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 (EGFRpos, EGFRneg) 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 EGFRpos and EGFRneg 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

Correlation of GATA1 occupancy with absolute RP expression levels in distinct stages of mouse and human terminal erythropoiesis.

<p>(A) The absolute mRNA expression levels of RP genes in morphologically distinct stages of mouse terminal erythroid differentiation are shown. GATA1 occupancy in early (Ter119-) and late (Ter119+) erythroid differentiation stages and PU.1 occupancy in mEsEPs cells is also included. (B) The quantification of absolute mRNA levels between GATA1 occupied (red) and non-GATA1 occupied (grey) RP genes consistently shows a significant association of GATA1 binding with higher expressed RP genes (red), despite the overall decline in RP gene expression with erythroid differentiation (grey). (C) The absolute mRNA expression levels of RPs in morphologically distinct stages of human terminal erythroid differentiation and GATA1 occupancy in fetal and adult derived human erythroid cells is shown. (D) The quantification of absolute mRNA levels between GATA1 occupied (red) and non occupied (grey) RPs again shows a consistently significant association of GATA1 binding with higher expressed RP genes (red), despite the overall decline in RP gene expression with erythroid differentiation (grey).</p

ChIP assays of GATA1 and PU.1 binding to the RPS19 proximal promoter region upon MEL cell differentiation.

<p>(A) GATA1 ChIP and (B) PU.1 ChIP of the RPS19 promoter (RPS19 prom) in MEL cells induced to terminally differentiate by treatment with 5mM HMBA. Controls include HS1 in the mouse GATA1 gene locus (GATA1+) and a negative control DNA region (GATA1-) that does not bind GATA1 based on MEL ChIPseq data. PU1+ and PU1- controls include a positive control region (PU.1+) corresponding to the Upstream Regulatory Element (URE) of the PU.1 gene locus and a negative control DNA region (PU.1-) which does not bind PU.1 on the basis of MEL ChIPseq data. No antibody ChIP controls are also shown. (C) Time course of fold-enrichment for GATA1 and PU.1 occupancies of the RPS19 promoter with MEL cell differentiation. Enrichment values for the negative control DNA regions for GATA1 (GATA1- ChIP) and PU.1 (PU1- ChIP) binding are also shown.</p

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

<div><p>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.</p></div

GATA1 and PU.1 ChIPseq occupancies or RP genes in erythroid cells and macrophages.

<p>GATA1 occupancies of large and small subunit RP genes (ChIPseq read density profiles, ±1.5kb around TSS is plotted) in (A) primary mouse fetal liver derived proerythroblasts (negative for Ter119 staining) and mature erythroid cells (positive for Ter119 staining)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref031" target="_blank">31</a>] and (B) in human fetal and adult erythroblasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref043" target="_blank">43</a>]. Note that for comparison, PU.1 occupancies of RP genes in macrophages with or without LPS stimulation are also shown in (A).</p

EMSA of GATA1 and PU.1 binding to the proximal promoter region of the mouse RPS19 gene.

<p>(A) Schematic representation of the mouse RPS19 gene. The translation initiation codon (ATG) was used to designate the transcription start site (TSS) and the transcription factor (TF) binding site positions. The dashed box upstream of the TSS indicates part of the sequenced RPS19 promoter region that is presented in greater detail below. The sequences for the GP (containing both the GATA1 and PU.1 sites) and P (including only the PU.1 site) EMSA probes are underlined, with the GATA1 and PU.1 consensus binding motifs indicated by italics. RPS19 promoter ChIP primers are indicated in bold. (B) EMSA showing PU.1 binding to the RPS19 promoter region. P: EMSA probe spanning the PU.1 predicted binding motif at position -653 of the RPS19 proximal promoter region. Ps: anti-PU.1 supershifted reaction; Pcom: addition of cold competitor probe; Pm: probe with PU.1 binding site mutated. (C) EMSA showing GATA1 and PU.1 binding to the RPS19 promoter region as two distinct protein complexes. GP: probe spanning the predicted PU.1 and GATA1 binding motifs at position -709bp of the RPS19 proximal promoter region. GsP: anti-GATA supershifted reaction; GPs: anti-PU.1 supershifted reaction; GmP: probe with GATA1 binding site mutated; GPm: probe with PU.1 binding site mutated; GmPm: probe with both GATA1 and PU.1 binding sites mutated.</p

GATA1 and PU.1 ChIP of selected mouse homologues of DBA-associated RP genes in MEL cells.

<p>ChIP assays in proliferating (A) and HMBA-differentiated (B) MEL cells. G: ChIP primers for assessing GATA1 binding; P: ChIP primers for assessing PU.1 binding; GP: ChIP primers for assessing GATA1 and PU.1 binding. ChIP primers were designed on the basis of ChIPseq data and their sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.s006" target="_blank">S2 Table</a>.</p

Gene expression fold change profiling of RP genes during human and mouse erythropoiesis in relation to GATA1 occupancy.

<p>(A) Gene expression fold change profiling of RP genes during sequential stages of human erythroid terminal differentiation with GATA1 occupancy in fetal and adult derived erythroid cells. (B) Gene expression fold change profiling of RP genes during sequential stages of mouse erythroid terminal differentiation and GATA1 occupancy in early (Ter119-) and late (Ter119+) differentiating mouse fetal liver cells. (C) Box plots of RP gene expression fold change comparing human and mouse differential expression in sequential erythroid differentiation stages, showing an overall steep decline in RP gene expression in early stages of mouse erythroid differentiation compared to human. The different stages of human and mouse erythroid differentiation were FACS purified and subjected to RNAseq as described in An et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref034" target="_blank">34</a>].</p

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

International audienceThe 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