Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia.

BACKGROUND: Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells. METHODS: Lactate dehydrogenase A levels were studied by quantitative RT-PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS. RESULTS: Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 µM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites. CONCLUSION: Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.

Ethnobotany of dye plants in Southern Italy, Mediterranean Basin: floristic catalog and two centuries of analysis of traditional botanical knowledge heritage.

BACKGROUND: Since ancient times, man has learned to use plants to obtain natural dyes, but this traditional botanical knowledge (TBK) is eroding. In the late, during, and the early 1800s, there was an increase in research related to dye species, and this allowed the development of industry and economy in rural contexts of Southern Italy. Today, dyes are mainly obtained from synthetic products, and this leads to risks for human health related to pollution. METHODS: Starting from the literature, three catalogs of the dyeing species (plants, algae, fungi, and lichens) used in the Mediterranean Basin and mainly in Southern Italy have been created. Percentages of parts used and colors extracted from species have been recorded and analyzed. The plant species present in the catalogs have been verified in the territories of Southern Italy, and the data have been registered. An ethnobotanical survey was conducted, in the region of Southern Italy, to verify the erosion level of traditional botanical knowledge, linked to the ethnobotanical dyeing, over time. RESULTS: A total of 524 species were recorded among plants, algae, fungi, and lichens, and related parts used and extracted pigments. Most uses concern the stems and leaves, and the most frequent color is yellow. From the on-field survey operations, 283 plant species have been verified. These represent 64.31% of the species reported in the flora of the dye plants produced. The results, from the ethnobotanical survey, show that only 8.6% of TBK remained in the collective memory. CONCLUSIONS: This catalog is among the largest in this sector and is the basis for studies related to the restoration of an eco-sustainable economy which would allow the development of marginal areas present throughout Southern Italy.

The effectiveness and efficiency of phytoremediation of a multicontaminated industrial site: Porto Marghera (Venice Lagoon, Italy).

The Venice Lagoon is worldwide considered as a typical example of the human impact on the surrounding ecosystem. The development of the industrial zone of Porto Marghera begun in 1917 as an extension of the Venice Port, in order to sustain activities related to oil and coal, as well as to exploit the railway system. Despite the recent decrease in the number of employees, Porto Marghera is still one of the most important chemical districts in Italy. This study reports early results from the ongoing in-situ phytoextraction of potentially toxic elements (Cd, Hg, Zn) within the industrial area of Porto Marghera. Two agronomic plant species with high annual biomass yield (Helianthus annuus L., Brassica juncea (L.) Czern.) were used. This paper also reports the microcosms and mesocosms tests to evaluate the efficacy of the treatments to be applied to the in-situ phytoextraction process of the polluted site. The combined use of EDTA and Ammonium Thiosulfate during phytoextraction increases the efficiency of Cd, Hg, Zn removal from contaminated soil.

Assessment of three approaches of bioremediation (Natural Attenuation, Landfarming and Bioagumentation - Assistited Landfarming) for a petroleum hydrocarbons contaminated soil.

Contamination with total petroleum hydrocarbons (TPH) subsequent to refining activities, is currently one of the major environmental problems. Among the biological remediation approaches, landfarming and in situ bioremediation strategies are of great interest. Purpose of this study was to verify the feasibility of a remediation process wholly based on biological degradation applied to contaminated soils from a decommissioned refinery. This study evaluated through a pot experiment three bioremediation strategies: a) Natural Attenuation (NA), b) Landfarming (L), c) Bioaugmentation-assisted Landfarming (LB) for the treatment of a contaminated soil with petroleum hydrocarbons (TPHs). After a 90-days trial, Bioagumentation - assistited Landfarming approach produced the best results and the greatest evident effect was shown with the most polluted samples reaching a reduction of about 86% of total petroleum hydrocarbons (TPH), followed by Landfarming (70%), and Natural Attenuation (57%). The results of this study demonstrated that the combined use of bioremediation strategies was the most advantageous option for the treatment of contaminated soil with petroleum hydrocarbons, as compared to natural attenuation, bioaugmentation or landfarming applied alone. Besides, our results indicate that incubation with an autochthonous bacterial consortium may be a promising method for bioremediation of TPH-contaminated soils.

Immunohistochemical localization of NPY, VIP and 5-HT in the thyroid gland of the lizard, Podarcis sicula.

The thyroid gland of the lizard Podarcis sicula was immunohistochemically studied in adult male specimens using specific antibodies against NPY, VIP and 5-HT and the avidin-biotin peroxidase complex (ABC) procedure to localize the three peptides. Fine beaded VIP-immunoreactive nerve fibers ran between the follicles, and VIP-immunoreactivity was evenly distributed in the apical cytoplasm of follicular cells. NPY-immunoreactive fibers were found around the follicles, and, in the cells, immunoreactivity was localizated only in the cellular apices. Immunoreactivity to 5-HT was observed in the colloid, with a concentration in the follicular lumen exceeding that in the follicular cells. In fact, most follicles showed immunoreactivity in the cytoplasmic bridges formed between the apical portion of the follicular cells and the colloid.

Inhibition of herpes simplex virus type 1 by aqueous extracts from leaves of Helichrysum litoreum Guss.

Helichrysum litoreum Guss, a Campania medicinal plant reported to have antibacterial properties, was evaluated for antiviral activity against herpes simplex virus type 1 (HSV-1) in vitro. The crude aqueous extract from leaves of Helichrysum litoreum at a concentration of 1.35 mg/ml (ww/v) showed significant antiviral activity on HSV-1 in human lung fibroblast as demonstrated by the absence of a cytopathic effect.

Molecular mechanisms and modulation of key pathways underlying the synergistic interaction of sorafenib with erlotinib in non-small-cell-lung cancer (NSCLC) cells.

Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.

Endocrinologic effects of the anaesthetic propofol.

Because of the similarities of the adrenal glands of mammals and of the lizard Podarcis s. sicula, the latter has already been the subject of various studies on the effects of Propofol and other anaesthetics. Because a relationship between the activities of the thyroid and adrenal glands of this species has been demonstrated, the authors administered Propofol to a species of lizard to investigate its effects on the thyroid gland. Propofol inhibited thyroid activity, promoted steroid synthesis, and caused the contemporaneous appearance of both adrenaline and noradrenaline granules in the cytoplasm of the chromaffin cells. These results suggest that inhibition of the activity of the thyroid gland is secondary to the action of Propofol on the adrenal gland.

Shift from noradrenaline to adrenaline production in the adrenal gland of the lizard, Podarcis sicula, after stimulation with vasoactive intestinal peptide (VIP).

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.

Immunocytochemical localization of substance P in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae): evidence for its involvement in the modulation of adrenal activity.

The occurrence of substance P (SP) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by ABC immunocytochemical technique: SP-immunoreactivity was present in both adrenaline and noradrenaline cells, in ganglion cells and nerve fibers in the connective capsule surrounding the gland. The involvement of substance P in the modulation of pituitary-interrenal axis was studied in vivo by intraperitoneal injections of SP. The effects were estimated by means of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone and catecholamines, adrenaline and noradrenaline. Substance P (0.07 mg/100 g body wt) decreased ACTH plasma levels and raised corticosterone release from steroidogenic tissue, that showed clear signs of stimulation. In the chromaffin tissue, the decrease in the number of noradrenaline cells, and the increase in the number of adrenaline cells, lowered numeric noradrenaline/adrenaline cell ratio. Moreover, an increase in adrenaline plasma level and a decrease in noradrenaline plasma level were found. The results suggest that (1) also in Reptiles as in other Vertebrates, SP may affect pituitary-adrenal axis activity, and (2) the chromaffin cells may be involved in the paracrine control of steroidogenic activity.

Annual variations of adrenal gland hormones in the lizard Podarcis sicula.

The adrenal gland regulates metabolism and maintains normal electrolyte balance. Adrenal hormones are equivalent in all vertebrates; the chromaffin tissue produces adrenaline and noradrenaline and the steroidogenic tissue produces most of the steroid hormones present in mammals. Podarcis sicula belongs to the Squamata family of lizards and it is the most abundant lizard species in southern Italy. This species shows a reproductive annual cycle and the presence of seasonal variations in the activity of the hypothalamus-hypophyseal-thyroid axis. To investigate the existence of an annual cycle of lizard adrenal gland, we have measured plasma concentrations of corticosterone, ACTH, noradrenaline and adrenaline. We have shown that corticosterone rapidly increased from January to March to reach a peak value that persisted until July, then, it slowly decreased until December. ACTH levels increased from January to May and slowly decreased from July to December. Noradrenaline levels were higher in March and then decreased until December. On the contrary, adrenaline levels increased from March to July and slowly decreased until December. Our results demonstrate the existence of an annual cycle of the lizard adrenal gland activity. This is very interesting because its activity is important to rightly regulate the reproductive status of the Podarcis sicula lizard.