Matrix metalloproteinases and their inhibitors in canine mammary tumors

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors

Resveratrol, a natural molecule against hypertension and cardiovascular diseases: development of prodrugs to enhance its bioavailability

Plant polyphenols are a vast family of natural compounds present in many foods and drinks. Many of them have noteworthy biological properties. Resveratrol, for example, can help prevent cardiovascular disease in humans, because it is able to lower blood pressure and lipid peroxidation, to induce vasodilatation and to reduce platelet aggregation. Moreover, resveratrol has antioxidant, anti-inflammatory, and anti-atherosclerotic properties, resulting in protection of the cardiovascular system, improvement of age-related cognitive decline and prevention and therapy of cancer. These potential positive effects are hampered by the low bioavailability of this compound and of polyphenols in general. As a result of a low level of absorption and a rapid metabolism in intestinal and liver cells, only small amounts of polyphenols are found in the bloodstream, and then mostly as metabolites. The major aim of my doctorate work has been the development of “pro-drugs”, resistant to metabolism during absorption and capable of regenerating the natural compound thanks to the action of ubiquitous enzymes. Resveratrol was chosen as model polyphenol. The project is carried out in collaboration with a group of organic chemists who synthesise the compounds and also contribute analytical know-how. Since polyphenols generally have a low solubility in water, and solubility is a key factor contributing to the bioavailability of a compound, a first resveratrol derivative was synthesised in which succinyl linkers connected resveratrol hydroxyls on one side and glucose residues on the other via carboxyester bonds. Pharmacokinetic studies with this compound showed that the levels and the composition of the metabolite mix in the bloodstream were the same as those obtained using resveratrol itself. This suggested that the compound was hydrolysed to resveratrol in the gastro-intestinal tract, before absorption. The carboxyester bond system thus turned out to be too labile in vivo, and therefore of limited usefulness. Other bond systems, such as the ether and sulfonate linkages, proved on the contrary to be too stable We therefore turned to other functionalities. In one approach the acetal bond system was used to link protective/solubilising groups to resveratrol hydroxyls. This type of protecting group is hydrolysed under acid conditions, but turned out to be rather too stable for use in vivo. Ex vivo and in vivo experiments with a series of acetal derivatives bearing short oligo-ethylene glycol chains however allowed us to evaluate and appreciate the positive effect this type of substituents can have on the absorption process. In the search for a protective functionality with ideal stability characteristics, we then turned to yet another, more labile, bond type, which cannot be specified here. To confer water solubility, polyhydroxylated moieties or PEG chains were introduced via this bond system. The work has so far led to satisfactory levels of absorption with two regioisomeric mono-substituted derivatives: uM levels of resveratrol-containing molecules were measured in blood samples, persisting for hours. However, the two free hydroxyl groups in these compounds are targets for glucuronosyltransferases, which largely modify them before the regeneration of resveratrol can be completed. A new approach has now been adopted, incorporating the positive aspects which have emerged from the investigation of acetal derivatives. In these new compounds short oligo-ethyleneglycol chains are linked to all three hydroxyl groups of resveratrol to improve absorption while offering protection. These constructs may be expected to be well absorbed (depending on chain length) and to hydrolyse with suitable kinetics. Only preliminary results with one compound are available at the time this thesis is submitted. During my doctorate I had the opportunity to spend a six-month period in the laboratory of prof. E. Mervaala at the Institute of Biomedicine of the University of Helsinki, Finland. There I performed a preliminary study on the possible positive effects of resveratrol and caloric restriction against sunitinib-induced cardiotoxicity and renal damage in spontaneously hypertensive rats, using normotensive WKY rats as control. Sunitinib is a tyrosine kinase inhibitor (TKI). The results did not reveal statistically significative differences between control and treated rats. Further experiments will be needed before a definite conclusion can be reached. Finally, it should be mentioned that in a side project I participated in studies on the metabolism of polyphenols by cultured cells. Work with monolayers of colonic Caco-2 cells revealed a remarkable heterogeneity in the expression of Phase II metabolism enzymes (sulfo- and glucuronosyl-transferases) within the same cell line. It was however possible to regenerate a uniform activity in the different populations by cultivating the cells with low concentrations of a xenobiotic compound (in our case quercetin).I polifenoli vegetali sono una vasta famiglia di composti naturali presenti in molti cibi e bevande. Molti di loro possiedono notevoli proprietà biologiche. Il resveratrolo per esempio, può aiutare a prevenire le malattie cardiovascolari, perchè è in grado di abbassare la pressione sanguigna, ridurre la perossidazione lipidica l’aggregazione piastrinica. Inoltre, il resveratrolo ha proprietà antiossidanti, anti infiammatorie e anti aterosclerotiche, risultanti nella protezione del sistema cardiovascolare, nel miglioramento del declino cognitivo legato all’età e nelle prevenzione e terapia del cancro. Questi potenziali effetti terapeutici trovano però un ostacolo nella scarsa biodisponibilità di questo composto e dei polifenoli in generale. Come risultato di un basso livello di assorbimento e di rapido metabolismo nelle cellule intestinali ed epatiche, nel sangue vengono ritrovate solo piccole quantità di polifenoli, e principalmente sotto forma di metaboliti. Lo scopo del mio progetto di dottorato è lo sviluppo di prodrugs di resveratrolo, il nostro polifenolo modello, resistenti al metabolismo durante l’assorbimento intestinale capaci di rigenerare il composto naturale grazie all’azione di enzimi ubiquitari. Il progetto è avvenuto in collaborazione con un gruppo di chimici organici che hanno sintetizzato i composti utilizzati in questa tesi. Dato che i polifenoli generalmente sono poco solubili in acqua, e la solubilità è un fattore determinante per la biodisponibilità di un composto, un primo precursore di resveratrolo è stato ottenuto funzionalizzando gli ossidrili con gruppi glucosio, attraverso un linker succinico (RGS); studi di farmacocinetica con questo composto mostrano che i livelli e la composizione dei metaboliti in circolo sono però del tutto analoghi a quelli ottenuti somministrando resveratrolo; questo suggerisce un’idrolisi del derivato a resveratrolo nel tratto intestinale, prima dell’assorbimento. Quindi, l’utilità del legame carbossiestereo in vivo è limitata. Sono stati successivamente sintetizzati altri precursori del resveratrolo, con un legame acetalico che lega gruppi protettivi/solubilizzanti agli ossidrili. Il legame acetalico è caratterizato da una bassa polarità e da uno scarso ingombro sterico, e si prevede quindi che favorisca il passaggio attraverso le bio-membrane. È suscettibile all’idrolisi in acido ma è comunque ancora troppo stabile per l’uso in vivo. Esperimenti ex vivo ed in vivo, con una serie di derivati acetalici recanti corte catene di oligo-etilene glicole utilizzando segmenti d’intestino, hanno permesso di determinare che la lunghezza della catena del gruppo solubilizzante influenza positivamente l’assorbimento. Alla ricerca di funzionalità protettive con ideali caratteristiche di stabilità, si è quindi passati ad un altro tipo di legame, più labile, che non può essere specificato qui. Tramite questo legame, allo scopo di conferire solubilità, sono state introdotte porzioni poliidrossilate o catene di PEG. Questa strategia ha portato a livelli di assorbimento soddisfacenti con due derivati (regioisomeri) monosostituiti: concentrazioni dell’ordine del uM di derivati del resveratrolo si ritrovano nel sangue, persistenti per ore. Tuttavia, i due idrossili liberi di questi composti sono substrati per le glucuronosiltrasferasi, e questa modificazione avviene prima che la rigenerazione di resveratrolo sia completa. Un nuovo approccio è stato ora adottato, incorporando gli aspetti positivi che sono emersi dagli esperimenti condotti con i derivati acetalici. In questi nuovi composti corte catene di oligo-etileneglicole sono legate ai tre idrossili del resveratrolo per aumentare l’assorbimento e nel contempo fornire protezione. Questi nuovi derivati dovrebbero essere ben assorbiti (in funzione della lunghezza della catena) e con un’adatta cinetica di idrolisi. Al momento della pubblicazione di questa tesi sono disponibili solo risultati preliminari. Durante il mio corso di dottorato ho avuto l’opportunità di trascorrere un periodo di sei mesi presso il gruppo di ricerca del Prof. Mervaala’s all’Università di Helsinki, dove ho condotto uno studio sui possibili effetti positivi del resveratrolo e della restrizione calorica sulla cardiotossicità e il danno renale indotti dal sunitinib in ratti sponteaneamente ipertesi, usando ratti normotesi come controllo. Il sunitinib è un inibitore delle tirosin-chinasi (TKI). I risultati preliminari non hanno permesso di evidenziare differenze statisticamente significative fra i controlli e i ratti trattati. Per trarre conclusioni maggiormente significative, sarà necessario condurre ulteriori esperimenti. Per finire, in durante lo stadio iniziale del progetto ho partecipato ad uno studio sul metabolismo di polifenoli in cellule in cultura. Gli studi di assorbimento/metabolismo con monostrati di cellule intestinali Caco-2 hanno messo in luce l’esistenza di eterogeneità nell’espressione degli enzimi metabolici di Fase II (solfo- e glucuronosil-trasferasi) all’interno della stessa linea cellulare. È comunque possibile uniformare nuovamente popolazioni con diversa attività metabolica coltivando le cellule in presenza di concentrazioni minime di xenobiotico (nel nostro caso quercetina)

Antidepressant hyperforin up-regulates VEGF in CNS tumour cells.

Vascular endothelial growth factor (VEGF) is a key player in neo-angiogenesis; it sustains the progression of solid neoplasias, brain tumours included. It has recently been demonstrated that the use of antidepressants correlates with increasing VEGF levels in the central nervous system (CNS). In order to elucidate whether the most used natural antidepressant [St. John's wort (SJW) extract] modulates VEGF expression, possible relationship between 64\u3bcM hyperforin (Hyp, the bioactive component in SJW) and VEGF in CNS tumours has been now examined in medulloblastoma and glioblastoma cells. Real-time PCR and ELISA revealed that under Hyp VEGF expression increased more than three fold in DAOY medulloblastoma cells; while, U87 glioblastoma cells - constitutively expressing high VEGF levels - showed no significant differences. Moreover, Hyp induced endothelial pro-angiogenic behaviour in a multi-parametric Matrigel colonisation assay, and down-modulation of pro-MMP-2 and pro-MMP-9 activities as measured by gelatin zymography. Should these results be confirmed in vivo for this and other types of CNS tumour, the antidepressant use of SJW extracts must be carefully re-considered, in particular for brain tumour patients

Antidepressant hyperforin up-regulates VEGF in CNS tumour cells

Vascular endothelial growth factor (VEGF) is a key player in neo-angiogenesis; it sustains the progression of solid neoplasias, brain tumours included. It has recently been demonstrated that the use of antidepressants correlates with increasing VEGF levels in the central nervous system (CNS). In order to elucidate whether the most used natural antidepressant [St. John's wort (SJW) extract] modulates VEGF expression, possible relationship between≤μM hyperforin (Hyp, the bioactive component in SJW) and VEGF in CNS tumours has been now examined in medulloblastoma and glioblastoma cells. Real-time PCR and ELISA revealed that under Hyp VEGF expression increased more than three fold in DAOY medulloblastoma cells; while, U87 glioblastoma cells - constitutively expressing high VEGF levels - showed no significant differences. Moreover, Hyp induced endothelial pro-angiogenic behaviour in a multi-parametric Matrigel colonisation assay, and down-modulation of pro-MMP-2 and pro-MMP-9 activities as measured by gelatin zymography. Should these results be confirmed in vivo for this and other types of CNS tumour, the antidepressant use of SJW extracts must be carefully re-considered, in particular for brain tumour patients

Heterogeneity and standardization of Phase II metabolism in cultured cells

Caco-2 cells are widely used for transepithelial transport and metabolism studies. We analysed the metabolites produced from quercetin (Q) during transport of this flavonoid across Caco-2 monolayers and by plastic-adhering cells. We found that the pattern of Phase II metabolic activity varies markedly depending on the particular cell clone, age of the cell culture, and stressful treatment such as freezing/thawing. Prolonged culturing and stress cause a decrease of "detoxifying" conjugating activity. This can be re-established by growing the cells with a low concentration of the transport/metabolism substrate for a few days. We suggest this metabolism-activating procedure be used to make studies with these cells more readily comparable