Fluorescence-activated Cell Sorter Assay Research Articles

MiR‑1271‑5p inhibits cell proliferation and induces apoptosis in acute myeloid leukemia by targeting ZIC2.

MicroRNAs (miRNAs) have been demonstrated to regulate the progression of numerous types of cancer, including acute myeloid leukemia (AML). Previous studies demonstrated that miR-1271-5p functions as a tumor suppressor; however, the roles of miR-1271-5p in AML remain unknown. In the present study, miR-1271-5p was significantly downregulated in AML tissues compared with normal tissues by reverse transcription-quantitative polymerase chain reaction analysis. Furthermore, the expression levels of miR-1271-5p in patients with AML may function as a prognostic marker. In addition, overexpression of miR-1271-5p significantly suppressed the proliferation and induced apoptosis of AML cells by Cell Counting kit-8 and fluorescence activated cell sorter assays; miR-1271-5p downregulation exhibited opposing effects. Additionally, transcription factor ZIC2 may be a direct target of miR-1271-5p in AML cells, which was demonstrated by a luciferase reporter assay and RNA pulldown assay. Overexpression of miR-1271-5p significantly reduced the mRNA and protein expression levels of ZIC2 in AML193 and OCI-AML2 cells by reverse transcription-quantitative polymerase chain reaction analysis and western blotting. Furthermore, an inverse correlation between miR-1271-5p and ZIC2 expression in AML samples was observed. In summary, ZIC2 was upregulated in AML tissues, and restoration of ZIC2 expression was able to promote the proliferation and reduce the apoptosis of AML cells transfected with miR-1271-5p mimics. The results of the present study demonstrated that miR-1271-5p inhibited the progression of AML by targeting ZIC2.

Erlotinib induces the human non-small-cell lung cancer cells apoptosis via activating ROS-dependent JNK pathways.

Although erlotinib (ERL) has drawn more and more attention toward its anticancer properties effect, the underlying mechanisms of ERL's anticancer properties effect remain unclear yet. So, the aim of this research was to explore the underlying anticancer mechanisms of ERL and to explore whether the reactive oxygen species (ROS)‐dependent c‐Jun N‐terminal kinase (JNK) pathway contributed to the anticancer properties provided by ERL. In our study, we used MTT assay to detect the anticell growth ability of ERL on human non–small‐cell lung cancer cell lines (A549). The extent of cell apoptosis was determined by Hoechst 33342 staining and fluorescence‐activated cell sorter (FACS) assay. Then, DCFH‐DA and JC‐1 staining were used to monitor intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Finally, the effect of ERL on phosphorylation state of JNK protein and downstream apoptosis concerned proteins were detected by western blotting assay. Results showed that ERL significantly suppressed the growth and reproduction of A549 cells with the concentration rising up in vitro. Hoechst 33342 staining and FACS assay also confirmed the proapoptosis effect of ERL on A549 cells with the concentration rising up. Furthermore, exposure of A549 cells to ERL increased the intracellular ROS production. As expected, intracellular ROS activated the proapoptotic JNK signaling pathway and inhibited the activation of EFGR signaling pathway. Our results also revealed that ERL could induce cell‐cycle arrest at G0/G1 period. Activation of JNK protein decreased MMP and downregulated content of antiapoptotic protein Bcl‐2 concomitant with the upregulated content of proapoptotic protein Bax in A549 cells. In addition, c‐Jun and cleaved caspase‐3 were also activated by the phosphorylated JNK induced by ERL. All of these proapoptosis effect of ERL was reversed by administration of N‐acetylcysteine (NAC), which performed as a ROS scavenger. Our results suggest that ERL induces A549 cells apoptosis via activating ROS‐dependent JNK pathways in human non–small lung cancer cells that provide a new experimental foundation for cancer therapy.

The effect of cyclooxygenase-2 on proliferation in gastric cancer cell line SGC7901

Objective To investigate the influence of cyclooxygenase-2 (COX-2) knock-down on cell proliferation and apoptosis in gastric cancer cell line SGC7901. Methods The mRNA levels of COX-2 in SGC7901 and gastric epithelium cells (GES) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The influence of COX-2 on the proliferation and apoptosis of cell line SGC7901 was evaluated with methyl thiazolyl tetrazolium (MTT) and fluorescence-activated cell sorter (FACS) assay. Results qRT-PCR assay indicated that the mRNA level of COX-2 in SGC7901 was significantly higher than that in GES (P<0.01). MTT and FACS assays showed that the proliferation was reversed by COX-2 knock-down in SGC7901 cells. Up-regulated Bax and down-regulated Bcl-2 can inhibit the expression of COX-2. Conclusions COX-2 could contribute to the proliferation of gastric cancer cell line SGC7901. Key words: RNA, small interfering; Cyclooxygenase 2/GE/ME; Stomach neoplasms/DT/GE/ME; Cell proliferation/DE; Apoptosis/DE

Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells.

BackgroundColon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated.MethodsIn present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot.ResultsWe found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.ConclusionsTherefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0228-7) contains supplementary material, which is available to authorized users.

Endothelium-Dependent Coronary Vasodilatation Requires NADPH Oxidase-Derived Reactive Oxygen Species

To determine the functional significance of physiological reactive oxygen species (ROS) levels in endothelium-dependent nitric oxide (NO)-mediated coronary vasodilatation. Endothelium-derived NO is important in regulating coronary vascular tone. Excess ROS have been shown to reduce NO bioavailability, resulting in endothelial dysfunction and coronary diseases. NADPH oxidase is a major source of ROS in endothelial cells (ECs). By using lucigenin-based superoxide production and dichlorfluorescein diacetate (DCFH-DA) fluorescence-activated cell sorter assays, we found that mouse heart ECs from NADPH oxidase-knockdown (p47(phox-/-)) animals have reduced NADPH oxidase activity (>40%) and ROS levels (>30%) compared with wild-type mouse heart ECs. Surprisingly, a reduction in ROS did not improve coronary vasomotion; rather, endothelium-dependent vascular endothelial growth factor-mediated coronary vasodilatation was reduced by greater than 50% in p47(phox-/-) animals. Western blots and L-citrulline assays showed a significant reduction in Akt/protein kinase B (PKB) and endothelial NO synthase phosphorylation and NO synthesis, respectively, in p47(phox-/-) coronary vessels and mouse heart ECs. Adenoviral expression of constitutively active endothelial NO synthase restored vascular endothelial growth factor-mediated coronary vasodilatation in p47(phox-/-) animals. Endothelium-dependent vascular endothelial growth factor regulation of coronary vascular tone may require NADPH oxidase-derived ROS to activate phosphatidylinositol 3-kinase-Akt-endothelial NO synthase axis.

Using the national cancer institute anticancer drug screen to assess the effect of MRP expression on drug sensitivity profiles.

The MRP gene contributes to one form of multidrug resistance. To identify drugs interacting with MRP, we measured MRP mRNA expression by quantitative PCR in 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Expression was detected in all cell lines (highest in lung carcinomas and central nervous system tumors) with a range of 14-fold. A mean graph of MRP mRNA levels was constructed to determine Pearson correlation coefficients (PCCs) with mean graphs of >40,000 compounds using the COMPARE analysis. Only 20 compounds had PCCs of >/=0.500. The PCCs for VP-16, doxorubicin, and vincristine were 0.008, 0.13, and 0.257, respectively. Initially, 36 compounds with PCCs of >/=0.428 were analyzed using two MRP-overexpressing cell lines; low levels of cross-resistance was demonstrated for 23 compounds (1.3-9.4-fold). Twenty-four compounds also were available for further studies. Using a fluorescence activated cell sorter assay to measure competition of calcein efflux from MRP-overexpressing cells, 10 compounds were found to increase calcein retention by >/=2-fold. Ten compounds also were able to reduce ATP-dependent [3H]LTC4 transport into vesicles from MRP-overexpressing cells. These results contrast with previous studies with MDR-1 in which high correlations were found and confirmed for a large number of compounds. Although other assays may be more revealing, in these unselected cell lines, MRP mRNA expression was a poor predictor of drug sensitivity. This raises the possibility that other factors, including conjugating enzymes, glutathione levels, or other transporters, confound the MRP effect.

Identification of a 40- to 42-kDa Attachment Polypeptide for Canine Parvovirus in A72 Cells

The attachment of canine parvovirus (CPV) to different cell lines was quantitated by a fluorescence-activated cell sorter assay. The viral attachment was observed to both permissive A72 and nonpermissive ST cells but not to nonpermissive MDBK cells. The binding of and infectivity for CPV to A72 cells was reduced upon prior treatment of cells with Vibrio cholerae neuraminidase or lectins, specific for sialic acid. Similarly, treatment of cells with any of several proteases reduced virus binding; however, phospholipase treatment had no effect indicating that one or more membrane glycoproteins were involved in virus binding. These proteins were characterized with a virus overlay protein blot assay. Virus bound to a protein with a molecular mass of 40 to 42 kDa in membranes prepared from A72 and ST cells and not from MDBK cells. The binding to this polypeptide was specific since increasing amounts of unlabeled virions competitively inhibited binding of radiolabeled virions in a dose-dependent manner. A polypeptide of similar molecular mass was immunoprecipitated from radiolabeled octyl glucoside (OG) extract of A72 cells using purified virions, virion-specific antiserum, and protein A. The binding to this polypeptide was decreased but not abolished upon prior treatment of the membrane with V. cholerae neuraminidase. CPV preferentially recognized a polypeptide of similar molecular size in the OG extract prepared from the biotinylated basolateral surface of polarized MDCK monolayer. Hence, we propose that the 40- to 42-kDa glycoprotein represents a specific attachment molecule for CPV in A72 cells.

Non-sequence-specific antimalarial activity of oligodeoxynucleotides

The effects of exogenously applied oligodeoxynucleotides on Plasmodium falciparum proliferation was investigated. A fluorescence-activated cell sorter assay was employed to measure parasitemia after administration of either phosphodiester or phosphorothioate oligodeoxynucleotides. We report sequence-independent antimalarial activity preferentially with phosphorothioate congeners with IC 50 values in the 1–2 μM range. Phosphorothioate oligodeoxynucleotides which were antisense, sense or nonsense to Plasmodium mRNA, as well as homopolymers (30-mers containing all A or T bases) were equally effective inhibitors of parasitemia. The antimalarial activity was dependent upon oligomer length, concentration, and time of addition to the cultures but was independent of the parasite strain tested. Four P. falciparum strains, including a multi-drug-resistant strain (MDR-K), a drug-sensitive strain (FCR-3), a erythrocyte membrane sialic acid-independent strain (7G8) and a strain isolated from a cerebral malaria patient (CM-87) were equally susceptible to treatment with a phosphorothioate oligomer. Inhibition of red cell invasion is primarily responsible for the observed decrease in proliferation as determined by a study of parasite maturation in the presence of a 30-mer nonsense phosphorothioate oligodeoxynucleotide.

Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus.

The attachment of lymphocytic choriomeningitis virus (LCMV) to murine and primate cell lines was quantitated by a fluorescence-activated cell sorter assay in which binding of biotinylated virus was detected with streptavidin-fluorescein isothiocyanate. Cell lines that were readily infected by LCMV (e.g., MC57, Rin, BHK, Vero, and HeLa) bound virus in a dose-dependent manner, whereas no significant binding was observed to lymphocytic cell lines (e.g., RMA and WIL 2) that were not readily infected. Binding was specific and competitively blocked by nonbiotinylated LCMV. It was also blocked by LCMV-specific antiserum and a neutralizing monoclonal antibody to the virus glycoprotein GP-1 but not by antibodies specific for GP-2, indicating that attachment was likely mediated by GP-1. Treatment of cells with any of several proteases abolished LCMV binding, whereas phospholipases including phosphatidylinositol-specific phospholipase C had no effect, indicating that one or more membrane proteins were involved in virus attachment. These proteins were characterized with a virus overlay protein blot assay. Virus bound to protein(s) with a molecular mass of 120 to 140 kDa in membranes from cell lines permissive for LCMV but not from nonpermissive cell lines. Binding was specific, since unlabeled LCMV, but not the unrelated enveloped virus herpes simplex virus type 1, competed with 125I-labeled LCMV for binding to the 120- to 140-kDa band. The proteinaceous nature of the LCMV-binding substance was confirmed by the lack of virus binding to proteinase K-treated membrane components. By contrast, glycosidase treatment of membranes did not abolish virus binding. However, in membranes treated with endoglycosidase F/N-glycosidase F, and/or neuraminidase and in membranes from cells grown in tunicamycin, the molecular mass of the LCMV-binding entity was reduced. Hence, LCMV attachment to rodent fibroblastic cell lines is mediated by a glycoprotein(s) with a molecular mass of 120 to 140 kDa, with complex N-linked sugars that are not involved in virus binding.