María F. Montenegro

The critical role of alpha-folate receptor in the resistance of melanoma to methotrexate

Although methotrexate (MTX) is an effective drug for several types of cancer, it is not active against melanoma. Experiments following methotrexate treatment indicated a reduced accumulation of the drug in the cytosolic compartment in melanoma cells, suggesting that the mechanisms that control the transport and retention of this drug could be altered in melanoma. For this reason, we analyzed the presence and function of folate receptor‐α (FRα) in melanoma cells. In this study, we have identified the presence of FRα in normal and pathological melanocytes and demonstrated that MTX is preferentially transported through this receptor in melanoma cells. FRα‐induced endocytic transport of MTX, together with drug melanosomal sequestration and cellular exportation, ensures reduced accumulation of this cytotoxic compound in intracellular compartments. The critical role of FRα in this mechanism of resistance and the therapeutic consequences of these findings are also discussed.

Cholinesterases are down-expressed in human colorectal carcinoma

Abstract.The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2A) and monomers (G1A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4H and PRiMA-containing G4A AChE forms, besides G4H, G4A and G1H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors.

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg−1, P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg−1, P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg−1). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg−1, P = 0.031). Glycosylphosphatidylinositol‐linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3′‐alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000‐fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10‐fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol‐anchored acetylcholinesterase explains the splicing to acetylcholinesterase‐H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.

Reactivation of the Tumour Suppressor RASSF1A in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation

Background Tumour suppressor genes are often transcriptionally silenced by promoter hypermethylation, and recent research has implicated alterations in chromatin structure as the mechanistic basis for this repression. In addition to DNA methylation, other epigenetic post-translational modifications that modulate the stability and binding of specific transcription factors to gene promoters have emerged as important mechanisms for controlling gene expression. The aim of this study was to analyse the implications of these mechanisms and their molecular connections in the reactivation of RASSF1A in breast cancer. Methods Compounds that modulate the intracellular concentration of adenosine, such as dipyridamole (DIPY), greatly increase the antiproliferative effects of 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin (TMCG), a synthetic antifolate derived from the structure of tea catechins. Quantitative real-time PCR arrays and MALDI-TOF mass spectrometry indicated that this combination (TMCG/DIPY) induced apoptosis in breast cancer cells by modulating the methylation levels of DNA and proteins (such as E2F1), respectively. Chromatin immunoprecipitation (ChIP) assays were employed to confirm that this combination induced chromatin remodelling of the RASSF1A promoter and increased the occupancy of E2F1 at the promoter of this tumour suppressor gene. Results The TMCG/DIPY combination acted as an epigenetic treatment that reactivated RASSF1A expression and induced apoptosis in breast cancer cells. In addition to modulating DNA methylation and chromatin remodelling, this combination also induced demethylation of the E2F1 transcription factor. The ChIP assay showed enhancement of E2F1 occupancy at the unmethylated RASSF1A promoter after TMCG/DIPY treatment. Interestingly, inhibition of E2F1 demethylation using an irreversible inhibitor of lysine-specific demethylase 1 reduced both TMCG/DIPY-mediated RASSF1A expression and apoptosis in MDA-MB-231 cells, suggesting that DNA and protein demethylation may act together to control these molecular and cellular processes. Conclusions/Significance This study demonstrates that simultaneous targeting of DNA and E2F1 methylation is an effective epigenetic treatment that reactivates RASSF1A expression and induces apoptosis in breast cancer cells.

Acetyl-and butyrylcholinesterase activities decrease in human colon adenocarcinoma

Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.

Melanoma coordinates general and cell-specific mechanisms to promote methotrexate resistance.

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current modalities of cancer therapy, including to the drug methotrexate. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mechanisms that contribute to the resistance of melanoma to methotrexate. In addition, other mechanisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This report elucidates how melanoma orchestrates these mechanisms to become extremely resistant to methotrexate, where both E2F1 and checkpoint kinase 1 (Chk1), two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of dihydrofolate in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate expression of dihydrofolate reductase and thymidylate synthase, two E2F1-target genes involved in folate metabolism and required for G(1) progression, favored dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1-mediated apoptosis in melanoma cells.

Melanoma activation of 3-o-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin to a potent irreversible inhibitor of dihydrofolate reductase.

Human melanoma is a significant clinical problem because it is resistant to treatment by most chemotherapeutic agents, including antifolates. It is therefore a desirable goal to develop a second generation of low-toxicity antifolate drugs to overcome acquired resistance to the prevention and treatment of this skin pathology. In our efforts to improve the stability and bioavailability of green tea polyphenols for cancer therapy, we synthesized a trimethoxy derivative of epicatechin-3-gallate, which showed high antiproliferative and proapoptotic activity against melanoma. This derivative, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), is a prodrug that is selectively activated by the specific melanocyte enzyme tyrosinase. Upon activation, TMECG generates a stable quinone methide product that strongly inhibits dihydrofolate reductase in an irreversible manner. The treatment of melanoma cells with TMECG also affected cellular folate transport and the gene expression of DHFR, which supported the antifolate nature of this compound. In addition, its pharmacological efficacy has been confirmed in a mouse melanoma model, in which tumor growth and metastasis were inhibited, significantly enhancing the mean survival of the treated groups. TMECG, therefore, shows a potential for clinical use in melanoma therapy.

Single Heteroatom Fine-Tuning of the Emissive Properties in Organoboron Complexes with 7-(Azaheteroaryl)indole Systems

The application of organoboron compounds as light-absorbing or light-emitting species in areas as relevant as organic electronics or biomedicine has motivated the search for new materials which contribute to the progress of those applications. This article reports the synthesis of four-coordinate boron complexes based on the unexplored 7-(azaheteroaryl)indole ligands. An easy synthetic approach has enabled the fine-tuning of the electronic structure of the organoboron species by modifying a heteroaromatic component in the conjugated system. Furthermore, a comprehensive characterization by X-ray diffraction, absorption and emission spectroscopy, both in solution and in the solid state, cyclic voltammetry, and computational methods has evidenced the utility of this simple strategy. Large Stokes shifts have been achieved in solid thin-films which show a range of emitted light from blue to orange. The synthesized compounds have been used as biocompatible fluorophores in cell bioimaging.

Comparison of a Pair of Synthetic Tea-Catechin-Derived Epimers: Synthesis, Antifolate Activity, and Tyrosinase-Mediated Activation in Melanoma

Despite bioavailability issues, tea catechins have emerged as promising chemopreventive agents because of their efficacy in various animal models. We synthesized two catechin‐derived compounds, 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐catechin (TMCG) and 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin (TMECG), in an attempt to improve the stability and cellular absorption of tea polyphenols. The antiproliferative and pro‐apoptotic activities of both compounds were analyzed with various cancer cell systems, and TMCG, which was easily synthesized in excellent yield, was more active than TMECG in both melanoma and non‐melanoma cell lines. TMCG was also a better inhibitor of dihydrofolate reductase and was more efficiently oxidized by tyrosinase, potentially explaining the difference in activity between these epimers.

Cholinesterase activity in brain of senescence‐accelerated‐resistant mouse SAMR1 and its variation in brain of senescence‐accelerated‐prone mouse SAMP8

The early‐onset, irreversible, severe deficits of learning and memory in the senescence‐accelerated mouse (SAM)‐prone/8 (SAMP8) support its use as an animal model for human dementias of early onset. Possible implication of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in cognitive dysfunction of SAMP8 mice was studied by comparing cholinesterase (ChE) expression in brains of SAMP8 mice and of their normal control, SAM‐resistant/1 (SAMR1) mice. The level of ChE mRNAs was the same in SAMP8 and SAMR1 brains, which agreed with their equal AChE activity (3.09 ± 1.45 vs. 3.07 ± 1.44 μmol·hr−1·mg protein−1, U/mg), but not with a doubled BuChE activity in SAMP8 brain (0.14 ± 0.05 vs. 0.07 ± 0.02 U/mg; P < 0.01). This great increase in neural BuChE activity may contribute to cognitive deficit of SAMP8 mice. Hydrophilic (G  4H , 8%) and amphiphilic (G  4A , 74%) AChE tetramers, besides dimers and monomers (G  2A + G  1A , 18%), were identified in SAMR1 brains. They also contained G  4H BuChE forms (18%) as well as G  4A (53%) and G  2A + G  1A (29%) species. Although SAMP8 brain displayed proportions of AChE and BuChE forms that were similar to those of SAMR1 brain, phenyl‐agarose chromatography with detergent‐free extracts showed a rise in the proportion of secretory G  4H BuChE from 35% in SAMR1 to 44% in SAMP8 brain. The strong immunolabelling of glial fibrillary acidic protein (GFAP), a marker of reactive gliosis, in SAMP8 brain and the consideration of BuChE as a marker of glial cells suggest a relationship between phenotypic changes in neuroglial cells and the excess of BuChE activity in SAMP8 brain.