Comparison of synthetic mycolactone A/B and its remote diastereomer.

<p>L929 fibroblasts and SW10 Schwann cells were cultured and treated with the same concentration of mycolactone A/B or mycolactone A/B remote diastereomer. Trypan blue staining and the TUNEL assay were performed. Synthetic mycolactone A/B (A, B) and its remote diastereomer (C, D) exerted identical cytotoxicity in both fibroblasts and Schwann cells at the same concentration.</p

Effect of mycolactone on cellular morphology.

<p>SW10 mouse Schwann cells and L929 mouse fibroblast cells were cultured for 24 hrs. Synthetic mycolactone A/B diluted to a final concentration of 3 ng/ml, 30 ng/ml, or 300 ng/ml was added to a cell culture and incubated for 12, 24, 48 and 72 hrs. Photomicrographs were taken by a phase-contrast microscope. Cells treated with 3 ng/ml of mycolactone showed no floating cells at 24, 48, or 72 hrs (A, B). Fibroblasts showed no changes until 48 hrs, but partial detachment began at 72 hrs with 30 ng/ml of mycolactone A/B (C). Schwann cells showed round shrinkage and floating at 24 hrs with 30 ng/ml of mycolactone A/B. Some of the cells remained adherent at 48 hrs, but all cells were detached at 72 hrs (D). Bar = 100 μm.</p

Detection of apoptosis by fluorescence microscopy.

<p>Fibroblasts and Schwann cells were cultured in chamber slides for 24 hrs. Synthetic mycolactone A/B with a final concentration of 3 ng/ml, 30 ng/ml, or 300ng/ml was added and further cultured for 12 and 24 hrs. Fixed cells were stained with fluorescent reagents. Red: cleaved caspase-3 (Rabbit anti-Cleaved Caspase-3 (1:1000)/Alexa Fluor 594 Goat Anti-Rabbit IgG); Blue: nuclear DNA (Hoechst 33342); and Green: intracellular actin (Alexa Fluor 488 Phalloidin). Cells were examined under a confocal laser scanning microscope (Olympus: FV10i-DOC Laser Scanning Microscope). As a positive control, actinomycin-D was added to the culture. Expression of cleaved caspase-3 was compared at 12 and 24 hrs after administration of mycolactone. Positive cell rate (number of cleaved caspase 3 positive cells/number of Hoechst 33342 positive cells, %) was calculated. (A and B) In the four conditions (12 and 24 hrs, 30 and 300 ng/ml mycolactone), the expression of cleaved caspase 3 was observed in the cytoplasm of SW10 Schwann cells (10–21%) and in some of L929 fibroblasts (2–3%). (C) Actinomycin-D showed the expression of cleaved caspase 3 to SW10 and L929 cells.</p

TUNEL assay in SW10 and L929 cells.

<p>L929 fibroblasts and SW10 Schwann cells were cultured on BD Falcon 2 well culture slides for 24 hrs (2.0x10⁴ cells/well) before synthetic mycolactone A/B was added. The cells were fixed with 4% paraformaldehyde-PBS and washed with PBS at 24 hrs and 48 hrs. The TUNEL assay and mild hematoxylin nuclear staining were performed. Total cell number and number of TUNEL-positive cells were counted using photomicrographs. At 24 hrs, 30 ng/ml of mycolactone induced less apoptosis (brown nuclear staining) in fibroblasts (A) than in Schwann cells (B). Three ng/ml of mycolactone did not show significant TUNEL reaction, while 300 ng/ml produced a strong TUNEL reaction in both fibroblasts and Schwann cells. In the quantitative analysis (C), fibroblasts in 3 ng/ml of mycolactone showed no apoptosis at 24 and 48 hrs, but 30 and 300 ng/ml of mycolactone induced apoptosis in a concentration-dependent and time-dependent manner. Schwann cells also showed no apoptosis at 24 and 48 hrs with 3 ng/ml of mycolactone; however, 30 and 300 ng/ml of mycolactone induced more apoptosis in Schwann cells (91% at 48 hrs, 300 ng/mg) than in fibroblasts (48% at 48 hrs, 300 ng/ml). Bar = 100 μm.</p

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

<div><p>MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5′-RCGTG-3′), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2′-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.</p> </div

Hypoxia enhances the recruitment of HIF1α to HRE and activates MUC17 transcription.

<p>(A) Binding of HIF1α to chromatin was confirmed by a ChIP assay. AsPC1 cells were cultured under normoxia or hypoxia for 24 h. PCR was performed with specific primers covering HRE. (B) The densities of the acquired bands in panel (A) were quantified using Image J (NIH) and normalized to Input included in each experiment. (C) To evaluate the transactivation activity of HIF1α through HRE, a dual luciferase assay was conducted. AsPC1 cells were transfected with wild-type or HRE mutant MUC17 promoter constructs under hypoxic conditions for 24 h. P values were determined using Student's <i>t</i>-test. ns, not significant. * P<0.01, ** P<0.001.</p

MUC17 expression is correlated with the hypomethylation of HRE within the MUC17 promoter in tissues from patients with PDAC.

<p>(A) MUC17 mRNA expression and the HRE methylation status in the normal pancreas (N) and pancreatic tumor tissues (T) was examined by RT-PCR and MSP, respectively. (B) Representative immunohistochemical staining data for MUC17 in a patient with PDAC. Scale bar, 100 µm. (C) Correlation of MUC17 mRNA expression and the HRE methylation status was analyzed by Spearman's test. The densities of the acquired bands were quantified using Image J, and the relative amount of unmethylation in each sample was calculated as an index of the aberrant unmethylation status using the equation (%) = U/(U+M).</p

Methylation status of the HRE site determines the sensitivity of MUC17 to HIF1α-induced transactivation.

<p>(A) Demethylation of CpG sites in the MUC17 promoter in BxPC3 and PANC1 cells after 1 µM 5-azadC treatment. MUC17-negative/low cell lines were treated with or without 5-azadC for 7 days. The methylation status of the MUC17 promoter harboring HRE was examined by MSP. The PCR products labeled M (methylated) were amplified by methylation-specific primers, and those labeled U (unmethylated) were amplified by primers specific for unmethylated DNA. (B) Restoration of MUC17 expression in pancreatic cancer cell lines by 5-azadC treatment. Cells were treated with or without 5-azadC for 7 days. During the last 24 h, each group was cultured under normoxic (N) or hypoxic (H) conditions. MUC17 expression was examined by Western blotting.</p

Hypoxic induction of MUC17 is dependent on HIF1α in AsPC1 cells.

<p>(A) After the transfection of HIF1A siRNAs, AsPC1 cells were cultured under normoxia (N) or hypoxia (H) for 24 h. The level of mRNA was measured by RT-PCR. (B) Cell lysates from AsPC1 cells treated with HIF1A siRNAs were immunoblotted with the indicated antibodies. α-tubulin served as a loading control. (C) The densities of the acquired bands from Western blotting analysis were quantified and expressed as relative fold increases compared with that obtained from mock cells under hypoxic culture conditions. ns, not significant. * P<0.05, ** P<0.005.</p

MUC17 expression is enhanced by hypoxia.

<p>(A) AsPC1 cells were cultured under hypoxic conditions (1% O₂) for the indicated times. MUC17 mRNA expression was examined by RT-PCR at each time point. (B) AsPC1 cells were cultured under normoxic or hypoxic conditions for the indicated times. Cell lysates were probed with anti-MUC17, HIF1α, and α-tubulin antibodies by Western blot analysis. The intensities of the bands were quantitated by densitometric scanning, and the ratio of MUC17 to α-tubulin expression is shown under each band as the relative intensity compared with that obtained in normoxic AsPC1 cells. (C) MUC17 promoter activity was measured by a Dual-Luciferase Reporter Assay. After transfection of the MUC17 reporter plasmid, AsPC1 cells were incubated under normoxic or hypoxic conditions for 24 h. Cell lysates were assayed using a luciferase assay kit in a Tristar multimode microplate reader LB941 (Berthold Technologies). Transformation efficiency was normalized on the basis of Renilla luciferase activity. The promoter activity under normoxic conditions was given a value of 1. P values were determined using the Student's <i>t</i>-test. * P<0.05.</p