In this mini-review, we focus on different strategies to bring nanotools specifically to cancer cells. We discuss about a better targeting of tumor, combining the characteristics of tumor environment, the increase in nanoparticles life time, the biomarkers overexpressed on cancer cells and different physical methods for non invasive therapies. Here we detail the necessity of a synergy between passive and active targeting for an actual specificity of cancer cells.

The association of estrogen receptor alpha (ERα) expression with differentiated breast tumors presenting a lower metastasis risk could be explained by the estrogen modulation of cell adhesion, motility and invasiveness. Since desmosomes play a crucial role in cell–cell adhesion and may interfere in tumor progression, we studied their regulation by estrogens in human breast cancer and normal mammary cells. Estrogens increased the formation of desmosomes in normal and malignant cells. Furthermore, four desmosomal proteins (desmocollin, γ-catenin, plakophilin and desmoplakin) appeared significantly up-regulated by estrogens in three ERα-expressing cancer cell lines and this effect was reversed by a pure antiestrogen. Finally, silencing of ERα or desmoplakin expression by specific siRNA revealed that estrogen-modulated desmosomal proteins are essential for the estrogenic control of intercellular adhesion. This estrogen modulation of desmosome formation could contribute to the lower invasiveness of ERα-positive tumors and to the integrity of epithelial layers in estrogen target tissues.

Earlier studies indicated that density-arrested cancer cells released an unidentified growth inhibitor whose secretion was prevented by overexpression of the lysosomal protease cathepsin D (cath D). In this study, this growth inhibitor was purified by affinity chromatography and identified as the heat shock cognate 70 protein (hsc70) based on its peptide microsequencing and specific antibody recognition. Among intracellular proteins, including other heat shock proteins, only constitutive hsc70 was secreted in response to the high-cell density. Moreover, hsc70 secretion from cancer cells was generated by serum deprivation, whereas its cellular concentration did not change. Prevention of Hsc70 secretion by cath D overexpression was associated with the formation of multilayer cell cultures, thus indicating a loss of contact inhibition. In addition, we showed that supplementing the culture medium with purified hsc70 inhibited cell proliferation in the nanomolar range. Conversely, removal of this extracellular hsc70 from the medium by either retention on ADP-agarose or competition at the Hsc70 binding site restored cell proliferation. Hsc70 appears active in human breast cancer cells and hypersecreted by direct cath D inhibition. These results suggest a new role of this secreted hsc70 chaperone in cell proliferation that might account for the higher tumor growth of cancer cells overexpressing cath D.

In the snow crab, Chionoecetes opilio (Brachyura: Majidae), LDH is present in rive isozymic forms, as revealed by the rive coloration zones in zymograms. This is characteristic of a tetrameric LDH enzyme formed by the random association between two distinct sub-units. Isozymes of C. opilio LDH show tissue specificity. No LDH activity is detected in the hepatopancreas. The LDH-B∗ locus is expressed in all other tissues examined, except the eye, in which LDH-A∗ is the only locus detected. Both loci are expressed in nervous, midgut and leg muscle tissues, the last showing individual variability. At least among males, the LDH-B∗ locus is expressed in leg muscle tissue at every developmental stage, while LDH-A∗ locus expression is initiated only in the VIIth post-larval stage, just before the onset of gametogenesis. Results suggest that LDH sub-units in C. opilio have distinct physiological roles. This is very well known in vertebrates, but is virtually undocumented in invertebrates.

The cation-independent mannose 6-phosphate receptor (CI-M6PR) is essential for the endocytosis of proteins bearing the mannose 6-phosphate (M6P) recognition marker. This study described the synthesis of M6P and M6S analogs presenting greater affinity for CI-M6PR than their natural compounds. Moreover, the finding of their lack of cytotoxicity for human cells and of their increased stability in human serum supports the high potential of these isosteric derivatives in therapies requiring CI-M6PR targeting.

Estrogen receptors (ERs) are overexpressed in human breast cancers (BCs) and associated with differentiated tumors and with a more favorable prognosis. Paradoxically, ERs mediate the mitogenic action of estrogens in human BC cells and the efficacy of antiestrogens in adjuvant therapy of primary tumors. The exact mechanism underlying the ER protection against cancer progression to metastasis remains to be investigated. Herein, we show that ERs decrease invasiveness of BC cells. Detailed studies revealed that the unliganded and the E-2-activated ERs decrease cancer cell invasion in vitro through two distinct mechanisms. In the presence of ligand, ER alpha inhibits invasion through a mechanism requiring the functional ER alpha domains involved in the transcriptional activation of target genes. Moreover, using different approaches, we found that cell-cell contacts were markedly increased by 17 beta-estradiol (E-2) treatment and decreased by the pure antiestrogen, ICI182,780. This cell-cell adhesion was associated with an increase of the major intercellular junctions, desmosomes. Conversely, in the absence of ligand, ER alpha also inhibits invasion through a distinct mechanism involving protein-protein interaction with the region of the first zinc finger of ER alpha. The relationship of these data with clinical studies and their potential therapeutic consequetices will be discussed.

Estrogens are mitogenic in human breast cancer cells, but the presence of estrogen receptor alpha (ER alpha) is associated with a favorable prognosis in primary tumors and the molecular basis for this paradoxical relationship remains unknown. Here we show that ER alpha and ER alpha mutants devoid of ligand and DNA-binding domains inhibit cell growth in three-dimensional matrix as well as tumor formation in nude mice. Using in vitro and intracellular approaches, we have found that ER alpha, via its amino acids 184-283, interacts with cyclin-dependent kinase inhibitor p21(WAF1). Both proteins exhibit mutual interactions in the absence of estrogens or in the presence of pure antiestrogen ICI(182,780), whereas estradiol treatment disrupts their interactions. Cross-linking experiments reveal that these proteins are present in a larger complex of approximately 200 kDa that also contains cdk2 and cyclin E. We further demonstrate that the unliganded full-length ER alpha or the variant having the p21(WAF1) interaction region significantly increases p21(WAF1) expression, whereas ER alpha silencing reduces p21(WAF1) levels and silencing of p21(WAF1) is sufficient to prevent ER alpha-induced growth inhibition. Taken together, our results point to an antiproliferative function of the unliganded ER alpha through its physical interactions with p21(WAF1) that may also explain the favorable prognosis of ER alpha-positive breast cancers.

Estrogens are mitogenic in human breast cancer cells, but the presence of estrogen receptor (ER) is associated with a favorable prognosis in pri- mary tumors and the molecular basis for this paradox- ical relationship remains unknown. Here we show that ER and ER mutants devoid of ligand and DNA- binding domains inhibit cell growth in three-dimen- sional matrix as well as tumor formation in nude mice. Using in vitro and intracellular approaches, we have found that ER, via its amino acids 184 -283, interacts with cyclin-dependent kinase inhibitor p21WAF1. Both proteins exhibit mutual interactions in the absence of estrogens or in the presence of pure antiestrogen ICI182,780, whereas estradiol treatment disrupts their interactions. Cross-linking experiments reveal that these proteins are present in a larger complex of 200 kDa that also contains cdk2 and cyclin E. We further demonstrate that the unliganded full-length ER or the variant having the p21WAF1 interaction region signifi- cantly increases p21WAF1 expression, whereas ER si- lencing reduces p21WAF1 levels and silencing of p21WAF1 is sufficient to prevent ER-induced growth inhibition. Taken together, our results point to an antiproliferative function of the unliganded ER through its physical interactions with p21WAF1 that may also explain the favorable prognosis of ER-positive breast cancers.—Maynadier, M., Ramirez, J.-M., Cath- iard, A.-M., Platet, N., Gras, D., Gleizes, M., Sheikh, M. S., Nirde, P., Garcia, M. Unliganded estrogen receptor alpha inhibits breast cancer cell growth through interaction with a cyclin-dependent kinase in- hibitor (p21WAF1). FASEB J. 22, 671- 681 (2007)

The cation-independent mannose 6-phosphate receptor is a multifunctional protein which binds at the cell surface to two distinct classes of ligands, the mannose 6-phosphate (M6P) bearing proteins and IGF-II. Its major function is to bind and transport M6P-enzymes to lysosomes, but it can also modulate the activity of a variety of extracellular M6P-glycoproteins (i.e., latent TGFbeta precursor, urokinase-type plasminogen activator receptor, Granzyme B, growth factors, Herpes virus). The purpose of this review is to highlight the synthesis and potential use of high affinity M6P analogues able to target this receptor. Several M6P analogues with phosphonate, carboxylate or malonate groups display a higher affinity and a stronger stability in human serum than M6P itself. These derivatives could be used to favour the delivery of specific therapeutic compounds to lysosomes, notably in enzyme replacement therapies of lysosomal diseases or in neoplastic drug targeting. In addition, their potential applications in preventing clinical disorders, which are associated with the activities of other M6P-proteins involved in wound healing, cell growth or viral infection, will be discussed.

There is still an ongoing debate concerning the cellular localization of BRCA1 protein in breast cancer. To address this question, we compared the localization of BRCA1 protein using several monoclonal (Ab-1) or polyclonal (C20, D20, I20) antibodies under different technical conditions on human breast cancer cell lines. We worked on the fixation and permeabilization conditions in order to preserve the morphological structures of the cells, as confirmed by transmission electron microscopy studies. As expected from the gene sequence analysis and the biochemical features, both nucleus and cytoplasmic BRCA1 protein staining were detected in cells fixed for 60 min in 4% paraformaldehyde and permeabilized with either 0.3% saponin or 0.02% Triton. In these conditions, the same results were obtained: (i) with the four antibodies tested, (ii) with several dilutions (up to tenfold) of the monoclonal antibody, and (iii) in all the tested breast cancer cell lines. In addition, we validated the functionality of these conditions by quantifying the effects of estrogens and their antagonists on the regulation of BRCA1 protein expression in the MCF7 cell line.

Mutation of a single amino acid in the ligand-binding domain (LBD) of the human androgen receptor (hAR) can induce functional abnormalities in androgen binding, stabilization of active conformation, or interaction with coactivators. The Gly708Ala and Gly708Val substitutions are associated with partial and complete androgen insensitivity syndromes, respectively. In this work, we introduced Ala, Val, and aromatic Phe mutations at position 708 on helix H3 of the hAR-LBD and tested the functional and structural consequences on hAR activity in the presence of steroidal or nonsteroidal agonists and antagonists. The residues involved in the specific recognition of these androgen ligands were identified and analyzed in the light of in vitro biological experiments and the 3D hAR-LBD structure. Our study demonstrated that the Gly708Ala mutation influenced the agonist versus antagonist activity of the ligands and confirmed the crucial role of this residue within the ligand-binding pocket (LBP) in the modulation of androgen agonists. The Gly708Ala mutation transformed the antiandrogen cyproterone acetate (CPA), a partial agonist, into a pure antiandrogen, and the pure nonsteroidal antiandrogen hydroxyflutamide in a partial agonist. From the docking studies, we suggest that CPA acts on AR through the novel mechanism called "passive antagonism".

In humans, spironolactone and mespirenone are well known antimineralocorticoids without C-11beta substituents. These compounds display antagonist properties by acting through the human androgen receptor (hAR). In contrast, we demonstrate here that synthetic mineralocorticoid antagonists bearing hydrophobic C-11beta substituents and C-17gamma-lactone are potent hAR agonists in vitro. The three-dimensional construction of both the ligand binding domain (LBD) of the hAR and the human mineralocorticoid receptor (hMR), based on the crystal structure of the LBD of the human progesterone receptor, revealed not only that the interactions with the steroidal A- and D-rings seemed to be crucial for stabilization of active hMR or hAR conformation, but that other steroidal substitutions could influence the agonist versus antagonist activity of ligands. The docking of synthetic compounds bearing C-11beta hydrophobic substituents within the ligand binding pocket of hAR demonstrated that precise positions of the steroid, such as C-11 and C-17, are in close contact with some residues on the receptor, C-11 with Gly 708 and C-17 with Asn705 and Thr877. These contacts are crucial for the stabilization of the active receptor conformation. Mutation of Asn705 by alanine altered the 11beta-substituted spirolactone-mediated trans-activation function of hAR, suggesting an anchoring of the C-17-lactone carbonyl group (C-22) with this residue. The stabilizing effect of the H12 helix in its active conformation is also induced by hydrophobic contacts between the Gly708 and C-11beta substituents, as recently observed with the A773G-hMR mutant in the presence of similar drugs. The study of the role of these substituents suggests efficient new directions for the drug design of selective androgen agonists.

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.

Although estrogen receptor (ER)-alpha is expressed in both benign and malignant ovarian tumors, the role of ER in ovarian carcinogenesis of epithelial tumors is still unknown, In view of the recent characterization of ER-beta, a second form of ER that seems to be highly expressed in ovaries, we reexamined this issue by studying the relative expression of ER-alpha and -beta in human ovarian tumor progression,We developed a competitive PCR assay based on coamplification of the two ERs in target nucleotide sequences displaying a high homology (exons 3 and 4). Coamplification experiments with varying amounts of plasmids containing ER-alpha and -beta cDNAs showed that this assay was reliable for discriminating as Little as a 2-fold difference in the initial ER-alpha:ER-beta cDNA ratio. The relative expression of ER-alpha compared with ER-beta mRNAs was studied in human ovarian cancer cell lines (n = 5) and in normal ovaries (n = 6), then in human benign and malignant tumor samples including ovarian cysts (n = 24), borderline tumors (n = 3), and cancers (n = 10), In normal ovaries, ER-beta mRNA was the predominant ER form, whereas in ovarian cancer cell lines ER-alpha mRNA was markedly increased as compared with ER-beta. In benign and borderline tumors, ER-beta mRNA was detected in 78% of tumors, whereas ER-alpha mRNA was detected in 29%, In ovarian carcinomas, both ER-alpha and -beta mRNAs were expressed in 80% of tumors. The ER-alpha:ER-beta mRNA ratio was >1 in only one cyst sample (4%), In contrast, the ER-alpha:ER-beta mRNA ratio was markedly increased in ovarian cancers because 60% showed an ER-alpha: ER-beta mRNA >1, In situ hybridization experiments showed overlapping tissular distribution of ER-beta and -alpha expression in cancers and cysts, with a main localization in the epithelium and only a low level of expression in stromal cells.In summary, we found an increase in the ER-alpha:ER-beta mRNA ratio in ovarian carcinomas as compared with normal ovaries and cysts. These data suggest that overexpression of ER-alpha relative to ER-beta mRNA may be a marker of ovarian carcinogenesis.