Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis.

Topical application of curcumin, the yellow pigment in turmeric and curry, strongly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase activity, DNA synthesis, and tumor promotion in mouse skin (Huang et al., Cancer Res., 48: 5941-5946, 1988). Chlorogenic acid, caffeic acid, and ferulic acid (structurally related dietary compounds) were considerably less active. In the present study, topical application of curcumin markedly inhibited TPA- and arachidonic acid-induced epidermal inflammation (ear edema) in mice, but chlorogenic acid, caffeic acid, and ferulic acid were only weakly active or inactive. The in vitro addition of 3, 10, 30, or 100 microM curcumin to cytosol from homogenates of mouse epidermis inhibited the metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) by 40, 60, 66, or 83%, respectively, and the metabolism of arachidonic acid to 8-HETE was inhibited by 40, 51, 77, or 85%, respectively [IC50 (concentration needed for 50% inhibition) = 5-10 microM]. Chlorogenic acid, caffeic acid, or ferulic acid (100 microM) inhibited the metabolism of arachidonic acid to 5-HETE by 36, 10, or 16%, respectively, and these hydroxylated cinnamic acid derivatives inhibited the metabolism of arachidonic acid to 8-HETE by 37, 20, or 10%, respectively (IC50 greater than 100 microM). The metabolism of arachidonic acid to prostaglandin E2, prostaglandin F2 alpha, and prostaglandin D2 by epidermal microsomes was inhibited approximately 50% by the in vitro addition of 5-10 microM curcumin. Chlorogenic acid, caffeic acid, and ferulic acid (100 microM) were inactive. In vitro rat brain protein kinase C activity was not affected by 50-200 microM curcumin, chlorogenic acid, caffeic acid, or ferulic acid. The inhibitory effects of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on TPA-induced tumor promotion in mouse epidermis parallel their inhibitory effects on TPA-induced epidermal inflammation and epidermal lipoxygenase and cyclooxygenase activities.

Surface properties of Sendai virus envelope.

Surface properties of Sendai virus envelope membrane have been measured, using both biological and biophysical techniques. Both normal and trypsin-treated virus were studied. SDS gel electrophoresis showed cleavage of the F protein exclusively by trypsin. The major activity change was observed in the hemolysing activity which is an expression of F protein. Hemolysis was reduced to less than 10% of its value for intact virus. 31P nuclear magnetic resonance studies of the envelope surface of the native virus showed a highly restricted phospholipid headgroup environment. Interestingly, this restriction was relieved by treatment with trypsin. Thus these data suggest a role of the F protein of Sendai virus in tightly organizing the surface of the viral envelope membrane.

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells.

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.

The interaction of Sendai virus glycoprotein-bearing recombinant vesicles with cell surfaces.

Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by beta-D-octylglucoside. The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN-phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins.

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C.

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.

Inhibitory effect of curcumin and some related dietary compounds on tumor promotion and arachidonic acid metabolism in mouse skin.

Topical application of curcumin, the major yellow pigment in turmeric and curry, has a potent inhibitory effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. The structurally related compounds chlorogenic acid, caffeic acid and ferulic acid are less potent inhibitors. Curcumin is a potent inhibitor of TPA-induced ornithine decarboxylase activity and inflammation in mouse skin whereas chlorogenic acid, caffeic acid and ferulic acid are only weakly active or inactive. Curcumin is a potent inhibitor of arachidonic acid-induced inflammation in vivo in mouse skin, and this compound is also a potent inhibitor of epidermal lipoxygenase and cyclooxygenase activity in vitro. Although chlorogenic acid is only weakly active as an inhibitor of epidermal lipoxygenase activity and TPA-induced ear inflammation, it is more active than caffeic acid and ferulic acid. The inhibitory effects of curcumin, chlorogenic acid, caffeic acid and ferulic acid on TPA-induced tumor promotion in mouse skin parallel their inhibitory effects on TPA-induced epidermal inflammation and epidermal lipoxygenase and cyclooxygenase activities. Examination of the structural features of curcumin required for its biological activity indicate that free hydroxyl groups on the benzene rings are not required for inhibition of TPA-induced ornithine decarboxylase activity and inflammation in mouse skin.

Cell-mediated cytotoxicity against targets bearing Sendai virus glycoproteins in the absence of viral infection.

The glycoproteins HN and F and the lipids were solubilized from Sendai virus envelopes by using the nonionic detergent beta-D-octylglucoside. When beta-D-octylglucoside was removed by dialysis, the glycoproteins and lipids reassociated to form vesicles. These vesicles displayed hemagglutinating, neuraminidase, and hemolysin activities comparable to those expressed by the intact virus. The vesicles were used as carriers to transfer the glycoproteins to the surface of P815 cells. The recipient cells were tested for the acquisition of the glycoproteins by demonstration of surface neuraminidase, hemadsorption activity, and antigens. The modified cells were used as targets for natural cell-mediated lysis and were found to be sensitive.

Inhibition of epidermal growth factor receptor tyrosine kinase activity in A431 human epidermoid cells following psoralen/ultraviolet light treatment.

One of the more prominent clinical treatments for skin diseases such as psoriasis and vitiligo involves the use of a combination of psoralens and UV light, a procedure referred to as PUVA chemotherapy. This drug regimen markedly alters epidermal cell growth and differentiation. In many cell types, an early cellular event following treatment of cells with PUVA is inhibition of binding of epidermal growth factor (EGF) to its receptor. To examine the mechanism underlying this effect, we used A431 cells, a human epidermal cell line known to express large numbers of EGF receptors. We found that exposure of A431 cells to PUVA caused a dramatic inhibition of EGF-stimulated EGF receptor tyrosine kinase activity. Inhibition required intact cells and did not appear to be mediated by protein kinase C, because this inhibition was apparent in cells in which the enzyme was down-regulated by phorbol ester pretreatment and in cells treated with inhibitors of protein kinase C. Inhibition of tyrosine kinase activity by PUVA was distinct from other inhibitors of EGF receptor function in that it was associated with a rapid increase in the amount of phosphate incorporated into serine residues of the EGF receptor. This suggested that PUVA-induced serine phosphorylation may mediate EGF receptor kinase activity. These results demonstrate that alterations in EGF receptor function may contribute to the therapeutic efficacy of PUVA in photo-chemotherapy.