Characterization of the infectivity of an Indonesian Zika virus strain in mammalian cell lines

Objective:To characterize the infection patterns and growth characteristics of the Zika virus(ZIKV)strain JMB-185 from Indonesia in various mammalian cell lines.Methods:ZIKV was grown in human(A549,HEK293,HepG2,Huh7,Jurkat,and THP-1)and non-human mammalian(RAW264.7,Vero,and Vero76)cell lines.Viral replication kinetics were measured using plaque assay,while intra-and extracellular viral RNA concentrations were assessed using RT-PCR.Flow cytometry was used to quantify the infected cells and cell viability was measured using an MTT assay.The ability of ZIKV to infect cell lines was visualized using a fluorescence immunostaining assay.Results:This ZIKV strain preferentially infected the lung,kidney,and liver cell lines A549,HEK293,Huh7,Vero,and Vero76,but not the immune cells Jurkat,RAW264.7,and THP-1.By contrast,the ZIKV showed no sign of infection in HepG2 cells,while maintaining viral titer over 3 days post-infection,with no infection recorded in immunostaining,no increase in viral RNA,and no indication of cell deterioration.Conclusions:The Indonesian ZIKV strain has a similar infection profile as other strains,except for its poor infectivity on HepG2 cells.Information on the growth characteristics of Indonesia ZIKV will help expand our understanding of the biology of ZIKV which will be useful for various applications including antiviral discovery.

GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines

BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.

Apoptotic activity of caged xanthones from Garcinia hanburyi in cholangiocarcinoma cell lines

AIM:To investigate the growth inhibitory mechanism of four caged xanthones from Garcinia hanburyi in cholangiocarcinoma(CCA) KKU-100 and KKU-M156 cells.METHODS:Four caged xanthones,selected on the basis of their anticancer potency and chemical structure diversities(i.e.isomorellin,isomorellinol,forbesione and gambogic acid) were used in this study.Growth inhibition of these caged xanthones was determined using the sulforhodamine B assay.Induction of apoptosis was assessed by observing cell morphology,ethidium bromide and acridine orange staining and DNA fragmentation assay.Levels of apoptotic-related gene and protein expressions were determined by a real-time reverse transcriptase polymerase chain reaction and Western blotting analysis,respectively.RESULTS:The compounds were found to inhibit growth of both cell lines in a dose-dependent manner and also showed selective cytotoxicity against the cancer cells when compared with normal peripheral blood mononuclear cells.Growth suppression by these compounds was due to apoptosis,as evidenced by the cell morphological changes,chromatin condensation,nuclear fragmentation,and DNA ladder formation.At the molecular level,these compounds induced down-regulation of Bcl-2 and survivin proteins with up-regulation of Bax and apoptosisinducing factor proteins,leading to the activation of caspase-9 and-3 and DNA fragmentation.The functional group variations did not appear to affect the anticancer activity with regard to the two CCA cell lines;however,at a mechanistic level,isomorellinol exhibited the highest potency in increasing the Bax/Bcl-2 protein expression ratio(120 and 41.4 for KKU-100 and KKU-M156,respectively) and in decreasing survivin protein expression(0.01 fold as compared to control cells in both cell lines).Other activities at the molecular level indicate that functional groups on the prenyl side chain may be important.CONCLUSION:Our findings for the first time demonstrate that four caged xanthones induce apoptosis in CCA cells which is mediated through a mitochondriadependent signaling pathway.

The diverse and contrasting effects of using human prostate cancer cell lines to study androgen receptor roles in prostate cancer

The androgen receptor （AR） plays an important role in the development and progression of prostate cancer （PCa）. Androgen deprivation therapy is initially effective in blocking tumor growth, but it eventually leads to the hormonerefractory state. The detailed mechanisms of the conversion from androgen dependence to androgen independence remain unclear. Several PCa cell lines were established to study the role of AR in PCa, but the results were often inconsistent or contrasting in different cell lines, or in the same cell line grown under different conditions. The cellular and molecular alteration of epithelial cells and their microenvironments are complicated, and it is difficult to use a single cell line to address this important issue and also to study the pathophysiological effects of AR. In this paper, we summarize the different effects of AR on multiple cell lines and show the disadvantages of using a single human PCa cell line to study AR effects on PCa. We also discuss the advantages of widely used epithelium-stroma co-culture systems, xenograft mouse models, and genetically engineered PCa mouse models. The combination of in vitro cell line studies and in vivo mouse models might lead to more credible results and better strategies for the study of AR roles in PCa.

STUDY ON THE EXPRESSION OF CYCLOOXYGENASE-2 IN HEPATOCELLULAR CARCINOMA CELL LINES AND ON THE GROWTH INHIBITION EFFECT OF NS-398

Objective： To investigate the expression of cyclooxygenase -2 （COX-2） in hepatocellular carcinoma cell lines and to explore the effect of NS-398, a selective inhibitor for COX-2, on HepG-2 cell line. Methods： lmmunohistochemistry and RT-PCR were used to investigate COX-2 expression in 6 HCC cell lines. MTT and Flowcytometry were used to evaluate the effect of the selective inhibitor of COX-2, NS-398, on HepG-2 cell lines. Results： All six HCC cell lines showed COX-2 expression at protein level. Five out of 6 cell lines showed COX-2 expression at mRNA level. NS-398 could suppress the growth of HepG-2 cell line, in a time and dose dependant manner. Conclusion： NS-398, a selective inhibitor of COX-2, showed inhibition effect on HepG-2 HCC cell line. The efficacy of inhibition was time and dose dependent, providing a new evidence for chemoprovention of hepatocellular carcinorma with COX-2 selective inhibitors.

The Construction of Marc-145 Cell Lines Expressing Nsp2 Gene of PRRSV and the Effects of Nsp2 Protein on PRRSV Replication

[Objective]The study aimed to investigate the effects of Nsp2 protein on porcine reproductive and respiratory syndrome virus （ PRRSV） replication. [Method]Through in vitro cloning,the Nsp2 gene of highly pathogenic PRRSV TJ and attenuated TJM were amplified by RT-PCR and cloned into the plasmid pEGFP-N1,which containing enhanced green fluorescent protein expression box. The constructed plasmids pEGFP-TJ Nsp2 and pEGFP-TJM Nsp2 were transfected into Marc-145 cells and screened by G418. Anti-G418 Marc-145-TJ Nsp2 and Marc-145-TJM Nsp2 cells were obtained,and the expression of Nsp2 protein in anti-G418 Marc-145-TJ Nsp2 and Marc-145-TJM Nsp2 cells was proved by PCR and RT- PCR. The Marc-145-TJ Nsp2 and Marc-145-TJM Nsp2 cells were infected by PRRSV,and TCID 50 was determined. [Result]The cells expressing Nsp2 gene of highly pathogenic PRRSV TJ and attenuated TJM,Marc-145-TJ Nsp2 and Marc-145-TJM Nsp2,were stable. PRRSV replication was fast in early stage on these cells. That is to say,Nsp2 protein played a positive role in early phase of PRRSV proliferation,and the effect of Nsp2 protein of highly pathogenic PRRSV TJ was more obvious. [Conclusion]The construction of Marc-145-Nsp2 cell lines provided data for the further discuss of PRRSV replication mechanism.

Establishment and Characterization of Four Long-Term Cultures of Neural Stem/Progenitor Cell Lines from the Japanese Flounder Paralichthys olivaceus

Neurogenesis is an important progress wherein the neural stem cells(NSCs) differentiate into functional neurons under conductive conditions. Neurogenesis occurs continuously in different areas of the central neural system in adult teleosts compared with adult mammals. Therefore, NSC cell lines must be established to offer a valuable in vitro system for studies on neurogenesis and other related functions. In this study, four cell lines designated as PoB1, PoB2, PoBf and PoBh were established from the brain of the Japanese flounder Paralichthys olivaceus. The cell lines were sub-cultured over 150 times and still grew well in DMEM/F12 medium at 24℃. PoB1, PoB2, PoBf and PoBh were identified as neural stem/progenitor cell lines on the basis of the mRNA expression of nestin and/or aldh111 or slc6 a4 and the formation of neurospheres. The cells transfected with the pEGFP-C1 plasmid showed fluorescent signals with distinct reagent dependencies. The established cell lines from the brain of P. olivaceus offer a valuable system in vitro for the study of neurogenesis, fish neural regulation and endocrinology-related functions.

The selection and stability analysis of stable and high Taxol-producing cell lines from Taxus cuspidata

In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell cultures. From 97 cell lines obtained from conditioned cultures, 10 cell lines with high Taxol content were selected. Stability analyses on solid and liquid B5 media were then used to obtain lines that stably produced high levels of Taxol. Fresh biomass and Taxol production of the ninth generation became stable. Taxol content of selected CL5, CL12, and CL21 samples was 0.0448, 0.0477, and0.0428% of dry mass（DW）, respectively. Proliferation of CL5, CL12 and CL21 was 346.3, 382.5, and 409.2%,respectively. From work over about 2 years, the three cell lines appear suitable for mass production of Taxol,promoting the industrialisation and commercial-scale production of Taxol using cell culture.

Effect of 80.55 MeV//u^(12)C^(6+) Ions on Radiosensitivity and Cell Cycle of Human Hepatoma Cell Lines

In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6＋ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 （survival fraction at 2 gray） of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6＋ ions.

Identification of differentially expressed genes in two new human bladder carcinoma cell lines

Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

THE EFFECTS OF ESTRADIOL ON BREAST CANCER CELL LINES

The estradiol effects on breast cancer cell lines including estrogen receptors (ER) positive and negative were studied with flow cytometry analysis, scanning and transmission electron microscopy (SEM and TEM), immunogold and immunofluorescence staining techniques. The results showed that estradiol markedly stimulated the division and proliferation of the ER( + ) MCF-7 cells at 10 nM, but had no marked effect on the cell cycle of the ER(-) H466B cells at the same concentration, and that tamo-xifen inhibited the stimulation of estradiol on MCF-7 cells. Estradiol obviously influenced the ultrastruc-ture of MCF-7 cells. Immunocytochemical localization of epidermal growth factor receptor (EGFR) on the MCF-7 cell membrane surface indicated that one of the mechanisms involving the growth of MCF-7 breast cancer cell and the stimulating effect on MCF-7 cells growth by estradiol is autocrine secretion.

Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

Summary： To investigate the expression of cyclooxygenase-2 （COX-2） in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.

TIME-AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

The effects of Sonic hedgehog(Shh) signaling pathway activation on S-type neuroblastoma(NB) cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components— Patched1(PTCH1) and Gli1 in 40 human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines(SK-N-AS,SK-N-SH and SHEP1),respectively.Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway.S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations.Cell viability was measured by using MTT method.Apoptosis rate and cell cycle were assayed by flow cytometry.The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice.High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines.Cyclopamine decreased the survival rate of the three cell lines while Shh increased it,and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment.Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase,while Shh induced the reverse effects and could partially prevent effects of cyclopamine.Cyclopamine could also inhibit the growth of NB in vivo.Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle,suggesting a new therapeutic approach to NB.

3-(Tetrazol-5-yl)-2-imino-coumarins Derivatives: Synthesis, Characterization, and Evaluation on Tumor Cell Lines

The first report of new 3-(tetrazol-5-yl)-2-iminocoumarin derivatives is described. The title compounds were prepared in two steps and were obtained in good yields (55-93%). They have been fully characterized by 1H, 13C NMR, FTIR, UV-Visible and HRMS. They were tested for their antiproliferative activities against six representative human tumor cell lines (Huh 7-D12, Caco2, MDA-MB231, HCT 116, PC3 and NCI-H727) and HaCat keratinocytes. Among them, compound 5e was active on HCT 116 (IC50 15 μM).

Expression of cyclooxygenase-2 mRNA in drug-sensitive cell and drug-resistant strains of ovarian cancer cell lines

Objective： To investigate the expression of cyclooxygenase-2 （COX-2） mRNA in drug-sensitive cell and drugresistant clones of ovarian cancer cell lines. Methods： RT-PCR and immunocytochemistry were used to investigate the expression of cyclooxygenase-2 in 3 clones drug-sensitive and 5 clones drug-resistant ovarian cancer cell. Results： Strong COX-2 mRNA expressions were detected in 3 clones of drug-sensitive cell and weak expressions were detected in 5 clones of drug-resistant cell. The protein expression of COX-2 in drug-sensitive cell was strongly positive reaction in immunocytochemistry stain and there was a weak positive reaction in 5 clones of drug-resistant cell. Conclusion： The expression of COX-2 mRNA in drug-sensitive cell strains is much higher than that in drugresistant strains of ovarian cancer cell lines, providing a basis of the chemoprevention for ovarian cancer.

In Vivo Animal Model Evaluation of a Powerful Oral Nanomedicine for Treating Breast Cancer in BALB/c Mice Using 4T1 Cell Lines without Chemotherapy

Nanopharmaceuticals containing quantum dot nanoparticles (Q-Dot NPs) for treating serious cancers such as breast cancer have made fantastic proposals. In this study, ZnO quantum dot NPs are formulated via ZnO@PVP nanopolymer as co-assistants coordinating with efficacious suitable wetting agents, PEG-binding compound, and W/O emulsifier for producing eco-friendly water-based nanodrug. Several characterization techniques containing SEM, TEM, FTIR, photoluminescence, zeta potential, and UV-Vis absorption were employed for ZnO Q-Dot NPs in nanodrug. This work aims to investigate the anti-tumor effects of such nanomedicine on the 4T1 breast cancer cell line in BALB/c mice, being elaborated through intraperitoneal, injection (IVP) and oral therapy. The impressive findings showed that ZnO nanodrug caused changes in blood factors, having the most effectiveness at 40 μg/ml concentration after two weeks of oral treatments. The significant increase in white blood cells (WBC) neutrophils and meaningful decreases in lymphocytes and especially cholesterol were powerful simultaneous impacts, successfully treating malignant breast cancer masses. In this significant animal model research for breast cancer, the sick mice recovered entirely and even had a safe space to mate. Histopathological results showed no evidence of breast tumor formation or metastasis in the group treated with nanodrug and their children. This nanomedicine has a therapeutic effect, and is ready to be applied for treating volunteer breast cancer patients. However, its prevention (inhibitory) effect can also be analyzed and added to current data in future studies.