Qinghua Xiong

Regulation of vascular endothelial growth factor expression by acidosis in human cancer cells

The influence of acidosis on the expression of the vascular endothelial growth factor (VEGF) gene was determined. FG human pancreatic adenocarcinoma cells were incubated for various time periods in media at a physiologically relevant pH level (6.7–7.4). The expression of VEGF mRNA and protein secretion was inversely correlated with pH in a pH- and time-dependent manner. Transient acidosis also activated the VEGF promoter/enhancer luciferase reporter, which was consistent with an increased VEGF gene transcription rate and VEGF mRNA half-life. These data indicated that acidosis transcriptionally and posttranscriptionally regulates VEGF expression, suggesting that an acidic tumor microenvironment contributes to tumor angiogenesis and progression.

A multispecialty approach to the diagnosis and management of pancreatic cancer

This article reviews recent developments in pancreatic cancer research and offers a multispecialty perspective on the diagnosis and management of this challenging disease. Current findings in the molecular biology of the disease and their implications for management are examined, as well as development in diagnostic techniques, including helical computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance cholangio-pancreatography (MRCP), and, particularly, endoscopic ultrasound–guided fine-needle aspiration. Surgical management, the role of adjuvant/neoadjuvant chemoradiation therapy, and the critical importance of accurate preoperative imaging are also addressed in this review. Palliative techniques, including endoscopic stenting for malignant obstructive jaundice and chemotherapy for locally advanced and metastatic disease, are discussed, and results of recent clinical trials in pancreatic cancer are summarized. Finally, future directions for research are identified.

Cooperation Between Transcription Factor AP-1 and NF-kappa B in the Induction of Interleukin-8 in Human Pancreatic Adenocarcinoma Cells by Hypoxia

The expression of interleukin-8 (IL-8) has been shown to play an important role in the growth and metastasis of human pancreatic cancer. In the present study, we investigated the regulation of IL-8 gene expression by hypoxic environments. Exposure of the human pancreatic cancer cells COLO357 and FG to hypoxia in culture resulted in a time-dependent increase in steady-state levels of IL-8 mRNA and IL-8 protein secretion. The induction of IL-8 expression was correlated with transcriptional activation of the IL-8 gene. Deletion and point mutation analyses of the IL-8 promoter revealed that both AP-1 and NF-kappaB binding sites were necessary for IL-8 induction by hypoxia. Consistently, hypoxia induced both AP-1 and NF-kappaB activity. These data suggest that hypoxic environments upregulate the IL-8 gene via cooperation of NF-kappaB and AP-1 and contribute to the progression and metastasis of human pancreatic cancer.

Molecular regulation of constitutive expression of interleukin-8 in human pancreatic adenocarcinoma

Recent studies have shown that interleukin-8 (IL-8) plays an important role in the growth and metastasis of human pancreatic cancer. In the present study, we determined the molecular regulation of constitutive IL-8 expression in human pancreatic cancer cells. Various human pancreatic cancer cell lines were incubated in vitro. Sixty-seven percent of the cell lines constitutively secreted high levels of IL-8, as determined using enzyme-linked immunosorbent assay. Consistently, these cells constitutively expressed high levels of IL-8 mRNA, as determined using Northern blot analysis. To determine the mechanisms of the high steady-state levels of IL-8 mRNA, the IL-8 half-life and transcription rate were measured. There was no significant difference in IL-8 half-life between cells expressing high and low levels of IL-8. However, higher transcription rates and increased IL-8 promoter activity were observed in the cells constitutively expressing high levels of IL-8. Detailed IL-8 promoter analysis using deletion mutation revealed that the region from -85 to -133 bp was essential for the constitutive IL-8 promoter activity. Also, point-mutation analysis indicated that mutation of NF-kappaB, AP-1, or NF-IL-6 binding sites significantly reduced or eliminated the constitutive IL-8 promoter activity. Consistent with the constitutive IL-8 transcription activity, high levels of constitutive NF-kappaB and AP-1 activity were detected in the cells overexpressing IL-8, as determined using electrophoretic mobility shift assay. In addition, transfection of a dominant-negative I-kappaBalpha expression vector (I-kappaBalphaM) inhibited constitutive NF-kappaB activity and IL-8 expression in pancreatic cancer cells. Collectively, our data demonstrated that constitutive NF-kappaB and AP-1 activation contributes to the overexpression of IL-8, which in turn plays an important role in tumor angiogenesis and contributes to the aggressive biology of human pancreatic cancer.

Regulation of Interleukin-8 Expression by Tumor-Associated Stress Factors

Tumor and host cells frequently express interleukin-8 (IL-8). IL-8 has been shown to be motogenic, mitogenic, and angiogenic and to play important roles in human tumor progression. IL-8 expression can be induced by numerous stress factors present in the tumor environment, such as hypoxia, acidosis, hyperglycemia, hyperosmotic pressure, high cell density, hyperthermia, radiation, and chemotherapeutic agents. Understanding the mechanisms of IL-8 expression and regulation will be helpful in designing potential therapeutic modalities targeting IL-8 to control tumor growth and metastasis.

Genetic disruption of host nitric oxide synthase II gene impairs melanoma-induced angiogenesis and suppresses pleural effusion

Our previous study showed that genetic disruption of nitric oxide (NO) synthase II (NOS II) expression inhibits the metastatic ability of non‐immunogenic B16 melanoma cells in syngeneic mice. In the present study, the mechanisms for this metastasis suppression were determined. B16‐BL6 and B16‐F10 murine melanoma cells were injected i.v. into syngeneic wild‐type (NOS II+/+) and NOS II‐null (NOS II–/–) C57BL/6 mice. Both melanoma cells produced less and smaller experimental pulmonary metastases in NOS II–/– mice than in NOS II+/+ mice. Moreover, less metastatic pleural effusion was observed in NOS II–/– mice than in NOS II+/+ mice. Immunohistochemical analyses indicated that absence of NOS II expression was correlated with decreased vascular endothelial growth factor expression and tumor‐associated vascular formation. After activation with lipopolysaccharide and IFN‐γ, neither melanoma cell line produced detectable levels of NO. Our data demonstrate that tumor‐induced expression of host NOS II enhances melanoma metastasis and pleural effusion, at least in part, through regulation of vascular formation and vascular permeability.

Regulation of interleukin-8 expression by cellular pH in human pancreatic adenocarcinoma cells.

The role of cellular pH in the expression and regulation of interleukin-8 (IL-8) in human tumor cell lines was determined. Transient exposure to pH ranging from 7.4 to 6.7 induced pH-dependent expression of IL-8 at both the mRNA and protein levels in three different human tumor cell lines, including COLO357 pancreatic adenocarcinoma cells, SW620 colon adenocarcinoma cells, and PC3 prostate adenocarcinoma cells. Investigation of the mechanisms of IL-8 induction in response to acidosis was carried out using the COLO357 human pancreatic cancer cell line. The increased steady-state level of mRNA correlated with an increased transcription rate and stability of IL-8 transcripts. Further experiments indicated that mild acidosis activated the transcription factors NF-kappaB and AP-1 and that the cooperation of these two factors appeared to be essential to the transactivation of the IL-8 gene. Our data demonstrated that low tumor pH contributes to the enhanced expression of IL-8 and plays an important role in tumor progression.

Regulation of Interleukin-8 Expression by Nitric Oxide in Human Pancreatic Adenocarcinoma

The regulation of interleukin-8 (IL-8) expression by nitric oxide (NO) was determined in human pancreatic cancer cell lines. CaPan-2 and FG human pancreatic adenocarcinoma cells were incubated for 24 h in medium alone or medium containing a cytokine mixture in the presence or absence of an NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (NMA). The NOS activity and level of IL-8 expression were determined. IL-8 expression was induced in the two cell lines. A low level of NOS activity was detectable only in CaPan-2 cells. Moreover, the presence of NMA did not reverse the induction of IL-8. The FG cells were then engineered to produce a physiologic level of NO and incubated in medium alone or medium containing 1 mM NMA. No significant IL-8 expression was induced in those producing a low level of NO, whereas IL-8 expression was induced in those producing a high level of NO. Inhibition of NO production by NMA reversed this effect. Incubation of FG cells with an NO donor, S-nitroso-D,L.-acetyl-penicillamine (SNAP), led to a concentration-dependent and time-dependent induction of IL-8 expression. This NO-mediated upregulation of IL-8 expression correlated with an increase in IL-8 gene transcription and mRNA stability. Our results indicate that NO is involved in the regulation of IL-8 expression in and contributes to the progression of human pancreatic cancer.

Genetic disruption of host interferon-γ drastically enhances the metastasis of pancreatic adenocarcinoma through impaired expression of inducible nitric oxide synthase

Synergistic induction of the inducible nitric oxide synthase (NOS II) gene requires a combination of interferon-γ (IFN-γ) and lipopolysaccharide (LPS). In this study, we determined whether the induction of IFN-γ was required for NOS II-mediated antitumor activity in vivo. Highly metastatic H7 murine pancreatic adenocarcinoma cells were implanted into the subcutis, footpad, and pancreas of syngeneic IFN-γ+/+ and IFN-γ−/− mice. These cells grew and produced metastases and ascites in IFN-γ+/+ mice. In sharp contrast, the same tumor cells grew much more aggressively, metastasized more extensively, and produced a larger amount of malignant ascites in IFN-γ−/− mice. Also, induction of IFN-γ correlated with NOS II gene expression and NO production in IFN-γ+/+ injected with the tumor cells but not in IFN-γ−/− mice or IFN-γ+/+ mice without tumor challenge. In vitro, only LPS plus IFN-γ induced a high level of NO production and cytotoxicity against H7 cells. These data suggested that the tumor cells stimulated IFN-γ secretion from host cells, which in turn stimulated NO production by host cells and suppressed tumor growth and metastasis.

A novel, clinically relevant animal model of metastatic pancreatic adenocarcinoma biology and therapy

SummaryIn this study, we report a metastatic model of Panc02 murine pancreatic adenocarcinoma. Parental Panc02 cells were orthotopically implanted into the pancreas of syngeneic C57BL/6 mice. Tumor cells were isolated from liver micrometastases 90 d after tumor implantation and established as a culture (Panc02-H1). The Panc02-H1 cells were then implanted into the pancreas of mice. Liver metastases were then collected and established as Panc02-H2 cells. This process was repeated until the Panc02-H7 cell line was established. These cells were extremely aggressive after implantation as manifested by progressive growth in the pancreas, peritoneal dissemination, and distant metastasis to multiple organs, including the liver and lungs. Moreover, Panc02-H7 cells expressed the inducible nitric oxide synthase gene at a very low level in culture and produced highly vascularized tumors having a large number of infiltrating macrophages. Collectively, this model system should be a valuable tool for investigating the molecular mechanisms governing pancreatic cancer growth and metastasis and exploring potential treatment modalities for this disease.