Scott M. Laster

Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis

The murine cell line C3HA has been used extensively in studies of the cytopathology that accompanies TNF‐induced cytolysis. This cell line undergoes an apoptic form of cell death characterized by plasma membrane blebbing and cytoplasmic boiling. Since plasma membrane blebs also appear on C3HA cells during mitosis, in this report we have compared these blebs with those that appear during apoptosis to determine whether they represent related structures. Our results reveal several differences. During mitosis, the blebs that appear are smaller and more heterogeneous in size than are those that appear during apoptosis. In addition, during mitosis bleb formation is preceded by the appearance of microvilli on the cell surface. No microvilli are observed during apoptosis. The staining pattern with rhodamine phalloidin also differed between mitotic and apoptic blebs, indicating a difference in their content of f‐actin. The blebs that form during mitosis stained in a bright, uniform manner, suggesting an association with f‐actin. In contrast, apoptic blebs were stained only at their base, the bleb itself being devoid of f‐actin‐associated staining. This difference may help explain why mitotic blebs are reintegrated into the cell surface, while the blebs that form during apoptosis are not.

Characterization of arachidonic acid-induced apoptosis

Tumor necrosis factor (TNF) can induce apoptosis in a number of different cell types. This response often depends on the activity of cytosolic phospholipase A2 (cPLA2), which catalyzes the release of arachidonic acid from the sn-2 position of membrane phospholipids. In this study, we investigate the ability of arachidonic acid itself to cause cell death. We show that in assays with 10% fetal bovine serum (FBS) arachidonic acid will not kill, nor does act synergistically with TNF. In contrast, by lowering the concentration of FBS to 2% it is possible to use arachidonic acid to induce cell death. Arachidonic acid-induced cell death was judged to be apoptotic based on morphology and the cleavage of poly (ADP) ribose polymerase. Arachidonic acid was able to kill all cell lines tested including two human melanoma-derived cell lines, and susceptibility to arachidonic acid was not influenced by adenovirus gene products that control susceptibility to TNF. Finally, we show that arachidonic acid is unique among 20 carbon fatty acids for its ability to induce apoptosis and that several other unsaturated, but not saturated fatty acids can also induce apoptosis.

The adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite inhibit the TNF-induced release of arachidonic acid.

In this report we show that the adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite, which have been defined previously as inhibitors of cytolysis, inhibit the tumor necrosis factor-alpha (TNF)-induced release of 3H-arachidonic acid from cycloheximide-sensitized C3HA fibroblasts. Since the A23187-induced release of 3H-a.a. was unaffected, our results suggest that these inhibitors provide resistance to lysis by selectively interfering with the lytic response pathway. Our results also show that heat and sodium arsenite can themselves induce the release of 3H-arachidonic acid. These results raise the possibility that stressor-induced resistance to TNF results from the selective desensitization of phospholipase A2.

Echinacea and its alkylamides: effects on the influenza A-induced secretion of cytokines, chemokines, and PGE2 from RAW 264.7 macrophage-like cells.

The goal of this study was to determine whether extracts and isolated alkylamides from Echinacea purpurea would be useful for prevention of the inflammatory response that accompanies infections with H1N1 influenza A. Seventeen extracts and 4 alkylamides were tested for the ability to inhibit production of cytokines, chemokines, and PGE₂ from RAW 264.7 macrophage-like cells infected with the H1N1 influenza A strain PR/8/34. The alkylamides undeca-2Z,4E-diene-8,10-diynic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, dodeca-2E,4E-dienoic acid isobutylamide, and undeca-2E-ene-8,10-diynoic acid isobutylamide suppressed production of TNF-α and PGE₂ from infected cells. Dodeca-2E,4E-dienoic acid isobutylamide was especially effective at inhibiting production of these mediators and also strongly inhibited production of G-CSF, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES. In contrast, the ethanol extracts (75%), which were prepared from dormant roots of E. purpurea grown in different locations throughout North Carolina, displayed a range of effects from suppression to stimulation of mediator production. Precipitation of the extracts with ethanol removed the stimulatory activity, however, even after precipitation; many of the extracts did not display any suppressive activity. Analysis of the extracts revealed slight variations in concentration of alkylamides, caftaric acid, and cichoric acid, but the activity of the extracts did not strongly correlate with concentrations of these compounds. Our in vitro experiments suggest that E. purpurea extracts have the potential for use in alleviating the symptoms and pathology associated with infections with influenza A; however, further study will be necessary to define procedures necessary to unmask the alkylamide activity in crude extracts.

Inhibition of H1N1 influenza A virus growth and induction of inflammatory mediators by the isoquinoline alkaloid berberine and extracts of goldenseal (Hydrastis canadensis)

In this study we tested whether the isoquinoline alkaloid berberine can inhibit the growth of influenza A. Our experiments showed strong inhibition of the growth of H1N1 influenza A strains PR/8/34 or WS/33 in RAW 264.7 macrophage-like cells, A549 human lung epithelial-derived cells and murine bone marrow derived macrophages, but not MDCK canine kidney cells. Studies of the mechanism underlying this effect suggest that berberine acts post-translationally to inhibit virus protein trafficking/maturation which in turn inhibits virus growth. Berberine was also evaluated for its ability to inhibit production of TNF-α and PGE(2) from A/PR/8/34 infected-RAW 264.7 cells. Our studies revealed strong inhibition of production of both mediators and suggest that this effect is distinct from the anti-viral effect. Finally, we asked whether berberine-containing ethanol extracts of goldenseal also inhibit the growth of influenza A and production of inflammatory mediators. We found strong effectiveness at high concentrations, although upon dilution extracts were somewhat less effective than purified berberine. Taken together, our results suggest that berberine may indeed be useful for the treatment of infections with influenza A.

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including Some That Originate from Endophytic Bacteria

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than

Calcium and Its Role in the Nuclear Translocation and Activation of Cytosolic Phospholipase A2 in Cells Rendered Sensitive to TNF-Induced Apoptosis by Cycloheximide

100 million annually. In an attempt to shed light on Echinaceas mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.

Terameprocol, a methylated derivative of nordihydroguaiaretic acid, inhibits production of prostaglandins and several key inflammatory cytokines and chemokines

In these experiments, we investigated the role of calcium as a second messenger in the apoptotic activation of cytosolic phospholipase A2 (cPLA2). As our model, we used a murine fibroblast cell line (C3HA) that was induced to undergo apoptosis by a combination of TNF and cycloheximide. Using fura 2 Ca2+ imaging, we found strong evidence for an intracellular calcium response after 1 h of treatment, which correlated with the onset of phosphatidylserine externalization, but preceded effector procaspase processing by several hours. The response was strongest in the perinuclear region, where mean levels rose 83% (144 ± 14 nM in untreated cells vs 264 ± 39 nM in treated), while cells displaying morphological evidence of apoptosis had the highest levels of calcium (250–1000 nM). Verapamil blocked this response, indicating an extracellular source for the calcium. Fluorescence microscopy revealed a pattern of nuclear translocation of cPLA2 during apoptosis, which was also blocked by verapamil, indicating an important role for calcium in this process. In addition, we found that verapamil prevented the release of [3H]arachidonic acid from C3HA cells induced to undergo apoptosis by the chemotherapeutic agents vinblastine, melphalan, and cis-platinum. Together, these data suggest that calcium is important for cPLA2 activation by diverse apoptotic stimuli.

Inhibition of poxvirus growth by Terameprocol, a methylated derivative of nordihydroguaiaretic acid

BackgroundExtracts of the creosote bush, Larrea tridentata, have been used for centuries by natives of western American and Mexican deserts to treat a variety of infectious diseases and inflammatory disorders. The beneficial activity of this plant has been linked to the compound nordihydroguaiaretic acid (NDGA) and its various substituted derivatives. Recently, tetra-O-methyl NDGA or terameprocol (TMP) has been shown to inhibit the growth of certain tumor-derived cell lines and is now in clinical trials for the treatment of human cancer. In this report, we ask whether TMP also displays anti-inflammatory activity. TMP was tested for its ability to inhibit the LPS-induced production of inflammatory lipids and cytokines in vitro. We also examined the effects of TMP on production of TNF-α in C57BL6/J mice following a sublethal challenge with LPS. Finally, we examined the molecular mechanisms underlying the effects we observed.MethodsRAW 264.7 cells and resident peritoneal macrophages from C57BL6/J mice, stimulated with 1 μg/ml LPS, were used in experiments designed to measure the effects of TMP on the production of prostaglandins, cytokines and chemokines. Prostaglandin production was determined by ELISA. Cytokine and chemokine production were determined by antibody array and ELISA.Western blots, q-RT-PCR, and enzyme assays were used to assess the effects of TMP on expression and activity of COX-2.q-RT-PCR was used to assess the effects of TMP on levels of cytokine and chemokine mRNA.C57BL6/J mice injected i.p. with LPS were used in experiments designed to measure the effects of TMP in vivo. Serum levels of TNF-α were determined by ELISA.ResultsTMP strongly inhibited the production of prostaglandins from RAW 264.7 cells and normal peritoneal macrophages. This effect correlated with a TMP-dependent reduction in levels of COX-2 mRNA and protein, and inhibition of the enzymatic activity of COX-2.TMP inhibited, to varying degrees, the production of several cytokines, and chemokines from RAW 264.7 macrophages and normal peritoneal macrophages. Affected molecules included TNF-α and MCP-1. Levels of cytokine mRNA were affected similarly, suggesting that TMP is acting to prevent gene expression.TMP partially blocked the production of TNF-α and MCP-1 in vivo in the serum of C57BL6/J mice that were challenged i.p. with LPS.ConclusionTMP inhibited the LPS-induced production of lipid mediators and several key inflammatory cytokines and chemokines, both in vitro and in vivo, raising the possibility that TMP might be useful as a treatment for a variety of inflammatory disorders.

Adenovirus Type 5 Exerts Multiple Effects on the Expression and Activity of Cytosolic Phospholipase A2, Cyclooxygenase-2, and Prostaglandin Synthesis

Terameprocol (TMP) is a methylated derivative of nordihydroguaiaretic acid, a phenolic antioxidant originally derived from creosote bush extracts. TMP has previously been shown to have antiviral and anti-inflammatory activities, and has been proven safe in phase I clinical trials conducted to evaluate TMP as both a topical and parenteral therapeutic. In the current study, we examined the ability of TMP to inhibit poxvirus growth in vitro, and found that TMP potently inhibited the growth of both cowpox virus and vaccinia virus in a variety of cell lines. TMP treatment was highly effective at reducing infectious virus yield in multi-step virus growth assays, but it did not substantially inhibit the synthesis of infectious progeny viruses in individual infected cells. These contrasting results showed that TMP inhibits poxvirus growth in vitro by preventing the efficient spread of virus particles from cell to cell. The canonical mechanism of poxvirus cell-to-cell spread requires morphogenesis of cell-associated, enveloped virions. The virions then trigger the formation of actin tails to project them from the cell surface. The number of actin tails present at the surface of poxvirus-infected cells was reduced dramatically by treatment with TMP. Whether TMP inhibits poxvirus morphogenesis, or subsequent events required for actin tail formation, remains to be determined. The results of this study, together with the clinical safety record of TMP, support further evaluation of TMP as a poxvirus therapeutic.