Bioactive Limonoids from the Leaves of Azaridachta indica (Neem)

Eight new limonoids (1−8) and one new phenol glycoside (9), along with six known compounds, were isolated from the leaves of Azaridachta indica. The structures of 1−9 were elucidated on the basis of spectroscopic data analysis. Compounds isolated were assayed for their cytotoxicity against different cancer cell lines. Moreover, their ability to interact with the molecular chaperone Hsp90, affecting its biological activity, was tested

The tumour microenvironment shapes dendritic cell plasticity in a human organotypic melanoma culture

Contains fulltext : 220729.pdf (publisher's version ) (Open Access)The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14(+) DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system

Nuclear and cytoplasmic expression of survivin in 67 surgically resected pancreatic cancer patients

Pancreatic cancer is one of the most aggressive gastrointestinal cancer with less than 10% long-term survivors. The apoptotic pathway deregulation is a postulated mechanism of carcinogenesis of this tumour. The present study investigated the prognostic role of apoptosis and apoptosis-involved proteins in a series of surgically resected pancreatic cancer patients. All patients affected by pancreatic adenocarcinoma and treated with surgical resection from 1988 to 2003 were considered for the study. Patients' clinical data and pathological tumour features were recorded. Survivin and Cox-2 expression were evaluated by immunohistochemical staining. Apoptotic cells were identified using the TUNEL method. Tumour specimen of 67 resected patients was included in the study. By univariate analysis, survival was influenced by Survivin overexpression. The nuclear Survivin overexpression was associated with better prognosis (P=0.0009), while its cytoplasmic overexpression resulted a negative prognostic factor (P=0.0127). Also, the apoptotic index was a statistically significant prognostic factor in a univariate model (P=0.0142). By a multivariate Cox regression analysis, both the nuclear (P=0.002) and cytoplasmic (P=0.040) Survivin overexpression maintained the prognostic statistical value. This is the first study reporting a statistical significant prognostic relevance of nuclear and cytoplasmic Survivin overexpression in pancreatic cancer. In particular, patients with high nuclear Survivin staining showed a longer survival, whereas patients with high cytoplasmic Survivin staining had a shorter overall survival

ZIC1 Is Downregulated through Promoter Hypermethylation, and Functions as a Tumor Suppressor Gene in Colorectal Cancer

The transcription factor, Zinc finger of the cerebellum (ZIC1), plays a crucial role in vertebrate development. Recently, ZIC1 has also been found to participate in the progression of human cancers, including medulloblastomas, endometrial cancers, and mesenchymal neoplasms. However, the function of ZIC1 in colon cancer progression has not been defined. In this study, we demonstrate ZIC1 to be silenced or significantly downregulated in colon cancer cell lines. These effects were reversed by demethylation treatment with 5-aza-2′-deoxycytidine (Aza). ZIC1 expression is also significantly downregulated in primary colorectal cancer tissues relative to adjacent non-tumor tissues (p = 0.0001). Furthermore, methylation of ZIC1 gene promoter is frequently detected in primary tumor tissues (85%, 34/40), but not in adjacent non-tumor tissues. Ectopic expression of ZIC1 suppresses cell proliferation and induces apoptosis, which is associated with MAPK and PI3K/Akt pathways, as well as the Bcl-xl/Bad/Caspase3 cascade. To identify target candidates of ZIC1, we employed cDNA microarray and found that 337 genes are downregulated and 95 genes upregulated by ectopic expression of ZIC1, which were verified by 10 selected gene expressions by qRT-PCR. Taken together, our results suggest that ZIC1 may potentially function as a tumor suppressor gene, which is downregulated through promoter hypermethylation in colorectal cancers

THE RAS SUPPRESSOR RSU-1, ENHANCES NGF-INDUCED DIFFERENTIATION OF PC12 CELLS AND INDUCES P21CIP EXPRESSION

The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21(WAF/CIP) expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells

Study of the interaction between nucleolin and 6,19-dihydroxy-ent-trachiloban-17-oic acid

The characterization of protein/plant molecule interactions in drug discovery, medicinal chemistry, biology and pharmacology is one of the new fields of interest in the last decades. 1 A target identification of bioactive plant molecules by a multidisciplinary approach represents an important goal of our research group. In particular, our research group are focused on the study of the interaction between plant molecules and proteins mainly involved in cancer and inflammation processes. One of our protein target is nucleolin (NCL). NCL is a multilocalized protein, found in nucleolus, nucleoplasm, cytoplasm as well as on the cell membrane.2 It is implicated in many physiological processes such as remodelling of chromatin structure, ribosome biogenesis, DNA transcription, and in cancer, inflammation and viral diseases.3 In order to study plant small molecules able to modulate nucleolin activity, a library of diterpenes was screened through the Celluar Thermal Shift Assay (CETSA). It allows us to monitor and quantify the extent to which a drug candidate reaches and directly binds to a protein target of interest within a cell. CETSA relies on the principle of thermodynamic stabilization inferred to the protein as a result of ligand binding.4 The screening, performed on HeLa cells, led us to obtain the diterpene 6,19-dihydroxy-ent-trachiloban-17-oic acid as main ligand of NCL. This molecule has been extracted from Psiadia punctulata (DC) Vatke (Asteraceae). In order to confirm the interaction, the apparent melting curve of nucleolin (Tm: 55°C), and the EC50 (5µM) of the complex has been determined by CETSA. Subsequently, a chemical proteomic cell-based approach of Drug Affinity Responsive Target Stability (DARTS) has been performed to validate the interaction.5 The DARTS results analized by Mass Spectrometry and Western Blot analysis confirmed the diterpene/NCL interaction

Identification of the ellagitannin geraniin as a novel Hsp90 inhibitor

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone assisting the proper protein folding and assembly, and targeting misfolded proteins to the proteolytic degradation.1 - 3 Inhibition of the Hsp90 activity incapacitates simultaneously multiple client proteins, resulting in a blockade of multiple signaling pathways and, ultimately, providing a combinatorial attack to cellular oncogenic processes.4 Because of the potential therapeutic use in multiple cancer indications, Hsp90 has emerged as an exciting new target for the development of antitumor agents. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90 similar to 17-(allylamino)-17-demethoxygeldanamycin (17AGG), one of the most potent known anti-Hsp90 agent. Geraniin was found to inhibit in a dose-dependent manner the Hsp90 ATPase activity, with an inhibitory efficiency similar to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90, monitored by the citrate synthase thermal induced aggregation. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, EGFR was strongly down-regulated in HeLa and Jurkat cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors

CONFRONTO DEI VALORI PLASMATICI DELLE METALLOPROTEASI DELLA MATRICE 2,3,9 IN PAZIENTI CON CARCINOMI E FIBROADENOMI MAMMARI.

Il processo di infiltrazione delle cellule tumorali nei tessuti limitrofi prevede il rimaneggiamento dello stroma peritumorale determinato da un’alta attività litica sulla matrice extracellulare da parte del fenotipo neoplastico invasivo. Tra i principali enzimi litici prodotti dalle cellule tumorali e principalmente coinvolti nel processo invasivo ci sono le metalloproteasi della matrice (MMPs). Lo scopo di questo lavoro è quello di confrontare i livelli plasmatici pre-operatori delle MMPs 2, 3, 9 in pazienti affette da carcinomi e fibroadenomi mammari per valutare un’eventuale differenza di espressione delle tre MMPs tra i due gruppi di pazienti. Sono stati quantizzati con ELISA test i valori delle MMPs 2, 3, 9 nel plasma prelevato 24 h prima dell’intervento chirurgico in 50 pazienti con carcinomi ed in 30 pazienti con fibroadenomi mammari. Il valore medio di MMP2 nelle pazienti con carcinoma è risultato essere significativamente superiore a quello nelle pazienti con fibroadenoma, mentre non è stata rivelata una differenza significativa per le altre due MMPs analizzate. Alla luce dei risultati ottenuti è possibile confermare il ruolo fondamentale svolto dalle MMPs nel fenomeno invasivo neoplastico. Inoltre, sulla base dei valori ottenuti per la MMP2, si potrebbe inserire la valutazione di questo enzima come marcatore a fianco ad altri parametri biologici e clinici nell’orientamento prognostico delle lesioni mammarie neoplastiche