Establishment and preliminary application of organoids in ovarian cancer / 中华妇产科杂志

Objective:To explore the establishment and application of ovarian cancer organoids.Methods:Fresh ovarian tumor tissues, obtaining from patients underwent surgery in the First Affiliated Hospital of Nanjing Medical University between October 2021 and March 2022, were collected, enzymatic degraded, digested, and embedded into matrigel to establish organoids. A total of 32 ovarian cancer samples were collected. Hematoxylin eosin (HE) staining and immunofluorescence (IF) procedure were used to verify the morphological structure of organoids and their expression of molecular markers. 3D cyto-live or dead assay was used to detecte the live or dead cells in organoids. Carboplatin with a concentration ranging from 5 to 80 μmol/L (5, 10, 20, 40, 80 μmol/L) was added to organoids to calculate the 50% inhibitory concentration (IC 50) in different organoids. Results:(1) Organoids from a total of 32 patients were established, of which 18 cases could be passaged stably in the long term in vitro, while 14 could be passaged in the short time. The average amplification time of long-term passage in vitro was over 3 months, and the longest reached 9 months. (2) In HE staining, significant nuclei atypia and local micropapillary structures were observed in organoids. IF staining revealed that ovarian cancer organoids expressed molecular markers similar to primary tumor tissues, such as Pan cytokeratin (Pan-CK), p53, paired box gene 8 (PAX8), and Wilms tumor gene 1 (WT1). (3) In 3D cyto-live or dead assay, a large number of apoptotic cells were observed inside and around the organoids after added carboplatin. The sensitivity to carboplatin varied in 18 organoids could amplify in the long term, with an average IC 50 of (29.5±15.8) μmol/L. Moreover, IC 50 values of 4 organoids derived from patients received neoadjuvant chemotherapy were much higher than the 14 organoids which did not received neoadjuvant chemotherapy [(48.7±11.3) μmol/L vs (24.0±12.1) μmol/L; t=3.429, P=0.022]. Conclusions:Organoids recapitulate ovarian cancers in vitro and could be stably passaged. Organoids derived from patients received neoadjuvant chemotherapy have higher resistance to carboplatin.

Correlation between neoadjuvant radiotherapy and chemotherapy efficacy and changes in peripheral blood T lymphocytes in patients with advanced mid-to-low rectal cancer / 中国基层医药

Objective:To correlate neoadjuvant radiotherapy and chemotherapy efficacy with changes in peripheral blood T lymphocytes in patients with advanced mid-to-low rectal cancer.Methods:A total of 106 patients with rectal cancer who received treatment in Jinhua Municipal Central Hospital from January 2019 to December 2020 were included in this study. Fasting venous blood was taken before neoadjuvant radiotherapy and chemotherapy and 7 days before surgery to measure the numbers of CD3 +, CD4 +, CD8 +, CD45RA + and CD45RO + cells using flow cytometry. The optimal cut-off point was determined using the receiver operating curve. The influential factors of tumor regression grade were analyzed using logistic regression analysis. Results:After neoadjuvant radiotherapy and chemotherapy, the numbers of CD3 +, CD4 +, CD8 + cells were (401.86 ± 138.65), (225.83 ± 87.17), and (155.84 ± 71.19) respectively, which were significantly decreased compared with before neoadjuvant radiotherapy and chemotherapy [(477.33 ± 141.74), (647.38 ± 203.19), (348.22 ± 113.75), t = 10.78, 11.17, 9.49, all P < 0.05]. There were no significant differences in the percentages of CD3 +, CD4 +, CD8 +, CD45RA + and CD45RO + cells between before and after treatment (all P > 0.05). The percentage of CD45RO + cells was significantly increased after neoadjuvant radiotherapy and chemotherapy. A higher percentage of CD45RO + cells led to a lower tumor regression grade ( P < 0.05). The receiver operating characteristic curve showed that the optimal cut-off point of the percentage of CD45RO + cells was 1.08. The area under the receiver operating characteristic curve was 0.774 ( P = 0.029), with a sensitivity of 82.5% and specificity of 69.6%. The logistic regression analysis revealed that the percentage of CD45RO + cells was significantly correlated with tumor regression grade ( P < 0.05). Conclusion:The percentage of CD45RO + cells in T lymphocyte subsets before and after neoadjuvant radiotherapy and chemotherapy is closely related to tumor regression grade. It can be used as an indicator to predict the sensitivity of neoadjuvant radiotherapy and chemotherapy. This study is of great innovation and science and provides a new idea for clinical practice.

Hepatobiliary tumor models and novel technology for accurate drug screening / 临床肝胆病杂志

Hepatobiliary tumor is a type of malignant tumor including primary liver cancer, cholangiocarcinoma, and gallbladder carcinoma. At present, hepatobiliary tumors have become the second leading cause of cancer-related death worldwide, while the treatment methods for such tumors cannot effectively meet clinical needs. Therefore, it is a key scientific problem in this field to explore and develop the experimental technology of accurate drug screening for hepatobiliary tumors, find new strategies and methods for clinical treatment, and provide new ideas for early diagnosis and comprehensive treatment of hepatobiliary tumors. This article introduces the latest research advances in the novel technologies for accurate drug screening for hepatobiliary tumor and their application potential by focusing on the construction of individualized pathological models of hepatobiliary tumor, drug screening technologies, the design and screening strategy of specific target drugs, and drug screening strategy based on artificial intelligence and big data analysis, as well as the directions for future development.

Multi-omics molecular subgrouping of hepatocellular carcinoma and its application in precision diagnosis and treatment / 临床肝胆病杂志

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality in China. In recent years, the application of targeted therapy and immunotherapy has improved the survival rate of HCC patients. However, a significant difference in treatment response is observed among HCC patients due to tumor heterogeneity and a lack of biomarkers to predict efficacy. The advance in proteogenomics-centered multi-omics studies and the development of high-throughput drug screening platforms will help to develop new clinical treatment strategies for HCC and new methods for predicting the efficacy of precision medication, thereby realizing personalized precision diagnosis and treatment.

Laboratory diagnosis of human T-lymphotropic virus in Brazil: assays, flowcharts, challenges, and perspectives

Abstract INTRODUCTION We present a data analysis and review of recent studies regarding the laboratory diagnosis of human T-lymphotropic virus 1 and 2 (HTLV-1/2) infections in Brazil. METHODS Target populations, available diagnostic serological assays (screening and complementary tests), molecular assays (in-house), causes of false-positive and false-negative results, and flowcharts were analyzed. RESULTS A table presents the target populations, two diagnostic flowcharts (depending on laboratory infrastructure and study population), and recent research that may improve how HTLV-1/2 is diagnosed in Brazil. CONCLUSIONS: Our results support the implementation of public policies to reduce HTLV-1/2 transmission and its associated diseases.

Specificity of HTLV screening tests and its impact on health care program costs: The perspective of antenatal screening in Brazil

Abstract INTRODUCTION: Brazil ranks first in the number of HTLV-1/-2-infected individuals worldwide. The high morbidity and mortality of HTLV-1-associated diseases, especially following infection in infancy, requires strong action to reduce vertical transmission. METHODS: To facilitate the appraisal of the implementation of the HTLV antenatal screening program by the Brazilian Ministry of Health, we determined the costs in distinct scenarios according to HTLV seroprevalence, specificity of the screening test, and type of confirmatory test. RESULTS: HTLV antenatal screening would cost R$ 55,777,012-R$ 77,082,123/year. Screening assays with high specificity reduce the need and cost of confirmatory assays by up to 25%. CONCLUSIONS: Careful selection of the screening assay is required to optimize the program.

In vitro Studies of Antitumor Activity of Vanadium Complexes with Orotic and Glutamic Acids / Estudos in vitro da Atividade Antitumoral de Complexos de Vanádio com Ácidos Órotico e Glutâmico / Estudios in vitro de la Actividad Antitumoral de Complejos de Vanadio con Ácidos Orótico y Glutámico

Introduction: Three vanadium complexes with orotic and glutamic acids, in their anion forms, were prepared and their in vitro cytotoxicity toward human lung fibroblasts (MRC-5), human hepatocellular carcinoma (HepG2) and human colorectal adenocarcinoma (Caco-2) are reported. Objective: Describe the synthesis and characterization of new vanadium complexes with orotic and glutamic acids, and test its antitumor activity against HepG2 and Caco-2. Method: The complexes were formulated as VO (oro), VO (α-glu) and VO (γ-glu) based on chemical, thermogravimetric analyses and infrared spectra. Results: Resazurin assay demonstrates its cytotoxicity against the HepG2 and Caco-2 cell lines with the IC50 ranging from 7.90 to 44.56 µmol.L-1. The cytotoxicity profiles indicate that the tumoral lines show more activity than the cells MRC-5, with selectivity indexes ranging from 1.58 to 8.96. Conclusion: The three complexes had better in vitro activity than cisplatin for both normal and cancer cell lines. The IC50 values are two to six times better for the cancer cell ines and five to seven times better for the normal cell lines. This study indicates that the complexes obtained are promising candidates for antitumor drugs.

Introdução: Foram preparados três complexos de vanádio com ácidos orótico e glutâmico, em suas formas aniônicas, e foi testada sua citotoxicidade in vitro para fibroblastos pulmonares humanos (MRC-5), carcinoma hepatocelular humano (HepG2) e adenocarcinoma colorretal humano (Caco-2). Objetivo: Descrever a síntese e caracterização de novos complexos de vanádio com ácidos orótico e glutâmico e testar sua atividade antitumoral contra HepG2 e Caco-2. Método: Os complexos foram formulados como VO (oro), VO (α-glu) e VO (γ-glu) com base em análises químicas, termogravimétricas e espectros no infravermelho. Resultados: O ensaio de resazurina demonstrou sua citotoxicidade contra as linhagens celulares HepG2 e Caco-2 com o IC50 variando de 7,90 a 44,56 µmol.L-1. Os perfis de citotoxicidade indicam que as linhas tumorais apresentam maior atividade que as células MRC-5, com índices de seletividade variando de 1,58 a 8,96. Conclusão: Os três complexos tiveram melhor atividade in vitro do que a cisplatina, tanto para linhagens celulares normais como cancerosas. Os valores de IC50 são de duas a seis vezes melhores para as linhagens celulares cancerosas e de cinco a sete vezes melhores para as linhagens celulares normais. Este estudo indica que os complexos obtidos são promissores candidatos a fármacos antitumorais.

Introducción: Tres complejos de vanadio con ácidos orótico y glutámico, en sus formas aniónicas, fueram preparados. Su citotoxicidad in vitro hacia los fibroblastos pulmonares humanos (MRC-5), el carcinoma hepatocelular humano (HepG2) y el adenocarcinoma colorrectal humano (Caco-2) son reportados. Objetivo: Los principales objetivos de este trabajo son describir la síntesis y caracterización de nuevos complejos de vanadio con ácidos orótico y glutámico y probar su actividad antitumoral contra el HepG2 y el Caco-2. Método: Los complejos fueron formulados como VO (oro), VO (α-glu) y VO (γ-glu) basados en análisis químicos, termogravimétricos y espectros infrarrojos. El ensayo de resazurina demuestra su citotoxicidad contra las líneas celulares HepG2 y Caco-2 con el IC50 que van de 7,90 a 44,56 µmol.L-1. Los perfiles de citotoxicidad indican que las líneas tumorales presentan mayor actividad que los MRC-5, con índices de selectividad que van de 1,58 a 8,96. Conclusión: Los tres complejos tuvieron mejor actividad in vitro que el cisplatino, tanto para líneas celulares normales como para líneas celulares cancerosas. Los valores del IC50 son de dos a seis veces mejores para las líneas celulares de cáncer y de cinco a siete veces mejores para las líneas celulares normales. Este estudio indica que los complejos obtenidos son candidatos prometedores para fármacos antitumorales.

Anticancer drug screening: standardization of in vitro wound healing assay / Triagem de drogas anticâncer: padronização do ensaio de ranhura in vitro

ABSTRACT Introduction: Gliomas are characterized by rapid proliferation and aggressive invasion into normal surrounding brain tissue. In medical laboratories, the in vitro wound healing assay stands out as a simple, easy, inexpensive and affordable method to evaluate cell migration and proliferation. Objective: To standardize the in vitro wound healing assay using antimicrotubule drugs as positive controls. Methods: U87MG glioma cells were seeded at different densities and, after 24 h, the monolayer was scratched using different micropipette tip size to create a gap with no cells. The cells were then treated with colchicine and paclitaxel in culture medium with the presence or absence of fetal bovine serum. The wound was photographed with the aid of an inverted microscope and the wound area was measured using the Image J software. Results: Better defined edges scratches and monolayer with approximately 90% confluence were obtained at 1.5 and 2 x 105 cells/well density. The width and area of the scratch were, respectively, 948 pm/2.193221 mm2; 964 pm/2.266 mm2 and 1448 pm/3.221 mm2 to 10. 200 and 1000 pl micropipette tips. Colchicine inhibited wound closure by 12.6% or 3.4%, both in the presence or absence of serum; paclitaxel 2.4 and 6.7% respectively. Conclusion: Under standardized conditions, colchicine and paclitaxel proved to be efficient positive controls into the in vitro wound healing assay.

RESUMEN Introducción: Gliomas se caracterizan por rápida proliferación e invasion agresiva del tejido cerebral normal circundante. En laboratorios médicos, el ensayo de cierre de herida - una prueba in vitro - se destaca por ser un método simple, fácil, de bajo costo y accesiblepara evaluar la migración y la proliferación celular. Objetivo: Estandarizar el ensayo de cierre de herida usando agentes anti-microtúbulos como control positivo. Métodos: Las células de glioma U87MG fueron sembradas en diferentes concentracionesy, después de 24 horas, la monocamadafue rayada con punteras de diferentes tamanos para crear una hendidura sin células. Las células fueron entonces tratadas con colchicina y paclitaxel, en medio con o sin suero fetal bovino. La ranura fue fotografiada con la ayuda de un microscopio invertido, y el área de la ranura fue medida mediante el programa Image J. Resultados: Ranuras con bordes más bien-definidos y monocamada con alrededor de 90% de confluencia se obtuvieron con 1,5 y 2 x 105 células/pozo. La anchura y el área de las ranuras obtenidas fueron, respectivamente, 948 p.m/2,193 mm2; 964p.m/2,266mm2; y 1448p.m/3,221 mm2 para las punteras de 10,200y 1000pl. La colchicina inhibió el cierre de las ranuras en 12,6% o 2,4%, tanto en presencia como en ausencia de suero; el paclitaxel, 3,4% y 6,7%, respectivamente. Conclusión: En condiciones estandarizadas, colchicina y paclitaxel pueden ser usados como control positivo en el ensayo de cierre de herida in vitro.

RESUMO Introdução: Gliomas são caracterizados por terem rápida proliferação e invasão agressiva no tecido cerebral circundante normal. Em laboratórios médicos, o ensaio de ranhura - um teste in vitro - destaca-se por ser um método simples, fácil, barato e acessível para avaliar a migração e a proliferação celular. Objetivo: Padronizar o ensaio de ranhura, utilizando drogas antimicrotúbulos como controles positivos. Métodos: As células de glioma U87MG foram semeadas em diferentes concentrações e, após 24 horas, a monocamada foi arranhada usando ponteiras de diferentes tamanhos para criar uma fenda sem células. As células foram então tratadas com colchicina e paclitaxel, com meio em ausência ou presença de soro fetal bovino. A ranhura foi fotografada com auxílio de microscópio invertido, e a área da ranhura foi medida por meio do programa Image J. Resultados: Ranhuras com bordas mais bem definidas e monocamada com aproximadamente 90% de confluência foram obtidas com 1,5 e 2 x 105 células/poço. A largura e a área das ranhuras obtidasforam, respectivamente, 948pm/2,193 mm2;964pm/2,266mm2; e 1448 pm/3,221 mm2 para as ponteiras de 10, 200 e 1000 pl. A colchicina inibiu o fechamento das ranhuras em 12,6% ou 2,4%, tanto na presença quanto na ausência de soro; o paclitaxel, em 3,4% e 6,7%, respectivamente. Conclusão: Em condições padronizadas, colchicina e paclitaxel podem ser usados como controles positivos eficientes no teste in vitro de ranhura.

High-throughput RNA sequencing for screening the circular RNA expression profiling associated with chemo-resistance inosteosarcoma / 中华骨科杂志

Objective To screen the expression profile of circular RNA associated with chemo-resistance in osteosarcoma,and to analyze and identify its possible molecular functions.Methods Three pairs of matched drug-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63,U2OS/DXR vs U2OS,KHOS/DXR vs KHOS) were first tested by CCK-8 assay for three commonly used osteosarcoma chemotherapy drugs (doxorubicin,cisplatin,and methotrexate);then,using next-generation high-throughput RNA sequencing technology,comparative analysis of circRNA expression was performed in three pairs of matched multidrug-resistant and sensitive human osteosarcoma cell lines;followed by real-time quantitative PCR (qRT-PCR) to confirm the reliability and accuracy of sequencing data in chemotherapy-resistant osteosarcoma cell lines and tissues.In addition,a variety of bioinformatics analyses,including GO,KEGG pathway analysis and the construction of circRNA-miRNA networks,were performed to predict potential molecular functions of differentially expressed circRNAs and to construct relevant regulatory pathways or networks.Results Three osteosarcoma-resistant cells (MG63/DXR,U2OS/DXR,KHOS/DXR) were significantly resistant to the three common osteosarcoma chemotherapy drugs compared with control cells (MG63,U2OS,KHOS),which laid a solid foundation for the further experiments.RNA sequencing revealed a total of 80 circRNAs differentially expressed between the two groups.Compared with drug-sensitive osteosarcoma cells,57 circRNAs were upregulated and 23 circRNAs weredownregulated in the drug-resistant osteosarcoma cell lines (fold change≥ 2 or ≤0.5,P ＜ 0.05).Tenrandomly selected circRNAs were verified by qRT-PCR and the results showed that 9 of the 10 circRNAswere consistent with the sequencing results.In addition,KEGG pathway analysis showed that 56 pathways were significantly enriched in differentially expressed circRNAs,including Glycolysis/gluconeogenesis,ABC transporter,VEGF signaling pathway,and so on.Moreover,thedifferently expressedcircRNA-hsa_circ_0004674 with the highestfold change was highly expressedin the chemotherapy-resistant osteosarcoma cells and tissues,associated with poor prognosis in osteosarcoma patients.Some potential endogenous competitive RNA (ceRNA) regulatory pathways associated with hsa_circ_0004674,such as hsa_circ_0004674-miR-490-3p-ABCC2 and hsa_circ_0004674-miR-1254-EGFR,were constructed by use of the authoritative databases (target scan and miRanda) and literature searching and the miRNA response element sequences (MREs) between miRNAs that have a potential ceRNA regulatory relationship with hsa_circ_0004674 were alsopredicted.Conclusion CircRNA is closely related to tumor progression and may play a role in ostoosarcoma chemo-resistance.Besides,hsa_circ_0004674 may be a potential candidate for reversing drug resistance of osteosarcoma.

Application of weighted gene co-expression network analysis technology in human tumor research / 肿瘤

Weighted gene co-expression network analysis (WGCNA) technology is a high-throughput gene expression data mining algorithm, which uses the idea of system biology to find the correlation of gene expression and to construct gene modules, so as to further discovery a high-throughput data mining algorithm with biological significance modules. In recent years, with the deep understanding of human diseases to gene level, WGCNA has been used increasingly in the researches of various diseases, especially in mining the highthroughput data about tumor-related genes. Moreover, with the continuous improvement of this technology, the research of this technology in disease pathogenesis, development and treatment etc has been developed from a single co-expression network analysis to the multiple technologies [such as genome-wide association study (GWAS) and support vector machine (SVM)] combined WGCNA or the innovative applicated WGCNA. As a high-throughput research method based on gene level, WGCNA is playing an important role.

Clinical value of an adenosine triphosphate-based chemotherapy response assay in resectable stage III colorectal cancer

PURPOSE: ATP-based chemotherapy response assay (ATP-CRA) is a well-documented and validated technology that can individualize chemotherapy. This study was undertaken to assess the usefulness of ATP-CRA in advanced colorectal cancer (CRC) patients receiving adjuvant chemotherapy. METHODS: A total of 136 patients with curative resection between January 2006 and April 2014 were evaluated using ATP-CRA. Patients received either the FOLFOX or Mayo clinic regimen chemotherapy following assay results. The sensitive-group (S-group) was defined as a drug-producing ≥ 40% reduction in ATP, and the resistant-group (R-group) as an ATP reduction of < 40%. These 2 groups were further subdivided to produce 4 subgroups: the FOLFOX sensitive subgroup (the FS subgroup [n = 65]), the Mayo sensitive subgroup (the MS subgroup [n = 40]), the FOLFOX resistant subgroup (the FR subgroup [n = 10]), and the Mayo resistant subgroup (the MR subgroup [n = 21]). Clinical responses and survival results were compared for both treatment regimens. RESULTS: The FS and MS subgroups showed a better disease-free survival rate (29% vs. 40%, 35% vs. 47.6%) and overall survival rate (92.3% vs. 80.0%, 87.5% vs. 76.2%) than FR and MR subgroups. The FS and MS subgroups showed a longer time to relapse (20.2 months vs. 9.5 months, 17.6 months vs. 16.4 months) than the FR and MR subgroups. CONCLUSION: ATP-CRA tailored-chemotherapy has the potential to provide a survival benefit in resectable advanced CRC.

Recent progress in chemosensitivity test of tumor / 肿瘤

Cancer is one of the major “killers” threatening human health, but so far there is not very effective treatment. Although chemotherapy is the most commonly used for the non-surgical treatment of cancer, due to the extensive presence of tumor heterogeneity, many cases of multidrug resistance and the failure in treatment of tumors can be found everywhere. In order to avoid the blindness of chemotherapy and improve the efficiency of chemotherapy, the personalized therapy of cancer based on chemosensitivity test has come into being. After more than half a century of evolution, the new methods of chemosensitivity test have been developed continuously, with a simple, rapid, accurate and reliable trend. A series of clinical studies have confirmed the importance of chemosensitivity test in guiding personalized therapy of cancer. However, the previous literatures have not distinguished the methods of culture and detection involved in chemosensitivity test, which is easy to cause conceptual confusion. Hence, the most commonly used methods of chemosensitivity test are reorganized in this article, with emphases on the introduction of three-dimensional cell culture, bioluminescence assay, fluorescence analysis and electric cell-substrate impedance sensing, to have a more comprehensive and in-depth understanding of chemosensitivity test.

Construction of in vitro high-throughput screening system for activin receptor-like kinases 4, 5 and 7 inhibitors / 中国药理学与毒理学杂志

OBJECTIVE To establish an in vitro screening system for activin receptor-like kinase 4,5 and 7 (ALK4,ALK5 and ALK7) inhibitors.METHODS The insect expression systems for kinase domain of ALK4,5,7 and Smad2/3 proteins were established using the Bac to Bac baculovirus expression system.The desired proteins were expressed in Sf9 insect cells and purified by GST affinity.The screening system was composed of the kinase,Smad3 protein,ATP as well as the compound.The impact of the compound on the activities of ALK kinase domains was examined by measuring the amount of remnant ATP in the system as ALKs catalyzed the phosphorylation of Smad3 protein and consumed ATP during the process.The screening conditions were optimized,and validation of the screening system was conducted using known ALKs inhibitors.RESULTS All the reconstructed Bacmids were identified to be correct by PCR and restriction enzyme digestion.All the proteins were expressed in Sf9 insect cells after transfection,and purified proteins were achieved by GST affinity purification.For the screening system,the optimized kinase concentration and Smad3 concentration were 10 mg· L-1 and the optimized ATP concentration was 10 nmol·L-1.The Z'factor for ALK4,ALK5,and ALK7 kinase inhibitors screening system was 0.71,0.51 and 0.74,respectively.The well-known ALK inhibitor SB431542 inhibited the catalytic activities of ALK4,ALK5,and ALK7 with IC50 values of 22,188 and 91 nmol· L-1,respectively.CONCLUSION The in vitro screening system for ALK4,ALK5 and ALK7 inhibitors is successfully established.

Charaterizing cytotoxicity of nitrogen mustard HN-3 based on cellular high content analysis / 中国药理学与毒理学杂志

OBJECTIVE To study the cytotoxic characteristics of nitrogen mustard HN-3 in different cell. METHODS Human epidermal keratinocytes-fetal (HEKf), human dermal fibroblasts-adult (HDFa) and human lung fibroblasts (HLF) cell lines were treated with HN-3100, 300 and 450μmol·L-1 for 0.5, 2, 4, 6, 12, 24 and 48 h, respectively. Multi-parameter analysis technology based on cell imaging was used to examine the effects of HN-3 on cell survival, cell cycle arrest, apoptosis, autophagy and oxidative stress, along with parameters concerning nucleus, cytoskeleton (actin and tubulin), lysosome, nuclear membrane permeability (NMP), mitochondrial membrane potential (MMP) and phosphohistone H 2AX (pH2AX). RESULTS HN-3 caused irreversible cellular damage by significantly decreasing the number of HEKf, HDFa and HLF cells in a time-dependent manner (P<0.01). Before the cell number was reduced robustly, the content of reactive oxygen species and pH2AX significantly increased, but the glutathione content decreased after cells were exposed to HN-3 for 0.5 h (P<0.01). In addition, the content of lyso-some was reduced in HEKf cells at 0.5 h, but increased in HDFa and HLF cells at 0.5 and 2 h respec-tively, accompanied by the increase in microtubule-associated protein 1 light chain 3B (LC3B) puncta.With the significant reduction of the cell number in HEKf cell line, the nuclear intensity increased, nuclear area decreased, the intensity and area of F-actin and α-tubulin decreased, MMP decreased (P<0.01) and lysosomal intensity increased. But the effects of HN-3 on HDFa and HLF cell lines were quite different. The nuclear area increased, the intensity and area of F-actin and a-tubulin increased, MMP increased (P<0.01) and the intensity of lysosome increased. In HLF cells, there was an increase in LC3B puncta (P<0.01). In all the three cell lines, NMP and manganese superoxide dismutase content were increased, and cell cycle arrested at G2 phase. HN-3 Induced early apoptosis in HDFa cells but late apoptosis in HEKf cells. CONCLUSION HN-3 causes DNA damage, oxidative stress and lysosome damage at an early stage, whereas at the late stage, the imbalance of MMP, increase in NMP, and G2 phage arrest are the major cytotoxic effects. Moreover, HN-3 specifically induces nuclear condensation, cytoskeleton protein aggregation and apoptosis in HEKf cell. HN-3 Induces nuclear swelling, and loose cytoskeleton in HDFa cells and HLF cells, eventually inducing early apoptosis in HDFa cells and autophagic death in HLF cells.

Chemical constituents from the heartwood of Trachycarpus fortunei (Hook.) H. Wendl / 军事医学

Objective To study the chemical constituents of the heartwood of Trachycarpus fortunei.Methods Compounds were isolated and purified by silica gel column , Sephadex LH-20 chromatography and recrystallization .Their structures were elucidated by physicochemical properties and spectral data .Results Ten compounds were isolated from the 95%ethanol extract of the heartwood of T.fortunei and identified as 1,3-dihydroxyanthraquinone (1),mollugin(2), 4-hydroxy-2, 6-dimethoxy-benzoic acid ( 3 ), 5-hydroxy-2-hydroxymethyl-γ-pyranone ( 4 ), 3, 5-dihydroxy-2-methyl-γ-pyranone(5),3,5-dihydroxy-γ-pyranone (6),daucosterol (7),4-hydroxybenzoic acid (8),pyrocatechol (9),β-sitosterol (10).Conclusion Compounds 1-7 have been obtained from this plant for the first time ,and 1-6 from the genus for the first time.The screening results of antitumor activity in vitro show that the half inhibitory concentration IC 50 of compound 2 for human osteosarcoma cells U20S and human neuroblastoma cells SY 5Y is 18.10 and 26.59 μg/ml respectively. Compound 5 can inhibit the growth of human breast cancer cells MCF-7 with a half inhibitory concentration IC 50 of 37.31μg/ml.

Effect of sorafenib combined with thalidomide on angiogenesis in chick chorioallantoic membrane / 临床肝胆病杂志

ObjectiveTo investigate the effect of sorafenib and/or thalidomide on angiogenesis in chick chorioallantoic membrane (CAM). MethodsWhite eggs incubated for 7 days were used to establish a CAM model. The model eggs were randomly divided blank control group, sorafenib group, thalidomide group, and sorafenib/thalidomide group. After treatment, each egg was incubated for another 2 days. The CAM samples were collected and fixed to take their pictures under a microscope, and the vascular coverage of each sample was calculated. Comparison between these groups was made by analysis of variance, and comparison between each two groups was made by SNK-q test. ResultsThe thalidomide group or sorafenib group had significantly lower vascular coverage than the blank control group (30.2%±2.9% or 26.5%±2.1% vs 38.3%±2.7%, P＜0.05). The sorafenib/thalidomide group had significantly lower vascular coverage than the thalidomide group or sorafenib group (12.6%±1.5% vs 30.2%±2.9% or 26.5%±2.1%, P＜0.05). ConclusionBoth sorafenib and thalidomide have a good anti-angiogenic effect on CAM, but a combination of the two drugs shows better efficacy.

In Vitro Adenosine Triphosphate-Based Chemotherapy Response Assay as a Predictor of Clinical Response to Fluorouracil-Based Adjuvant Chemotherapy in Stage II Colorectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We evaluated the usefulness of the in vitro adenosine triphosphate-based chemotherapy response assay (ATP-CRA) for prediction of clinical response to fluorouracil-based adjuvant chemotherapy in stage II colorectal cancer. MATERIALS AND METHODS: Tumor specimens of 86 patients with pathologically confirmed stage II colorectal adenocarcinoma were tested for chemosensitivity to fluorouracil. Chemosensitivity was determined by cell death rate (CDR) of drug-exposed cells, calculated by comparing the intracellular ATP level with that of untreated controls. RESULTS: Among the 86 enrolled patients who underwent radical surgery followed by fluorouracil-based adjuvant chemotherapy, recurrence was found in 11 patients (12.7%). The CDR ≥ 20% group was associated with better disease-free survival than the CDR < 20% group (89.4% vs. 70.1%, p=0.027). Multivariate analysis showed that CDR < 20% and T4 stage were poor prognostic factors for disease-free survival after fluorouracil-based adjuvant chemotherapy. CONCLUSION: In stage II colorectal cancer, the in vitro ATP-CRA may be useful in identifying patients likely to benefit from fluorouracil-based adjuvant chemotherapy.

Cytotoxic effect of geniposide and its metabolite genipin on HepG2 cells and mechanism / 中国药理学通报

Aim To compare the cytotoxicity of geni-poside (GS)and its metabolite genipin (GP)on hu-man hepatocelluar HepG2 cells and explore the sub-stance and mechanism of hepatotoxicity induced by Fructus Gardeniae.Methods The cytotoxic effect of GS and GP was analyzed by MTT method;the antioxi-dant enzyme activities of manganese superoxide dis-mutase (Mn-SOD),catalase (CAT)and levels of glu-tathione (GSH)were detected by respective kits;the change of intracellular reactive oxygen species (ROS ) was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)staining method;multiparameter cytotox-icity analysis (cell loss,nuclear size and morphologi-cal changes,DNA content,cell membrane permeabili-ty,mitochondrial membrane potential changes,cyto-chrome C release ) were measured simultaneously by high content screening (HCS)assays.Results No cytotoxicity was found in GS (20~1 000 μmol·L-1 ) groups (P>0.05 ),but GP was found to exert obvi-ous cytotoxic effect,and 50μmol·L-1 GP could obvi-ously inhibit HepG2 cell proliferation (P0. 0 5 ),GP could significantly decrease the activity of Mn-SOD,CAT and the level of GSH,and obviously increase the content of ROS (P0.05 ). Conclusion GP is the direct substance of hepatotox-icity induced by Fructus Gardeniae,and the mecha-nism might be associated with oxidative stress,mito-chondria injury and apoptosis.

Pharmacodynamic screening of anticancer agents used as c-Met inhibitor in vitro and in vivo / 天津医药

Objective To screen 8 series of LY compounds, c-Met tyrosine kinase inhibitors, and evaluate their anti-tumor effects in vitro and in vivo. Methods Preliminary screening was carried out by detecting the c-Met kinase phosphor?ylation inhibition activity of the compounds. CCK-8 assay was adopted for secondary anti-tumor screen of the selected com?pounds using MKN-45, U87MG, Caki-1 and PC-3 cell lines in vitro. The transplanted tumor model of U87MG cells in nude mice was established to evaluate the antitumor activity in vivo. Results Four compounds (LY22, LY25, LY28 and LY32) with better activities were selected by HTRF method, in which LY28 had better inhibitory effect on c-Met than that of Crizo?tinib. The above active compounds showed different degrees of inhibition on the four kinds of target cells (MKN-45, U87MG, Caki-1 and PC-3) detected by CCK-8 method, and the inhibitory effect of LY28 showed the most obvious. Antitumor activi?ty in vivo showed that LY28 can significantly inhibited tumor growth in a dose-dependent manner. The tumor inhibitory rate in high-dose of LY28 was 78.13%. Conclusion The compound LY28 has good antitumor activity in vitro and in vivo, which will be a new tyrosine kinase inhibitor.

High-throughput screening of single Chinese plant extracts on specific anti-hepatoma drugs / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the effect of single traditional Chinese medicine(TCM) herb extracts on hepatoma and normal fibroblast cells using high-throughput screening in order to obtain extracts with specific anti-hepatoma effect. METHODS 242 commonly used TCM herbs were extracted by petroleum ether,ethanol and water,respectively. The total number of TCM extracts was 554. The cyto?toxicity of samples was evaluated by MTT in human hepatoma cells Bel7402 and mice normal fibroblasts NIH3T3. RESULTS 7.4%of the total extracts had an inhibitory effect greater than 50%for Bel7402,but 14.8% for fibroblasts NIH3T3 cells. Extracts with an inhibitory effect above 50% on both Bel7402 and NIH3T3 cells accounted for 4.4%of the total extracts. Our results showed that the sample DF173 had preferable cytotoxicity effect on hepatoma carcinoma cells in a good dose-effect relationship. DF173 is an ethanol extract from Stephania tetrandra,which is a commonly used herb in TCM. The cytotoxic IC50 of DF173 against Bel7402 was 8.27 mg·L-1,and 19.48 mg·L-1 on NIH3T3. CONCLUSION The components of TCM herbs are highly complicated. The combination of tumor cells with normal fibroblast cells to evaluate the cytotoxicity effect during anti-tumor drug screening will contribute much to the discovery of TCM drugs with high anti-tumor efficiency and lower toxicity.