The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

Background: The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6- methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity. Methods: Tinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments. Results: We demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity. Conclusions: Our data suggest that tinostamustine deserves further investigation in patients with glioblastoma

The possible prognostic role of histone deacetylase and transforming growth factor β/Smad signaling in high grade gliomas treated by radio-chemotherapy: a preliminary immunohistochemical study

Glioblastoma (GBM) is the most common and aggressive tumor of the central nervous system. Unfortunately, patients affected by this disease have a very poor prognosis, due to high level of invasiveness and resistance to standard therapies. Although the molecular profile of GBM has been extensively investigated, the events responsible for its pathogenesis and progression remain largely unknown. Histone Deacetylases (HDAC) dependent epigenetic modifications and transforming growth factor (TGF)-β/Smad pathway seem to play an important role in GBM tumorigenesis, resistance to common therapies and poor clinical outcome. The aim of this study was to evaluate the involvement and the possible interaction between these two molecular cascades in the pathogenesis and prognosis of GBM. Immunohistochemistry (IHC) was performed on microdissected GBM samples, collected from 14 patients (6 men and 8 women) ranging in age from 43 to 74 years. The patients were previously divided, on the basis of their overall survival (OS), into two groups: short and long OS. Patients with poor prognosis showed hyperexpression of HDAC4 and HDAC6, an activation of the TGF-β/Smad pathway, with high levels of IL-13, Smad2, PDGF and MMP3 expression, compared to the long survivors. The short OS group exhibits a decrease in Smad 7 expression and also low levels of p21 immunostaining, which represents a common target of the two pathways. The IHC data was confirmed by quantitative analysis and Immunoblotting. Our preliminary results suggest that both HDAC4 and HDAC6 together with the TGF-β/Smad pathway may be involved in progression of GBM and this cross talking could be a useful prognostic marker in this deadly disease

Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer

Background and aims: Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon. Material and methods: Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives. Results and conclusions: We show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients

Dual PI3 K/mTOR inhibition reduces prostate cancer bone engraftment altering tumor-induced bone remodeling:

Morbidity in advanced prostate cancer patients is largely associated with bone metastatic events. The development of novel therapeutic strategies is imperative in order to effectively treat this incurable stage of the malignancy. In this context, Akt signaling pathway represents a promising therapeutic target able to counteract biochemical recurrence and metastatic progression in prostate cancer. We explored the therapeutic potential of a novel dual PI3 K/mTOR inhibitor, X480, to inhibit tumor growth and bone colonization using different in vivo prostate cancer models including the subcutaneous injection of aggressive and bone metastatic (PC3) and non-bone metastatic (22rv1) cell lines and preclinical models known to generate bone lesions. We observed that X480 both inhibited the primary growth of subcutaneous tumors generated by PC3 and 22rv1 cells and reduced bone spreading of PCb2, a high osteotropic PC3 cell derivative. In metastatic bone, X480 inhibited significantly the growth and osteolytic activit..

Features of intestinal lesions in the clinical course of Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are chronic, progressive and relapsing inflammatory disorders of unknown etiology. UC is characterized by inflammation of the large bowel mucosa and submucosa, whereas in CD inflammation is trans-mural and may involve various sites of the gastrointestinal tract. Superficial mucosal lesions are most prone to heal, whereas deep ulcers or transmural fissures may heal with more difficulty and may be followed by the development of fibrosis and strictures requiring surgery. Inflammation appears to be necessary to trigger the onset of the fibrotic process, but subsequently plays a minor role in its progression. In IBD, anti-inflammatory treatment does not prevent evolution of fibrosis once the process has started. Therefore, the mechanisms that regulate fibrosis appear to be distinct from those regulating inflammation. Intestinal fibrosis is due to an abnormal accumulation of extracellular matrix proteins producted by activated intestinal myofibroblasts. Increased evidence indicate that a number of molecules are involved in the development of the disease and a crosstalk between TGFβ/Smads pathway and αvβ6 integrin, mTOR and PPARγ could play a crucial role in the development of intestinal fibrosis. Animal models represent a useful tool to investigate the molecular and cellular mechanisms of intestinal inflammation and fibrosis and to test the effectiveness of novel therapeutic strategies for the prevention and treatment of intestinal fibrosis that still remain the major cause of surgical intervention

The antiinflammatory and antifibrotic effect of olive phenols and Lactiplantibacillus plantarum IMC513 in dextran sodium sulfate–induced chronic colitis

After a chronic intestinal injury, several intestinal cells switch their phenotype to activated myofi- broblasts, which in turn release an abnormal amount of extracellular matrix proteins, leading to the onset of the fibrotic process. To date, no resolutive pharmacological treatments are available, and the identification of new therapeutic approaches represents a crucial goal to achieve. The onset, maintenance, and progression of inflammatory bowel disease are related to abnormal intestinal immune responses to environmental factors, including diet and intestinal microflora components. This study aimed to evaluate the potential antiinflam- matory and antifibrotic effect of a biologically debittered olive cream and its probiotic oral administration in an experimental model of dextran sodium sulfate (DSS)induced chronic colitis. Methods: Chronic colitis was induced in mice by three cycles of oral administration of 2.5% DSS (5 d of DSS followed by 7 d of tap water). Mice were randomly divided into five groups: 10 control mice fed with stan- dard diet (SD), 20 mice receiving SD and DSS (SD+DSS), 20 mice receiving an enriched diet (ED) with olive cream and DSS (ED+DSS), 20 mice receiving SD plus probiotics (PB; Lactiplantibacillus plantarum IMC513) and DSS (SD+PB+DSS), and 20 mice receiving ED plus PB and DSS (ED+ PB+DSS). Clinical features and large bowel macroscopic, histologic, and immunohistochemical findings were evaluated. Results: The simultaneous administration of ED and PB induced a significant reduction in macroscopic and microscopic colitis scores compared with the other DSS-treated groups. In addition, ED and PB led to a signif- icant decrease in the expression of inflammatory cytokines and profibrotic molecules. Conclusions: The concomitant oral administration of a diet enriched with biologically debittered olive cream and a specific probiotic strain (Lactiplantibacillus plantarum IMC513) can exert synergistic antiinflammatory and antifibrotic action in DSS-induced chronic colitis. Further studies are needed to define the cellular and molecular mechanisms modulated by olive cream compounds and by Lactiplantibacillus plantarum IMC513

HDACs expression in glioblastoma: an immunohistochemical study

Glioblastoma is the most common and lethal primary malignant brain tumor. Although standard treatments have been improving, the clinical outcome remains unacceptably poor. Several genetic alterations are supposed to be involved in the eti- ology of different grades of astrocytoma, including epimutations. Histone deacety- lases (HDACs) are involved in the post-translational modification on the lysines of histone tails. For this reason HDACs are recognized as promising targets for cancer treatment (1). In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis (2). Aim of our study was to evaluate with an immunohistochemi- cal and immunoblotting analyses the expression of different classes of HDACs (Class I: HDAC 1-2-3-8; class II: HDAC 4-6) in microdissected glioblastoma. Tumor sam- ples were taken from 14 patients (n.8 men and n.6 women) ranging in age from 43 to 74 years. HDAC1 and HDAC3 expression was not significantly different between the two proteins and was predominantely located at cytoplasmic level of cancer cells with different intensity of immureaction from mild to moderate whereas HDAC2 staining was localized to the nucleous of neoplastic cells. The pattern of HDAC4 immureactivity was always cytoplasmatic and showed a marked and diffuse increase of immunostaining in neoplastic areas. HDAC8 was always absent in cancer cells and the only positivity was located in the endothelial cells of the vessels. HDAC6 was often absent and, if present, showed a very low cytoplasmic immunopositivity in cancer cells. HDAC1, HDAC2 and HDAC3 levels were not significantly different in immunoblotting results; HDAC4 showed a marked increase while HDAC6 and HDAC8 expression was poor, confirming the IHC data. These previous results dem- onstrate a different pattern of HDAC expression and could suggest a more addressed therapeutical use of HDACis in glioblastoma.

DDS-induced colorectal fibrosis in mice: anti-fibrotic effects of GED 0507-34 levo, a novel PPARγ ligand

Intestinal fibrosis is a progressive process characterized by de novo synthesis and uncontrolled deposition of extracellular matrix components (ECM) following a tissue chronic inflammation mainly regulated by Transforming Growth Factor (TGF)β/ Smads pathway. Frequently associated to Inflammatory Bowel Disease (IBD), intestinal fibrosis may lead to stenosis and obstructions that require surgery up to 75% of patients as drugs currently used in IBD are unable to improve fibrostenosis lesions (1). Peroxisome proliferator-activated receptor (PPAR)-γ is able to antagonize (TGF) β/Smads and could be an crucial target to develop novel antifibrotic therapeutic strategies (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED 0507-34 levo, in colonic fibrosis in mice. Immunohistochemistry and immunoblotting evaluations, TGFβ1, CTGF, Collagen types I-III, Smad3, α-SMA, were performed in in three groups of C57BL/6 mice: Dextran Sulphate Sodium (DSS) colitis group, DSS+GED group and controls. Evident macroscopic and microscopic lesions in the most of colons of DSS treated mice were observed compared to DSS+GED mice and controls. The tissue levels of the main markers of fibrosis resulted significantly increased in DSS mice and restored by administration of GED. GED seems to prevents ECM colonic deposition and to improve the intestinal fibrotic lesions by its ability in controlling TGFβ/Smads pathway signalling activation

Anti-fibrotic effect of a novel PPAR-γ ligand, GED-0507-34 LEVO, in DSS induced colonic fibrosis in mice

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) in which chronic inflammation leads to abnormal deposition of extracellular matrix components (ECM) causing obstruction and loss of function of the intestinal tract involved (1). Fibrogenesis is mainly regulates by trasforming growth factor (TGF) β/Smad pathway and a key antagonist of this signaling is represented by peroxisome proliferator-activated receptor (PPAR)-γ. As the anti-inflammatory drugs currently used in IBD are unable to improve intestinal fibrosis the exploration of new therapeutical approaches has now became crucial (2). Aim of this study is to evaluate the antifibrotic action of a novel PPARγ agonist, GED-0507-34-LEVO (GED), in colon fibrosis in mice. Chronic colitis and fibrosis were induced in C57BL/6 mice by administration of 2,5% (w/v) dextrane sulphate sodium (DSS) in drinking water for 5 days followed by 7 days of water for 3 cycles. Mice were divided into 3 groups: DSS, DSS+GED and control. 30mg/Kg/mice of GED was daily administrated by oral gavage starting from the second DSS cycle. Samples from colon were excised and processed to assess macroscopic lesions, histological and morphometrical aspects and immunohistochemical and immunoblotting analysis for TGFβ1, CTGF, collagen types I-III, Smad3,α-SMA. Evident shortening and dilation in the most of colons of DSS treated mice were observed. Macroscopic and microscopic findings were significantly improved in DSS mice+GED compared with control mice. The tissue levels of collagen and α-SMA, specific markers of fibrosis, resulted significantly increased in mice receving DSS compared to control mice, as well as the expression of TGFβ1, CTGF, Smad3. DSS+GED group showed reduced expression of all markers involved. GED significantly improves the intestinal fibrotic lesions in DSS chronic colitis murine model and controls the pivotal molecular events leading to fibrosis