Pancreatic cancer is an aggressive malignancy that is generally refractory to chemotherapy, thus posing experimental and clinical challenges. In this study, the antiproliferative effect of the triterpenoid compound cucurbitacin B was tested in vitro and in vivo against human pancreatic cancer cells. Dose-response studies showed that the drug inhibited 50% growth of seven pancreatic cancer cell lines at 10 À7 mol/L, whereas clonogenic growth was significantly inhibited at 5 Â 10 À8 mol/L. Cucurbitacin B caused dose-and timedependent G 2 -M-phase arrest and apoptosis of pancreatic cancer cells. This was associated with inhibition of activated JAK2, STAT3, and STAT5, increased level of p21 WAF1 even in cells with nonfunctional p53, and decrease of expression of cyclin A, cyclin B1, and Bcl-XL with subsequent activation of the caspase cascade. Interestingly, the combination of cucurbitacin B and gemcitabine synergistically potentiated the antiproliferative effects of gemcitabine on pancreatic cancer cells. Moreover, cucurbitacin B decreased the volume of pancreatic tumor xenografts in athymic nude mice by 69.2% (P < 0.01) compared with controls without noticeable drug toxicities. In vivo activation of JAK2/STAT3 was inhibited and expression of Bcl-XL was decreased, whereas caspase-3 and caspase-9 were up-regulated in tumors of drug-treated mice. In conclusion, we showed for the first time that cucurbitacin B has profound in vitro and in vivo antiproliferative effects against human pancreatic cancer cells, and the compound may potentate the antiproliferative effect of the chemotherapeutic agent gemcitabine. Further clinical studies are necessary to confirm our findings in patients with pancreatic cancer.

BACKGROUND & AIMS Alterations in methylation of protein-coding genes are associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Dys-regulation of noncoding RNAs occurs during carcinogen-esis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.

Objectives Long non-coding RNAs (IncRNA) have been shown to play important roles in the development and progression of cancer. However, functional IncRNAs and their downstream mechanisms are largely unknown in the molecular pathogenesis of oesophageal adenocarcinoma (EAC) and its progression. Design IncRNAs that are abnormally upregulated in EACs were identified by RNA-sequencing analysis, followed by quantitative RT-PCR (qRTPCR) validation using tissues from 25 EAC patients. Cell biological assays in combination with small interfering RNA-mediated knockdown were performed in order to probe the functional relevance of these IncRNAs. Results We discovered that a IncRNA, HNF1A-AS1, is markedly upregulated in human primary EACs relative to their corresponding normal oesophageal tissues (mean fold change 10.6, p<0.01). We further discovered that HNF1A-AS1 knockdown significantly inhibited cell proliferation and anchorage-independent growth, suppressed S-phase entry, and inhibited cell migration and invasion in multiple in vitro EAC models (p<0.05). A gene ontological analysis revealed that HNF1A-AS1 knockdown preferentially affected genes that are linked to assembly of chromatin and the nucleosome, a mechanism essential to cell cycle progression. The well known cancer-related IncRNA, H19, was the gene most markedly inhibited by HNF1A-AS1 knockdown. Consistent to this finding, there was a significant positive correlation between HNF1A-AS1 and H19 expression in primary EACs (p<0.01). Conclusions We have discovered abnormal upregulation of a IncRNA, HNF1A-AS1, in human EAC. Our findings suggest that dysregulation of HNF1A-AS1 participates in oesophageal tumorigenesis, and that this participation may be mediated, at least in part, by modulation of chromatin and nucleosome assembly as well as by H19 induction.