Non-canonical roles of Bcl-2 and Bcl-xL proteins: Relevance of BH4 domain

Bcl-2 protein family is constituted by multidomain members originally identified as modulators of programmed cell death and whose expression is frequently misbalanced in cancer cells. The lead member Bcl-2 and its homologue Bcl-xL proteins are characterized by the presence of all four conserved BH domain and exert their antiapoptotic role mainly through the involvement of BH1, BH2 and BH3 homology domains, that mediate the interaction with the proapoptotic members of the same Bcl-2 family. The N-terminal BH4 domain of Bcl-2 and Bcl-xL is responsible for the interaction with other proteins that do not belong to Bcl-2 protein family. Beyond a classical role in inhibiting apoptosis, BH4 domain has been characterized as a crucial regulator of other important cellular functions attributed to Bcl-2 and Bcl-xL, including proliferation, autophagy, differentiation, DNA repair, cell migration, tumor progression and angiogenesis. During the last two decades a strong effort has been made to dissect the molecular pathways involved the capability of BH4 domain to regulate the canonical antiapoptotic and the non-canonical activities of Bcl-2 and Bcl-xL, creating the basis for the development of novel anticancer agents targeting this domain. Indeed, recent evidences obtained on in vitro and in vivo model of different cancer histotypes are confirming the promising therapeutic potential of BH4 domain inhibitors supporting their future employment as a novel anticancer strategy

Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation

Anti-inflammatory reprogramming of microglia cells by metabolic modulators to counteract neurodegeneration; a new role for Ranolazine

Microglia chronic activation is a hallmark of several neurodegenerative diseases, including the retinal ones, possibly contributing to their etiopathogenesis. However, some microglia sub‐populations have anti‐inflammatory and neuroprotective functions, thus making arduous deciphering the role of these cells in neurodegeneration. Since it has been proposed that functionally different microglia subsets also rely on different metabolic routes, we hypothesized that modulating microglia metabolism might be a tool to enhance their anti‐inflammatory features. This would have a preventive and therapeutic potential in counteracting neurodegenerative diseases. For this purpose, we tested various molecules known to act on cell metabolism, and we revealed the anti‐inflammatory effect of the FDA‐approved piperazine derivative Ranolazine on microglia cells, while confirming the one of the flavonoids Quercetin and Naringenin, both in vitro and in vivo. We also demonstrated the synergistic anti‐inflammatory effect of Quercetin and Idebenone, and the ability of Ranolazine, Quercetin and Naringenin to counteract the neurotoxic effect of LPS‐activated microglia on 661W neuronal cells. Overall, these data suggest that using the selected molecules ‐also in combination therapies‐ might represent a valuable approach to reduce inflammation and neurodegeneration while avoiding long term side effects of corticosteroids

Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4’-chlorophenyl)thiazol-2-yl] hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC

BCL-XL overexpression promotes tumor progression-associated properties article

By using human melanoma and glioblastoma cell lines and their derivative BCL-XL overexpressing clones, we investigated the role of BCL-XL in aggressive features of these two tumor histotypes. We found that in both models, BCL-XL overexpression increased in vitro cell migration and invasion and facilitated tumor cells to form de novo vasculogenic structures. Furthermore, BCL-XL overexpressing cells exhibited higher tumors sphere formation capacity and expressed higher levels of some stem cell markers, supporting the concept that BCL-XL plays essential roles in the maintenance of cancer stem cell phenotype. BCL-XL expression reduction by siRNA, the exposure to a BCL-XL-specific inhibitor and the use of a panel of human melanoma cell lines corroborated the evidence that BCL-XL regulates tumor progression-associated properties. Finally, the vascular markers and the vasculogenic mimicry were up-regulated in the BCL-XL overexpressing xenografts derived from both tumor histotypes. In conclusion, our work brings further support to the understanding of the malignant actions of BCL-XL and, in particular, to the concept that BCL-XL promotes stemness and contributes to the aggressiveness of both melanoma and glioblastoma

Zebrafish modeling reveals that SPINT1 regulates the aggressiveness of skin cutaneous melanoma and its crosstalk with tumor immune microenvironment

Background Skin cutaneous melanoma (SKCM) is the most lethal form of skin cancer and while incidence rates are declining for most cancers, they have been steadily rising for SKCM. Serine protease inhibitor, kunitz-type, 1 (SPINT1) is a type II transmembrane serine protease inhibitor that has been shown to be involved in the development of several types of cancer, such as squamous cell carcinoma and colorectal cancer. Methods We used the unique advantages of the zebrafish to model the impact of Spint1a deficiency in early transformation, progression and metastatic invasion of SKCM together with in silico analysis of the occurrence and relevance of SPINT1 genetic alterations of the SKCM TCGA cohort. Results We report here a high prevalence of SPINT1 genetic alterations in SKCM patients and their association with altered tumor immune microenvironment and poor patient survival. The zebrafish model reveals that Spint1a deficiency facilitates oncogenic transformation, regulates the tumor immune microenvironment crosstalk, accelerates the onset of SKCM and promotes metastatic invasion. Notably, Spint1a deficiency is required at both cell autonomous and non-autonomous levels to enhance invasiveness of SKCM. Conclusions These results reveal a novel therapeutic target for SKCM

Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.Results: In NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.Conclusions: Overall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC

Before-During-After Biomonitoring Assessment for a Pipeline Construction in a Coastal Lagoon in the Northern Adriatic Sea (Italy)

During 2006–2008, a pipeline was buried in Vallona lagoon in the Northern Adriatic Sea (Italy). A Before-During-After environmental monitoring programme was scheduled to monitor possible alterations. Bioaccumulation of metal(loid)s, BTs (butyltins) and HMW-PAHs (High Molecular Weight Polycyclic Aromatic Hydrocarbons), and biological responses (Condition index, air Survival –LT50, Acetylcholinesterase, Micronuclei –MN, acyl-CoA oxidase, catalase, malondialdehyde –MDA, and the total oxyradical scavenging capacity-TOSCA) were investigated in Manila clams (Ruditapes philippinarum) from November 2005 to June 2015. In opera (IO) results showed higher levels of HMW-PAHs (73 ± 13 ng/g), BTs (90 ± 38 ng Sn/g) and increasing levels of Pb (6.7 ± 0.7 mg/kg) and Zn (73.6 ± 6.08 mg/kg) probably linked to works. Other contaminant alterations, especially metal(loid)s, before (AO) and after (PO) the burial, were attributed to a general condition of the area and mostly unrelated to works. In addition, LT50, MN and TOSCA showed alterations, probably due to hotspots occurring in IO. TOSCA and MDA increases, right after the burial, were considered delayed responses of IO, whilst other biological responses detected later were connected to the general condition of the area. Comparisons between results of Principal Component Analyses (PCAs) highlighted partial overlapping of AO and IO, whilst PO differed only for contaminants. Visual correlations between PCAs highlighted the biomarkers’ latter response

Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90