Targeting of multiple myeloma-related angiogenesis by miR-199a-5p mimics: in vitro and in vivo anti-tumor activity

Multiple myeloma (MM) cells induce relevant angiogenic effects within the human bone marrow milieu (huBMM) by the aberrant expression of angiogenic factors. Hypoxia triggers angiogenic events within the huBMM and the transcription factor hypoxia-inducible factor-1α (HIF-1α) is over-expressed by MM cells. Since synthetic miR-199a-5p mimics negatively regulates HIF-1α, we here investigated a miRNA-based therapeutic strategy against hypoxic MM cells. We indeed found that enforced expression of miR-199a-5p led to down-modulated expression of HIF-1α as well as of other pro-angiogenic factors such as VEGF-A, IL-8, and FGFb in hypoxic MM cells in vitro. Moreover, miR-199a-5p negatively affected MM cells migration, while it increased the adhesion of MM cells to bone marrow stromal cells (BMSCs) in hypoxic conditions. Furthermore, transfection of MM cells with miR-199a-5p significantly impaired also endothelial cells migration and down-regulated the expression of endothelial adhesion molecules such as VCAM-1 and ICAM-1. Finally, we identified a hypoxia\AKT/miR-199a-5p loop as a potential molecular mechanism responsible of miR-199a-5p down-regulation in hypoxic MM cells. Taken together our results indicate that miR-199a-5p has an important role for the pathogenesis of MM and support the hypothesis that targeting angiogenesis via a miRNA/HIF-1α pathway may represent a novel potential therapeutical approach for this still lethal diseas

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells

Il linfonodo sentinella nei tumori del colon

Introduzione. I tumori maligni possono dare metastasi seguendo il sistema linfatico in modo sequenziale. In ogni catena linfatica al primo linfonodo che drena la regione dove si è sviluppato il tumore viene dato il nome di “linfonodo sentinella’’ (LS). Obiettivo dello studio. L’obiettivo principale del presente studio è la determinazione del valore predittivo della metodica del linfonodo sentinella nella stadiazione del cancro colico non metastatico. Pazienti e metodi. Abbiamo effettuato uno studio prospettico arruolando pazienti con adenocarcinoma del colon che soddisfacessero i seguenti criteri: - età minima di 18 anni; - stadiazione con colonoscopia, Rx torace, ecografia o TC addome completo per selezionare pazienti con adenocarcinoma del colon T2-T3 senza metastasi linfonodali ed epatiche; - rischio anestesiologico ASA 1-3; - consenso informato. A seguito della resezione colica con linfadenectomia è stata eseguita un’iniezione sottomucosa di colorante vitale (patent blue) che ha permesso di identificare il linfonodo sentinella. I linfonodi sono stati sottoposti ad esame istologico con ematossilina-eosina e successivamente con tecnica immunoistochimica. Risultati. Dal gennaio a dicembre 2008, 26 pazienti sono stati arruolati in questo studio prospettico. Di questi sono stati considerati elegibili per il nostro studio solamente 14 pazienti. L’esame con ematossilina - eosina dei linfonodi ha evidenziato: a) in 4 casi su 14 (28,57%) erano presenti metastasi sui linfonodi contenuti nel mesocolon, b) in 10 casi su 14 (71,42%) erano assenti metastasi sui linfonodi contenuti nel mesocolon. Nei casi in cui non erano presenti metastasi, all’esame con ematossilina-eosina, nei linfonodi del mesocolon è stato eseguito l’esame istologico dei linfonodi sentinella con tecnica immunoistochimica; in 2 casi è stata evidenziata la presenza di micrometastasi. In un caso sono state identificate linee aberranti di drenaggio mesenterico (skip metastasis); il linfonodo sentinella (negativo all’esame con ematossilina eosina) è stato studiato con tecnica immunoistochimica che non ha evidenziato la presenza di micrometastasi. Conclusioni. È possibile affermare che l’esame del linfonodo sentinella è fattibile con la metodica ex vivo. Nel 20% dei casi da noi studiati a livello dei LS sono presenti micrometastasi non evidenziate al classico esame con ematossilina-eosina. Lo studio dei linfonodi sentinella con sezioni multiseriate e tecniche immunoistochimiche consente un miglioramento della stadiazione patologica

The abdominal compartment syndrome and the importance of decompressive laparotomy

Premessa. La Sindrome Compartimentale Addominale (ACS) è una complicanza la cui insorgenza è sempre più riconosciuta sia nei pazienti medici che in quelli chirurgici. La World Society of the Abdominal Compartment Syndrome definisce Ipertensione Intra-Addominale (IAH) la presenza di una Pressione Intra-Addominale (IAP) ? 12mmHg e definisce la ACS una condizione caratterizzata da una IAP ? 20mmHg (con o senza una pressione di perfusione addominale 60 mmHg) associata alla disfunzione o al danneggiamento di uno o più organi non presenti precedentemente. La IAH contribuisce alla disfunzione d'organo nei pazienti con trauma addominale e sepsi e porta alla formazione di ACS. Obiettivo. In questo studio si è cercato di valutare la reale incidenza della sindrome compartimentale addominale nei pazienti sottoposti a laparotomie d’urgenza e si è valutato il ruolo della re-laparotomia decompressiva. Pazienti e metodi. Lo studio include 10 pazienti, 4 uomini e 6 donne con un'età media di 68 anni (range, 38-86), sottoposti a laparotomia dal gennaio 2007 al settembre 2008. In accordo alle indicazioni dettate dalla WSACS (World Society of the Abdominal Compartment Syndrome) abbiamo misurato la IAP in maniera indiretta attraverso l'uso di un catetere vescicale di Foley. Risultati. Dei 10 pazienti , in 8 la pressione intraaddominale rientrava nei valori compresi tra 8 mmHg e 20 mmHg e, non essendo stati riscontrati sintomi clinici significativi correlati all’aumento della IAP, non è stato necessario eseguire una re-laparotomia decompressiva. Nei 2 pazienti in cui la pressione intra-addominale era superiore ai 20 mmHg la sintomatologia era caratterizzata da tensione della parete addominale, instabilità emodinamica, oligo/anuria, modificazioni respiratorie e squilibrio acidobase per cui sono sati sottoposti a re-laparotomia decompressiva d’urgenza. Conclusioni. In base alla nostra esperienza ed ai risultati della letteratura riteniamo indispensabile il monitoraggio della pressione intraaddominale nei pazienti sottoposti a re-laparotomia addominale. Nelle re-laparotomie decompressive eseguite d’urgenza la sutura solo cutanea ha permesso una rapida chiusura dell’addome in pazienti instabili ed a rischio

Synthetic miR-34a mimics as a novel therapeutic agent for Multiple Myeloma: in vitro and in vivo evidence

PURPOSE: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. EXPERIMENTAL DESIGN: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. RESULTS: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. CONCLUSIONS: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a-based treatment strategies in MM patients

The sentinel lymph node mapping in colon cancer.

Malignant tumors of the colon can metastases along the lymphatic system in a sequential way, which means that there will be a first node to be involved and then from this disease will pass to another node and so gradually. The sentinel lymph node is the first lymph node or group of nodes reached by metastasizing cancer cells from a tumor. The present work aims to determine the predictive value of the sentinel lymph node procedure in the staging of non-metastatic colon cancer. In this prospective study joined up only 26 patients with adenocarcinoma of the colon T2-T3, without systemic metastases, and with these criteria for inclusion: a) minimum age: 18 years old; b) staging by total colonoscopy, chest X-ray and CT scan; c) patients classified as ASA 1-3; d) informed consent. Within 20 minutes from the colic resection, the bowel was cut completely along the antimesenteric margin and is performed submucosal injection of vital dye within 5 mm from the lesion at the level of the four cardinal points; then the lymph nodes are placed in formalin and sent to the pathologist. The lymph nodes were subjected to histological examination with haematoxylin-eosin and with the immunohistochemistry technique. From January to December 2008 only 26 patients joined up in this prospective study. From the study were excluded the 4 patients with T4 and M1 tumour. Also 7 patients with stenotic lesions were excluded. Patients considered eligible for our study were only 14. The histopathological examination of haematoxylin-eosin revealed: a) in 4 cases were detected mesocolic lymph node metastases; b) in 10 cases were not detected mesocolic lymph node metastases. In cases there were no metastases, the mesocolic sentinel lymph nodes lymph nodes were examined with immunohistochemical technique; in 2 cases were revealed the presence of micrometastases. In one case was identified aberrant lymphatic drainage patterns (skip metastasis); the sentinel lymph node (negative examination wit eaematoxylin-eosin) was studied with immunohistochemical technique that has not revealed the presence of micrometastases. Examination of the sentinel node is feasible with the ex vivo method. Using the immunohistochemical technique we detect micrometastasis in 20% of the cases, not revealed with the classical haematoxylin-eosin examination. The study of sentinel lymph node with multilevel microsections and immunohistochemical techniques allow a better histopathological staging

In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p=0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM