Isolation of a polyamine transport deficient cell line from the human non-small cell lung carcinoma line NCI H157

In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N¹,N¹²-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCIH157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidine/spermine N¹-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylomithine (DFMO) induced growth inhibition in the mutant cells. © 1996 Wiley-Liss, Inc.     

Cytokine expression in placenta-derived mesenchymal stem cells in patients with pre-eclampsia and normal pregnancies

Objectives: The aim of this study was to investigate the levels of cytokines in placenta-derived mesenchymal stem cells (MSCs) in normal pregnancies and those with pre-eclampsia. Materials and methods: C5a, CD40 Ligand, G-CSF, GM-CSF, GROα, I-309, sICAM-1, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32α, IP-10, I-TAC, MCP-1, MIF, MIP-1α, MIP-1β, Serpin E1, RANTES, SDF-1, TNFα, and sTREM-1 were measured in mesenchymal stem cells using the human cytokine array panel A. The soluble intracellular adhesion molecule-1 (sICAM-1), stromal-derived factor-1 (SDF-1) and monocyte chemotactic protein-1 (MCP-1) were measured by real-time PCR and confirmed by Western blot analysis. Results: MSCs derived from the deciduas of normal pregnancies had significantly elevated levels of sICAM (p = 0.000) and SDF-1 (p = 0.011), compared to the pregnancies with pre-eclampsia. The level of MCP-1 in the decidua-derived MSCs was not significantly different. No significant difference was observed between normal and pre-eclamptic pregnancies for the amnion-derived MSCs. Conclusions: The decreased levels of sICAM and SDF-1 found in the decidua-derived MSCs from pre-eclamptic pregnancies might be associated with some of the immunological alterations in pre-eclampsia.     

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRAS^G12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRAS^G12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.     

Enhanced clonogenic survival induced by protein tyrosine phosphatase (PTP) inhibition after Cr(VI) exposure is mediated by c-Raf and Ras activity

Our recent studies showed that maintenance of protein tyrosine phosphorylation by PTP inhibition enhanced cell growth, clonogenic survival, and mutagenesis after a single low-level Cr(VI) exposure, thereby suggesting that tyrosine phosphorylation-dependent signaling may govern inappropriate survival in human lung fibroblasts (HLFs). Our goal is to identify specific phospho-tyrosine regulator(s)/ downstream effectors involved in enhanced survival after Cr(VI) exposure and PTP inhibition. Phosphotyrosine profiling array showed that PTP inhibition following Cr(VI) exposure increased tyrosine phosphorylation of specific proteins, such as FGR and ABL, which are upstream regulators of both Erk and Akt pathways. To explore the roles of these pathways in the PTP-induced increase in clonogenic survival after Cr(VI) exposure, we examined the effect of combined Akt1 and Erk1/2 knockdown via siRNA technology. Akt1 and/or Erk1/2 silencing had no effect on the PTP inhibitor-induced increase in survival following Cr(VI) exposure, suggesting the presence of non-Akt/non-Erk-mediated survival signaling. Interestingly, geldanamycin, an HSP90 inhibitor and non-specific Raf inhibitor, abrogated the PTP inhibitor-mediated increase in survival following Cr(VI) exposure and abolished the expression/activity of c-Raf and activity of Mek. These findings prompted us to explore upstream regulators of Erk, i.e., Ras, c-Raf and Mek for their potential roles in clonogenic survival. GW5074, a specific c-Raf kinase inhibitor did not alter the effect of the PTP inhibitor but decreased Cr(VI)-mediated clonogenic lethality, potentially though Mek hyperactivation. A genetic approach with a c/a Mek1 mutant also showed that Mek activity was not directly associated with the PTP inhibitor effect. Finally, a genetic approach with d/n or c/a Ras and c-Raf mutants, showed that Ras and c-Raf activities play a substantive role in enhancing clonogenic survival by PTP inhibition following Cr(VI) insult. In conclusion, these studies highlight a novel pro-survival mechanism for clonogenic survival in the face of genotoxic stress in the presence of PTP inhibition via an Erk/Mek-independent and Ras/c-Raf-dependent regulation in normal human lung fibroblasts.     

Quantitative analysis of chondroitin sulfate in raw materials, ophthalmic solutions, soft capsules and liquid preparations

We performed the quantitative analysis of chondroitin sulfate (CS) obtained from raw materials and various pharmaceutical preparations. To quantify CS content in raw materials and in an ophthalmic solution, each test sample and the authentic CS were first digested by chondroitinase ABC. The CS disaccharides produced were analyzed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC) and CS content was quantified by calculating the total peak areas of the disaccharides derived from a CS calibration curve. In the case of soft capsules, CS was first extracted with hexane followed by phenol-chloroform to remove oil and protein ingredients. The extracted CS samples were depolymerized by chondroitinase ABC and CS content was determined. Quantitative analysis of the disaccharides derived from raw materials and an ophthalmic solution showed the CS contents (%) were 39.5+/-0.1 to 105.6+/-0.1 and 103.3+/-1.2, respectively. In case of CS analysis in soft capsules and liquid preparations, the overall recovery (%) of the spiked CS was 96.79+/-0.53-103.54+/-1.78 and 97.10+/-1.82 to 103.17+/-2.34, respectively. In conclusion, the quantitative analysis of the disaccharides produced by enzymatic digestion can be used in the direct quantitation of CS containing pharmaceutical formulations.     

Effects of metabolites and analogs of amiodarone on alveolar macrophages: structure-activity relationship

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono-N-desethylamiodarone (MDEA), di-N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1). MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2). dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3). the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.     

IL-12 priming during in vitro antigenic stimulation changes properties of CD8 T cells and increases generation of effector and memory cells

Antigenic and costimulatory signals trigger a developmental program by which naive CD8 T cells differentiate into effector and memory cells. However, initial cytokine signals that regulate the generation of effector and memory CD8 T cells are not well understood. In this study, we show that IL-12 priming during in vitro antigenic stimulation results in the significant increase of both primary and memory CD8 T cell population in mice after adoptive transfer of activated cells. The effect of IL-12 priming is closely associated with qualitative changes in CD8 T cells, such as reduced MHC I tetramer binding and CD69 expression, altered distribution of lipid rafts, decreased cytolytic activity, and less susceptibility to apoptosis. Furthermore, exogenous IL-12 priming improved the intrinsic survival properties of memory CD8 T cells, leading to better protective immunity and vaccine-induced memory CD8 T cell responses. However, the experiments with IL-12p40- and IL-12Rbeta1-deficient mice showed similar levels of primary and memory CD8 T cell responses compared with wild-type mice, implying that endogenous IL-12 and/or IL-12R signaling in vivo is not critical for CD8 T cell immunity. Together, our results suggest that IL-12 can serve as an important, but dispensable regulatory factor for the development of CD8 T cells, and IL-12 priming could be useful in many medical applications.     

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

Identification of peptides that antagonize formyl peptide receptor-like 1-mediated signaling

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1's agonists (Trp-Lys-Tyr-Met-Val-D-Met-CONH(2) (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH(2) (WRWWWW (WRW(4))) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW(4) inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW(4) to the FPR family, we observed that WRW(4) specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid beta42 (Abeta42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW(4) on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Abeta42 peptide in human neutrophils. Abeta42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW(4), which also completely inhibited the internalization of Abeta42 peptide in human macrophages. WRW(4) is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.