Lipophilic Metabolites from Five-Needle Pines, Pinus armandii and Pinus kwangtungensis, Exhibiting Antibacterial Activity.

Lipophilic extractive metabolites from needles and defoliated twigs of Pinus armandii and P. kwangtungensis were studied by GC/MS. Needles of P. armandii contained predominantly 15-O-functionalized labdane type acids (anticopalic acid), fatty acids, nonacosan-10-ol, sterols, nonacosan-10-ol and sterol saponifiable esters, and acylglycerols, while P. kwangtungensis needles contained no anticopalic acid, but more trinorlabdane (14,15,16-trinor-8(17)-labdene-13,19-dioic acid) and other labdane type acids, nonacosan-10-ol and its saponifiable esters. The major compounds in the P. armandii defoliated twig extract were abietane and isopimarane type acids, fatty acids, sterols, labdanoids (cis-abienol), cembranoids (isocembrol and 4-epi-isocembrol), saponifiable sterol esters, and acylglycerols. The same extract of P. kwangtungensis contained larger quantities of fatty acids, caryophyllene oxide, serratanoids, sterols, saponifiable sterol esters, and acylglycerols, but lesser amounts of abietane and isopimarane type acids, cis-abienol, and lacked cembranoids. Both twig and needle extracts of P. armandii and P. kwangtungensis, as well as the extracts' fractions, significantly inhibited the growth of Gram-negative bacteria Serratia marcescens with MIC of 0.1 mg ml-1 , while in most cases they slightly stimulated the growth of Gram-positive bacteria Bacillus subtilis at the same concentrations. Thus, lipophilic extractive compounds from the needles and defoliated twigs of both pines are prospective for the development of antiseptics against Gram-negative bacteria.

Composition and Bioactivity of Lipophilic Metabolites from Needles and Twigs of Korean and Siberian Pines (Pinus koraiensis Siebold & Zucc. and Pinus sibirica Du Tour).

Lipophilic extractive metabolites in different parts of the shoot system (needles and defoliated twigs) of Korean pine, Pinus koraiensis, and Siberian pine, Pinus sibirica, were studied by GC/MS. Korean pine needles comprised mainly bornyl p-coumarate, heterocyclic 15-O-functionalized labdane type acids (lambertianic acid), 10-nonacosanol, sterols and their esters. While Siberian pine needles contained less bornyl p-coumarate, lambertianic acid, sterols and their esters, but were richer in other 15-O-functionalized labdane type acids. The major components of the twig extract of P. koraiensis were lambertianic acid, abietane and isopimarane type acids, cembrane type alcohols, 8-O-functionalized labdanoids, sterols, sterol esters, and acylglycerols. The same extract of P. sibirica differed in larger amounts of other 15-O-functionalized labdane type acids and pinolenic acid glycerides, but in less quantities of cembranoids and 8-O-functionalized labdanoids. The labdane type pinusolic acid was detected for the first time in Korean pine. P. koraiensis was found to be unique in the genus for an ability to synthesize phyllocladane diterpenoids. The content of bound Δ5 -unsaturated polymethylene-interrupted fatty acids in the twig extracts of the both pines was similar or superior to that in their seed oil. Among the pines' metabolites tested isocembrol was strongest in inhibition of both α-glucosidase (IC50 2.9 µg/ml) and NO production in activated macrophages (IC50 3.6 µg/ml).

Endothelial plasticity governs the site-specific leukocyte recruitment in hepatocellular cancer.

The correct programming of the endothelial cell phenotype is crucial for efficient leukocyte recruitment to tumor tissue. It has been previously described that T cells infiltrated hepatocellular cancer (HCC) tissue mainly in peritumoral, stromal and tumor border areas. In the current study, phenotype features of tumor endothelial cells and their potential impact on leukocyte recruitment were analyzed in murine tissue of HCC. In the murine model, proinflammatory stimulation with IL-1ß induced leukocyte recruitment in the blood vessels of peripheral tumor areas and in nonmalignant liver tissue, but not in deeper tumor blood vessels. Furthermore, peripheral tumor endothelium, but not deeper tumor blood vessels exhibited a "normalized" hepatic sinusoidal endothelial cell (HSEC)-like phenotype with regard to the expression of adhesion molecules and liver sinusoidal endothelial markers. When tumor endothelial cells were isolated and incubated in vitro, their phenotype rapidly changed and became almost identical to normal hepatic endothelial cells. Interestingly, cytokine production in HCC was strongly dysregulated as compared to normal liver, with IL-1RN exhibiting the most prominent elevation. Experiments with isolated hepatic endothelial cells showed that IL-1RN effectively antagonized the activating action of IL-1ß on the expression of adhesion molecules and T cell attachment. These novel insights indicate that tumor endothelium of HCC represents a plastic system that is susceptible to microenvironmental changes. The peritumoral and tumor border areas have distinct endothelial cell phenotype, which promotes leukocyte recruitment to HCC tissue.

The role of ß2-integrins and CD44 in intrahepatic leukocyte sequestration.

BACKGROUND: Intrahepatic leukocyte sequestration is a component of the systemic inflammatory response, and can be triggered by systemic immune dysfunction during sepsis. METHODS: To examine leukocyte sequestration over time during endotoxemia, its influence on liver function, and the role of specific cell adhesion molecules, endotoxemia was induced in mice by intraperitoneal application of lipopolysaccharides. Leukocyte sequestration was measured at different times after induction using fluorescence microscopy. Liver injury was evaluated by measuring liver enzymes and tissue histology. RESULTS: Endotoxin induces a strong leukocyte sequestration in the liver microvasculature. This was associated with an induction of liver injury, as reflected by an increase in enzyme levels and histomorphologic changes. Intrahepatic leukocyte sequestration was reduced in CD44(-/-), but not in intercellular adhesion molecule-1 (ICAM-1)(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) antigen mice. Leukocyte sequestration dropped in ICAM-1(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) mice in later stages, but remained stable in wild-type and CD44(-/-) animals. Reduced leukocyte sequestration in CD44(-/-) mice was accompanied by a significant decrease in transferase levels. CONCLUSIONS: Endotoxemia induces stable intra-sinusoidal leukocyte sequestration, which contributes to liver injury. At the initial stage of the endotoxemia, leukocyte sequestration depends on CD44 but is independent of ICAM-1 and ß2-integrins. Intercellular adhesion molecule-1 and ß2-integrins, but not CD44, stabilize leukocyte sequestration during the later stage of endotoxemia. The molecular modulation of intrahepatic leukocyte sequestration may have important therapeutic implications in sepsis, reducing liver injury, and improving immune defense capabilities.

Lipophilic extracts from needles and defoliated twigs of Pinus pumila from two different populations.

Hexane extracts of needles and defoliated twigs of Pinus pumila (Pall.) Regel from two distant populations, located in the southwest and the east (i.e., Lake Baikal region and Sakhalin Island) of the species distribution range were studied by GC/MS analysis. Composition and retention indices of major components were determined. A drastic composition divergence for the extracts of P. pumila needles and defoliated twigs, depending on growth location, was established. Needle extracts from the eastern population sample contained mainly labdane-type acids (anticopalic acid derivatives), whereas the predominant components of needle extracts from the other population sample were abietane-type acids (abietic, neoabietic acids) and isopimarane-type diterpenoids (sandaracopimaric acid, sandaracopimaradien-3ß-ol). The main components of defoliated twig extracts from Sakhalin Island population sample were abietane-type acids and cembrane-type diterpenoids, while content of these compounds in the extracts of the southwestern marginal population sample was remarkably lower.

αL ß2 integrin is indispensable for CD8+ T-cell recruitment in experimental pancreatic and hepatocellular cancer.

Recruitment of activated leukocytes from peripheral blood into the tumor tissue is a crucial step of the immune response, which is controlled by the interaction between specific adhesion molecules such as endothelial ICAM-1 and leukocyte ß(2) -integrins. Although attenuated expression of adhesion molecules on tumor endothelium has been proposed to represent a mechanism, which suppresses the intratumoral leukocyte infiltration, the relevance of adhesion molecules for leukocyte recruitment in tumor tissue is poorly understood. The present study is the first investigation of the role of ICAM-1 and ß(2) -integrins in leukocyte recruitment in pancreatic and hepatocellular cancer in vivo, which was studied using knockout mice, intravital time-lapse microscopy and immunohistochemistry. We found that tumor tissue of both pancreatic and hepatocellular cancer was infiltrated with numerous active lymphoid and myeloid leukocytes, although the leukocyte extravasation rate in tumor blood vessels was very low. The knockout of LFA-1 (also known as α(L) ß(2) integrin) strongly suppressed recruitment of CD8(+) T cells whereas no significant differences of leukocyte adhesion and infiltration were found in ICAM-1(-/-) and Mac-1(-/-) mice. Analysis of the interstitial leukocyte migration demonstrated that intratumoral leukocytes used haptokinetic type of migration, however, no significant differences of leukocyte migration between any knockout strains were found. We concluded that leukocyte recruitment in pancreatic and hepatocellular cancer is a slow-going process whose dynamics clearly contrasts to a high-speed leukocyte recruitment during acute inflammation. In contrast to acute inflammatory reaction, only LFA-1 controls recruitment of CD8(+) T-cells in both pancreatic and hepatocellular cancer, whereas ICAM-1 and Mac-1 are dispensable.

Stability Study, Quantification Method and Pharmacokinetics Investigation of a Coumarin-Monoterpene Conjugate Possessing Antiviral Properties against Respiratory Syncytial Virus.

The stability of a new coumarin derivative, agent K-142, bearing α-pinene residue and possessing antiviral activity against respiratory syncytial virus (RSV) was studied in whole mice blood in vitro, and a method for its quantification in this matrix was developed and validated. The sample preparation method was precipitation of whole blood with a mixture of 0.2 M ZnSO4 with MeOH (2:8 v/v) containing 2-adamantylamine hydrochloride as an internal standard (IS). Analysis was carried out by HPLC-MS/MS using reversed phase chromatography and a triple quadrupole mass spectrometer 6500 QTRAP (SCIEX) in multiple reaction monitoring (MRM) mode. The transitions 351.2 → 217.1 Da and 152.2 → 93.1/107.2 Da were monitored for K-142 and the IS, respectively. The method was validated in terms of selectivity, calibration curve, LLOQ, accuracy and precision, stability, recovery and carry over. The developed method was used for a pharmacokinetics study of the compound after its oral administration to mice at a dose of 20 mg/kg.

Rapamycin inhibits proliferation and migration of hepatoma cells in vitro.

BACKGROUND: Systemic immunosuppression represents the major factor for cancer recurrence after orthotopic liver transplantation for hepatocellular carcinoma (HCC). Rapamycin is an immunosuppressant with unique antitumoral properties. Although rapamycin has been successfully used in HCC patients after liver transplantation, the detailed mechanisms of rapamycin action on tumor cells are poorly understood. METHODS: Two HCC cell lines (PLC5 and HuH7) were used to evaluate the effect of rapamycin. Tumor cell proliferation was analyzed using cell counting and BrdU incorporation assay. Expression of phosphorylated Akt was studied using enzyme linked immunosorbent assay. Digital time-lapse microscopy was utilized to measure tumor cell migration in vitro. RESULTS: Rapamycin induced a strongly dose-dependent inhibition of tumor cell proliferation in both HCC cell lines. Additionally, rapamycin inhibited activation of Akt phosphorylation and tumor cell migration after prolonged treatment. CONCLUSIONS: Rapamycin suppresses tumor progression due to inhibition of phosphorylated Akt, cell proliferation, and migration. The data of the present study strengthen clinical implications of rapamycin after liver transplantation with HCC.

Low-volatile lipophilic compounds in needles, defoliated twigs, and outer bark of Pinus thunbergii.

Despite a long history of the use of Pinus thunbergii for technical, medicinal, agricultural, and other purposes, the composition of low-volatile metabolites in the used parts of the plant has been poorly investigated. We report here on the distribution of lipophilic extractive compounds in different parts of the shoot system (needles, defoliated twigs, outer bark) of P. thunbergii studied by GC/MS. The highest and lowest contents of lipophilic substances were found in defoliated twigs and in outer bark correspondingly. Acid compounds in the extract of needles comprised mainly labdane type diterpenoids (trans-communic acid), while in the extracts of defoliated twigs and outer bark the acids were represented predominantly by abietane type compounds (neoabietic, dehydroabietic, abietic, levopimaric and palustric acids). The major neutral components of the extract of needles were 10-nonacosanol, labdanoids (18-hydroxy-1 3-epi-manoyl oxide, trans-communol), and beta-sitosterol. In the case of the extract of defoliated twigs, labdanoids (18-hydroxy-13-epi-manoyl oxide, trans-communol, 13-epi-torulosol), serratane triterpenoids (3beta-methoxyserrat-14-en-21-one), and beta-sitosterol were the main neutral constituents, whereas serratanoids (3beta-methoxyserrat-14-en-21-one) alone dominated among the neutral compounds of the outer bark extract. Most of the neutral components and the labdane type acids were detected for the first time in organs and tissues of P. thunbergii. The distribution of lipophilic metabolites in the studied parts of P. thunbergii shoot system may be applied for chemotaxonomy purposes. Diversified accumulation of extractive substances in different organs of the plant should be taken into account for isolation of specific components from the pine raw material.

Hypoxia induces EMT in low and highly aggressive pancreatic tumor cells but only cells with cancer stem cell characteristics acquire pronounced migratory potential.

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC(low)) and highly aggressive CSC-like cells (CSC(high)) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC(high) cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC(low) cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC(high) cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC(high) cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.

New gene-immunotherapy combining TRAIL-lymphocytes and EpCAMxCD3 Bispecific antibody for tumor targeting.

PURPOSE: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA. EXPERIMENTAL DESIGN: Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells. RESULTS: Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced. CONCLUSIONS: This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.