thymosin and Neuroblastoma

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by β-amyloid (Aβ) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin β4 (Tβ4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tβ4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aβ. Overexpression of Tβ4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tβ4 overexpression group compared to control. Furthermore, overexpression of Tβ4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tβ4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aβ-treated SH-SY5Y cells. We found that Tβ4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tβ4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tβ4 in AD which may open up new therapeutic applications in AD treatment.

Topics: Aged; Alzheimer Disease; Apoptosis; Cell Line, Tumor; Humans; Neuroblastoma; Neuroprotection; Oxidative Stress; Receptor, Serotonin, 5-HT1A; Signal Transduction; Thymosin

Retinoids are an important component of neuroblastoma therapy at the stage of minimal residual disease, yet 40-50% of patients treated with 13-cis-retinoic acid (13-cis-RA) still relapse, indicating the need for more effective retinoid therapy. Vorinostat, or Suberoylanilide hydroxamic acid (SAHA), is a potent inhibitor of histone deacetylase (HDAC) classes I & II and has antitumor activity in vitro and in vivo. Fenretinide (4-HPR) is a synthetic retinoid which acts on cancer cells through both nuclear retinoid receptor and non-receptor mechanisms. In this study, we found that the combination of 4-HPR + SAHA exhibited potent cytotoxic effects on neuroblastoma cells, much more effective than 13-cis-RA + SAHA. The 4-HPR + SAHA combination induced caspase-dependent apoptosis through activation of caspase 3, reduced colony formation and cell migration in vitro, and tumorigenicity in vivo. The 4-HPR and SAHA combination significantly increased mRNA expression of thymosin-beta-4 (Tβ4) and decreased mRNA expression of retinoic acid receptor α (RARα). Importantly, the up-regulation of Tβ4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tβ4 knockdown in neuroblastoma cells increased cell migration and blocked the effect of 4-HPR + SAHA on cell migration and focal adhesion formation. In primary human neuroblastoma tumor tissues, low expression of Tβ4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tβ4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Resistance, Neoplasm; Fenretinide; Humans; Hydroxamic Acids; Neuroblastoma; Thymosin; Vorinostat

Neuroblastoma is the most common malignant solid cancers in early childhood. Overexpression of the proto-oncogene, N-myc, has been reported to be correlated with more malignant course of the disease. Prothymosin alpha, a cellular proliferation-associated gene, is reported to be a target of myc and elevated in several malignant cells and tissues. Expression of prothymosin alpha and N-myc messenger RNAs were evaluated by real-time reverse transcription polymerase chain reaction (RT-PCR) assay in 18 tumor samples from neuroblastoma using LightCycler. The data was analyzed in reference to clinicopathological factors. There was a tendency that higher prothymosin alpha transcripts levels in the tumor samples from younger patients (<1year.) when compared to the older group (>1 year.) (P=0.0845). There was no relationship between prothymosin alpha gene expression and gender (P=0.3029), mass screening case or not (P=0.3007), or stage. The prothymosin alpha mRNA expression levels were correlated with N-myc mRNA levels (P=0.006). Thus we suggest that prothymosin alpha plays an active role as a target of N-myc in neuroblastoma.

Topics: Age Factors; Child; Child, Preschool; Female; Gene Expression; Genes, myc; Humans; Infant; Male; Neuroblastoma; Prognosis; Protein Precursors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thymosin

The myc proto-oncogenes are transcription factors that directly regulate the expression of other genes, by binding to the specific DNA sequence, CACGTG. Among the target genes for c-Myc regulation are ECA39, p53, ornithine decarboxylase (ODC), alpha-prothymosin and Cdc25A. In this study we examined the involvement of c-Myc target genes in human oncogenesis induced by c-myc or N-myc. In MCF-7 breast cancer cells, the induction of c-myc expression by estrogen was followed by the induction of all the Myc targets that we examined, indicating that those genes can serve as c-Myc targets in human oncogenesis. Moreover, in breast tumors exhibiting c-myc overexpression, several Myc targets were also overexpressed. A clear correlation between the expression of c-myc and its targets was also detected in Burkitt's lymphomas, which involve a specific translocation of c-myc gene, but not in other lymphoma cells. Yet, in cells derived from a neuronal origin the pattern of expression of Myc targets was more complex. In a neuroepithelioma cell line that overexpresses c-myc, only some targets were expressed. In addition in neuroblastomas, in which N-myc is amplified and overexpressed, only ODC was overexpressed in all cell lines, while all other target genes were expressed in only some of the cell lines. The more complex expression pattern found for the Myc targets in neuroblastomas suggests that genes that were identified originally as targets for c-Myc regulation may be regulated by N-Myc, but other cell specific factors are also needed for transcription of the target genes.

Topics: Breast Neoplasms; Burkitt Lymphoma; cdc25 Phosphatases; Cell Transformation, Neoplastic; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Ornithine Decarboxylase; Protein Precursors; Protein Tyrosine Phosphatases; Proteins; Proto-Oncogene Proteins c-myc; Thymosin; Transaminases

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.

Topics: Base Sequence; Brain; Calcium-Binding Proteins; Carrier Proteins; DNA Primers; Estradiol; Fetus; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neuroblastoma; Polymerase Chain Reaction; Protein Precursors; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Thymosin; Transcription, Genetic; Tumor Cells, Cultured

To elucidate the contribution of the N-Myc protein to neuroblastomas we have used a synthetic inducible expression system on the basis of the tetracycline repressor of E coli to reversibly express N-myc in a human neuroblastoma cell line in which expression of endogenous N-myc is barely detectable. Like the c-Myc protein, N-Myc up-regulates the expression of both alpha-prothymosin and ornithine decarboxylase. Induction of N-myc increases both the rate of DNA-synthesis and the proliferation rate, and shortens the G1 phase of the cell cycle. A comparison of cell populations in which the presence of N-Myc protein was restricted to different parts of G(zero)/G1 revealed that N-Myc is rate-limiting for cell cycle progression during the first 5 h after serum stimulation of quiescent cells providing direct evidence that Myc-proteins act early after mitogenic stimulation of quiescent cells.

Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; Biopolymers; Cell Nucleus; DNA-Binding Proteins; G1 Phase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mitogens; Neuroblastoma; Ornithine Decarboxylase; Protein Precursors; Proto-Oncogene Proteins c-myc; Resting Phase, Cell Cycle; S Phase; Thymosin; Transcription Factors; Tumor Cells, Cultured

1. Investigations have demonstrated that the gene encoding thymosin beta 10 (a 43-amino acid member of a family of related proteins originally described in the rat immune system) is a target for morphogenic retinoids in both human and rat neuroblastoma cells. 2. Structure-activity studies revealed that the stimulatory actions of retinoids upon the thymosin beta 10 gene reflect the differing affinities of retinoid analogues for a retinoic acid receptor. 3. To examine further the possibility that the trophic actions of retinoic acid upon expression of the thymosin beta 10 gene involved retinoid receptors, neuroblastoma cells were transiently transfected with an expression vector encoding the nuclear retinoic acid receptor (alpha) protein. 4. Northern blot and slot-blot analyses revealed that neuronal cells overexpressing RAR alpha-mRNA exhibited an enhanced sensitivity to exogenous and endogenous retinoic acid in terms of thymosin beta 10 mRNA. Although the RAR-alpha gene was expressed (at low levels) a priori in these neuroblastoma cells, retinoic acid (2 x 10(-7) M for 3 days) slightly stimulated RAR-alpha-mRNA accumulation. 5. Collectively, these findings indicate the retinoic acid receptor (alpha) is regulated by retinoid acid and that the developmentally regulated, retinoid-responsive thymosin beta 10 gene is a target for this nuclear transcription factor in cells derived from the neural crest.

Topics: Animals; Base Sequence; Carrier Proteins; Gene Expression Regulation, Neoplastic; Humans; Liposomes; Molecular Sequence Data; Neoplasm Proteins; Neuroblastoma; Rats; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Retinoids; RNA, Messenger; RNA, Neoplasm; Structure-Activity Relationship; Thymosin; Transfection; Tumor Cells, Cultured

Cyclic AMP can profoundly influence the growth and differentiation of neuronal cells in culture. In this study, the relationship between this second messenger signal transduction pathway, cell differentiation, and the expression of a retinoid-responsive, thymosin beta-10 gene was examined. Thymosin beta-10 and cognate mRNA were expressed at high levels in actively proliferating rat B104 neuroblastoma cells cultured in medium containing 10% FCS. These cells were induced to differentiate in the presence of the cAMP analog N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2-cAMP) (1 mM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 microM). Expression of thymosin beta-10 mRNA was markedly inhibited (greater than 90% and 70%, respectively) by these compounds. Addition of sodium butyrate (NaB, 1 mM) indicated that at least part of the inhibitory actions of Bt2-cAMP were due to esterase-induced release of butyrate from this compound. Adenosine (50 microM), a metabolic precursor to endogenous cyclic AMP, also inhibited accumulation of thymosin beta-10 mRNA (to less than 70% of control levels). The inhibitory action of Bt2-cAMP upon thymosin beta-10 mRNA levels was time dependent; levels were inhibited by greater than 50% 24 hours after addition of the cAMP analog and by greater than 90% after 72 hours. Serum starvation (0.2% FCS for seven days) provoked a marked increase in neurite out-growth; this morphological change was also accompanied by a modest inhibition of thymosin beta-10 mRNA accumulation. These findings together with previous observations imply that both cyclic AMP-dependent and retinoid-responsive mechanisms coordinate thymosin beta-10 gene expression during neuroembryogenesis.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Amino Acid Sequence; Animals; Blood Physiological Phenomena; Bucladesine; Butyrates; Butyric Acid; Cell Differentiation; Cholera Toxin; Cyclic AMP; DNA; Gene Expression Regulation, Neoplastic; Incidence; Molecular Sequence Data; Neuroblastoma; Rats; Retinoids; Second Messenger Systems; Thymosin; Tumor Cells, Cultured

A small acidic polypeptide, termed thymosin beta 10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin beta 10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin beta 10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin beta 10, as well as its homologue gene product, thymosin beta 4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin beta 10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time- and dose-dependent increase in the content of thymosin beta 10, but not that of thymosin beta 4. The increase in thymosin beta 10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin beta 4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on beta-thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin beta 10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin beta 10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.

Topics: Animals; Bucladesine; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Kinetics; Neuroblastoma; Nucleic Acid Hybridization; Rats; RNA, Messenger; Thymosin; Tretinoin; Tumor Cells, Cultured

A cloned thymosin beta-10 cDNA was used to study modulation of thymosin beta-10 mRNA levels in the rat B104 neuroblastoma cell line in response to retinoic acid. Northern blot analysis revealed the presence of a single greater than 600-nucleotide thymosin beta-10 mRNA species that was constitutively expressed in proliferating neuroblastoma cells. Addition of retinoic acid to the culture medium induced a dose- and time-dependent increase in thymosin beta-10 mRNA abundance. Additional studies showed that although thymosin beta-4 and beta-10 are coexpressed in this cell line, the stimulatory action of retinoic acid is specific for the thymosin beta-10 gene. Serum was found to augment the stimulatory action of retinoic acid. Blockade of protein synthesis with cycloheximide abrogated the stimulatory action of retinoic acid upon thymosin beta-10 mRNA accumulation; this observation suggests that activation of the thymosin beta-10 gene in this cell line by retinoic acid is dependent upon the de novo synthesis of a labile protein. Collectively, these findings demonstrate that the developmentally regulated thymosin beta-10 gene is a target for morphogenic retinoids in cells derived from the neural crest.

Topics: Animals; Blood; Blotting, Northern; Cattle; Chromatography, High Pressure Liquid; Culture Media; Cycloheximide; DNA Probes; Gene Expression Regulation, Neoplastic; Kinetics; Neuroblastoma; RNA, Messenger; RNA, Neoplasm; Thymosin; Tretinoin

Human fetal brain expresses high levels of a polypeptide identified by protein biochemistry and molecular cloning as thymosin beta 10. Within the first 18 months after birth, the thymosin beta 10 content of human brain falls to undetectable levels. In order to establish the molecular basis of this process we screened a number of human tumor cell lines derived from the nervous system for the presence of thymosin beta 10. All of the cell line expressed authentic thymosin beta 10. However, in the HTB-10 neuroblastoma, retinoic acid caused a reduction in the level of thymosin beta 10. This effect of the retinoid was conditional upon its continual presence in the tissue culture medium and was not evident in the other cell lines examined. These results suggest that the thymosin beta 10 gene may be a target for retinoids in the developing nervous system.

Topics: Amino Acid Sequence; Base Sequence; Brain; Brain Chemistry; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes; Humans; Infant; Infant, Newborn; Molecular Sequence Data; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Thymosin; Tretinoin; Tumor Cells, Cultured