Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells.

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-ß-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not p-STAT3 (Y705) and p-STAT3 (S727), in the nucleus of HK-1 cells. Pre-treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl-xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p-STAT3 in the poorly differentiated C666-1 cells. Pharmacological inhibition of STAT3 or Bcl-2/xL resulted in a decrease in endoreduplication of C666-1 cells. Taken together, the expression of p-STAT5 and p-STAT3 was upregulated in 2ME2-induced endoreduplicating HK-1 and C666-1 cells, respectively. Combination of 2ME2 with Bcl-2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.

Ginsenoside-Rb1 promotes adipogenesis through regulation of PPARγ and microRNA-27b.

Ginsenoside-Rb1 (Rb1), one of the bioactive components in ginseng extract, is recently reported to be able to promote adipogenesis and peroxisome proliferator-activated receptor gamma (PPARγ) expression. Meanwhile, microRNA-27b (miR-27b) is also identified to regulate adipogenesis by targeting PPARγ2. In the present study, we attempted to link up the Rb1-promoted adipogenesis with PPARγ binding and miR-27b regulation. First, we demonstrated that GW9662, an antagonist of PPARγ, could block Rb1-induced 3T3-L1 differentiation with little toxicity towards cell proliferation. Then, expression levels for both of miR-27b and its primary transcript, pri-mir-27b, were found to decrease upon Rb1 treatment. Again, GW9662 could attenuate the inhibitory effect of Rb1 on both miR-27 and pri-mir-27b expression. Since Rb1 was demonstrated to have binding activity towards PPARγ, we thus speculate that Rb1 may act though PPARγ to downregulate mir-27b gene transcription and mature miR-27b activity, which in turn promotes PPARγ expression and adipogenesis. Enhancement on adipogenesis of adipose tissues is expected to prevent lipotoxicty in nonadipose tissues. Our data may give a better illustration to explain the antidiabetic effect of Rb1 and provide a hint on treatment of lipid related metabolic diseases in the future.

Role of mitogen-activated protein kinase in Zn-BC-AM PDT-induced apoptosis in nasopharyngeal carcinoma cells.

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn-BC-AM was found to effectively induce apoptosis in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 cell line. Sustained activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) as well as a transient increase in activation of extracellular signal-regulated kinase (ERK) were observed immediately after Zn-BC-AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT-induced apoptosis of HK-1 cells. PD169316 also prevented the loss of Bcl-2 and Bcl-xL in PDT-treated HK-1 cells. However, inhibition of JNK with SP600125 had no effect on Zn-BC-AM PDT-induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn-BC-AM PDT-induced apoptosis. Further study showed that knockdown of the p38beta isoform with siRNA also increased Zn-BC-AM PDT-induced apoptosis, indicating that the anti-apoptotic effect of PD169316 in PDT-treated HK-1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38beta and ERK may enhance the therapeutic efficacy of Zn-BC-AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution.

Ecotoxicological study on sediments of Mai Po marshes, Hong Kong using organisms and biomarkers.

Sediments from Mai Po Ramsar site, Hong Kong were in general shown to be highly toxic based on the results of four toxicity tests (Microtox solid-phase test, Daphnia mortality test, algal [Microcystis aeruginosa] growth inhibition test and ryegrass [Lolium perenne] seed germination/root elongation test). Sediment of the mudflat (which is open to Deep Bay, i.e., the pollution source) was the most toxic while sediment of gei wai 24g (an enclosed freshwater pond) was the least toxic. Results of biomarker studies (tilapia hepatic metallothionein; glutathione (GSH) and EROD activity using H4IIE rat hepatoma cell) were also concordant with those in the toxicity tests. Significant liner relationships (p<0.01) were found between GSH contents in the rat hepatoma cells and PAHs, OCPs contents in the sediment extracts. It is recommended that the present suite of bioassays is useful and is biologically relevant for future ecotoxicological studies focusing on similar wetlands.

MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation.

Benzo[a]pyrene (BaP) is a potentially genotoxic and cytotoxic environmental pollutant. Previous studies showed that exposure of HepG(2) cells to BaP causes necrotic cell death [Lin, T., Yang, M.S., 2007b. Cell death induced by benzo[a]pyrene in the HepG(2) cells is dependent on PARP-1 activation and NAD depletion. Toxicology 245, 147-153]. In the present study, the signaling pathways associated with this response was studied. BaP induced accumulation and activation of p53 in HepG(2) cells, which occurred as early as 12h after exposure. Activation of p53 was evidenced by its phosphorylation at serine 15 (Ser15) and acetylation at lysine 382 (Lys382). Chemical inhibition and siRNA-mediated knockdown of p53 expression suppressed its phosphorylation as well as cell death. BaP also activated p38 MAPK and ERK, but not JNK, at 6h after exposure. SB203580 and PD98059, specific inhibitors of p38 MAPK and ERK, respectively, suppressed phosphorylation of p53 at Ser15, but the accumulation of p53 was only moderately reduced. Acetylation of p53 at Lys 382 was not affected by these inhibitors, suggesting that acetylation stabilizes p53 in response to DNA damage. SB203580 and PD98059 prevented downstream energy failure and BaP-induced cell death. Similar results were obtained with siRNA against two isoforms of p38 MAPK, p38alpha and p38beta. Wortmannin, selective inhibitor of DNA-PK and ATM/ATR, abolished p53 phosphorylation, indicating an involvement of multiple pathways of p53 phosphorylation upon exposure to BaP. In summary, the current study demonstrated that both MAPK and p53 activation are required for BaP-induced necrotic cell death. The results also provide a novel model for studying the regulation between p53 and p38 MAPK in the progression of cellular necrosis.

Angiomodulatory and neurological effects of ginsenosides.

Panax ginseng C.A. Meyer, one of the most popular and valued herbs, has been used extensively in traditional Chinese medicine for thousands of years. More than thirty ginsenosides, the pharmacologically active ingredients in ginseng, have been identified with various sugar moieties attached at the C-3, C-6 and C-20 positions of the steroidal skeleton. We herein review the current literature on the pharmacological effects of ginsenosides on the modulation of angiogenesis, dysregulations of which contribute towards many pathological conditions. Regarding the adaptogenic property of ginseng, the effects of ginsenosides on central nervous system are also discussed. Recent researches have pointed to the steroid hormone receptors as the target molecules to elicit the diverse cellular and physiological activities of ginseng. We believe that understanding the interaction between ginsenosides and various steroid hormone receptors may provide clues to unravel the secret of ginseng.

Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity.

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinson's disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth.

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen beta receptor.

BACKGROUND AND PURPOSE: Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside-Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti-angiogenic protein, pigment epithelium-derived factor (PEDF), is involved in regulating the anti-angiogenic effects of Rb1. EXPERIMENTAL APPROACHES: Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER). KEY RESULTS: Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERbeta. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERbeta, but not ERalpha. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERbeta siRNA, indicating ERbeta dependence. CONCLUSIONS AND IMPLICATIONS: Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERbeta. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy.

Studies on the anti-tumor activities of the soy isoflavone daidzein on murine neuroblastoma cells.

Daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone) are two major isoflavones found predominantly in soy beans, as well as in certain traditional Chinese medicinal herbs and tea leaves. In the past decade, there have been extensive studies on the anti-tumor effects of genistein on cancers of the breast, prostate and colon in humans. However, the anti-tumor effects of daidzein on neuronal cancer cells and its action mechanisms remain poorly understood. In this study, daidzein was shown to inhibit the proliferation of a number of murine and human neuroblastoma cell lines in vitro. Using the murine neuroblastoma Neuro-2a (BU-1) cells as the cell model, daidzein was also found to prevent the cell cycle progression to G2/M phase and induced apoptosis of the neuronal tumor cells, as measured by flow cytometry and gel electrophoresis for fragmented DNA respectively. Taken together, our results showed that daidzein could exert pleiotropic effects on the murine neuroblastoma cells, including inhibition of cell proliferation, modulation of cell cycle check point regulation, and triggering of neuronal cell apoptosis.

The angiosuppressive effects of 20(R)- ginsenoside Rg3.

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.

Bifunctional modulating effects of an indigo dimer (bisindigotin) to CYP1A1 induction in H4IIE cells.

In this study, we measured and characterized the bifunctional effects of a newly identified natural compound-bisindigotin (SLY-1), isolated from leaf extracts of Isatis indigotica, to CYP1A1/EROD activities in H4IIE cells. The compound, SLY-1 (1muM) elicited a transitory and significant induction of CYP1A1 RNA/protein levels and EROD activities in the cells. Maximum levels of CYP1A1 expression and EROD induction were attained at 8 and 12h of post-treatment, respectively. Thereafter the induction decreased significantly. Similar profile of CYP1A2 and CYP1B1 mRNA induction was observed. In contrast TCDD elicited CYP1A1/EROD induction was persistent. The transitory effect by SLY-1 is most likely due to the clearance of SLY-1 by cellular metabolism. Taken together the observation indicated that SLY-1 is an Ah receptor agonist for CYP1A1/CYP1A2/CYP1B1/EROD induction. Interestingly in the TCDD/SLY-1 cotreatment study, although synergistic effects on CYP1A1 expression and EROD induction were observed at 4-8h, significant inhibitory effects to TCDD induced CYP1A1 protein and EROD activity were detected at 12-24h of post-treatment. Because there was no significant reduction of CYP1A1, CYP1A2 or CYP1B1 transcript levels between TCDD- and TCDD/SLY-1 treated cells, the data pointed to the translational and/or post-translational inhibitory effect. The cellular signal transduction system may be modulated following exposure to SLY-1. To investigate the possible mechanisms involved, various specific kinase inhibitors or activators (chelerythrin, PD98059, U0126, ZM336372, SB202190, PKA inhibitor PKI (6-22) amide, and dbcAMP) were used for the assessment. Chelerythrine, PD98059 or dbcAMP treatment in TCDD induced cells showed significant inhibitory effects on CYP1A1 mRNA/protein expressions and EROD activities. U0126 had no observable EROD inhibitory effect. ZM336372 or SB202190 showed inhibition only at EROD activities. The results indicated that the SLY-1 inhibitory effect was possibly not mediated by the cAMP/PKA, PKC or MEK pathways. Nevertheless our results indicate that SLY-1 is not only an inducer of the CYP1A1 system, but also a potent inhibitor of CYP1A1 enzyme.

Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells.

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2µM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and beta-chemokines (MIP-1ß, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

Energy and redox States in the c6 glioma cells following acute exposure to zn, se(+4), and se(+6) and the correlation with apoptosis.

Changes in cellular energy and redox states in the C6 glioma cells exposed to increasing concentrations of either Zn or Se were studied to examine whether different elements cause different patterns of changes in cellular metabolism. Following a 3-h exposure, both Zn and Se(+4) caused dose-dependent decreases in cell viability and total adenosine nucleotides (TAN = ATP + ADP + AMP). In addition, Zn caused a dose-dependent increase in cellular ATP/TAN and a decrease in the ADP/TAN and AMP/TAN. These changes resulted in a significant increase in energy charge potential (ECP = [ATP + 0.5ADP]/TAN). Se(+4), on the other hand, caused a dose-dependent decrease in ATP/TAN but an increase in both ADP/TAN and AMP/TAN, resulting in a dose-dependent decrease in ECP. Both Zn and Se(+4) caused a dose-dependent decrease in GSH/GSSG and an increase in GSH + GSSG when compared to TAN. In contrast to Zn and Se(+4), the nontoxic Se(+6) caused no significant changes in cellular energy states but reduced the GSH/GSSG ratio from 3.14 +/- 0.49 to 2.05 +/- 0.29, which could be explained by the effect of Se on enzymes responsible for GSH metabolism. As the cellular ATP level has been considered an important element that mediates the mode of cell death, it was suggested that a significant increase in ATP/TAN upon exposure to Zn would indicate that cell death occurred via apoptosis, while Se(+4) caused a different pattern of cell death. This was confirmed by the appearance of cells with fragmented nucleus in cells treated with Zn, but not Se(+4) and Se(+6). The results demonstrated that different chemicals caused different patterns of metabolic changes. The correlation between metabolic changes and the mode of cell death was discussed.

Detection of differential expression of mouse interferon-alpha subtypes by polymerase chain reaction using specific primers.

Specific primers for nine mouse interferon-alpha (IFN-alpha) subtypes, namely, IFN-alpha1, IFN-alpha1-9, IFN-alpha2, IFN-alpha4, IFN-alpha5, IFN-alpha7, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB, were designed and evaluated on Poly(I).Poly(C)-induced and influenza virus-infected L929 cells. Specificity of the primers was confirmed in a cross-polymerase chain reaction (cross-PCR). IFN-alpha1, IFN-alpha1-9, IFN-alpha4, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB were found to be induced in L929 cells 6-9 h after Poly(I).Poly(C) treatment. The amplification of a particular subtype was not biased in the presence of excess of other templates. Differential expression of the IFN-alpha subtypes was observed in influenza A/NWS/33- and B/Lee/40-infected L929 cells. A/NWS/33 virus was found to upregulate the gene expression of IFN-alpha1, IFN-alpha4, IFN-alpha6/8, IFN-alpha11, and IFN-alphaB in L929 cells as early as 6 h after infection. In B/Lee/40-infected L929 cells, only IFN-alpha4 was upregulated. Our results suggest that the designed primers will serve as a useful tool in analyzing the expression of IFN-alpha subtypes in various systems and hence for the evaluation of their function.

Distinguishing between mouse IL-3 and IL-3 receptor-like (IL-5/GM-CSF receptor converter) mRNAs using the polymerase chain reaction method.

A set of primers (MF43, MF44 and MF45) were designed and used in the polymerase chain reaction to distinguish between the expression of mouse IL-3 receptor and mouse IL-3 receptor-like mRNAs. Primers MF43 and MF45 were specific for IL-3 receptor mRNA while the primers MF44 and MF45 were specific for IL-3 receptor-like mRNA. Primers MF44 and MF45 could not amplify IL-3 receptor cDNA even at an annealing temperature of 46 degrees C which is 20 degrees C below the melting temperature of the primers, or at high template concentrations (up to 100 ng cDNA). The optimal range of Mg2+ concentrations for the two pairs of primers MF43, MF45 and MF44, MF45, were essentially the same and this permits comparisons of the expression level of these two mRNAs under identical PCR conditions. Both the IL-3 receptor and IL-3 receptor-like mRNAs could be detected in normal bone marrow cells and IL-3-dependent cell lines (FDC-P1 and 32D cl-23), as well as in the IL-3 independent cell lines P388D1 and WEHI-3B, the latter being a constitutive producer of IL-3. In contrast, neither species of mRNA was detected in the T lymphoma cell line (EL-4). The ratio of IL-3 receptor-like mRNA to IL-3 receptor mRNA was usually greater than 1, except in 32D cl-23 cells where it was 0.66.

Cellular uptake, subcellular localization and photodamaging effect of temoporfin (mTHPC) in nasopharyngeal carcinoma cells: comparison with hematoporphyrin derivative.

Temoporfin (meta-tetra (hydroxyphenyl)chlorin; mTHPC) potentiated a 100-fold higher cytotoxic effect than hematoporphyrin derivative (HPD) on two nasopharyngeal carcinoma cell lines (HK1 and CNE2) in terms of the overall photodynamic therapy (PDT) dose. The cellular uptake, evaluated by flow cytometry and spectrophotometry demonstrated that mTHPC exhibited higher uptake ability than HPD. Confocal laser scanning microscopy detection for both the sensitizer and mitochondria probe on the same cell images revealed that both drugs accumulated diffusely in the cytoplasm and that mitochrondria is a target organelle. Photo-activation ruptured the mitochrondria, with more pronounced mitochondrial damage being observed in mTHPC-PDT course. This correlated well with the cell photokilling efficiency of mTHPC.

Effects of dibutyryl cAMP on stanniocalcin and stanniocalcin-related protein mRNA expression in neuroblastoma cells.

Stanniocalcin is a polypeptide hormone that was first reported in fish as a regulator of mineral metabolism. Its recent identification in mammals has opened a new area of investigation in basic and clinical endocrinology. In the present study, regulation of the stanniocalcin (STC) and stanniocalcin related protein (STCrP) genes were investigated in mouse neuroblastoma cells (Neuro-2A) in relation to neuronal cell differentiation. Neuro-2A is an undifferentiated cell line that contains measurable levels of STCrP mRNA, but undetectable levels of STC mRNA. Treatment of the cells with either dbcAMP (1-4 mM) or 50 microM euxanthone (PW1) resulted in extensive differentiation and neurite outgrowth. However, only neurites of dbcAMP-treated cells developed varicosities, a phenotypic marker of axon formation. Furthermore, following differentiation induced by dbcAMP, there was an upregulation of STC and downregulation of STCrP mRNA levels. In the first 24 and 48 h of treatments, there was a maximum twofold induction and 1.5-fold reduction in STC and STCrP mRNAs respectively. Following 96 h of treatment, an additional 14-fold STC induction and 1.2-fold STCrP reduction were observed. The increase in STC mRNA levels was accompanied by a concomitant increase in axon-specific low molecular form microtubule-associated protein (MAP-2c) mRNA and varicosities on the neurites, suggesting a possible role for STC in axonogenesis. There was no induction of STC mRNA levels when PW1 was added into the culture media, whereas ionomycin (1-10 microM) had no observable effects on cell differentiation or STC/STCrP mRNA. Immunocytochemical staining of dbcAMP-treated cells revealed abundant levels of immunoreactive STC, particularly in the varicosities, with only weak staining in control, untreated cells. Antisense oligodeoxynucleotides transfection studies indicated that the expression of STC was a cause of varicosity formation and a consequence of cell differentiation. Our findings lend further support to the notion that STC is involved in the process of neural differentiation.