Qing-You Kong

c-Myc downregulation: a critical molecular event in resveratrol-induced cell cycle arrest and apoptosis of human medulloblastoma cells

The correlation of c-Myc expression with resveratrol-induced turnover of medulloblastoma cells was investigated in this study by checking (1) c-Myc expression in medulloblastoma tissues and cell lines (UW228-2 and UW228-3), (2) the in vitro effect of resveratrol on c-Myc expression and (3) the influences of c-Myc inhibition in cell growth and survival. Immunohistochemical staining of human medulloblastomas and noncancerous cerebellar tissues revealed that 8 out of 11 tumor tissues (72.7%) expressed c-Myc, in which 4 cases (50%) showed intensified nuclear labeling. RT-PCR, Western blotting, immunocytochemical and immunofluorescence stainings revealed c-Myc downregulation accompanied with growth suppression and apoptosis. Flow cytometry analysis showed S phase arrest in resveratrol-treated cell populations. Transfection of c-Myc directed antisense oligonucleotides to the cultured medulloblastoma cells could reduce c-Myc expression, inhibit cell growth and arrest the cell cycle at S phase. Our results thus for the first time demonstrate that c-Myc downregulation is a critical molecular event of resveratrol-mediated anti-medulloblastoma activity, which is closely associated with growth suppression, cell cycle arrest and apoptosis of medulloblastoma cells.

Differential Notch1 and Notch2 Expression and Frequent Activation of Notch Signaling in Gastric Cancers

CONTEXT The biologic effects of Notch1 and Notch2 vary with cancer types and their potential role(s) in gastric cancers (GCs) remains largely unknown. OBJECTIVES This study aimed to address the previously mentioned issue by checking the expression of Notch1, Notch2, and Notch target gene Hes1 in GCs, premalignant gastric lesions, and noncancerous endoscopic gastric mucosa and by inhibiting Notch signal transduction in GC cells. DESIGN The status of Notch1, Notch2, and Hes1 expression in 74 GC surgical specimens, 10 endoscopic samples, and 4 human GC cell lines was evaluated by tissue microarray-based immunohistochemical staining, Western blotting, and reverse transcription-polymerase chain reaction, and the importance of Notch signaling was elucidated by treating 2 GC cell lines with 2 γ-secretase inhibitors. RESULTS Notch1 was undetectable in noncancerous gastric mucosa but was expressed with nuclear translocation in 16.7% (4 of 24) of chronic gastritis, 50.0% (9 of 18) of intestinal metaplasia, 54.2% (26 of 48) of intestinal GC, and 23.1% (6 of 26) of diffuse GC, showing distinct differences of Notch1 detection rates between either intestinal metaplasia and chronic gastritis or intestinal GCs and diffuse GCs (P  =  .03; P  =  .005, respectively). Notch2 nuclear translocation frequencies were 10.0% (1 of 10) in noncancerous endoscopic mucosa, 71.4% (30 of 42) in premalignant lesions, and 97.3% (72 of 74) in GC tissues, demonstrating a correlation of Notch2 expression with both intestinal GC and diffuse GC formation (P < .001). The rates of nuclear-Hes1 labeling were 1 of 10 among noncancerous, 42.9% premalignant, and 81.1% cancer tissues, which were closely correlated with Notch2 (P < .001) rather than Notch1 (P  =  .42) nuclear translocation. Only Notch2 was expressed accompanied with Hes1 nuclear labeling in the 4 GC cell lines established from diffuse GC cases. Inhibition of Notch signaling with γ-secretase inhibitors, L-685,458 and DAPT, prevented Hes1 nuclear translocation but neither suppressed growth nor induced cell death. CONCLUSIONS This study demonstrated a close correlation of Notch2 expression with GC formation and the potential link of Notch1 upregulation with intestinal-like phenotypes of gastric lesions. Although inhibition of Notch activity failed to achieve anti-GC effects, the activated Notch signaling may reflect a potential GC risk.

Short-term resveratrol exposure causes in vitro and in vivo growth inhibition and apoptosis of bladder cancer cells.

Conventional adjuvant chemotherapies for bladder transitional cell carcinomas (TCCs) may cause strong systemic toxicity and local irritation. Non-toxic resveratrol inhibits TCC cell growth but its feasibility in clinical management of TCCs remains obscure. This study aimed to evaluate the safety and anti-TCC efficacy of resveratrol, using the experimental models closer to the clinical treatment condition. Human TCC EJ cells were exposed to 100 µM, 150 µM and 200 µM resveratrol respectively for 1 hour and 2 hours to mimic intravesical drug instillation and the cell responses were analyzed by multiple experimental approaches. An orthotopic TCC nude mouse model was established by injecting EJ cells into the sub-urothelial layer and used for short-term intravesical resveratrol instillation. The safety of resveratrol instillation was evaluated and compared with that of MCC. The results revealed that 2 h 150 µM or 200 µM resveratrol treatment leaded to remarkable S phase arrest and apoptosis at 72 h time-point, accompanied with attenuated phosphorylation, nuclear translocation and transcription of STAT3, down-regulation of STAT3 downstream genes (survivin, cyclinD1, c-Myc and VEGF) and nuclear translocations of Sirt1 and p53. The importance of STAT3 signaling in cell growth was confirmed by treating EJ cells with JAK2 inhibitor tyrphostin AG490. The efficacy and safety of resveratrol instillation were proved by the findings from nude mouse orthotopic xenograft models, because this treatment caused growth suppression, distinctive apoptosis and STAT3 inactivation of the transplanted tumors without affecting normal urothelium. Our results thus suggest for the first time the practical values of resveratrol as a safe and effective agent in the post-operative treatment of TCCs.

Frequent loss of membranous E-cadherin in gastric cancers : A cross-talk with Wnt in determining the fate of β-catenin

The potential correlation of E-cadherin reduction and Wnt2 up-regulation in determining the intracellular distribution of β-catenin in gastric cancers was investigated by the methods of frozen tissue array-based immunohistochemistry, Western blot and RT-PCR analysis. It was revealed that membranous E-cadherin was reduced frequently in the two major subtypes of gastric cancer (intestinal gastric cancer, i-GC and diffuse gastric cancer, d-GC) and closely correlated with the risk of lymphoid node metastasis (P < 0.05). The reduction of membranous E-cadherin was paralleled with cytosolic and nuclear accumulation of β-catenin and the increased Wnt2 expression. These results indicate that the reduced E-cadherin is a common genetic phenotype of GCs and plays beneficial roles in tumor metastasis. Altered β-catenin distribution may result from the imbalance of E-cadherin production and Wnt expression, which confers on gastric cancer cells more aggressive behaviors.

CRABP-II methylation: A critical determinant of retinoic acid resistance of medulloblastoma cells

Medulloblastoma cells exhibit varied responses to therapy by all‐trans retinoic acid (RA). The underlying mechanism for such diverse effects however remains largely unclear. In this study, we attempted to elucidate the molecular basis of RA resistance through the study of RA signaling components in both RA‐sensitive (Med‐3) and RA‐resistant (UW228‐2 and UW228‐3) medulloblastoma cells. The results revealed that RARα/β/γ and RXRα/β/γ were found in the three cell lines. Expression of CRABP‐I and CRABP‐II was seen in Med‐3 cells, up‐regulated when treated with RA, but was absent in UW228‐2 and UW228‐3 cells regardless of RA treatment. Bisulfite sequencing revealed 8 methylated CG sites at the promoter region of CRABP‐II in UW228‐2 and UW228‐3 but not in Med‐3 cells. Demethylation by 5‐aza‐2′‐deoxycytidine recovered CRABP‐II expression. Upon restoration of CRABP‐II expression, both UW228‐2 and UW228‐3 cells responded to RA treatment by forming neuronal‐like differentiation, synaptophysin expression, β‐III tubulin upregulation, and apoptosis. Furthermore, CRABP‐II specific siRNA reduced RA sensitivity in Med‐3 cells. Tissue microarray‐based immunohistochemical staining showed variable CRABP‐II expression patterns among 104 medulloblastoma cases, ranging from negative (42.3%), partly positive (14.4%) to positive (43.3%). CRABP‐II expression was positively correlated with synaptophysin (rs = 0.317; p = 0.001) but not with CRABP‐I expression (p > 0.05). In conclusion, aberrant methylation in CRABP‐II reduces the expression of CRABP‐II that in turn confers RA resistance in medulloblastoma cells. Determination of CRABP‐II expression or methylation status may enable a personalized RA therapy in patients with medulloblastomas and other types of cancers.

Correlative analyses of notch signaling with resveratrol-induced differentiation and apoptosis of human medulloblastoma cells.

Altered Notch signaling seems linked with medulloblastoma (MB) formation and resveratrol exhibits anti-medulloblastoma effects. However, the influence of resveratrol in Notch signaling of MB cells has not been described. This issue was addressed here by checking Notch1 and Notch2 statuses in three MB cell lines with and without resveratrol treatment. Notch1 and Notch2 were detected in the cytoplasm of three cell lines under normal condition, which were up-regulated by resveratrol along with differentiation, apoptosis and enhanced Hes1 nuclear translocation. Nevertheless, blockage of Notch enzymatic cleavage with gamma-seacretase inhibitors, DAPT and L-685,458, neither interrupted resveratrol-caused cellular events nor affected MB cell growth. These results demonstrate that Notch signaling has little relevance with resveratrol-induced differentiation and apoptosis and may not be a universal critical factor of MB cells.

Metabolic patterns and biotransformation activities of resveratrol in human glioblastoma cells: relevance with therapeutic efficacies.

Background Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s). Methodology/Principal Findings Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours. Conclusions/Significance LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.

Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells.

Cancer preventive reagent trans-resveratrol is intracellularly biotransformed to different metabolites. However, it is still unclear whether trans-resveratrol exerts its biological effects directly or through its metabolite(s). This issue was addressed here by identifying the metabolic pattern and the bioactive form of resveratrol in a resveratrol-sensitive human medulloblastoma cell line, UW228-3. The cell lysates and condition media of UW228-3 cells with or without 100 microM resveratrol treatment were analyzed by HPLC and LC/MS which revealed (1) that resveratrol was chemically unstable and the spontaneous generation of cis-resveratrol reduced resveratrols anti-medulloblastoma efficacy and (2) that resveratrol monosulfate was the major metabolite of the cells. To identify the bioactive form of resveratrol, a mixture-containing approximately half fraction of resveratrol monosulfate was prepared by incubating trans-resveratrol with freshly prepared rat brain lysates. Medulloblastoma cells treated by 100 microM of this mixture showed attenuated cell crisis. The overall levels of the three brain-associated sulfotransferases (SULT1A1, 1C2 and 4A1) were low in medulloblastoma cells in vivo and in vitro in comparison with that in human noncancerous and rat normal cerebella; resveratrol could more or less up-regulate the production of these enzymes in UW228-3 cells but their overall level was still lower than that in normal cerebellum tissue. Our study thus demonstrated for the first time that trans-resveratrol is the bioactive form in medulloblastoma cells in which the expression of brain-associated SULTs was down-regulated, resulting in the increased intracellular bioavailability and anti-medulloblastoma efficacy of trans-resveratrol.

Methylation-associated silencing of S100A4 expression in human epidermal cancers

Abstract:  S100A4 appears important for cancer metastasis and its overexpression is common in a variety of human malignancies, but its status in epidermal cancers remains lesser known. Likewise, E‐cadherin downregulation and Wingless (Wnt) activation are frequent cancer‐associated alterations, whereas their potential correlations with S100A4 expression in skin lesions have not been characterized. These issues were addressed in the present study using tissue microarray‐based immunohistochemical staining, reverse transcriptase polymerase chain reaction and western blotting. Meanwhile, the underlying epigenetic mechanism leading to the altered S100A4 expression in epidermal tumors was elucidated. Immunohistochemistry revealed that S100A4 expression frequencies were 100% (8/8) in normal epidermis, 80.6% (25/31) in tumor‐surrounding non‐cancerous epidermis, 66.7% (10/15) in premalignant diseases, 8.3% (1/11) in Bowen’s disease and 7.7–26.3% in different cancer tissues. The incidence of S100A4 detection in the normal and non‐cancerous epidermis was significantly different from that of epidermal cancers (P = 0.000). Accordingly, human immortalized keratinocyte line HaCat but not skin squamous cell carcinoma (SCC) line colo16 was positive in S100A4 expression. S100A4 downregulation, E‐cadherin reduction and Wnt activation coexisted in most of epidermal cancers but unnecessarily overlapped. Methylation DNA sequencing revealed methylation of four critical (cytosine and guanine separated by a phosphate or ‐C‐phosphate‐G‐) CpG sites within S100A4 intron first in S100A4‐negative colo16 cells and skin SCCs, and demethylator/5‐aza‐2′‐deoxycytidine treatment efficiently recovered S100A4 expression in colo16 cells. Our findings demonstrate that S100A4 downregulation, as the consequence of DNA methylation, is closely correlated with skin tumor formation. Wnt activation and E‐cadherin reduction and S100A4 down‐regulation are paralleled molecular events in skin tumors, which may serve as the biomarkers for predicting epidermal cancer risk.

Expression of seven gastric cancer-associated genes and its relevance for Wnt, NF-κB and Stat3 signaling†

The aim of the current study was to profile c‐Myc, standard CD44 (CD44s), CD44v6, cyclin D1, survivin, MMP‐7 and VEGF expression patterns in different gastric samples and to elucidate their relevance for Wnt, NF‐κB and/or Stat3 activation using multiple experimental approaches. The results revealed that 87.1% (27/31) of gastric cancers and 8.7% (2/23) of noncancerous lesions (chronic gastritis and intestinal metaplasia) showed Wnt activation (Wnt+) that was closely related to the expression of the seven genes. Some Wnt− noncancerous lesions also expressed the above‐mentioned genes, higher frequencies of survivin (7/8), VEGF (7/8), cyclin D1 (6/8) and c‐Myc (5/8) but not CD44s (2/8), CD44v6 (3/8) and MMP‐7 (2/8) being detected in the NF‐κB+ samples. Stat3 was activated in 37/54 gastric tissues, and in 3/4 VEGF, 4/6 c‐Myc, 4/8 survivin, 2/4 MMP‐7, 1/2 CD44v6, and 4/9 cyclin D1+ but Wnt−/NF‐κB− samples. These findings showed a close correlation in GCs between Wnt, NF‐κB and Stat3 signaling and expression of the seven genes, the importance of NF‐κB and Stat3 activation in regulating c‐Myc, survivin, cyclin D1 and VEGF in noncancerous lesions, and the potential coordinative effects of these three signalings on GC formation presumably by promoting the transcription of their common target genes.