Nicoletta Fortunati

Valproic acid enhances tubulin acetylation and apoptotic activity of paclitaxel on anaplastic thyroid cancer cell lines

The introduction of paclitaxel into multimodal therapy for anaplastic thyroid carcinoma has failed to improve overall survival. Toxicity rules out the high doses required, especially in older patients. The search for strategies to enhance paclitaxel antineoplastic activity and reduce its side effects is thus advisable. The study aimed to determine whether the histone deacetylase (HDAC) inhibitor valproic acid (VPA) improves the anticancer action of paclitaxel and elucidate the mechanisms underlying the effects of combined treatment. We examined the effect of VPA on the sensitivity to paclitaxel of two anaplastic thyroid carcinoma cell lines (CAL-62 and ARO), and the ability of the drug to determine tubulin acetylation and enhance paclitaxel-induced acetylation. The addition of as little as 0.7 mM VPA to paclitaxel enhances both cytostatic and cytotoxic effects of paclitaxel alone. Increased apoptosis explains the enhancement of the cytotoxic effect. The mechanism underlying this effect is through inhibition of HDAC6 activity, which leads to tubulin hyperacetylation. The results suggest a mechanistic link between HDAC6 inhibition, tubulin acetylation, and the VPA-induced enhancement of paclitaxel effects, and provide the rationale for designing future combination therapies.

The androgen receptor CAG repeat: a modifier of carcinogenesis?

The first exon of the human androgen receptor (AR) contains a translated CAG (poly-glutamine) repeat. The repeat length is polymorphic in the normal population ranging from 8 to 35 repeats. Expansions to over 40 repeats lead to spinal bulbar muscular atrophy (SBMA), a late onset neurodegenerative disease. The repeat is located between the two parts of a bipartite amino-terminal transactivation function and the repeat length, also within in the normal range, is inversely correlated to the transactivation power of the receptor. P160 type co-activators bind more strongly to shorter repeats. A correlation between AR CAG repeat length and total risk, age at diagnosis, recurrence after surgery and aggressive growth has been reported for tumors of classical androgen target tissues. In the prostate, where androgens exert a mitogenic effect, the cancer risk increases with decreasing AR-CAG repeat length. In contrast, in the breast, where the hormone probably acts as anti-mitogen, a higher risk and earlier onset of breast cancer has been reported for carriers of BRCA1 mutations who also have long CAG repeats in the receptor gene. Somatic alterations during carcinogenesis appear to be frequent in endometrial and in colon cancer. In the endometrium the AR CAG repeat prevalently undergoes expansions consistent with the putative protective function of androgens in this tissue. Frequent repeat reductions during colon carcinogenesis would be consistent with a mitogenic effect of androgens. Analysis of AR protein expression by Western blot reveals expression of the AR in healthy and neoplastic colon tissues. Normal mucosa of the colon expresses both AR-isoforms of 110 and 87 kDa, while the tumor samples have lost the expression of the 110-kDa isoform. The 87-kDa isoform is devoid of the amino-terminal portion of the receptor molecule that also contains the poly-glutamine tract. The temporal and causal relation between isoform switch and somatic repeat reductions during colon carcinogenesis is as yet unclear, but the two events could both enhance p160 mediated androgen signaling. The recent finding that smad3 interacts with the AR in a way similar to p160 links the AR to TGFbeta signaling. Interruption of this signaling pathway is a frequent event in colon carcinogenesis.

OXYTOCIN INHIBITS THE PROLIFERATION OF MDA-MB231 HUMAN BREAST-CANCER CELLS VIA CYCLIC ADENOSINE MONOPHOSPHATE AND PROTEIN KINASE A

Oxytocin (OT) inhibits the proliferation of breast‐cancer cells in vitro via a specific G‐coupled receptor. To elucidate the intracellular mechanism involved in this biological effect, different G‐coupled receptor mediators have been investigated in untreated and OT‐treated MDA‐MB231 breast‐carcinoma cells. In these cells, after OT treatment, a significant cAMP increase was observed using a radioimmunoassay procedure, whereas the Ca2+ (determined with the fluorescent probe fura‐2) and the inositol phosphate (determined after cell labeling with myo(2‐3H)‐inositol) concentrations were not modified, contrary to what has been observed in myometrial and myo‐epithelial cells. The PKA inhibitor PKI (6‐22) amide reverted the effect of OT, indicating that the anti‐proliferative effect of the peptide is strictly related to the cAMP–PKA pathway. OT treatment did not modify tyrosine phosphorylation either. Our results indicate that in breast epithelial cells devoid of contractile activity, cAMP is the intracellular mediator of OT action, whereas the Ca2+‐phosphoinositide system is not involved. Int. J. Cancer 72:340–344, 1997.

The receptor-mediated action of sex steroid binding protein (SBP, SHBG): Accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment

The interaction of sex steroid binding protein (SBP) with its specific receptor in MCF-7 cell (estrogen-sensitive human breast cancer cells), followed by the binding of estradiol (E2) to the complex SBP-receptor, induced a significant accumulation of intracellular cAMP. SBP alone as well as E2 alone did not elicit any modification of the nucleotide. The maximal increase in cAMP was observed with 1 nM SBP + 1 nM E2. Increasing doses of both SBP and E2, even raising cAMP levels with respect to basal, did not give any higher response. Both testosterone and dihydrotestosterone, used instead of E2, were not able to induce any significant modification of cAMP. E2-induced MCF-7 cell proliferation was significantly reduced by 8Br-cAMP. MDA-MB 231 cells (estrogen-insensitive breast cancer cells) were not shown to bind SBP, or to respond to SBP + E2 as far as both their proliferation and cAMP content are concerned. In summary, the present study provides evidence that the SBP receptor is part of the G-protein receptor family, and that SBP can act as modulator of E2 action at cell site through the second messenger cAMP.

Modulation by cytokines of glucocorticoid action.

ABSTRACT: Glucocorticoids (GC) are potent modulators of the inflammatory response. Their effects serve to down‐regulate the inflammatory response and are mediated by genomic pathways that follow the interaction with specific receptors (glucocorticoid receptors, GR). Interleukin (IL)‐1, IL‐2, and IL‐6 are able to increase GC secretion by enhancing synthesis and release of CRH and ACTH. Cytokine effects upon steroidogenesis also occur at the adrenal level. The role of cytokines as modulators of GR has received scarce attention. IL‐1 has been shown to up‐regulate GR mRNA expression in hypothalamic CRH secreting cells. On the other hand, macrophage migration inhibitory factor (MIF), a T‐cell product inducible by inflammatory substances including other cytokines, counterregulates GC action within the immune system. Besides immunocytes and neurons, bone cells are a sensitive target for GC and cytokines. We have found that IL‐2 and IL‐6 up‐regulate remarkably the number of GR binding sites and the expression of GR mRNA in peripheral blood mononuclear cells and in osteoblast‐like Saos‐2 cells. Available data suggest that inflammatory cytokines have both direct and indirect effects on GC action at the target level. Autocrine‐induced transcription of GR in immunocytes and/or osteoblasts could be a mechanism that restrains excess cytokine production.

Presence and significance of oxytocin receptors in human neuroblastomas and glial tumors

To determine whether oxytocin (OT) could be added to the list of growth factors acting on neoplastic cells of nervous origin, we investigated the presence of oxytocin receptors (OTR) in human primary neuroblastomas and glioblastomas and related cell lines. OTR were demonstrated both at mRNA level (using a RT‐PCR procedure) and at protein level (using immunocytochemical and immunofluorescence procedures). In order to clarify whether OT exerts any biological effect on these tumors through OTR, we also studied cell proliferation in 3 human neuroblastoma cell lines (SK‐N‐SH, SH‐SY5Y, IMR‐32) and one human anaplastic astrocytoma cell line (MOG‐G‐UVW) treated with OT 1 nM to 100 nM for 48 and 96 hr. At these doses, a dose‐dependent inhibitory effect on cell proliferation was demonstrated. This inhibition was accompanied by a significant increase in the intracellular concentration of cAMP, which we have reported to be the intracellular mediator of the OT anti‐proliferative effect in breast‐carcinoma cell lines. Our data indicate that specific OTR are present in human neuroblastomas and glioblastomas. Through these receptors, OT could inhibit cell proliferation and modulate tumor growth. Int. J. Cancer 77:695–700, 1998.

Sex Hormone-Binding Globulin (SHBG), estradiol and breast cancer

The human serum Sex Hormone-Binding Globulin (SHBG) plays an important role in breast cancer pathophysiology and risk definition, since it regulates the bioavailable fraction of circulating estradiol. We here summarize data reported over the years concerning the involvement of SHBG and SHBG polymorphisms in the definition of breast cancer risk. We also report what is known about the direct action of SHBG in breast cancer cells, illustrating its interaction with these cells and the subsequent initiation of a specific intracellular pathway leading to cross-talk with the estradiol-activated pathway and, finally, to the inhibition of several effects of estradiol in breast cancer cells. In conclusion, as a result of its unique property of regulating the estrogen free fraction and cross-talking with the estradiol pathways, by inhibiting estradiol-induced breast cancer cell growth and proliferation, SHBG is associated with a reduced risk of developing the neoplasm after estrogen exposure.

Estrogen receptor-β is expressed in stromal cells of fibroadenoma and phyllodes tumors of the breast

An estrogen dependency has been suggested for the growth of fibroadenomas: however, thus far, none of the steroid hormone receptors acting on breast tissues has been demonstrated in the stroma of breast fibroepithelial lesions. In this study, the expression of estrogen receptor (ER)-α and -β was investigated by immunohistochemistry in 33 fibroadenomas and in 30 benign, three borderline and seven malignant phyllodes tumors, all with spindle cell growth and in one distant metastasis. In addition, the presence of ER-β mRNA and its variants was evaluated by RT-PCR in microdissected stroma. The possible correlation between hormone receptor expression and differentiation processes of stromal cells was investigated by smooth muscle actin and calponin immunostaining. ER-β was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-β was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-β-positive stromal cells showed expression of smooth muscle actin and/or calponin, as demonstrated by double immunostaining. In addition, the mean age at diagnosis was significantly lower in patients with ER-β-positive vs ER-β-negative fibroadenomas. In contrast, in phyllodes tumors, ER-β expression was higher in older patients. In conclusion, (i) only ER-β is detected in the stroma of fibroadenomas and phyllodes tumors; (ii) its expression correlates with the expression of smooth muscle markers and suggests a role of ER-β in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-β-positive stroma cells, may further indicate a hormone-receptor mechanism involved in regulating the growth of fibroadenomas. Conversely, the older age of patients with ER-β-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.

Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells.

Sex hormone-binding globulin, the plasma carrier for sex steroids, inhibits the estradiol-induced proliferation of breast cancer cells. Estradiol induces cell proliferation triggering multiple mechanisms. Besides regulating growth factors, it activates Erk-1/-2, thus inhibiting apoptosis. In the present study, we investigated the effect of SHBG on estradiol-mediated anti-apoptotic effect in MCF-7 breast cancer cells. As expected, estradiol reduced the number of cells undergoing apoptosis. Although no modification of estradiol action was observed in cells treated contemporarily with estradiol and SHBG, pre-incubation with SHBG before estradiol treatment contrasted the anti-apoptotic effect completely. A mutant form of SHBG, lacking the O-linked oligosaccharide in Thr(7), displayed no such effect. Moreover, SHBG prevented the estradiol-induced phosphorylation of Erk-1/-2, whereas it had no effect on estradiol-induced transcription. Taken together, our observations suggest that the interaction of SHBG with MCF-7 cell membranes causes inhibition of the anti-apoptotic effect of estradiol which might account for SHBGs inhibitory effect on breast cancer cell growth.

Estrogen Receptor α Is a Novel Marker Expressed by Follicular Dendritic Cells in Lymph Nodes and Tumor-Associated Lymphoid Infiltrates

During routine assessment of the hormonal phenotype of breast carcinomas, we detected expression of the estrogen receptor (ER) in the germinal centers of reactive lymphoid follicles surrounding malignant foci. To confirm and extend this finding, we compared ER-alpha, progesterone receptor (PR), and androgen receptor (AR) immunostaining in hyperplastic or metastatic lymph nodes obtained from patients with various pathology, disease location, gender and age. Irrespective of these parameters, we found that: 1) ER-alpha-positive cells were located prevalently in germinal centers, 2) the PR was weakly expressed by cells within and surrounding germinal centers, and 3) the androgen receptor was undetectable. Transcripts for ER-alpha and PR were also detected by reverse transcription-polymerase chain reaction on laser-microdissected lymph node germinal centers. Morphologically, the ER-positive cells resemble dendritic cells and by double immunostaining were found to express both CD21 and CD23, which is characteristic of follicular dendritic cells. Finally, we assessed the effects of Tamoxifen treatment by comparing the numbers of ER-positive follicular dendritic cells in lymph nodes obtained from breast cancer patients before and after treatment. The results show that Tamoxifen treatment generated larger germinal centers with more abundant ER(+)/CD21(+)/CD23(+) cells. Taken together, these results open new perspectives on the effects of sex steroids and their antagonists on the human response in cancer and inflammation.