The thyroid hormone-αvß3 integrin axis in ovarian cancer: regulation of gene transcription and MAPK-dependent proliferation.

Ovarian carcinoma is the fifth common cause of cancer death in women, despite advanced therapeutic approaches. αvß3 integrin, a plasma membrane receptor, binds thyroid hormones (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3) and is overexpressed in ovarian cancer. We have demonstrated selective binding of fluorescently labeled hormones to αvß3-positive ovarian cancer cells but not to integrin-negative cells. Physiologically relevant T3 (1 nM) and T4 (100 nM) concentrations in OVCAR-3 (high αvß3) and A2780 (low αvß3) cells promoted αv and ß3 transcription in association with basal integrin levels. This transcription was effectively blocked by RGD (Arg-Gly-Asp) peptide and neutralizing αvß3 antibodies, excluding T3-induced ß3 messenger RNA, suggesting subspecialization of T3 and T4 binding to the integrin receptor pocket. We have provided support for extracellular regulated kinase (ERK)-mediated transcriptional regulation of the αv monomer by T3 and of ß3 monomer by both hormones and documented a rapid (30-120 min) and dose-dependent (0.1-1000 nM) ERK activation. OVCAR-3 cells and αvß3-deficient HEK293 cells treated with αvß3 blockers confirmed the requirement for an intact thyroid hormone-integrin interaction in ERK activation. In addition, novel data indicated that T4, but not T3, controls integrin's outside-in signaling by phosphorylating tyrosine 759 in the ß3 subunit. Both hormones induced cell proliferation (cell counts), survival (Annexin-PI), viability (WST-1) and significantly reduced the expression of genes that inhibit cell cycle (p21, p16), promote mitochondrial apoptosis (Nix, PUMA) and tumor suppression (GDF-15, IGFBP-6), particularly in cells with high integrin expression. At last, we have confirmed that hypothyroid environment attenuated ovarian cancer growth using a novel experimental platform that exploited paired euthyroid and severe hypothyroid serum samples from human subjects. To conclude, our data define a critical role for thyroid hormones as potent αvß3-ligands, driving ovarian cancer cell proliferation and suggest that disruption of this axis may present a novel treatment strategy in this aggressive disease.

P53 in blind subterranean mole rats--loss-of-function versus gain-of-function activities on newly cloned Spalax target genes.

A tumor suppressor gene, p53, controls cellular responses to a variety of stress conditions, including DNA damage and hypoxia, leading to growth arrest and/or apoptosis. Recently, we demonstrated that in blind subterranean mole rats, Spalax, a model organism for hypoxia tolerance, the p53 DNA-binding domain contains a specific Arg174Lys amino acid substitution. This substitution reduces the p53 effect on the transcription of apoptosis genes (apaf1, puma, pten and noxa) and enhances it on human cell cycle arrest and p53 stabilization/homeostasis genes (mdm2, pten, p21 and cycG). In the current study, we cloned Spalax apaf1 promoter and mdm2 intronic regions containing consensus p53-responsive elements. We compared the Spalax-responsive elements to those of human, mouse and rat and investigated the transcriptional activity of Spalax and human Arg174Lys-mutated p53 on target genes of both species. Spalax and human-mutated p53 lost induction of apaf1 transcription, and increased induction of mdm2 transcription. We conclude that Spalax evolved hypoxia-adaptive mechanisms, analogous to the alterations acquired by cancer cells during tumor development, with a bias against apoptosis while favoring cell arrest and DNA repair.

Vasoactive intestinal peptide and related molecules induce nitrite accumulation in the extracellular milieu of rat cerebral cortical cultures.

Nanomolar concentrations of vasoactive intestinal peptide (VIP), picomolar concentrations of stearyl-norleucine17-VIP (SNV) and femtomolar concentrations of NAPVSIPQ (NAP), an 8-amino-acid peptide derived from the VIP-responsive activity-dependent neuroprotective protein, provide broad neuroprotection. In rat cerebral cortical cultures, 10(-16)-10(-7) M NAP increased intracellular cyclic guanosine monophosphate (cGMP) (2.5-4-fold) and 10(-10) M NAP increased extracellular nitric oxide (NO) by 60%. In the same culture system, VIP and SNV (at micromolar concentrations) increased extracellular NO by 45-55%. The NAP dose required for cGMP increases correlated with the dose providing neuroprotection. However, the concentrations of NAP, SNV and VIP affecting NO production did not match the neuro-protective doses. Thus, NO may mediate part of the cell-cell interaction and natural maintenance activity of VIP/SNV/NAP, while cGMP may mediate neuroprotection.

SNV, a lipophilic superactive VIP analog, acts through cGMP to promote neuronal survival.

The current study explored whether the neuroprotective effects of vasoactive intestinal peptide (VIP) and its analog Stearyl-Nle17-VIP (SNV) were mediated through cGMP. SNV, was previously found to be 100-fold more potent than VIP in providing neuroprotection. Neuronal survival was assessed in rat cerebral cortical cultures. A cGMP antagonist (RP-8-pCPT-cGMPS, 10(-12)-10(-9) M) reduced the number of surviving neurons (40-60%), this decline was spared in the presence of SNV (10(-13)M). A cGMP agonist (Sp-8-pCPT-cGMPS, 10(-14)-10(-8)M) and SNV (10(-16)-10(-8)M) both provided significant neuroprotection against 10(-12) M of the cGMP antagonist. Immunoassays indicated that SNV induced increases in cGMP (two-threefold) in these cultures, whereas VIP was 1000-fold less potent. These results implicate cGMP as a second messenger for VIP/SNV-mediated effects on neuronal survival.

Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: neuroprotective drug design.

The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar-picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N-terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr-Leu-NH2 (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against beta-amyloid toxicity in vitro. Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer's disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases.

Identification of VIP/PACAP receptors on rat astrocytes using antisense oligodeoxynucleotides.

Vasoactive intestinal peptide (VIP) has been shown to be a potent promoter of neuronal survival. Pituitary adenylate cyclase-activating peptide (PACAP), a homologous peptide, shares activity and receptor molecules with VIP. The neuroprotective effects of VIP have been shown to be mediated via astroglial-derived molecules. Utilizing a battery of antisense oligodeoxynucleotides directed against the multiple cloned VIP-preferring (VIP receptors 1 and 2) or PACAP-preferring receptors (six splice variants derived from the same gene transcript), the authors have demonstrated the existence of a specific PACAP receptor splice variant (PACAP4 or hop2) on astrocytes as well as a VIP type2 receptor. The identification of the receptors was achieved by incubation of the cells in the presence of the specific antisense oligodeoxynucleotide followed by radiolabeled VIP binding and displacement. Polymerase chain reaction (PCR) coupled to direct sequencing identified the expression of the PACAP4-hop2 receptor splice variant in astrocytes. Neuronal survival assays were conducted in mixed neuronal-glial cultures derived from newborn rat cerebral cortex. When these cultures were exposed to the battery of the antisense oligodeoxynucleotides, in serum-free media, only the PACAP-specific ones (e.g., hop2-specific) had an effect in decreasing neuronal cell counts. Thus, the VIP neuronal survival effect is mediated, at least in part, via a specific PACAP receptor (containing a unique insertion of 27 amino acids--the hop2 cassette). These data indicate that a hop2-like PACAP/VIP receptor is the receptor that mediates neurotropism.