expression, whereas enalapril restored IL-6 levels to Sham levels; both atenolol and enalapril normalized IFN-γ levels. I/R mice showed upregulation of ß1-adrenoreceptors, adenylyl cyclase, PKA and noradrenaline. Renal I/R increased cardiac levels of AT1R, which decreased after losartan or enalapril treatment. Renin expression also increased, with the upregulation returning to Sham levels after treatment with SNS and RAS blockers. Angiotensinogen/Ang II levels in heart were unaffected by renal I/R, but they were significantly decreased after treatment with losartan and enalapril, whereas increase in renin levels decreased. CONCLUSION: Renal I/R-induced cardiac inflammatory events provoked by the simultaneous upregulation of SNS and RAS in the heart, possibly underpin the mechanism involved in the development of cardiorenal syndrome.

Twist1 Correlates With Epithelial-Mesenchymal Transition Markers Fibronectin and Vimentin in Adrenocortical Tumors.

BACKGROUND/AIM: Although the knowledge regarding adrenocortical carcinomas (ACC) tumorigenesis has significantly improved during the last decade, it still remains to be completely determined. Epithelial-mesenchymal transition (EMT) is a well described transcription factor induced process, postulated as an essential step toward cancer progression and metastasis development. In this context, Twist1 has been described as the EMT master-regulator. The aim of this study was to assess the association among Twist1, fibronectin, vimentin and E-cadherin gene expression in adrenocortical tumor samples. MATERIALS AND METHODS: Twist1, fibronectin, vimentin and E-cadherin gene expression in 18 adrenal adenomas, 18 ACC, and 24 childhood onset adrenocortical tumors were assessed in formalin-fixed paraffin-embedded tissues. The fold expression was calculated according to the 2ΔCt method. RESULTS: A significant correlation between mRNA levels of Twist1, fibronectin and vimentin was evident. Although their expression was inversely proportional, no association was observed between Twist1 and E-cadherin expression. CONCLUSION: The expression of Twist1, the major regulator of EMT, is directly correlated to the expression of mesenchymal markers fibronectin and vimentin in ACC samples.

A Rat Eye Lens Model of Cataract Formation.

This chapter describes the use of lenses obtained from rats as a model of cataractogenesis. At the molecular level, this is visualized as reduced activity of oxidative reductive enzymes such as aldose reductase and increased proteolysis of lens structural proteins including vimentin. In this chapter, protocols for assessment of these two pathways are presented. Specifically, this analysis shows a comparison of aldose reductase activity and vimentin cleavage in male and female rat lenses. This is because female rats are more susceptible to cataract formation compared to males.

Vimentin, osteocalcin and osteonectin expression in canine primary bone tumors: diagnostic and prognostic implications.

Canine primary bone tumors have a plastic radiographic image, demanding histopathological confirmation. Bone tumors are characterized by the type and amount of extracellular matrix produced what cannot be easily recognized, especially in biopsy samples. Identifying cellular markers that could aid diagnosis has supported various studies in oncological pathology. This study aimed to evaluate 22 canine primary bone neoplasms, establishing their histopathological diagnosis and evaluated vimentin, osteonectin and osteocalcin expression and their implication in diagnosis and prognosis. There were 12 productive osteoblastic osteosarcomas, six minimally productive osteoblastic osteosarcoma, two chondrosarcomas, one fibrosarcoma and one hemangiosarcoma. Immunostaining was cytoplasmatic in all cases, with average percentage of 87.9% for vimentin, 98.0% for osteonectin and 99.9% for osteocalcin. In this last case, only osteosarcomas were considered. Intensity was higher in vimentin labeling (+++), followed by osteonectin (++) and osteocalcin (+). One osteosarcoma showed negative immunostaining for vimentin and of samples submitted to anti-osteocalcin immunostaining, three osteosarcomas and one fibrosarcoma had negative staining. Besides identifying mesenchymal origin, vimentin elevated expression in canine bone tumors can be related to epithelial-mesenchymal transition, leading to more aggressive tumoral phenotypes and metastasis development. Similarly, high osteonectin expression is implicated in neoplastic cell invasion and is also related to metastasis spread. Decreased osteocalcin expression was found in some osteosarcoma samples and can be related to poor prognosis, as in human osteosarcomas. Our findings suggest that vimentin, osteonectin and osteocalcin not only aid diagnosis but can be related to prognosis in canine primary bone tumors, especially osteosarcomas and its osteoblastic subtype.

Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Upregulation of PIR gene expression induced by human papillomavirus E6 and E7 in epithelial oral and cervical cells.

The hallmark of high-risk human papillomavirus (HR-HPV)-related carcinogenesis is E6 and E7 oncogene overexpression. The aim of this work was to characterize epithelial oral and cervical cancer cells that express HR-HPV E6 and E7 oncoproteins. Transcriptomic assay using DNA microarrays revealed that PIR gene expression was detected in oral cells in an HR-HPV E6/E7-dependent manner. In addition, PIR was overexpressed in HPV-positive SiHa and Ca Ski cells, whereas it was undetectable in HPV-negative C33A cells. The PIR expression was dependent on functional HR-HPV E6 and E7 oncoproteins even though the E7 oncoprotein had higher activity to induce PIR overexpression in comparison with E6. In addition, using an siRNA for PIR silencing in oral cells ectopically expressing HR-HPV E6/E7, there was a significant increase in E-cadherin transcripts and a decrease in Vimentin, Slug, Zeb and Snail transcripts, suggesting that HR-HPV-induced PIR overexpression is involved in epithelial-mesenchymal transition. Furthermore, migration of PIR-silenced cells was significantly decreased. Finally, using inhibitors of some specific pathways, it was found that EGFR/ERK and PI3 K/AKT signalling pathways are important for E7-mediated PIR overexpression. It can be concluded that PIR gene expression is highly dependent on the expression of HR-HPV oncoproteins and is important for EMT regulation.

Molecular iodine impairs chemoresistance mechanisms, enhances doxorubicin retention and induces downregulation of the CD44+/CD24+ and E-cadherin+/vimentin+ subpopulations in MCF-7 cells resistant to low doses of doxorubicin.

One of the most dreaded clinical events for an oncology patient is resistance to treatment. Chemoresistance is a complex phenomenon based on alterations in apoptosis, the cell cycle and drug metabolism, and it correlates with the cancer stem cell phenotype and/or epithelial-mesenchymal transition. Molecular iodine (I2) exerts an antitumor effect on different types of iodine-capturing neoplasms by its oxidant/antioxidant properties and formation of iodolipids. In the present study, wild-type breast carcinoma cells (MCF-7/W) were treated chronically with 10 nM doxorubicin (DOX) to establish a low-dose DOX-resistant mammary cancer model (MCF-7/D). MCF-7/D cells were established after 30 days of treatment when the culture showed a proliferation rate similar to that of MCF-7/W. These DOX-resistant cells also showed increases in p21, Bcl-2 and MDR-1 expression. Supplementation with 200 µM I2 exerted similar effects in both cell lines: it decreased the proliferation rate by ~40%, and I2 co-administration with DOX significantly increased the inhibitory effect (to ~60%) and also increased apoptosis (BAX/Bcl-2 index), principally by inhibiting Bcl-2 expression. The inhibition by I2 + DOX was also accompanied by impaired MDR-1 induction as well as by a significant increase in PPARγ expression. All of these changes could be attributed to enhanced DOX retention and differential down-selection of CD44+/CD24+ and E-cadherin+/vimentin+ subpopulations. I2 + DOX-selected cells showed a weak induction of xenografts in Foxn1nu/nu mice, indicating that the iodine supplements reversed the tumorogenic capacity of the MCF-7/D cells. In conclusion, I2 is able to reduce the drug resistance and invasive capacity of mammary cancer cells exposed to DOX and represents an anti-chemoresistance agent with clinical potential.

Cellular stress response in human Müller cells (MIO-M1) after bevacizumab treatment.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, is widely used in the treatment of retinal vascular diseases. However, due to the essential role Müller cell derived-VEGF plays in the maintenance of retinal neurons and glial cells, cell viability is likely to be affected by VEGF inhibition. We therefore evaluated the effect of bevacizumab-induced VEGF inhibition on Müller cells (MIO-M1) in vitro. MIO-M1 cells were cultured for 12 or 24 h in media containing bevacizumab at 0.25 or 0.5 mg/mL. Controls were cultured in medium only. Cell viability was determined with the trypan blue exclusion test and MTT assay. Caspase-3, beclin-1, glial fibrillary acidic protein (GFAP) and vimentin content were quantified by immunohistochemistry. Gene expression was evaluated by real-time quantitative PCR. Treatment with bevacizumab did not reduce MIO-M1 cell viability, but increased metabolic activity at 24 h (0.5 mg/mL) and induced apoptosis and autophagy, as shown by the increased caspase-3 levels at 12 h (0.25 and 0.5 mg/mL) and the increased beclin levels at 24 h (0.5 mg/mL). Caspase-3 mRNA was upregulated at 12 h and downregulated at 24 h in cells treated with bevacizumab at 0.25 mg/mL. Bevacizumab treatment was also associated with structural protein abnormalities, with decreased GFAP and vimentin content and upregulated GFAP and vimentin mRNA expression. Although bevacizumab did not significantly affect MIO-M1 cell viability, it led to metabolic and molecular changes (apoptosis, autophagy and structural abnormalities) suggestive of significant cellular toxicity.

Type-III interferons and rheumatoid arthritis: Correlation between interferon lambda 1 (interleukin 29) and antimutated citrullinated vimentin antibody levels.

AIM: To assess serum type III or lambda (λ) interferons (IFN) levels and its clinical and laboratory associations in rheumatoid arthritis (RA). METHODS: A cross-sectional study including 43 patients with RA (86% females; age 45.3 ± 10.3 years) and 43 healthy individuals was performed. Clinical data including disease activity, acute-phase reactants, rheumatoid factor and anticyclic citrullinated peptide (anti-CCP) antibodies were collected. Serum IFNλ1, IFNλ2, IFNλ3, CXCL8 and anti-mutated citrullinated vimentin (anti-MCV) antibody levels were measured. RESULTS: Patients with RA had higher IFNλ1 (113.5 ± 118.6 pg/mL versus 55.9 ± 122.3 pg/mL; p < 0.0001) and IFNλ2 (245.4 ± 327.7 pg/mL versus 5.1 ± 11.0 pg/mL; p = 0.009) levels than controls, but not IFNλ3 levels. Notably, IFNλ1 levels were found to be higher in both patients with active disease (124.9 ± 135.9 pg/mL; p < 0.001) and quiescent disease (99.0 ± 93.7 pg/mL; p < 0.01), while IFNλ2 levels were higher only in patients with active disease (264.0 ± 356.1 pg/mL; p = 0.02). A noteworthy association between serum IFNλ1 levels and anti-MCV antibody titers (Spearman's rho coefficient 0.36, 95% CI 0.36 to 0.61; p = 0.02) was observed. CONCLUSION: Serum IFNλ1 and IFNλ2 levels are abnormally elevated in patients with RA and the former are linearly associated with circulating anti-MCV antibody levels. These results may place type-III IFN as an attractive new therapeutic target in RA.

Effects of miRNA-143 and the non-coding RNA MALAT1 on the pathogenesis and metastasis of HeLa cells.

Cervical cancer is a common female malignancy of global dimensions. MicroRNAs (miRNAs) play crucial roles in the development, differentiation, proliferation, and apoptosis of tumors. The non-coding RNA MALAT1 participates in various physiological processes that are important for proper functioning of the body. Here, we analyzed the expression of miRNA-143 and MALAT1 in HeLa cells to evaluate their roles in the occurrence and metastasis of cervical cancer. HeLa cells were divided into five groups depending on the treatment conditions, namely, transfected with miRNA-143, MALAT1, miRNA-143 inhibitor and the MALAT1 inhibitor, and the untreated control. Reverse transcription-polymerase chain reaction was used to analyze the expression of miRNA-143 and MALAT1, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess proliferation, the trans-well assay to study cell invasion and migration, and western blot to analyze the levels of E-cadherin and vimentin. The proliferation of HeLa cells increased upon treatment with the miRNA-143 inhibitor and decreased when treated with the MALAT1 inhibitor, compared to the proliferation of the groups that were transfected with miRNA-143 and MALAT1, respectively (P < 0.05). Thus, miRNA-143 decreased cell invasion and migration potency, downregulated vimentin and upregulated E-cadherin expression, while MALAT1 had the opposite effects. In conclusion, the low expression of miRNA-143 and high expression of MALAT1 in cervical cancer cells could possibly potentiate cell invasion/migration and alter the levels of vimentin and E-cadherin.