Autophagic-Related anticancer effect of grapes extract and tomatoes extract: Ex-Vivo study

Cells undergo autophagy to save themselves from injury, but progressive autophagy can cause cell death. This study characterized and compared the effect of grape (resveratrol) and tomato (lycopene) extracts and their combination on modulating autophagy-related miRNA and its target gene in squamous cell carcinoma cell line. Docking analysis for extracts and selected genes was performed. Methyl Thiazol Tetrazolium assays were used to assess the cytotoxicity of extracts and their combination toward HEp-2 cells. qRT-PCR was used to quantify changes in gene expression. Data were statistically analyzed. miRNA-20a was identified as a potential effector in laryngeal cancer, and sequestosome-1 (SQSTM1) was its target gene. Docking analysis showed that resveratrol interacted with miRNA-20a and showed less affinity toward SQSTM1. Hydrogen bonds and hydrophobic interactions were predicted. In contrast, lycopene showed less affinity toward miRNA-20a than resveratrol. Increasing doses of resveratrol, lycopene, and their combination induced a statistically significant reduction in mean percent viability and mean fold changes of miRNA-20a and SQSTM1 expression in treated HEp-2 cells. Pearson's correlation showed a statistically significant positive correlation between miRNA-20a and SQSTM1 (R=0.812, p≤0.001). Grape and tomato extracts and their combination display promising cytotoxicity against HEp-2 cells in a dose- and time-dependent fashion. Both extracts reduce the expression of miRNA-20a and SQSTM1 with subsequent inhibition autophagy and promotion of apoptosis in HEp-2 cells.

Las células se someten a autofagia para salvarse de lesiones, pero la autofagia progresiva puede provocar la muerte celular. Este estudio caracterizó y comparó el efecto de los extractos de uva (resveratrol) y tomate (licopeno) y su combinación en la modulación de miARN relacionado con la autofagia y su gen diana en la línea celular de carcinoma de células escamosas. Se realizó análisis de acoplamiento para extractos y genes seleccionados. Se utilizaron ensayos de metil tiazol tetrazolio para evaluar la citotoxicidad de los extractos y su combinación frente a las células HEp-2. qRT-PCR se utilizó para cuantificar los cambios en la expresión génica. Los datos fueron analizados estadísticamente. El miARN-20a se identificó como un efector potencial en el cáncer de laringe y el secuenciasoma-1 (SQSTM1) fue su gen diana. El análisis de acoplamiento mostró que el resveratrol interactuaba con miRNA-20a y mostraba menos afinidad hacia SQSTM1. Se predijeron enlaces de hidrógeno e interacciones hidrofóbicas. Por el contrario, el licopeno mostró menos afinidad hacia el miARN-20a que el resveratrol. El aumento de las dosis de resveratrol, licopeno y su combinación indujo una reducción estadísticamente significativa en el porcentaje medio de viabilidad y los cambios medios en la expresión de miRNA- 20a y SQSTM1 en las células HEp-2 tratadas. La correlación de Pearson mostró una correlación positiva estadísticamente significativa entre miRNA-20a y SQSTM1 (R=0,812, p≤0,001). Los extractos de uva y tomate y su combinación muestran una citotoxicidad prometedora contra las células HEp-2 de forma dependiente de la dosis y el tiempo. Ambos extractos reducen la expresión de miRNA-20a y SQSTM1 con la posterior inhibición de la autofagia y promoción de la apoptosis en células HEp-2.

The influence of demography and referral medical specialty on the detection of autoantibodies to HEP-2 cells in a large sample of patients

Abstract Background: The prevalence of anti-cell autoantibodies detected by indirect immunofluorescence assay on HEp-2 cells (HEp-2-IIFA) increases with age and is higher in female sex. The number of medical specialties that use HEp-2-IIFA in the investigation of autoimmune diseases has increased lately. This study aimed to determine the prevalence and patterns of autoantibodies on HEp-2-IIFA according to demographics variables and referring medical specialties. Methods: A retrospective analysis of the HEp-2-IIFA carried out between January and June of 2017 was performed. The International Consensus on Antinuclear Antibodies Patterns (ICAP) and the Brazilian Consensus on Autoantibodies were used for patterns definition on visual reading of the slides. Anti-cell (AC) codes from ICAP and Brazilian AC codes (BAC) were used for patterns classification. Results: From 54,990 samples referred for HEp-2-IIF testing, 20.9% were positive at titer ≥ 1/80. HEp-2-IIFA positivity in females and males was 24% and 12%, respectively ( p < 0.0001). The proportion of positive results in the 4 age groups analyzed: 0-19, 20-39, 40-59, and ≥ 60 years was 23.3, 20.2, 20.1, and 22.8%, respectively ( p < 0.0001). Considering all positive sera (n = 11,478), AC-4 nuclear fine speckled (37.7%), AC-2 nuclear dense fine speckled (21.3%), BAC-3 nuclear quasi -homogeneous (10%) and mixed/composite patterns (8.8%) were the most prevalent patterns. The specialties that most requested HEp-2-IIFA were general practitioner (20.1%), dermatology (15%), gynecology (9.9%), rheumatology (8.5%), and cardiology (5.8%). HEp-2-IIFA positivity was higher in patients referred by rheumatologists (35.7% vs. 19.6%) ( p < 0.0001). Moderate (46.4%) and high (10.8%) titers were more observed in patients referred by rheumatologists ( p < 0.0001). We observed a high proportion of mixed and cytoplasmic patterns in samples referred by oncologists and a high proportion of BAC-3 (nuclear quasi -homogeneous) pattern in samples referred by pneumologists. Conclusions: One-fifth of the patients studied were HEp-2-IIFA-positive. The age groups with more positive results were 0-19 and ≥ 60 years. AC-4, AC-2, BAC-3 and mixed/composite patterns were the most frequent patterns observed. Rheumatologists requested only 8.5% of HEp-2-IIFA. Positive results and moderate to high titers of autoanti-bodies were more frequent in patients referred by rheumatologists.

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma / Diagnóstico e tratamento de células HEp-2 contaminadas por micoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

Rheumatologist perspective of the Brazilian consensus for detection of auto antibodies in HEp-2 CELLS

Abstract Objective: To evaluate the perception of rheumatologists regarding the recommendations of the Brazilian Consensus for detection of Autoantibodies (BCA) on HEp-2 Cells by Indirect Immunofluorescence assay (IFA) and how BCA recommendations help in clinical practice. Methodology: A structured questionnaire regarding the BCA recommendations for detection and interpretations of autoantibodies in HEp-2 cells was applied to randomly selected rheumatologists. The results were tabulated using the Microsoft® Excel program, expressed as a simple percentage and the dichotomous data were analyzed using the Chi-square test and the Epi Info® program. Results: Four hundred fuorteen rheumatologists participated in the study: 70% of them considered their knowledge of the HEp-2 IFA test satisfactory or excellent, and 43% said they knew the BCA recommendations in general, without distinguishing the edition of the BCA to which they refer. The Revista Brasileira de Rheumatologia/ Advances in Rheumatology was the means of dissemination most consulted by specialists (50%). According to the rheumatologists' opinion, the most relevant pattern was the homogeneous nuclear (78%) and 65% stated they were satisfied with the BCA recommendations at a level of satisfaction greater than or equal to 80%. There was no significant difference in the perception of rheumatologists from the several Brazilian geographic regions. Conclusion: Brazilian rheumatologists are aware of the BCA guidelines and most are satisfied with the content published, considering that the BCA recommendations assist positively in the clinical practice. Most rheumatologists recognize the patterns associated with rheumatic autoimmune diseases and have used BCA recommendations to interpret the results of the HEp-2 IFA test.

Perspectivas en investigación: células HEp-2 infectadas con EB's de Chlamydia trachomatis serovar 2 (VR-902B) / Chlamydia trachomatis: Research Perspectives. HEp-2 Cells Infected with EB's from Chlamydia trachomatis serovar 2 (VR-902B): Research Perspectives

Resumen Chlamydia trachomatis (C. trachomatis) es una bacteria Gram negativa inmóvil, caracterizada por ser un microorganismo intracelular obligado y por poseer un ciclo reproductivo en el que puede distinguirse una forma infecciosa extracelular metabólicamente inerte (cuerpo elemental - EB's), y una forma no infecciosa intracelular y activa (cuerpo reticulado - RB's). C trachomatis se caracteriza por causar infección en humanos, está relacionada con enfermedades de transmisión sexual e infecciones oculares; por lo que puede conllevar a secuelas de interés, si no se da un tratamiento oportuno. El objetivo de este estudio fue optimizar el modelo de infección de C. trachomatis en células HEp-2 con cuerpos elementales (EB's) de C. trachomatis serovar L2. Inicialmente, se establecieron las condiciones para el crecimiento adecuado de las células HEp-2 en tiempo y con una confluencia del 90%, para continuar con la optimización de un protocolo de infección. La infección fue confirmada a partir de la coloración con Giemsa permitiendo evaluar características morfológicas tanto de las células HEp-2 sin infectar e infectadas, y así mismo, de los cuerpos elementales de C. trachomatis. Finalmente, se corroboró la infección con la técnica de inmunofluorescencia directa que detecta la proteína de membrana MOMP de C. trachomatis. Tras los ensayos realizados se evidenció la presencia de cuerpos elementales próximos y dentro del citoplasma celular, así como células vacuoladas y daño celular causado por la infección.

Abstract Chlamydia trachomatis (C. Trachomatis) is a Gram negative unmoving bacterium, characterized by being an obligate intracellular microorganism and having a reproductive cycle in which a metabolically inactive extracellular infectious form (elementary body - EB's) can be distinguished from an intracellular active and non-infectious form (reticulated body - RB's). C trachomatis is characterized by causing infection in humans, is related to sexually transmitted diseases and eye infections, so it can lead to sequelae of interest if timely treatment is not given. The objective of this study was to optimize the infection model of C. trachomatis in HEp-2 cells with elementary bodies (EB's) of C. trachomatis serovar L2. Initially, the conditions for the adequate growth of HEp-2 cells were established in time and with a confluence of 90%, to continue with the optimization of an infection protocol. The infection was confirmed from the staining with Giemsa allowing to evaluate morphological characteristics of both uninfected and infected HEp-2 cells and also of the elementary bodies of C. trachomatis. Finally, the infection was corroborated with the direct immunofluorescence technique, that detects the C. trachomatis MOMP membrane protein. After the tests were performed, the presence of elementary bodies nearby and within the cellular cytoplasm was evidenced, as well as vacuolated cells and cellular damage caused by the infection.

Inhibitory effect of miR-3195 on the proliferation of human laryngeal carcinoma Hep-2 cells and its mechanism / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of miR-3195 on the proliferation of laryngeal carcinoma Hep-2 cells and its molecular mechanism. Methods: From January 2008 to August 2012, the laryngeal cancer tissues and their corresponding paracancerous tissues from 29 patients with laryngeal cancer who were admitted to the Department of Otorhinolaryngology, Chenzhou First People's Hospital Affiliated to teaching hospital of University of South China were selected for this study. qPCR was used to detect the expression of miR-3195 in laryngeal carcinoma and the paracancerous tissues; Hep-2 cell line with stable and high expression of miR-3195 was constructed. The proliferation of miR-3195 over-expressed Hep-2 cells and the control cells was observed by MTT method. A nude mouse xenograft model was established to observe the proliferation of miR-3195 overexpressed Hep-2 cells in nude mice. Bioinformatics tools were used to predict the target gene of miR-3195; the luciferase vector of TBX1 3'UTR was constructed, and its luciferase activity was examined with dual luciferase detection system; Western blotting was used to detect the TBX1 protein expression in miR-3195 over-expressed cells and control cells. Results: The expression of miR-3195 in laryngeal carcinoma tissues was significantly lower than that in paracancerous tissues (P<0.01); miR-3195 up-regulation could inhibit the proliferation of Hep-2 cells (P<0.01) and significantly inhibit the growth of transplanted tumors in nude mice (P<0.05); The results of the Dual luciferase reporter gene assay indicated that miR-3195 might targetedly bind to TBX1 (P<0.05), and Western blotting proved that miR-3195 could inhibit the expression of TBX1 protein (P<0.05). Conclusion: miR-3195 has a significant inhibitory effect on the proliferation of Hep-2 cells, and its molecular mechanism may be related to the negative regulation of TBX1 expression.

Effect of miR-340-5p on proliferation of laryngeal cancer Hep2 cells and its intrinsic molecular mechanism / 临床耳鼻咽喉头颈外科杂志

Objective@#To study the effect of miR-340-5p on the proliferation of laryngeal cancer Hep2 cells and explore its intrinsic molecular mechanism, so as to screen potential biomarkers and targets for the diagnosis and treatment of laryngeal cancer. @*Method@#The expression of miR-340-5p in laryngeal cancer tissues, paracancerous tissues, laryngeal cancer cell lines Hep2 and normal bronchial HBE cell lines was quantitatively analyzed by qRT-PCR; The double luciferase reporter vector was constructed to verify whether STAT3 was a potential target gene of microRNA-340-5p; The miR-340-5p mimics/inhibitor was transfected into Hep2 cells by liposome and verified by qRT-PCR; The CCK-8 method and Annexin V/PI method were used to analyze the proliferation and apoptosis of transfected cells; and Western Blot was used to detect the expression of STAT3 and Wnt/β-catenin pathway-related proteins after transfection. @*Result@#The results of qRT-PCR showed that the level of miR-340-5p in laryngeal cancer tissues and Hep2 cells was significantly lower than that in adjacent tissues and HBE cells, and the expression of miR-340-5p was significantly increased or decreased after overexpression or inhibition; Luciferase activity showed that miR-340-5p directly interacted with target gene STAT3 3'-UTR and negatively regulated its expression; Cell proliferation and apoptosis analysis showed that up-regulation of microRNA-340-5p could significantly inhibit the proliferation and induce apoptosis of Hep2 cells in vitro, and vice versa; Western Blot results showed that the levels of STAT3 and β-catenin, c-Myc, TCF-4, CyclinD1 and ROCK1 in Hep2 cells were significantly lower than those in the control group after over-expression of miR-340-5p, and vice versa. @*Conclusion@#The expression of miR-340-5p is abnormally low in laryngeal cancer tissues and Hep2 cells. It can be used as a potential biological target for diagnosis and treatment of laryngeal cancer by targeting STAT3 gene to negatively regulate Wnt/β-catenin signaling pathway.

Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

Effect of miR-340-5p on proliferation of laryngeal cancer Hep2 cells and its intrinsic molecular mechanism / 临床耳鼻咽喉头颈外科杂志

To study the effect of miR-340-5p on the proliferation of laryngeal cancer Hep2 cells and explore its intrinsic molecular mechanism, so as to screen potential biomarkers and targets for the diagnosis and treatment of laryngeal cancer. The expression of miR-340-5p in laryngeal cancer tissues, paracancerous tissues, laryngeal cancer cell lines Hep2 and normal bronchial HBE cell lines was quantitatively analyzed by qRT-PCR; The double luciferase reporter vector was constructed to verify whether STAT3 was a potential target gene of microRNA-340-5p; The miR-340-5p mimics/inhibitor was transfected into Hep2 cells by liposome and verified by qRT-PCR; The CCK-8 method and Annexin V/PI method were used to analyze the proliferation and apoptosis of transfected cells; and Western Blot was used to detect the expression of STAT3 and Wnt/β-catenin pathway-related proteins after transfection. The results of qRT-PCR showed that the level of miR-340-5p in laryngeal cancer tissues and Hep2 cells was significantly lower than that in adjacent tissues and HBE cells, and the expression of miR-340-5p was significantly increased or decreased after overexpression or inhibition; Luciferase activity showed that miR-340-5p directly interacted with target gene STAT3 3'-UTR and negatively regulated its expression; Cell proliferation and apoptosis analysis showed that up-regulation of microRNA-340-5p could significantly inhibit the proliferation and induce apoptosis of Hep2 cells in vitro, and vice versa; Western Blot results showed that the levels of STAT3 and β-catenin, c-Myc, TCF-4, CyclinD1 and ROCK1 in Hep2 cells were significantly lower than those in the control group after over-expression of miR-340-5p, and vice versa. The expression of miR-340-5p is abnormally low in laryngeal cancer tissues and Hep2 cells. It can be used as a potential biological target for diagnosis and treatment of laryngeal cancer by targeting STAT3 gene to negatively regulate Wnt/β-catenin signaling pathway.

miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of human laryngeal carcinoma Hep-2 cells by down-regulation of SOX2 / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of miR-135a on the malignant biological behaviors of human laryngeal carcinoma epithelial Hep-2 cells and its sensitivity to oxaliplatin. Methods: Samples of laryngeal carcinoma tissues and para-cancerous tissues were collected from 10 patients who underwent laryngectomy in Nanyang Hospital Affiliated to Zhengzhou University-Nanyang City Center Hospital from January 2018 to June 2018. The expression of miR-135a in laryngeal carcinoma tissues and Hep-2 cells was detected by qPCR.After being transfected with miR-135 inhibitor, cell proliferation viability of Hep-2 cells was measured by CCK-8 assay, cell colony formation ability was detected by colony formation assay, and cell proliferation invasion and migration abilities were detected by Transwell analysis, and the expression of SOX2 protein in Hep-2 cells was detected by WB. Hep-2 cells transfected with miR-135 inhibitor were further treated with various concentrations (0.5, 1.0, 1.5 and 2.0 μmol/L) of oxaliplatin, and the cell proliferation viability was detected by CCK-8 while cell apoptosis was detected by Annexin-V-FITC/PI double staining flow cytometry. miR-135a inhibitor plasmid, control pcDNA empty vector (SOX2-Con) plasmid, and pcDNA-SOX2 (SOX2-OE) plasmid were transfected into Hep-2 cells to construct the miR-135a inhibitor+SOX2-Con group and miR-135a inhibitor+SOX2-OE group, and the cell viability, cell colony formation ability, cell invasion and migration ability in two groups were detected. Results: Compared with para-cancerous tissues, miR135a expression in laryngeal cancer tissues was significantly increased (P<0.01). Compared with normal NHP cells, miR-135a expression in Hep-2 cells was significantly increased (P<0.01). miR-135a inhibitor significantly reduced the expression level of miR-135a in Hep-2 cells (P<0.01). miR-135a knockdown significantly reduced the cell proliferation viability, cell colony number, migration, invasion and SOX2 expression in Hep-2 cells (all P <0.01), but significantly enhanced the sensitivity of Hep-2 cells to oxaliplatin (P<0.01). Compared with miR-135a inhibitor+SOX2-Con group, the cell proliferation viability, cell colony number, migration and invasion of Hep-2 cells in miR-135a inhibitor+SOX2-OE group were significantly increased (P<0.01); Meanwhile, the cells of the 2 groups were treated with different concentrations of oxaliplatin, and the results of CCK-8 assay showed that, compared with the miR-135a inhibitor+ SOX2-Con group, the cell proliferation viability of Hep-2 cells in miR-135a inhibitor+SOX2-OE group was significantly increased (P< 0.01). Conclusion: miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of Hep-2 cells possibly by inhibiting the expression of the transcription factor SOX2.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

Abstract Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

Effect of MiR-1264 on the proliferation and migration of Hep2 cell line / 医学研究生学报

Objective Previous study shows that the expression of mir-1264 is down-regulated in laryngeal carcinoma. This study aimed to investigate the effect of miR-1264 on the biological function of laryngeal carcinoma Hep2 cells and verify whether miR-1264 could down-regulate the expression of tumor suppressor gene la-ryngeal carcinoma related gene 1(LCRG1)in laryngeal carcinoma. Methods Real-time quantitative PCR was used to assess the miR-1264 expression in human laryngeal cancer tissues. There are three groups in the experiment:miR-1264 mimic/inhibitor group,mimic/inhibitor NC group and untransfected group. MTT and Transwell were applied to observe the effect of miR-1264 on the proliferation,migration and invasion capabilities of Hep2 cells. Luciferase experiment was used to verify the combination of miR-1264 and LCRG1 3'UTR.RT-PCR and Western blot were used to test the expression of miR-1264 and LCRG1 protein respectively. Results Compared with the adjacent laryngeal tissue,miR-1264 expression in human laryn-geal cancer tissues was significantly increased(P<0.05).Compared with NC control group and blank group,the proliferation,migration and invasion capabilities of Hep2 cells were significantly enhanced after they were transiently transfected with miR-1264 mimic(P<0.05);however,after Hep2 cells were transiently transfected with miR-1264 inhibitor,their proliferation,migration and invasion ca-pabilities were significantly inhibited(P<0.05). Luciferase experiment showed miR-1264 mimic could significantly decrease LCRG1 3'UTR luciferase activity,which was of statistical significance. After the verification of successful transient transfection,the results of further Western blot on LCRG1 protein expression showed that there was no significant difference between the experimental group and the mimic NC control group as well as the blank group(P>0.05). Conclusion miR-1264 is highly expressed in laryngeal cancer tissues. Though miR-1264 may not participate in down-regulating the expression of tumor suppressor gene LCRG1 in laryngeal carcino-ma,it can promote the proliferation,migration and invasion capabilities of Hep2 cells.

HEp-2 cell line as an experimental model to evaluate genotoxic effects of pentavalent inorganic arsenic / HEp-2 como modelo experimental para evaluar los efectos genotóxicos del arsénico inorgánico pentavalente

Early detection of toxic events induced by xenobiotics is necessary for a proper assessment of human risk after the exposure to those agents. The aim of this work was to evaluate the cell line HEp-2 as an experimental model to determine the genotoxic effects of sodium arsenate. To this end, we determined the metabolic activity cells by the MTT test on seven concentrations of arsenate that range from 27 to 135,000 μM, obtaining the median lethal concentration (LC50), the lowest observed effect concentration (LOEC), and the not observed effect concentration (NOEC) of sodium arsenate at 24 h of exposition. According to the cytotoxic response obtained, we evaluated the genotoxic effect of the 27 and 270 μM concentrations by using the micronucleus assay and chromosomal aberrations test. We found a statistically significant increase (p<0.05) in the frequency of micronuclei between control cultures and those exposed to the highest concentration of sodium arsenate. Furthermore, the frequencies of nucleoplasmic bridges and tripolar mitosis were significantly higher in cell cultures exposed to the above concentrations compared to the control cultures (p<0.05). The participation of the glutathione system as response to the arsenate exposition was also analyzed, and a statistically significant increase in the glutathione content was found in those cells exposed to 27 μM of arsenate. The Glutathione S-transferase activity did not increase in the exposed cells compared to control cells, suggesting that the arsenate reduction involved other metabolic pathways in the HEp-2 cells. These results confirm that, under the conditions carried out in this study, sodium arsenate is genotoxic for HEp-2 cells. Therefore, we suggest that this cell line would be a good model for the assessment of the cytotoxic and genotoxic effects of xenobiotics on human cells.

La detección temprana de eventos tóxicos inducidos por xenobióticos es necesaria para una adecuada evaluación del riesgo humano ante la exposición a dichos agentes. El objetivo de este trabajo fue evaluar a la línea celular HEp-2 como modelo experimental para determinar los efectos genotóxicos del arseniato de sodio. Para ello, se determinó la actividad metabólica de las células mediante el ensayo de MTT, en siete concentraciones de arseniato de sodio en el rango 27-135.000 μM, determinando la concentración letal media (LC50), la menor concentración de efecto observado (LOEC) y la mayor concentración de efecto no observado (NOEC) de arseniato de sodio para una exposición de 24 h. Teniendo en cuenta los datos de citotoxicidad, se evaluó el efecto genotóxico a las concentraciones 27 y 270 μM por medio del ensayo de micronúcleos y aberraciones cromosómicas, encontrando un aumento estadísticamente significativo en la frecuencia de micronúcleos entre el control y la mayor concentración arseniato de sodio ensayada. Además, la presencia de puentes nucleoplasmáticos y mitosis tripolar fue significativamente mayor en ambas concentraciones estudiadas con respecto al control. Se analizó la participación del sistema de glutatión como respuesta a la exposición al arseniato, encontrándose un aumento estadísticamente significativo en el contenido de glutatión en la concentración de arseniato de 27 μM. La actividad de la glutatión S-transferasa no aumentó, lo que sugiere que la reducción del arseniato implicó otra vía metabólica en las células HEp-2. Estos resultados confirman que el arseniato de sodio induce genotoxicidad en células HEp-2 en las condiciones realizadas en este estudio y por lo tanto este tipo de línea celular es un buen modelo para ensayos de citotoxicidad y genotoxicidad en los cuales se quiere evaluar el riesgo humano.

Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma

Abstract Contamination of primary and cell cultures by mycoplasmas is one of the main economic and biological pitfalls in basic research, diagnosis and manufacture of biotechnological products. It is a common issue which may be difficult to conduct surveillance on. Mycoplasma presence may affect several physiological parameters of the cell, besides being considered an important source of inaccurate and/or non-reproducible scientific results. Each cell type presents characteristical symptoms, mainly morphological, that indicate a contamination by mycoplasma. HEp-2 cells originate from carcinoma of the larynx and are, therefore, part of the respiratory tract, which is one of mycoplasma habitats. Despite the importance these cells in several biological research (evaluation of cell proliferation and migration, apoptosis, antiviral and antitumor compounds), the alterations induced by mycoplasma contamination in HEp-2 cells have not yet been described. Here, we describe the progressive morphological alterations in culture of HEp-2 cells infected with mycoplasma, as well as the-diagnosis of the infection and its treatment. Mycoplasma contamination described within this work led to cytoplasm elongation, cell-to-cell spacing, thin plasma membrane projections, cytoplasmic vacuoles, fusion with neighboring cells, and, finally, cell death. Contamination was detected by fluorescence imaging (DAPI) and PCR reactions. The cultures were treated with BM-Cyclin antibiotic to eliminate contamination. The data presented here will be of relevance to researchers whose investigations involve cell culture, especially respiratory and HEp-2 cells.

Resumo A contaminação de culturas primárias e celulares por micoplasmas é uma das principais armadilhas econômicas e biológicas da pesquisa básica, diagnóstico e fabricação de produtos biotecnológicos. Trata-se de uma contaminação rotineira, mas de difícil acompanhamento. A presença de micoplasma pode afetar vários parâmetros fisiológicos da célula, além de ser considerada uma importante fonte de resultados científicos imprecisos e/ou não reprodutíveis. Cada tipo de célula apresenta sintomas característicos, principalmente morfológicos, que indicam uma contaminação por micoplasma. As células HEp-2 são originárias do carcinoma da laringe e, portanto, fazem parte do trato respiratório, um dos habitats do micoplasma. Apesar da importância destas células em diversas pesquisas biológicas (avaliação da proliferação e migração celular, apoptose, compostos antivirais e antitumorais), as alterações decorrentes da contaminação por micoplasma nestas células ainda não foi descrita. Aqui, descrevemos as alterações morfológicas progressivas na cultura de células HEp-2 infectadas por micoplasma, bem como o diagnóstico da infecção e seu tratamento. A contaminação por micoplasma descrita neste trabalho resultou em alongamento citoplasmático, espaçamento entre células, projeções delgadas da membrana plasmática, vacúolos citoplasmáticos, fusão de células vizinhas e, finalmente, morte celular. A contaminação foi detectada por imagens de fluorescência (DAPI) e reações de PCR. As culturas foram tratadas com antibiótico BM-Cyclin para eliminar a contaminação. Os dados aqui apresentados serão de relevância para pesquisadores cujas investigações envolvem cultura celular, principalmente células respiratórias e HEp-2.

Influence of RhoC on Apoptosis and Cancer-stem-cell-marker-ALDH1A1 and Morphology in Hep2 Laryngeal Squamous Carcinoma Cell / 医学研究杂志

Objective To investigate the effects of RhoC on biological behavior in the laryngeal squamous carcinoma.Methods By delivering exogenous gene into Hep2 laryngeal squamous carcinoma cell line,we alternatively repressed and strengthened the expression of RhoC.We tested the apoptosis of Hep2 tumor cell line with TUNEL,visualized tumor cells shape by staining cell skeleton with Alexa fluor phalloidin,measured the mRNA of CSC marker ALDH1A1 with QPCR.Results After repressing the expression of RhoC in Hep2 cell line,the apoptosis of cancer cells was elevated,the expression of CSC marker ALDH1A1 was significantly decreased.RhoC impacted the shapes of Hep2.Conclusion RhoC havd a positive role in LSCC metastasis.RhoC is a promising target of anti-metastases in LSC.

Impact of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep2 / 中国免疫学杂志

Objective:To study the effect of miR-122a on inhibition proliferation of laryngeal carcinoma cell line Hep-2.Meth-ods:The oligomucleotide of miR-122a was transfected into laryngeal carcinoma cell line Hep 2 cells,which were devided into three groups of A(miR-122a transfection),B(miR-122a inhibitor),C(miR-122a-NC inhibitor) and group of D(blank control).The expres-sion of miR-122a was defected by RT-PCR,and relevant protein expression was evaluated by Western blot .The cell proliferation and cell cycle were determined by MTT assy and flow cytometry ,respectively.Results:Compared to group D,miR-122a expression in Hep2 cells was obviously elevated atter miR-122a-transfected.The proliferation of Hep2 cells in group A was significantly inhibited and the cell cycle arrested at G1/G0 phase.The protein expression of CDC42 was downregulated with decreased expressions of CDK 4 and cyclin D1 in group A.Conclusion:miR-122a inhibits the proliferation activity of Hep 2 cells,suggesting that miR-122a can be taken as a po-tential candidate for gene therapy of laryngeal carcinoma .

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

Objective To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. Methods The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells. Results Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P < 0.01), the cell apoptosis rate was significantly increased (P < 0.01), the abilities of cell migration and invasion were significantly reduced (P < 0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P < 0.01). Conclusions The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells

OBJECTIVE@#To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells.@*METHODS@#The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells.@*RESULTS@#Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P < 0.01), the cell apoptosis rate was significantly increased (P < 0.01), the abilities of cell migration and invasion were significantly reduced (P < 0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P < 0.01).@*CONCLUSIONS@#The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

Fluid shear stress regulates epithelial-mesenchymal transition (EMT) in Hep2 cells / 医用生物力学

Objective To examine the effects of fluid shear stress (FSS) on epithelial-mesenchymal transition (EMT) in Hep2 cells. Methods Hep2 cells were exposed to 140 mPa FSS. The morphologic changes of Hep2 cells exposed to FSS at different durations were observed using inverted microscope. The migration ability of Hep2 cells after FSS loading was investigated using scratch wound assay. The distribution and expression of cytoskeleton protein F-actin were examined by confocal microscope. The expression of the EMT marker proteins were detected by Western blotting. Results Most of Hep2 cells changed their morphology from polygon to elongated spindle with well-organized F-actin under FSS. After removing FSS, Hep2 cells recovered their initial morphology with flat polygon. FSS regulated Hep2 cells to enhance their migration capacity in a time-dependent manner. FSS promoted the rearrangement of cytoskeletal protein F-actin，which enhanced the migration behavior of Hep2 cells. In addition, FSS induced a time regularity of expression of the EMT marker proteins in Hep2 cells. Conclusions FSS as an important physical factor can induce EMT in Hep2 cells.

The research on apoptosis of human laryngeal cell line Hep-2 induced by 1,4-bis[2-(benzylselanyl) ethoxy] anthracene / 中国癌症杂志

Background and purpose:Selenium is one of the essential trace elements for human activities, and plays an incomparable role in maintaining human health. It was reported that selenium compound 1,4-bis[2-(benzylse-lanyl)ethoxy] (BSEA) anthracene has antiseptic and antiphlogistic effects. However, the mechanisms underlying anti-cancer effects of BSEA are rarely reported. BSEA-induced apoptosis in human laryngeal carcinoma Hep-2 cells and its mechanisms were studied.Methods:Methyl thiazolyl tetrazolium (MTT) assay was used to determine inhibition ratio of Hep-2 cells 24 hours after Hep-2 cells were treated with different concentrations of BSEA. Fluorescence microscope was used to observe the morphology change of apoptosis in Hep-2 cells. The apoptosis was detected by Annexin Ⅴ-FITC. Mi-tochondrial membrane potential was assayed by JC-1. Microplate reader detected the activity of caspase-3 and caspase-8. The mRNA and protein levels of Bax and XIAP were measured by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot.Results:The results showed that BSEA caused a dose-dependent inhibition of the growth of human laryngeal carcinoma cell line Hep-2in vitro, andIC50 was 35.74μmol/L. The apoptotic bodies were distinctly observed at a concentration of 80μmol/L of BSEA by AO fluorescence staining. This study found that the eversion of phosphatidyl serine intensified, and mitochondrial membrane potential also began to decline. The activity of caspase-3 appeared the tendency of dependence on dosage, while the activity of caspase-8 did not change significantly. The mRNA and protein expression level of Bax increased, whereas the mRNA and protein expression level of XIAP de-creased.Conclusion:Therefore, BSEA could obviously inhibit human laryngeal carcinoma Hep-2 cells proliferation and induce apoptosis via the mitochondrial pathway.