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Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.

Abstract Background The diagnosis of lymph node metastasis in esophageal cancer by the presence and number of metastatic lymph nodes is an extremely important prognostic factor. In addition, the indication of non-surgical therapy is gaining more attention. Vascular endothelial growth factor C (VEGF-C) is potentially lymphangiogenic and selectively induces hyperplasia of the lymphatic vasculature. In this study, we investigated the expression of VEGF-C and whether it correlated with various clinico-pathologic findings. Methods KYSE series of esophageal cancer cell lines and 106 patients with primary esophageal squamous cell carcinomas who had undergone radical esophagectomy were analyzed. VEGF-C mRNA expression was determined by quantitative RT-PCR. Results High expression of VEGF-C was detected in most of the KYSE cell lines, especially KYSE410, yet, in an esophageal normal epithelium cell line, Het-1A, VEGF-C was not detected. In the clinical specimen, the expression of VEGF-C in the cancerous tissue was higher than in the corresponding noncancerous esophageal mucosa (p = 0.026). The expression of VEGF-C was found to be higher in Stage2B-4A tumors than in Stage0-2A tumors (p = 0.049). When the patients were divided into two groups according to their expression levels of VEGF-C (a group of 53 cases with high expression and a group of 53 cases with low expression), the patients with high VEGF-C expression had significantly shorter survival after surgery than the patients with low expression (p = 0.0065). Although univariate analysis showed that high expression of VEGF-C was a statistically significant prognostic factor, this was not shown in multivariate analysis. In the subgroup of patients with Tis and T1 tumors, the expression of VEGF-C was higher in N1 tumors than in N0 tumors (p = 0.029). The survival rate of patients from the high expression group (n = 10) was lower than that in the low expression group (n = 11), and all the patients in the low VEGF-C expression group survived. Conclusions The expression of VEGF-C correlates with lymph node metastasis and poor prognosis. In patients with Tis and T1 esophageal tumors, the expression of VEGF-C may be a good diagnostic factor for determining metastasis of the lymph node.

Abstract Background β-catenin is a multifunctional protein involved in two apparently independent processes: cell-cell adhesion and signal transduction. β-catenin is involved in Wnt signaling pathway that regulates cellular differentiation and proliferation. In this study, we investigated the expression pattern of β-catenin and cyclin D1 using immunohistochemistry and searched for mutations in exon 3 of the β-catenin gene and Axin gene in esophageal squamous cell carcinoma. Materials and methods Samples were obtained from 50 esophageal cancer patients. Immunohistochemical staining for β-catenin and cyclin D1 was done. Mutational analyses of the exon3 of the β-catenin gene and Axin gene were performed on tumors with nuclear β-catenin expression. Results Four (8%) esophageal cancer tissues showed high nuclear β-catenin staining. Overexpression of cyclin D1 was observed in 27 out of 50 (54%) patients. All four cases that showed nuclear β-catenin staining overexpressed cyclin D1. No relationship was observed between the expression pattern of β-catenin and cyclin D1 and age, sex, tumor size, stage, differentiation grade, lymph node metastasis, response to chemotherapy, or survival. No mutational change was found in β-catenin exon 3 in the four cases with nuclear β-catenin staining. Sequencing analysis of the Axin cDNA revealed only a splicing variant (108 bp deletion, position 2302–2409) which was present in the paired normal mucosa. Conclusion A fraction of esophageal squamous cell carcinomas have abnormal nuclear accumulation of β-catenin accompanied with increased cyclin D1 expression. Mutations in β-catenin or axin genes are not responsible for this abnormal localization of β-catenin.

Xantohumol, a prenylated chalcone from hops (Humulus lupulus L.), has been shown to inhibit proliferation in some cancers. However, little is known regarding the effects of xanthohumol in pancreatic cancer. We have previously reported that activation of the transcription factor nuclear factor‐κB (NF‐κB) plays a key role in angiogenesis in pancreatic cancer. In this study, we investigated whether xanthohumol inhibited angiogenesis by blocking NF‐κB activation in pancreatic cancer in vitro and in vivo. We initially confirmed that xanthohumol significantly inhibited proliferation and NF‐κB activation in pancreatic cancer cell lines. Next, we demonstrated that xanthohumol significantly suppressed the expression of vascular endothelial growth factor (VEGF) and interleukin‐8 (IL‐8) at both the mRNA and protein levels in pancreatic cancer cell lines. We also found that coculture with BxPC‐3 cells significantly enhanced tube formation in human umbilical vein endothelial cells, and treatment with xanthohumol significantly blocked this effect. In vivo, the volume of BxPC‐3 subcutaneous xenograft tumors was significantly reduced in mice treated with weekly intraperitoneal injections of xanthohumol. Immunohistochemistry revealed that xanthohumol inhibited Ki‐67 expression, CD31‐positive microvessel density, NF‐κB p65 expression, and VEGF and IL‐8 levels. Taken together, these results showed, for the first time, that xanthohumol inhibited angiogenesis by suppressing NF‐κB activity in pancreatic cancer. Accordingly, xanthohumol may represent a novel therapeutic agent for the management of pancreatic cancer. [ABSTRACT FROM AUTHOR]