Pharmacological Development of Target-Specific Delocalized Lipophilic Cation-Functionalized Carboranes for Cancer Therapy.

PURPOSE: Tumor cell heterogeneity and microenvironment represent major hindering factors in the clinical setting toward achieving the desired selectivity and specificity to malignant tissues for molecularly targeted cancer therapeutics. In this study, the cellular and molecular evaluation of several delocalized lipophilic cation (DLC)-functionalized carborane compounds as innovative anticancer agents is presented. METHODS: The anticancer potential assessment of the DLC-carboranes was performed in established normal (MRC-5, Vero), cancer (U-87 MG, HSC-3) and primary glioblastoma cancer stem (EGFR(pos), EGFR(neg)) cultures. Moreover, the molecular mechanism of action underlying their pharmacological response is also analyzed. RESULTS: The pharmacological anticancer profile of DLC-functionalized carboranes is characterized by: a) a marked in vitro selectivity, due to lower concentration range needed (ca. 10 fold) to exert their cell growth-arrest effect on U-87 MG and HSC-3, as compared with that on MRC-5 and Vero; b) a similar selective growth inhibition behavior towards EGFR(pos) and EGFR(neg) cultures (>10 fold difference in potency) without, however, the activation of apoptosis in cultures; c) notably, in marked contrast to cancer cells, normal cells are capable of recapitulating their full proliferation potential following exposure to DLC-carboranes; and, d) such pharmacological effects of DLC-carboranes has been unveiled to be elicited at the molecular level through activation of the p53/p21 axis. CONCLUSIONS: Overall, the data presented in this work indicates the potential of the DLC-functionalized carboranes to act as new selective anticancer therapeutics that may be used autonomously or in therapies involving radiation with thermal neutrons. Importantly, such bifunctional capacity may be beneficial in cancer therapy.

Desmoglein-3/γ-catenin and E-cadherin/ß-catenin differential expression in oral leukoplakia and squamous cell carcinoma.

OBJECTIVE: The purpose of this study was to investigate gene/protein expression alterations of intercellular connections' components in oral leukoplakia (OLs) and squamous-cell carcinoma (OSCCs). MATERIALS AND METHODS: Expression of desmogleins-2,3 (Dsg2/Dsg3), E-cadherin, and their cytoplasmic ligand, ß/γ-catenins were quantitatively assessed in HSC-3 cells growing as monolayer cultures (ML)/multicellular aggregates (MCAs), using RT-PCR/Western blot, whereas their localization was detected by immunofluorescence. Furthermore, their expression was semi-quantitatively investigated in tissues from 25 OLs/25 OSCCs, using automated immunohistochemistry. RESULTS: The steady-state levels of Dsg3 RNA transcripts increased as HSC-3 cells enter their exponential phase of growth, before a dramatic decrease to be observed as cells reached their plateau phase especially in MCAs. Upon the same period of time, Dsg2 levels have been increased. The expression of γ-catenin but not that of ß-catenin was increased after 48 h in both MLs and MCAs. In clinical samples, Dsg3, Ε-cadherin, ß/γ-catenin down-regulation was observed to be associated with the grade of OLs-dysplasia and OSCCs. Importantly, a membrane-to-cytoplasmic switch of expression and strong perinuclear aggregation of Dsg3/γ-catenin was seen in both HSC-3 cells and OLs/OSCCs. CONCLUSIONS: The altered expression of Dsg3/γ-catenin and E-cadherin/ß-catenin, in vitro and in ODs/OSCC imply their involvement in growth regulation and phenotype of dysplastic/malignant oral epithelial cells, contributing to the better understanding of epithelial dysplasia and OSCCs. CLINICAL RELEVANCE: The observed alterations of their expression suggest a role of Dsg3 and γ-catenin (additionally to E-cadherin/ß-catenin) as biomarkers of malignant transformation risk of oral dysplasia and the biological behavior (aggressiveness) of oral cancer, respectively.

Possible interaction between B1 retrotransposon-containing sequences and ß(major) globin gene transcriptional activation during MEL cell erythroid differentiation.

Repetitive sequences consist of >50% of mammalian genomic DNAs and among these SINEs (short interspersed nuclear elements), e.g. B1 elements, account for 8% of the mouse genome. In an effort to delineate the molecular mechanism(s) involved in the blockade of the in vitro differentiation program of MEL (murine erythroleukaemia) cells by treatment with methylation inhibitors, we detected a DNA region of 559 bp in chromosome 7 located downstream of the 3'-end of the ß(major) globin gene (designated B1-559) with unique characteristics. We have fully characterized this B1-559 region that includes a B1 element, several repeats of ATG initiation codons and consensus DNA-binding sites for erythroid-specific transcription factors NF-E2 (nuclear factor-erythroid-derived 2), GATA-1 and EKLF (erythroid Krüppel-like factor). Fragments derived from B1-559 incubated with nuclear extracts form protein complexes in both undifferentiated and differentiated MEL cells. Transient reporter-gene experiments in MEL and human erythroleukaemia K-562 cells with recombinant constructs containing B1-559 fragments linked to HS-2 (hypersensitive site-2) sequences of human ß-globin gene LCR (locus control region) indicated potential cooperation upon erythropoiesis and globin gene expression. The possible interaction between the B1-559 region and ß(major) globin gene transcriptional activation upon execution of erythroid MEL cell differentiation programme is discussed.

Comparison of different zeolite framework types as carriers for the oral delivery of the poorly soluble drug indomethacin.

Microporous zeolites of distinct framework types, textural properties and crystal morphologies namely BEA, ZSM and NaX, have been employed as carriers to assess their effect on modulating the dissolution behavior of a BCS II model drug (indomethacin). Preparation of the loaded carriers via the incipient wetness method induced significant drug amorphization for the BEA and NaX samples, as well as high drug payloads. The stability of the amorphous drug content was retained after stressing test evaluation of the porous carriers. The dissolution profile of loaded indomethacin was evaluated in simulated gastric fluid (pH 1.2) and simulated intestinal fluids FaSSIF (fasted) and FeSSIF (fed state) conditions and was found to be dependent on the aluminosilicate ratio of the zeolites and the degree of crystalline drug content. The feasibility of the zeolitic particles as oral drug delivery systems was appraised with cytocompatibility and cellular toxicity studies in Caco-2 cultures in a time- and dose-dependent manner by means of the MTT assay and flow cytometry analysis, respectively. Intracellular accumulation of the zeolite particles was observed with no apparent cytotoxic effects at the lower concentrations tested, rendering such microporous zeolites pertinent candidates in oral drug delivery applications.

PLGA/DPPC/trimethylchitosan spray-dried microparticles for the nasal delivery of ropinirole hydrochloride: in vitro, ex vivo and cytocompatibility assessment.

In the present study we investigated polymer-lipid microparticles loaded with ropinirole hydrochloride (RH) for nasal delivery. RH microparticles were further evaluated by means of scanning electron microscopy (SEM), ζ-potential measurements, Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). In vitro release studies were performed in simulated nasal electrolyte solution (SNES) pH5.5 at 35°C. Ex vivo permeation studies were conducted across sheep nasal mucosa. Cytocompatibility was tested in cultured human airway epithelial cells (Calu-3). SEM studies revealed spheroid microparticles in the range of 2.09µm to 2.41µm. The presence of trimethylchitosan (TMC) induced a slight shift towards less negative ζ-potential values. Surface chemistry (XPS) revealed the presence of dipalmitoylphospatidylcholine (DPPC) and poly(lactic-co-glycolic acid) (PLGA) onto microparticles' surface, further corroborating the FT-IR and XRD findings. In vitro release studies showed that the microparticle composition can partly modulate the release of RH. Ex vivo studies demonstrated a 2.35-folded enhancement of RH permeation when RH was co-formulated with TMC of low molecular weight, compared to the control. All formulations tested were found to be non-toxic to cells. The results suggest that polymer-lipid microparticles may be a promising carrier for the nasal delivery of RH.

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies.

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies.

Development of new drug delivery system based on ordered mesoporous carbons: characterisation and cytocompatibility studies.

Ordered mesoporous carbons that encapsulate the poorly soluble compounds ibuprofen and indomethacin were systematically studied by means of X-ray diffraction (XRD), differential scanning calorimetry (DSC) and X-ray photon electron spectroscopy (XPS). The results showed marked differences in the release profiles of the two drug molecules in simulated gastric fluids. In vitro toxicity profiles appear to be compatible with potential therapeutic applications bringing them to the forefront as carriers of poorly water soluble drugs.

Cloning and characterization of polyA- RNA transcripts encoded by activated B1-like retrotransposons in mouse erythroleukemia MEL cells exposed to methylation inhibitors.

We have previously identified a DNA silent region located downstream of the 3'-end of the ß(major) globin gene (designated B1-559) that contains a B1 retrotransposon, consensus binding sites for erythroid specific transcription factors and shares the capacity to act as promoter in hematopoietic cells interacting with ß-globin gene LCR sequences in vitro. In this study, we have cloned four new non-polyA RNA transcripts being detected upon blockade of murine erythroleukemia (MEL) cell differentiation to erythroid maturation by methylation inhibitors and demonstrated that two of them share high structural homology with sequences of B1 element found within the B1-559 region. Although it is not clear yet whether and how these RNAs interfere with induction of erythroid maturation, these data provide evidence for the first time showing that methylation inhibitors can activate silent repetitive DNA sequences in MEL cells and may have implications in cancer chemotherapy using demethylating drugs as antineoplastic agents.

Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring.

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines (13-17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19-22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24-27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35-38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound.