The antimicrobial potential of adarotene derivatives against Staphylococcus aureus strains.

Multidrug-resistant (MDR) pathogens are severely impacting our ability to successfully treat common infections. Here we report the synthesis of a panel of adarotene-related retinoids showing potent antimicrobial activity on Staphylococcus aureus strains (including multidrug-resistant ones). Fluorescence and molecular dynamic studies confirmed that the adarotene analogues were able to induce conformational changes and disfunctions to the cell membrane, perturbing the permeability of the phospholipid bilayer. Since the major obstacle for developing retinoids is their potential cytotoxicity, a selected candidate was further investigated to evaluate its activity on a panel of human cell lines. The compound was found to be well tolerated, with IC50 5-15-fold higher than the MIC on S. aureus strains. Furthermore, the adarotene analogue had a good pharmacokinetic profile, reaching a plasma concentration of about 6 µM after 0.5 h after administration (150 mg/kg), at least twice the MIC observed against various bacterial strains. Moreover, it was demonstrated that the compound potentiated the growth-inhibitory effect of the poorly bioavailable rifaximin, when used in combination. Overall, the collected data pave the way for the development of synthetic retinoids as potential therapeutics for hard-to-treat infectious diseases caused by antibiotic-resistant Gram-positive pathogens.

The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Novel adamantyl retinoid-related molecules with POLA1 inhibitory activity.

Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

A Herbal Mixture from Propolis, Pomegranate, and Grape Pomace Endowed with Anti-Inflammatory Activity in an In Vivo Rheumatoid Arthritis Model.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by the production of inflammatory factors. In order to overcome the side effects of currently used anti-inflammatory drugs, several attempts have been made to identify natural products capable of relieving RA symptoms. In this work, a herbal preparation consisting of propolis, pomegranate peel, and Aglianico grape pomace (PPP) extracts (4:1:1) was designed and evaluated for its effect on a murine collagen-induced arthritis (CIA) model. Firstly, the chemical contents of four different Italian propolis collected in the Campania region (Italy) were here reported for the first time. LC-MS analyses showed the presence of 38 constituents, identified in all propolis extracts, belonging to flavonoids and phenolic acids classes. The Pietradefusi extract was the richest one and thus was selected to design the PPP preparation for the in vivo assay. Our results highlight the impact of PPP on RA onset and progression. By using in vivo CIA models, the treatment with PPP resulted in a delayed onset of the disease and alleviated the severity of the clinical symptoms. Furthermore, we demonstrated that early PPP treatment was associated with a reduction in serum levels of IL-17, IL-1b, and IL-17-triggering cytokines.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Antitumor activities of the synthetic retinoid ST1926 in two-dimensional and three-dimensional human breast cancer models.

Despite recent advances in chemotherapy, aggressive and metastatic breast cancers remain refractory to targeted therapy and the development of novel drugs is urgently needed. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is disease relapse. Therefore, synthetic retinoids, specifically ST1926, have emerged as potent anticancer agents. Given the importance of the microenvironment in modulating the response of cancer cells to chemotherapeutic drugs, we investigated the antitumor activities of ST1926 in two-dimensional (2D) and different three-dimensional (3D) human breast cancer models and compared them with ATRA. We have shown that in 2D cell culture models, ATRA-resistant MCF-7 and MDA-MB-231 cells were sensitive to ST1926 at submicromolar concentrations that spared the 'normal-like' breast epithelial cells. ST1926 induced apoptosis and S-phase arrest, caused DNA damage, and downregulated the Wnt/ß-catenin pathway in breast cancer cells in 2D and 3D cell culture models. ST1926-mediated growth inhibition was independent of the retinoid receptor-signaling pathway. Long-term treatments with low submicromolar ST1926 concentrations reduced the anchorage-independent growth and decreased the sphere-forming ability of breast cancer progenitor cells in the sphere formation assay. Furthermore, ST1926 potently induced cell death of breast cancer cells under 3D conditions and spared the lumen-forming ability of normal-like breast epithelial cells. In tested 3D models, ATRA had minimal effects on the growth of breast cancer cells compared with ST1926. In summary, our results highlight the therapeutic potential of ST1926 in breast cancer and warrant its further clinical development.

The synthetic retinoid ST1926 as a novel therapeutic agent in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children. Despite multiple attempts at intensifying chemotherapeutic approaches to treatment, only moderate improvements in survival have been made for patients with advanced disease. Retinoic acid is a differentiation agent that has shown some antitumor efficacy in RMS cells in vitro; however, the effects are of low magnitude. E-3-(4'-hydroxyl-3'-adamantylbiphenyl-4-yl) acrylic acid (ST1926) is a novel orally available synthetic atypical retinoid, shown to have more potent activity than retinoic acid in several types of cancer cells. We used in vitro and in vivo models of RMS to explore the efficacy of ST1926 as a possible therapeutic agent in this sarcoma. We found that ST1926 reduced RMS cell viability in all tested alveolar (ARMS) and embryonal (ERMS) RMS cell lines, at readily achievable micromolar concentrations in mice. ST1926 induced an early DNA damage response (DDR), which led to increase in apoptosis, in addition to S-phase cell cycle arrest and a reduction in protein levels of the cell cycle kinase CDK1. Effects were irrespective of TP53 mutational status. Interestingly, in ARMS cells, ST1926 treatment decreased PAX3-FOXO1 fusion oncoprotein levels, and this suppression occurred at a post-transcriptional level. In vivo, ST1926 was effective in inhibiting growth of ARMS and ERMS xenografts, and induced a prominent DDR. We conclude that ST1926 has preclinical efficacy against RMS, and should be further developed in this disease in clinical trials.

Preclinical efficacy of the synthetic retinoid ST1926 for treating adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 oncoprotein Tax plays an important role in ATL pathogenesis. ATL carries a poor prognosis due to chemotherapy resistance, stressing the need for alternative therapies. Here, we investigate the preclinical efficacy of the synthetic retinoid ST1926 in ATL and peripheral T-cell lymphomas. Clinically achievable concentrations of ST1926 induced a dramatic inhibition of cell proliferation in malignant T-cell lines and primary ATL cells with minimal effect on resting or activated normal lymphocytes. ST1926 induced apoptosis, DNA damage, and upregulation of p53 proteins in malignant T cells, whereas it caused an early downregulation of Tax proteins in HTLV-1-positive cells. In murine ATL, oral treatment with ST1926 prolonged survival and reduced leukemia cell infiltration, white blood cell counts, and spleen mass. In spleens of ST1926-treated animals, p53 and p21 proteins were upregulated, poly (ADP-ribose) polymerase was cleaved, and Tax transcripts were reduced. These results highlight the promising use of ST1926 as a targeted therapy for ATL.

ST1926, an orally active synthetic retinoid, induces apoptosis in chronic myeloid leukemia cells and prolongs survival in a murine model.

The tyrosine kinase inhibitor, imatinib, is the first line of treatment for chronic myeloid leukemia (CML) patients. Unfortunately, patients develop resistance and relapse due to bcr-abl point mutations and the persistence of leukemia initiating cells (LIC). Retinoids regulate vital biological processes such as cellular proliferation, apoptosis, and differentiation, in particular of hematopoietic progenitor cells. The clinical usage of natural retinoids is hindered by acquired resistance and undesirable side effects. However, bioavailable and less toxic synthetic retinoids, such as the atypical adamantyl retinoid ST1926, have been developed and tested in cancer clinical trials. We investigated the preclinical efficacy of the synthetic retinoid ST1926 using human CML cell lines and the murine bone marrow transduction/transplantation CML model. In vitro, ST1926 induced irreversible growth inhibition, cell cycle arrest and apoptosis through the dissipation of the mitochondrial membrane potential and caspase activation. Furthermore, ST1926 induced DNA damage and downregulated BCR-ABL. Most importantly, oral treatment with ST1926 significantly prolonged the longevity of primary CML mice, and reduced tumor burden. However, ST1926 did not eradicate LIC, evident by the ability of splenocytes isolated from treated primary mice to develop CML in untreated secondary recipients. These results support a potential therapeutic use of ST1926 in CML targeted therapy.

Investigation on the ZBG-functionality of phenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase inhibitors.

A series of alternative Zn-binding groups were explored in the design of phenyl-4-yl-acrylohydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors. Most of the synthesized compounds were less effective than the parent hydroxamic acid. However, the profile of activity shown by the analog bearing a hydroxyurea head group, makes this derivative promising for further investigation.

7-Azaindole-1-carboxamides as a new class of PARP-1 inhibitors.

7-Azaindole-1-carboxamides were designed as a new class of PARP-1 inhibitors. The compounds displayed a variable pattern of target inhibition profile that, in part, paralleled the antiproliferative activity in cell lines characterized by homologous recombination defects. A selected compound (1l; ST7710AA1) showed significant in vitro target inhibition and capability to substantially bypass the multidrug resistance mediated by Pgp. In antitumor activity studies against the MX1 human breast carcinoma growth in nude mice, the compound exhibited an effect similar to that of Olaparib in terms of tumor volume inhibition when used at a lower dose than the reference compound. Treatment was well tolerated, as no deaths or significant weight losses were observed among the treated animals.

RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients.

BACKGROUND: A major obstacle in treating colorectal cancer (CRC) is the acquired resistance to chemotherapeutic agents. An important protein in the regulation of cancer cell death and clinical outcome is Raf kinase inhibitor protein (RKIP). In contrast, activated signal transducer and activator of transcription 3 (STAT3) is a protein that promotes tumor cell survival by inhibiting apoptosis and has an important role in cancer progression in many of cancer types. The aim of this study was to evaluate the regulation of RKIP and STAT3 after treatment with clinically relevant chemotherapeutic agents (camptothecin (CPT) and oxaliplatin (OXP)) and the cytokine interleukin-6 (IL-6) in HCT116 colon cancer cells as well as evaluate the association between RKIP and STAT3 with clinical outcome of Stage II colon cancer patients. METHODS: HCT-116 colon cancer cells were treated with CPT, OXP, and IL-6 separately or in combination in a time and dose-dependent manner and examined for phosphorylated and non-phosphorylated RKIP and STAT3 via Western blot analysis. STAT3 transcriptional activity was measured via a luciferase reporter assay in HCT116 cells treated with CPT, IL-6 or transfected with JAK 1, 2 separately or in combination. We extended these observations and determined STAT3 and RKIP/ pRKIP in tumor microarrays (TMA) in stage II colon cancer patients. RESULTS: We demonstrate IL-6-mediated activation of STAT3 occurs in conjunction with the phosphorylation of RKIP in vitro in human colon cancer cells. OXP and CPT block IL-6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130. We determined that STAT3 and nuclear pRKIP are significantly associated with poor patient prognosis in stage II colon cancer patients. CONCLUSIONS: In the analysis of tumor samples from stage II colon cancer patients and the human colon carcinoma cell line HCT116, pRKIP and STAT3, 2 proteins potentially involved in the resistance to conventional treatments were detected. The phosphorylation of pRKIP and STAT3 are induced by the cytokine IL-6 and suppressed by the chemotherapeutic drugs CPT and OXP. Therefore, these results suggest that STAT3 and pRKIP may serve as prognostic biomarkers in stage II colon cancer patients and may improve chemotherapy.

Development and Challenges of Synthetic Retinoid Formulations in Cancer.

Retinoids represent a class of chemical compounds derived from or structurally and functionally related to vitamin A. Retinoids play crucial roles in regulating a range of crucial biological processes spanning embryonic development to adult life. These include regulation of cell proliferation, differentiation, and cell death. Due to their promising characteristics, retinoids emerged as potent anti-cancer agents, and their effects were validated in vitro and in vivo preclinical models of several solid and hematological malignancies. However, their clinical translation remained limited due to poor water solubility, photosensitivity, short half-life, and toxicity. The development of retinoid delivery formulations was extensively studied to overcome these limitations. This review will summarize some preclinical and commercial synthetic retinoids in cancer and discuss their different delivery systems.

Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor phenotype.

Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype.

The biflavonoid amentoflavone inhibits neovascularization preventing the activity of proangiogenic vascular endothelial growth factors.

The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low µm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.

Enhanced cell cycle perturbation and apoptosis mediate the synergistic effects of ST1926 and ATRA in neuroblastoma preclinical models.

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Camptothecins in tumor homing via an RGD sequence mimetic.

A RGD peptide mimetic was conjugated to four camptothecins, with the purpose to improve their therapeutic index. The conjugate derivatives were evaluated against two tumor cell lines, one overexpressing integrins (human ovarian carcinoma, A2780) and a second one with a low integrin expression (human prostate cancer, PC3). The in vitro screening was completed with the adhesion behavior to vitronectin. Compound 8 (ST7456CL1) was selected for the in vivo investigation after stability tests over 24h, in PBS solution and in rat plasma, and compared to irinotecan. The former showed a prolonged half-life.

New retinoid derivatives as back-ups of Adarotene.

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models.

Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants.

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs.