In vitro differentiation of human neuroblastoma cells induced by sodium phenylacetate

doi:10.1016/0304-3835(93)90069-L

Cancer Letters

Volume 70, Issues 1–2, 15 June 1993, Pages 15-24

https://doi.org/10.1016/0304-3835(93)90069-L Get rights and content

Abstract

Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.

References (27)

T.R. Chen
In situ detection of mycoplasma contamination of cell cultures by fluorescent Hoechst 33258 stain
Exp. Cell Res.
(1977)
K.E. Nichols et al.
Essential amino acid deprivation induces monocytic differentiation of the human HL-60 myeloid leukemia cell line
Blood
(1989)
S. Pahlman et al.
Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbol ester-induced differentiation
Cell Differ.
(1984)
K. Pavelic et al.
Nerve growth factor induced differentiation of human neuroblastoma cells
Int. J. Biochem.
(1987)
J.A. Robson et al.
Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid
Neuroscience
(1985)
D. Sandquist et al.
Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone
Exp. Cell Res.
(1978)
N. Sidell et al.
Effects of retinoic acid on the growth and phenotypic expression of several human neuroblastoma lines
Exp. Cell Res.
(1983)
A. Adam et al.
Ganglioneuroblastoma of the posterior mediastinum: a clinicopathologic review of 80 cases
Cancer
(1981)
R.P. Bolande
Spontaneous regression and cytodifferentiation of cancer in early life: the oncogenic grace period
Surv. Synth. Pathol. Res.
(1985)
J.E. Bottenstein
Differentiated properties of neuronal cell lines

S.W. Brusilow et al.

Treatment of episodic hyperammonemia in children with inborn errors of urea synthesis

N. Engl. J. Med.

(1984)

J. Cinatl et al.

Differentiation arrest in neuroblastoma cell culture

J. Cancer Res. Clin. Oncol.

(1990)

T.C. Everson

Spontaneous regression of cancer

Ann. N.Y. Acad. Sci.

(1964)

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For the evaluation of novel therapies, and for initial in vitro testing of potential in vivo graft-versus-tumour-effects (GvT), cytotoxicity of effector cells against target tumour cells needs to be determined in a reliable fashion. Recently Zimmermann et al. [Zimmermann, S.Y., Esser, R., Rohrbach, E., Klingebiel, T., Koehl, U., 2005. A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemia cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J. Immunol. Methods. 296(1–2), 63–76] introduced a single platform, no-wash flow cytometric assay to quantify natural killer (NK) cell cytotoxicity against leukaemia cells. Here we have optimised this method introducing a novel five-colour flow cytometric assay for the evaluation of NK cell activity against adherent tumour cells, in particular neuroblastoma cells (NB cells). Beside an enhanced cytotoxic activity corresponding to increasing effector/target (E:T) ratios, we could demonstrate an increasing cytotoxicity in a time-dependent manner over a time period of 8 h. The usefulness of this novel method was also confirmed with human tumour cells lines of various other origin including breast and ovarian carcinoma and Wilms tumour cells freshly isolated from a patient after surgery. In addition to flow cytometric analysis, we monitored NK-cell-mediated induction of target cell apoptosis via the caspase cascade in attacked NB cells by fluorescence microscopy after immunofluorescence staining of activated Caspase-3 (Casp-3) in combination with detection of CD45⁺ and CD9⁺ for discrimination between NK and NB cells. In summary, this novel flow cytometric cytotoxicity assay enables efficient quantification of the phenotype of both, effector and adherent target tumour cells, and therefore represents a useful tool for research on immunotherapies that rely on cytotoxic effector cells.
Pharmacological activity of DTPA linked to protein-based drug carrier systems
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Citation Excerpt :
HCMV strain AD169 (ATCC, Manassass, USA) was propagated in confluent HFF maintained in EMEM with 4% FBS. Neuroblastoma cells (UKF-NB-3) [24] and breast adenocarcinoma cells (MCF-7, ATCC, Manassass, USA) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS that contained 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. Cytotoxicity assay.
The chelating agent diethylenetriaminepentaacetic acid (DTPA) inhibits human cytomegalovirus replication. Since chelating agents are known to exhibit anti-cancer effects, DTPA-induced cytotoxicity was evaluated in breast cancer cells (MCF-7) and neuroblastoma cells (UKF-NB-3). DTPA inhibited cancer cell growth in threefold lower concentrations compared to human foreskin fibroblasts (HFF). Antiviral and anti-cancer activity of chelating agents is caused by intracellular complexation of metal ions. DTPA, an extracellular chelator, was covalently coupled to human serum albumin (HSA) molecules, HSA nanoparticles (HSA-NP), gelatin type B (GelB) molecules, and GelB nanoparticles (GelB-NP) to increase cellular uptake. Coupling of DTPA to drug carrier systems increased its cytotoxic and antiviral activity by 5- to 8-fold. Confocal laser scanning microscope examination revealed uptake of DTPA-HSA-NP in UKF-NB-3 cells and HFF. Therefore, coupling of DTPA to protein-based drug carrier systems increases its antiviral and anti-cancer activity probably by mediating cellular uptake.
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Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G₁ arrest in CH27 cells. Western blot analysis of G₁ phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21^CIP1/WAF1. Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G₁-associated Cdks and cyclins and upregulation of p53 and p21^CIP1/WAF1 may contribute to SCK6-mediated G₁-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.
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We investigated the effects of valproic acid (VPA) on the growth and survival of human leukemia cell lines. VPA induced cell death in all of the nine cell lines tested in a dose dependent manner. VPA-treatment induced apoptotic changes in MV411 cells including DNA fragmentation, phosphatidylserine externalization, cytochrome c release from mitochondria, and activation of caspases-3, -8, and -9. A caspase inhibitor, zVAD-FMK, inhibited the DNA fragmentation induced by VPA but not cell death. These findings suggest that VPA exerts an anti-leukemic effect by both caspase-dependent and -independent apoptotic signaling pathways.
Phenylacetate inhibits isoprenoid biosynthesis and suppresses growth of human pancreatic carcinoma
1998, Surgery
Background: Phenylacetate is growth inhibitor for a variety of tumors at concentrations that have been safely achieved in human beings. This antitumor effect is related to inhibition of the isoprenoid synthetic pathway by blocking the enzyme, mevalonate pyrophosphate (MVAPP) decarboxylase. The purpose of this study was to evaluate the effects of phenylacetate on human pancreatic carcinoma. Methods: For in vitro studies, six human pancreatic carcinoma cell lines (BxPc, AsPc, MIAPaCa-2, Panc-1, CFPAC, and HS 766T) were studied. For in vivo studies, nude mice were inoculated with pancreatic cells (BxPc and MIA PaCa-2) and randomized to receive phenylacetate or saline control. Results: Phenylacetate produces reversible in vitro growth arrest at doses of 2.5 to 10 mmol. The antiproliferative effect is cytostatic, producing accumulation of cells in G1, and is not associated with cell toxicity. Systemic treatment of nude mice bearing heterotopic human pancreatic carcinoma results in growth inhibition of tumors without host toxicity. Phenylacetate blocks the processing of mevalonate to isopentenyl-pyrophosphate by inhibiting MVAPP and exhibits suppression of biosynthetic pathways requiring isoprenoids, including cholesterol and dolichol biosynthesis, protein glycosylation, and isoprenylation of proteins. Conclusions: These results indicate that phenylacetate has cytostatic activity in pancreatic carcinoma and support the conclusion that suppression of multiple biosynthetic pathways requiring isoprenoids is contributing to the drug's antiproliferative action. The safety profile and efficacy of phenylacetate make it an attractive agent for the treatment of pancreatic cancer. (Surgery 1998;124:541-50.)
Interferon-γ and retinoic acid down-regulate N-myc in neuroblastoma through complementary mechanisms of action
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The N-myc oncogene plays a key role in the biology of neuroblastoma and the differentiation process. N-myc expression is associated with metastatic disease, as well as the undifferentiated state of normal neuroblasts migrating from the neural crest during embryogenesis. Its down-regulation is a pivotal event in the differentiation of neuroblastoma cells by retinoic acid (RA). Our previous work has shown that RA works synergistically with other agents, such as interferon-γ (IFN-γ), to down-regulate N-myc expression and induce differentiation. The present study demonstrates that IFN-γ, like RA, decreases N-myc transcription. However, functional analysis of N-myc upstream regulatory sequences using 5′ deletion mutants of a promoter-CAT construct containing germ line sequences from nucleotide position −887 to +151 showed that IFN-γ and RA act through different sites on the N-myc promoter. In addition to its transcriptional effect, IFN-γ was also found to shorten the half-life of N-myc mRNA. Taken together, these findings provide a mechanistic basis for the synergistic action of IFN-γ and RA in inducing neuroblastoma differentiation and a rationale for the possible development of combination differentiation therapy for clinical use.

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In vitro differentiation of human neuroblastoma cells induced by sodium phenylacetate

Abstract

Exp. Cell Res.

Blood

Cell Differ.

Int. J. Biochem.

Neuroscience

Exp. Cell Res.

Exp. Cell Res.

Ganglioneuroblastoma of the posterior mediastinum: a clinicopathologic review of 80 cases

Cancer

Spontaneous regression and cytodifferentiation of cancer in early life: the oncogenic grace period

Surv. Synth. Pathol. Res.

Differentiated properties of neuronal cell lines

Treatment of episodic hyperammonemia in children with inborn errors of urea synthesis

N. Engl. J. Med.

Differentiation arrest in neuroblastoma cell culture

J. Cancer Res. Clin. Oncol.

Spontaneous regression of cancer

Ann. N.Y. Acad. Sci.