UV-B induced alteration in purinergic receptors and signaling on HaCaT keratinocytes

Although there are a number of recognized risk factors resulting in cutaneous malignancies, very little is known about the exact mechanism. In keratinocytes different purinergic receptors have been implicated to play essential roles in deciding the fate of the cells through regulating proliferation and differentiation. While P2Y receptors seem to control the former, P2X receptors, among which the P2X(7) receptor is associated with the induction of apoptosis, are likely to be responsible for the latter. Forty mJ/cm(2) UV-B irradiation decreased the number of viable cells as assessed using MTT assay. This irradiation decreased the amount of both P2X(1) and P2Y(2) receptors and essentially destroyed the P2X(7) receptors in surviving cells. Morphology of ATP-induced Ca(2+) transients were altered in irradiated cells compared to control. The amplitude and the rate of rise of the transients were decreased and the return to resting [Ca(2+)](i) prolonged. This observation is consistent with the finding that in control cells mostly ionotropic, while in irradiated cells mostly metabotropic receptors were underlying the response to ATP. These alterations in the expression pattern of purinergic receptors and in the Ca(2+) transients could explain the observed decreased tendency for ATP-induced apoptosis and possibly contribute to the malignant transformation of keratinocytes.     

Inhibition of UVB-mediated oxidative stress and markers of photoaging in immortalized HaCaT keratinocytes by pomegranate polyphenol extract POMx

In recent years there has been an increase in use of botanicals with antioxidant properties as skin photoprotective agents. Pomegranate (Punica granatum L.) fruit possesses strong antioxidant and antiinflammatory properties. Recently, we have shown that pomegranate-derived products rich in anthocyanidins and ellagitannins inhibit UVB-mediated activation of nuclear factor kappa B and modulate UVA-mediated cell proliferation pathways in normal human epidermal keratinocytes. In this study, we evaluated the effect of polyphenol-rich pomegranate fruit extract (POMx) on UVB-induced oxidative stress and photoaging in human immortalized HaCaT keratinocytes. Our data show that pretreatment of HaCaT cells with POMx (10-40 microg mL(-1)) inhibited UVB (15-30 mJ cm(-2))-mediated (1) decrease in cell viability, (2) decrease in intracellular glutathione content and (3) increase in lipid peroxidation. Employing immunoblot analysis we found that pretreatment of HaCaT cells with POMx inhibited UVB-induced (1) upregulation of MMP-1, -2, -7 and -9, (2) decrease in TIMP-1, (3) phosphorylation of MAPKs and (iv) phosphorylation of c-jun, whereas no effect was observed on UVB-induced c-fos protein levels. These results suggest that POMx protects HaCaT cells against UVB-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products.     

The synthesis of prostaglandin E1 and related substances

The authors describe in detail the conversion of 4 isometric exo-substituted bicyclo (3.1.0) hexanones 1a, 1b, 2a and 2b (R=CH3) to 4 prostaglandins of the E series, including crystalline dl-prostaglandin E1 (PGE1). Oxidative solvolysis of the keto esters 1a and 2a and of the desired keto acids 1a and 2a (R=H) according to the method of Just and Simonovitch resulted in a good yield of the mixture of vic-glycols of unrearranged carbon skeleton. The presence of dl-PGE1, or dl-8-iso PGE1 or their methyl esters was not detected in either case. Analysis of the unalkylated ketones 5a and 5b under the same and other conditions yielded unrearranged vic-glycols 6 as essentially the only product. A reaction sequence produced dl-8-isoprostaglandin E1, the 15-epimers of these prostaglandins, and dl-PGA1 and dl-PGB1 methyl esters.     

Interleukin-1beta stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP-2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.     

Total synthesis of the ethyl ester of the major urinary metabolite of prostaglandin E(2)

The preparation of the ethyl ester of the major urinary metabolite of prostaglandin E(2) 3 is described. The key step is the kinetic opening of the TBS-protected bicyclic ketone 7 with thiophenol.     

The structure of the major urinary metabolite of prostaglandin E2 in man

This letter reports the structure of the major urinary metabolite formed from prostaglandin E2 (PGE2) in humans. Radiolabeled PGE2 was injected intravenously into male subjects. 50% of the radioactivity was recovered in urine during the first 5 hours and less than 3% during the following 12 hours. The urine was acidified, and this extract was subjected to reversed-phase partition chromatography. Formation of the major metabolite (depicted stereochemically in the text) from PGE2 involved 4 steps of reactions: 1) dehydrogenation of the alcohol group in the side chain; 2) reduction of the trans double bond; 3) 2 steps of beta oxidation; and 4) delta oxidation.     

The effect of photofrin on DNA strand breaks and base oxidation in HaCaT keratinocytes: a comet assay study

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. alpha-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 microM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical micro-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.     

Preparation of chiral, highly functionalized cyclopentanes and its application to the synthesis of prostaglandin E1

This paper describes the preparation of the polyfunctionalized cyclopentane ((+/-)-5) by silica gel-catalyzed air-oxidation, and its kinetic resolution by means of microbial reduction with Rhodotorula rubra to afford optically pure (-)-5 (greater than 99% ee). Starting with (-)-5, a new route to prostaglandin E1 was established.     

Hibiscus sabdariffa synthesized gold nanoparticles ameliorate aluminum chloride induced memory deficits through inhibition of COX-2/BACE-1 mRNA expression in rats

Alzheimer’s disease (AD) is a major health challenge worldwide, especially among the elderly. The disease is associated with cognitive and memory deficits. This study investigated the effect of Hibiscus sabdariffa synthesized-gold nanoparticles (HS-AuNPs) on AlCl₃-induced memory deficits in rats. Forty-two male Wistar rats were divided into six groups (n = 7). Group I served as control. Rats in group II - V were exposed to AlCl₃ (100 mg/kg) to induce AD. Group III - V rats were treated with 5 mg/kg donepezil, 5 and 10 mg/kg HS-AuNPs, respectively, for 14 days. Behavioral tests were carried out on the rats on day 28 and 42. At the end of animal experiment, rats were sacrificed and used for various biochemical assays and gene expression. The AD rats showed memory and learning impairment, and these conditions were ameliorated by HS-AuNPs. Significant (p < 0.05) elevation in the activities of acetylcholinesterase, monoamine oxidase and adenosine deaminase, as well as malondialdehyde levels was noted. A significant reduction in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) noted in AlCl₃-induced rats were ameliorated by the 5 and 10 mg/kg b.w. doses of HS-AuNPs. In addition, the increased mRNA expression of cyclooxygenase-2 (COX-2) and beta-secretase 1 (BACE-1) caused by AlCl₃ were assuaged by the HS-AuNPs treatment. Based on the activities of HS-AuNPs against AlCl₃-induced AD, HS-AuNPs could be considered a potential therapeutic agent for managing AD.     

Computational analysis of ligand recognition mechanisms by prostaglandin E2 (subtype 2) and D2 receptors

The eight members of the prostanoid receptor family belong to the class A G protein-coupled receptors. We investigated the evolutionary relationship of the eight members by a molecular phylogenetic analysis and found that prostaglandin E₂ receptor subtype 2 (EP₂) and prostaglandin D₂ receptor (DP) were closely related. The structures of the ligands for the two receptors are similar to each other but are distinguished by the exchanged locations of the carbonyl oxygen and the hydroxy group in the cyclopentane ring. The ligand recognition mechanisms of the receptors were examined by an integrated approach using several computational methods, such as amino acid sequence comparison, homology modeling, docking simulation, and molecular dynamics simulation. The results revealed the similar location of the ligand between the two receptors. The common carboxy group of the ligands interacts with the Arg residue on the seventh transmembrane (TM) helix, which is invariant among the prostanoid receptors. EP₂ uses a Ser on TM1 to recognize the carbonyl oxygen in the cyclopentane ring of the ligand. The Ser is specifically conserved within EP₂. On the other hand, DP uses a Lys on TM2 to recognize the hydroxy group of the ?? chain of the ligand. The Lys is also specifically conserved within DP. The interaction network between the D(E)RY motif and TM6 was found in EP₂. However, DP lacked this network, due to the mutation in the D(E)RY motif. Based on these observations and the previously published mutational studies on the motif, the possibility of another activation mechanism that does not involve the interaction between the D(E)RY motif and TM6 will be discussed.     

Low-dose UVB irradiation stimulates matrix metalloproteinase-1 expression via a BLT2-linked pathway in HaCaT cells

Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX- 2) and its product, prostaglandin E(2) (PGE((2))), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B((4)) (LTB((4))) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB((4)) and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS- ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB- irradiated keratinocytes and thus potentially contributes to photo-aging.