The Role of Ion Channels and Chemokines in Cancer Growth and Metastasis: A Proposed Mode of Action Using Peptides in Cancer Therapy.

Metastasis (Met) largely contributes to the major cause of cancer deaths throughout the world, rather than the growth of the tumor mass itself. The present report brings together several of the pertinent contributors to cancer growth and metastatic processes from an activity standpoint. Such biological activities include the following: (1) cell adherence and detachment; (2) cell-to-cell contact; (3) contact inhibition; (4) the cell interfacing with the extracellular matrix (ECM); (5) tumor cell-to-stroma communication networks; (6) chemotaxis; and (7) cell membrane potential. Moreover, additional biochemical factors that contribute to cancer growth and metastasis have been shown to comprise the following: (a) calcium levels in the extracellular matrix and in intracellular compartments; (b) cation voltage and ATP-regulated potassium channels; (c) selective and non-selective cation channels; and (d) chemokines (cytokines) and their receptors, such as CXCL12 (SDF-1) and its receptor/binding partner, CXCR4. These latter molecular components represent a promising group of an interacting and synchronized set of candidates ideal for peptide therapeutic targeting for cancer growth and metastasis. Such peptides can be obtained from naturally occurring proteins such as alpha-fetoprotein (AFP), an onco-fetal protein and clinical biomarker.

Alpha-fetoprotein is an autoantigen in hepatocellular carcinoma and juvenile Batten disease.

Failure of immune tolerance leads to production of autoantibodies to self-antigens. The repertoire of autoantibodies detected in cancer patients can indicate the presence of autoimmune disease. Alpha-fetoprotein (AFP) autoantibodies have been found in patients with hepatocellular carcinoma (HCC) and in juvenile Batten disease (BD), a neurodegenerative condition involving autoimmunity. Variant conformational forms of AFP together with exposed occult antigenic determinant sites on the AFP polypeptide resemble the features of a disordered protein which can impair central immune tolerance. These aberrant structural protein forms can lead to the persistence of autoantibody production by immune sensitized B-lymphocytes. Thus, it is not surprising that AFP, a self-antigen, can induce autoimmune responses in humans. Herein, we discuss the molecular and antigenic properties of AFP which make it a disordered protein, and its ability to induce autoantibody production to AFP cryptic epitopes in both HHC and BD patients. Such insights might aid in the future design of AFP-based vaccines and to discovery of novel pathogenic mechanisms of autoimmune diseases which demonstrate the presence of denatured intermediate forms of AFP.

Breast cancer and amyloid bodies: is there a role for amyloidosis in cancer-cell dormancy?

Breast cancer and Alzheimer's disease (AD) are major causes of death in older women. Interestingly, breast cancer occurs less frequently in AD patients than in the general population. Amyloidosis, the aggregation of amyloid proteins to form amyloid bodies, plays a central role in the pathogenesis of AD and other human neuropathies by forming intracellular fibrillary proteins. Contrary to popular belief, amyloidosis is a common occurrence in mammalian cells, and has recently been reported to be a natural physiological process in response to environmental stress stimulations (such as pH and temperature extremes, hypoxia, and oxidative stress). Many proteins contain an intrinsic "amyloid-converting motif", which acts in conjunction with a specific noncoding RNA to induce formation of proteinaceous amyloid bodies that are stored in intracellular bundles. In cancer cells such as breast and prostate, the process of amyloidosis induces cells to enter a dormant or resting stage devoid of cell division and proliferation. Therefore, cancer cells undergo growth cessation and enter a dormant stage following amyloidosis in the cell; this is akin to giving the cell AD to cease growth.

Alpha-fetoprotein and Fanconi Anemia: Relevance to DNA Repair and Breast Cancer Susceptibility.

Elevations of serum alpha-fetoprotein (sAFP) have been reported in fetal and infant states of anemia. Fanconi anemia (FA) belongs to a family of genetic instability disorders which lack the capability to repair DNA breaks. The lesion occurs at a checkpoint regulatory step of the G2 to mitotic transition, allowing FA cells to override cell-cycle arrest. FA DNA repair pathways contain complementation groups known as FANC proteins. FANC proteins form multi-protein complexes with BRCA proteins and are involved in homologous DNA repair. An impaired cascade in these events imparts an increased breast cancer susceptibility to female FA patients. Elevations of sAFP have availed this fetal protein to serve as a biomarker for FA disease. However, the origin of the synthesis of sAFA has not been determined in FA patients. We hypothesize that hematopoietic multipotent progenitor stem cells in the bone marrow are the source of sAFP production in FA patients.

Review of the Third Domain Receptor Binding Fragment of Alphafetoprotein (AFP): Plausible Binding of AFP to Lysophospholipid Receptor Targets.

Alpha-fetoprotein (AFP) is a 69 kD fetal- and tumor-associated single-chain glycoprotein belonging to the albuminoid gene family. AFP functions as a carrier/transport molecule as well as a growth regulator and has been utilized as a clinical biomarker for both fetal defects and cancer growth. Lysophospholipids (LPLs) are plasma membrane-derived bioactive lipid signaling mediators composed of a small molecular weight single acyl carbon chain (palmitic, oleic acid) attached to a polar headgroup; they range in molecular mass from 250-750 daltons. The LPLs consist of either sphingosine-1-phosphate or lysophosphatidic acid, and mostly their choline, ethanolamine, serine or inositol derivatives. They are present only in vertebrates. These bioactive paracrine lipid mediators are ubiquitously distributed in tissues and are released from many different cell types (platelets, macrophages, monocytes, etc.) involved in developmental, physiological, and pathological processes. The LPLs bind to four different classes of G-protein coupled receptors described herein which transduce a multiple of cell effects encompassing activities such as morphogenesis, neural development, angiogenesis, and carcinogenesis. The identification of potential binding sites of LPL receptors on the AFP third domain receptor binding fragment was derived by computer modeling analysis. It is conceivable, but not proven, that AFP might bind not only to the LPL receptors, but also to LPLs themselves since AFP binds medium and long chain fatty acids. It is proposed that some of the activities ascribed to AFP in the past might be due in part to the presence of bound LPLs and/or their receptors.

Does alpha-fetoprotein contribute to the mortality and morbidity of human hepatocellular carcinoma? A commentary.

The fifth most common cancer worldwide is hepatocellular carcinoma (HCC), while being the third leading cause of global cancer-related deaths. Although HCC incidence is less frequent in North America, it is a common malignancy in Asia and Africa associated with a high rate of mortality and morbidity due to ineffective therapies against cancer growth, invasion, and metastasis. It is well established that serum alpha-fetoprotein (AFP) is the "gold standard" biomarker for liver cancer; however, less known are the biological activities of AFP regarding carcinogenesis, growth, proliferation, and metastasis. Clinicians are well aware that increasing AFP serum levels parallel disease progression of HCC patients, but many are less knowledgeable in the lethal growth-promoting properties of AFP as an autocrine stimulator of hepatoma cell proliferation. This commentary addresses the mortality and morbidity concerning AFP in the genesis, growth, progression, and spread of HCC and emphasizes the perilous consequences of AFP-supported growth in human liver cancer even after liver resection and transplantation. Thus, AFP is not just a biomarker for HCC but also an ardent promoter of liver cancer growth and progression.

Route of antigen delivery impacts the immunostimulatory activity of dendritic cell-based vaccines for hepatocellular carcinoma.

BACKGROUND: Dendritic cells (DC) are uniquely equipped to capture, process, and present antigens from their environment. The context in which an antigen is acquired by DC helps to dictate the subsequent immune response. Cancer vaccination promotes antitumor immunity by directing an immune response to antigens expressed by tumors. We have tested the tumor-associated antigen alpha-fetoprotein (AFP) as an immunotherapy target. The majority of hepatocellular carcinomas (HCC) upregulate and secrete this oncofetal antigen. METHODS: To develop cancer vaccines for HCC capable of promoting potent tumor-specific T cell responses, we tested adenovirally-encoded synthetic AFP, with or without its signal sequence, as well as protein forms of AFP and compared intracellular routing and subsequent antigen-specific CD8+ and CD4+ T cell responses. RESULTS: Surprisingly, the secreted form of antigen was superior for both CD4+ and CD8+ T cell activation. We also examined the mechanism through which AFP protein is endocytosed and trafficked in human DC. We identify the mannose receptor (MR/CD206) as the primary uptake pathway for both normal cord blood-derived AFP (nAFP) and tumor-derived AFP (tAFP) proteins. While in healthy donors, nAFP and tAFP were cross-presented to CD8+ T cells similarly and CD4+ T cell responses were dependent upon MR-mediated uptake. In HCC patient cells, tAFP was more immunogenic, and CD4+ T cell responses were not MR-dependent. CONCLUSIONS: Secreted, cytoplasmically retained, and endocytosed forms of AFP utilize unique uptake and processing pathways, resulting in different immunologic responses from the induced antigen-specific CD4+ and CD8+ T cells and between healthy donors and HCC patients. Collectively, these data elucidate pathways of spontaneous and induced anti-tumor immunity in HCC patients to this secreted antigen.

Engineering α-fetoprotein-based gene vaccines to prevent and treat hepatocellular carcinoma: review and future prospects.

Activation of a patient's immune system offers an attractive approach to prevent and treat hepatocellular carcinoma (HCC). However, the antitumor efficacy of current HCC vaccines was weak owing to insufficient immune activation of targeting self/tumor antigens. We recently found that epitope-optimized α-fetoprotein effectively activated CD8 T cells and generated potent antitumor effects in the carcinogen-induced autochthonous HCC mouse model. We predict that the same antigen engineering approach of epitope-optimization will enable us to develop effective human vaccines to prevent HCC recurrence after liver resection. The engineered human HCC vaccines may also allow us to identify high-affinity T-cell receptors and antibodies that can be used to reprogram T cells to treat HCC tumors via adoptive transfer.

Cancer during pregnancy: what is the role of maternal serum and placental biomarkers? A review and commentary.

Cancer during pregnancy, referred to as gestational cancer (GC), is infrequent but can occur in 1.0% of pregnant women. Hepatocellular carcinoma (HCC) is often lethal and is the fifth most common cancer worldwide, while breast adenocarcinoma (breast cancer) is the most common cancer seen during pregnancy. Liver and breast carcinomas are two examples of cancer types that present challenges to the obstetrician due to late and/or delayed diagnosis during pregnancy. Delays in diagnosis limit choices available to physicians regarding surgery, radiation, and chemotherapy. In view of such clinical situations, a role for maternal serum and placental biomarker (MSPB) screening results contributing to cancer diagnosis should be recognized; overlooking such data in GC could result from a lack of knowledge and understanding of MSPB biology, chemistry, and physiology. In this report, obstetricians and perinatologists seeking a diagnosis are urged to take advantage of available results from MSPB screening programs obtained from first- and second-trimester patient data. Using liver and breast cancer as examples, the present review and commentary seeks to demonstrate that MSPB levels, profiles, patterns, and cellular responses could provide foundational data in planning invasive or noninvasive methods and procedures (biopsy, imaging, scans, surgery) to attain a diagnosis as soon as possible in pregnancy. Finally, MSPB epidemiological and cancer risk studies could aid in providing baseline information for decisions regarding GC diagnosis from knowledge of their proposed roles in reducing lifetime risk of malignancies such as breast cancer.

Newborn screening for autism: in search of candidate biomarkers.

BACKGROUND: Autism spectrum disorder (ASD) represents a wide range of neurodevelopmental disorders characterized by impairments in social interaction, language, communication and range of interests. Autism is usually diagnosed in children 3-5 years of age using behavioral characteristics; thus, diagnosis shortly after birth would be beneficial for early initiation of treatment. AIM: This retrospective study sought to identify newborns at risk for ASD utilizing bloodspot specimens in an immunoassay. MATERIALS & METHODS: The present study utilized stored frozen specimens from ASD children already diagnosed at 15-36 months of age. The newborn specimens and controls were analyzed by immunoassay in a multiplex system that included 90 serum biomarkers and subjected to statisical analysis. RESULTS: Three sets of five biomarkers associated with ASD were found that differed from control groups. The 15 candidate biomarkers were then discussed regarding their association with ASD. CONCLUSION: This study determined that a statistically selected panel of 15 biomarkers successfully discriminated presumptive newborns at risk for ASD from those of nonaffected controls.

Review of the putative cell-surface receptors for alpha-fetoprotein: identification of a candidate receptor protein family.

The identification of a receptor for alpha-fetoprotein (AFP) has long been sought in the field of medicine. The uptake and endocytosis of AFP by rat tumor cells in 1984 sparked a series of confirmatory reports and the original studies were then extended to include multiple tumor types in rats, mice, and humans. The following year, French investigators partially characterized the binding properties of the AFP receptor, but they were not able to purify the receptor. It was not until 1991-1992 that an AFP receptor was partially purified and characterized from both human monocytes and breast cancer cells. By 1993, a monoclonal antibody had been raised against the AFP receptor produced from a breast cancer extract with claims that the receptor was a widespread (universal) oncofetal biomarker for cancer. However, that receptor has yet to be cloned and/or purified due to its complex multimeric binding interactions and associations. The present report will review the literature of the multiple putative AFP receptors described to date, the cellular uptake and endocytosis of AFP, and the biochemical characterization of these putative cell-surface proteins. In addition, evidence derived from computer modeling, proteolytic degradation patterns, and amino acid sequence analysis will be presented in a proposed identification of a family of multi-ligand binding receptors; this family fits many, if not most, of the criteria required for an AFP receptor. The purposed receptor protein family is tentatively identified as the Scavenger receptors which comprise several classes of single- and double-pass integral transmembrane proteins. Present data do not support the concept that the AFP receptor is a "universal" tumor receptor and/or biomarker.

Mechanism of Cancer Growth Suppression of Alpha-Fetoprotein Derived Growth Inhibitory Peptides (GIP): Comparison of GIP-34 versus GIP-8 (AFPep). Updates and Prospects.

The Alpha-fetoprotein (AFP) derived Growth Inhibitory Peptide (GIP) is a 34-amino acid segment of the full-length human AFP molecule that inhibits tumor growth and metastasis. The GIP-34 and its carboxy-terminal 8-mer segment, termed GIP-8, were found to be effective as anti-cancer therapeutic peptides against nine different human cancer types. Following the uptake of GIP-34 and GIP-8 into the cell cytoplasm, each follows slightly different signal transduction cascades en route to inhibitory pathways of tumor cell growth and proliferation. The parallel mechanisms of action of GIP-34 versus GIP-8 are demonstrated to involve interference of signaling transduction cascades that ultimately result in: (1) cell cycle S-phase/G2-phase arrest; (2) prevention of cyclin inhibitor degradation; (3) protection of p53 from inactivation by phosphorylation; and (4) blockage of K+ ion channels opened by estradiol and epidermal growth factor (EGF). The overall mechanisms of action of both peptides are discussed in light of their differing modes of cell attachment and uptake fortified by RNA microarray analysis and electrophysiologic measurements of cell membrane conductance and resistance. As a chemotherapeutic adjunct, the GIPs could potentially aid in alleviating the negative side effects of: (1) tamoxifen resistance, uterine hyperplasia/cancer, and blood clotting; (2) Herceptin antibody resistance and cardiac (arrest) arrhythmias; and (3) doxorubicin's bystander cell toxicity.

Alpha-fetoprotein (AFP)-derived peptides as epitopes for hepatoma immunotherapy: a commentary.

The various immunological roles of human alpha-fetoprotein (HAFP), and its correlation with hepatomas, that is, hepatocellular carcinomas (HCCs), are not often addressed together in biomedical reports considering that HAFP is an established biomarker for hepatomas. Studies reporting measurement of HAFP serum levels in hepatoma patients in basic/clinical research settings has greatly increased over the years. Recent reports have now expanded our base knowledge in the mounting of an immune response against AFP, a self antigen, during hepatoma tumorigenesis. Advances in the detection and identification of AFP-derived peptide epitopes are opening new vistas of knowledge regarding the immunological role of AFP-peptides as T cell stimulating antigens in the course of hepatoma growth and progression. The present commentary addresses HAFP-derived peptides as immunologic responders in HCC and their use in the study and generation of AFP-peptide sensitized T cells directed against hepatoma cells. Attempts were further made to relate the AFP-derived peptide epitopes to T cell activities during the course of hepatoma immunotherapies and to profile the traits and properties of the peptides themselves. Hence, the present commentary was divided into two sections; (1) the characterization, properties, and traits of AFP peptide epitopes, and (2) the use of AFP-derived peptides in the therapeutic induction of T cells primed against hepatoma cells using both in vivo and in vitro models.

Physiology of alpha-fetoprotein as a biomarker for perinatal distress: relevance to adverse pregnancy outcome.

The many physiologic roles of human alpha-fetoprotein (HAFP) and its correlation with perinatal distress/pregnancy outcome are rarely addressed together in the biomedical literature, even though HAFP has long been used as a biomarker for fetal birth defects. Although the well being of the fetus can be monitored by the measurement of gestational age-dependent HAFP in biologic fluid levels (serum, amniotic fluid, urine, and vaginal fluids) throughout pregnancy, the majority of clinical reports reflect largely second trimester and (less likely) first trimester testing due to regulatory clinical restrictions. However, reports of third-trimester and pregnancy term measurement of HAFP levels performed in clinical research and/or investigational settings have gradually increased over the years and have expanded our base knowledge of AFP-associated pregnancy disorders during these stages. The different structural forms of HAFP (isoforms, epitopes, molecular variants, etc.) detected in the various biologic fluid compartments have been limited by antibody recognition of specific epitopic sites developed by the kit manufacturers based on antibody specificity, sensitivity, and precision. Concomitantly, the advances in elucidating the various biologic actions of AFP are opening new vistas toward understanding the physiologic roles of AFP during pregnancy. The present review surveys HAFP as a biomarker for fetal distress during the perinatal period in view of its structural and functional properties. An attempt is then made to relate the AFP fluid levels to adverse pregnancy complications and outcomes. Hence, the present review was divided into two major sections: (I) AFP structure and function considerations and (II) the relationship of AFP levels to the distressed fetus during the third trimester and at term.

The alpha-fetoprotein-derived growth inhibitory peptide 8-mer fragment: review of a novel anticancer agent.

This review describes the antigrowth and anticancer activities of the alpha-fetoprotein (AFP)-derived growth inhibitory peptide (GIP) 8-mer fragment. The 8-amino acid peptide (GIP-8) comprises the carboxy-terminal portion of a 34-amino acid peptide (GIP-34) previously identified as an occult epitopic segment of the full-length human AFP molecule. The GIP-8 segment has been chemically synthesized, purified, characterized, and bioassayed. The purified 8-mer segment was characterized as a random coil (disordered) structure extending from a C-terminal beta-hairpin that forms a horseshoe-shaped partially cyclic octapeptide; this structure can be formulated into a fully cyclic form by the addition of asparagine or glutamine residues. The pharmacophore of the octo- and nanopeptide forms is largely composed of a PXXP motif known to interact with Src-3 (SH3) domains of serine/theronine kinases. The GIP-8 has been shown to be growth-suppressive largely in estradiol (E2)-dependent neonatal and tumor-cell proliferation models and to inhibit tumor-cell adhesion to extracellular matrices. The 8-mer GIP displays antigrowth properties in immature mouse uterine cells and anticancer cell proliferation traits in estrogen receptor positive (ER(+)), but not (ER()) negative breast tumor cells. Even though its mechanism of action has not been fully elucidated, GIP-8 has been shown by computer modeling to dock with the extracellular loops of G-coupled seven transmembrane helical-like receptors, which could possibly interfere with signal transduction through MAP kinase pathways. It was apparent that the GIP-8 derived from the 34-mer GIP fragment of HAFP represented an E2-sensitive growth inhibitory motif, which allows the participation in cellular events, such as receptor binding, contact inhibition, extracellular matrix adhesion, angiogenesis, and T-cell activation. Thus, it was proposed that the 8-mer fragment derived from GIP could potentially serve as a lead compound for targeted cancer therapeutic agents of the biologic-response modifier type.

Maternal serum transformed alpha-fetoprotein levels in women with intrauterine growth retardation.

OBJECTIVE: To evaluate maternal serum transformed alpha-fetoprotein (t-AFP) levels in women with intrauterine growth retardation (IUGR). METHODS: 60 pregnant women in two groups were studied: 30 with IUGR and 30 healthy pregnant women as a control group. t-AFP concentrations were determined by ELISA assay. RESULTS: Maternal serum t-AFP levels were higher in women with IUGR than in the control group: 15.39 (10.81-24.01) ng/ml vs. 8.66 (6.22-13.45) ng/ml (p = 0.003). t-AFP levels were even higher in those with fetal hemodynamic redistribution 21.08 (16.02-40.85) ng/ml than in those without 12.15 (10.48-17.45) ng/ml (p = 0.03). CONCLUSIONS: Maternal serum t-AFP is increased in women with IUGR and this elevation is marked in those with fetal hemodynamic redistribution.

Review and proposed action of alpha-fetoprotein growth inhibitory peptides as estrogen and cytoskeleton-associated factors.

The (H) human growth-promoting factor, alpha-fetoprotein (AFP), has been reported to possess a growth inhibitory motif as an occult epitope in the compactly folded circulating form of the protein. Intermediate unfolded forms of the human HAFP molecule induced by stress, shock, and high ligand concentrations have revealed the presence of an encrypted growth-suppressive segment on the third domain of HAFP. A purified linear synthetic 34-mer segment termed the "growth inhibitory peptide" (GIP) exhibits various oligomeric forms with complex aggregation behaviors, in which dominant trimeric forms were found to be suppressive in assays of estrogen-induced growth. While several amino acid analogs of the cysteines of the GIP retained inhibitory activity, heavy metal binding and pre-incubation of the peptides with a variety of cations and hormone ligands were found to influence the outcomes of growth bioassays. Smaller segments of the original 34-mer were each found to display growth activities of their own, with the middle segment (P149b) also showing hydrophobic dye-binding properties. Studies of amino acid sequence identity further revealed that the GIP sequences displayed identity/similarity matches to both cytoplasmic and nucleus-cytoskeleton-associated proteins, and experimental evidence served to support these findings. That is, the peptide was capable of modulating tubulin polymerization, cell shape, and cell-surface aggregation phenomena reminiscent of a microtubule-associated protein. Immunofluorescence studies further pinpointed the localization of the GIP to cytoplasmic regions of high cytoskeletal density in the cell. Because of the involvement of the GIP in experimental models of the estrogen receptor/cytoskeleton, a mechanism of action is forwarded in which the linear GIP is proposed to be a G-coupled receptor binding ligand that is translocated across the plasma membrane via receptor-mediated endocytosis. Thus, it was predicted that the linear GIP and possibly its peptidic segments serve as decoy ligands to cell-surface receptors in order to gain access to the cytoplasmic compartment of the cell.