Zhimin Tong

CXCL8/IL-8 and CXCL12/SDF-1α Co-operatively Promote Invasiveness and Angiogenesis in Pancreatic Cancer

CXC‐chemokines are involved in the chemotaxis of neutrophils, lymphocytes and monocytes. However, role of these chemokines in tumorigenesis, especially with regard to interaction between tumor and its microenvironment, has not been clearly elucidated. The purpose of this study was to analyze the co‐operative role of CXCL8 and CXCL12 in the tumor‐stromal interaction in pancreatic cancer (PaCa). Using enzyme‐linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT‐PCR), we initially confirmed the expression of ligands and receptors, respectively, of CXC‐chemokines in PaCa and stromal cells. We examined the co‐operative role of CXCL8 and CXCL12 in proliferation/invasion of PaCa and human umbilical vein endothelial cells (HUVECs), and in HUVEC tube‐formations through tumor‐stromal interaction by MTS, Matrigel invasion, and angiogenesis assays, respectively. We detected expression of CXCR4, but not CXCR2, in all PaCa cells and fibroblasts. PaCa cells secreted CXCL8, and fibroblast cells secreted CXCL12. CXCL8 production in PaCa was significantly enhanced by CXCL12, and CXCL12 production in fibroblasts was significantly enhanced by co‐culturing with PaCa. CXCL8 enhanced proliferation/invasion of HUVECs but did not promote proliferation/invasion of PaCa. Both recombinant and PaCa‐derived CXCL8 enhanced tube formation of HUVECs that were co‐cultured with fibroblast cells. CXCL12 enhanced the proliferation/invasion of HUVECs and the invasion of PaCa cells but had no effect on tube formation of HUVEC. We showed that PaCa‐derived CXCL8 and fibroblast‐derived CXCL12 cooperatively induced angiogenesis in vitro by promoting HUVEC proliferation, invasion, and tube formation. Thus, corresponding receptors CXCR2 and CXCR4 are potential antiangiogenic and antimetastatic therapeutic targets in PaCa.

Neutrophil Gelatinase–Associated Lipocalin: A Novel Suppressor of Invasion and Angiogenesis in Pancreatic Cancer

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa secreted acute phase protein, which is also up-regulated in multiple cancers, including breast, lung, and pancreas. Recently, NGAL has been proposed as an early biomarker in pancreatic cancer (PaCa). However, its biological role in PaCa is unknown. In this study, we examined in vitro and in vivo the functional role of NGAL in PaCa. Well- to moderately differentiated PaCa cells (AsPC-1, BxPC-3, and Capan-2) expressed high levels of NGAL but moderately to poorly differentiated PaCa cells (PANC-1 and MIAPaCa-2) expressed undetectable NGAL levels. Immunohistochemistry of untreated tissue microarray showed specific NGAL staining in resected PaCa specimens (P = 0.0167). Stable NGAL overexpression (MIAPaCa-2 and PANC-1) significantly blocked PaCa cell adhesion and invasion in vitro and vice versa with stable PaCa clones (BxPC-3 and AsPC-1). Moreover, NGAL overexpression reduced focal adhesion kinase (FAK) tyrosine-397 phosphorylation in PaCa cells. Furthermore, NGAL overexpression potently decreased angiogenesis in vitro partly through reduced vascular endothelial growth factor (VEGF) production and vice versa. Stable NGAL overexpression or underexpression had no effect on PaCa cell survival, viability, and response to chemotherapeutic drugs. Finally, MIAPaCa-2 cells overexpressing NGAL reduced tumor volume (P = 0.012), local and distant metastasis (P = 0.002), and angiogenesis (P = 0.05) with no effect on K-67 proliferation index (P > 0.1) in an orthotopic nude mouse PaCa model. Collectively, our results suggest that NGAL reduces adhesion/invasion partly by suppressing FAK activation and inhibits angiogenesis partly by blocking VEGF production in PaCa cells. Thus, NGAL is a potential suppressor of invasion and angiogenesis in advanced PaCa.

CXC-chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer

Angiogenesis is essential for tumor growth and metastasis. Although ELR+‐CXC‐chemokines and their corresponding receptor, CXC‐receptor 2 (CXCR2), are known mediators of angiogenesis, little is known about their role in pancreatic cancer (PaCa). The aim of our study was to determine the role of ELR+‐CXC‐chemokine/CXCR2 biological axis in promoting PaCa angiogenesis. We prospectively collected secretin‐stimulated exocrine pancreatic secretions (SSEPS) from normal individuals (NP) and PaCa patients. We showed that summed concentrations of ELR+‐CXC‐chemokines in SSEPS from PaCa patients were significantly higher than in those from NP (p = 0.002). We measured ELR+‐CXC‐chemokine levels in supernatants from multiple PaCa cell lines and confirmed that BxPC‐3, Colo‐357 and Panc‐28 had significantly higher expression compared with an immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR+‐CXC‐chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on human umbilical vein endothelial cells (HUVEC). Both recombinant ELR+‐CXC‐chemokines and co‐culturing with BxPC‐3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (p < 0.05). These biological effects were significantly inhibited by treatment with a neutralizing antibody against CXCR2 (anti‐CXCR2 Ab) (p < 0.05). Finally, anti‐CXCR2 Ab significantly reduced tumor volume (p < 0.05), Ki‐67 proliferation index (p = 0.043) and Factor VIII+ microvessel density (p = 0.004) in an orthotopic nude mouse PaCa model. Our results show that ELR+‐CXC‐chemokines promote PaCa tumor‐associated angiogenesis through CXCR2, suggesting that CXCR2 is an anti‐angiogenic target in PaCa.

γ-Tocotrienol Inhibits Pancreatic Tumors and Sensitizes Them to Gemcitabine Treatment by Modulating the Inflammatory Microenvironment

Pancreatic cancers generally respond poorly to chemotherapy, prompting a need to identify agents that could sensitize tumors to treatment. In this study, we investigated the response of human pancreatic cells to γ-tocotrienol (γ-T3), a novel, unsaturated form of vitamin E found in palm oil and rice bran oil, to determine whether it could potentiate the effects of gemcitabine, a standard of care in clinical treatment of pancreatic cancer. γ-T3 inhibited the in vitro proliferation of pancreatic cancer cell lines with variable p53 status and potentiated gemcitabine-induced apoptosis. These effects correlated with an inhibition of NF-κB activation by γ-T3 and a suppression of key cellular regulators including cyclin D1, c-Myc, cyclooxygenase-2 (COX-2), Bcl-2, cellular inhibitor of apoptosis protein, survivin, vascular endothelial growth factor (VEGF), ICAM-1, and CXCR4. In an orthotopic nude mouse model of human pancreatic cancer, p.o. administration of γ-T3 inhibited tumor growth and enhanced the antitumor properties of gemcitabine. Immunohistochemical analysis indicated a correlation between tumor growth inhibition and reduced expression of Ki-67, COX-2, matrix metalloproteinase-9 (MMP-9), NF-κB p65, and VEGF in the tissue. Combination treatment also downregulated NF-κB activity along with the NF-κB-regulated gene products, such as cyclin D1, c-Myc, VEGF, MMP-9, and CXCR4. Consistent with an enhancement of tumor apoptosis, caspase activation was observed in tumor tissues. Overall, our findings suggest that γ-T3 can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing NF-κB-mediated inflammatory pathways linked to tumorigenesis.

A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth In vitro and In vivo

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-κB activation; and abrogated the expression of NF-κB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 μmol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67–positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive apoptotic cells (P < 0.05), and abrogated the expression of NF-κB–dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136–46. ©2010 AACR.

Protein kinase D1 promotes anchorage-independent growth, invasion, and angiogenesis by human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases. Novel molecularly targeted therapies are urgently needed. Here, we extended our studies on the role of protein kinase D1 (PKD1) in PDAC cell lines. Given that Panc‐1 express moderate levels of PKD1, we used retroviral‐mediated gene transfer to create a Panc‐1 derivative that stably over‐expresses PKD1 (Panc‐1‐PKD1). Reciprocally, we used shRNA targeting PKD1 in Panc‐28 to produce a PKD1 under‐expressing Panc‐28 derivative (Panc‐28‐shPKD1). Our results demonstrate that Panc‐1‐PKD1 cells exhibit significantly increased anchorage‐independent growth in soft agar and increased in vitro invasion compared with Panc‐1‐mock. Reciprocally, Panc‐28‐shPKD1 cells show a significant decrease in anchorage‐independent growth and invasiveness, as compared with Panc‐28‐mock cells. The selective PKD family inhibitor CRT0066101 markedly decreased colony‐forming ability and invasiveness by either Panc‐1‐PKD1 or Panc‐28‐mock cells. Secretion of the pro‐angiogenic factors vascular endothelial growth factor (VEGF) and CXC chemokines (CXCL8) was significantly elevated by PKD1 over‐expression in Panc‐1 cells and reduced either by depletion of PKD1 via shRNA in Panc‐28 cells or by addition of CRT0066101 to either Panc‐1‐PKD1 or Panc‐28‐mock cells. Furthermore, human umbilical vein endothelial cell (HUVEC) tube formation was significantly enhanced by co‐culture with Panc‐1‐PKD1 compared with Panc‐1‐mock in an angiogenesis assay in vitro. Conversely, PKD1 depletion in Panc‐28 cells decreased their ability to induce endotube formation by HUVECs. PDAC‐induced angiogenesis in vitro and in vivo was markedly inhibited by CRT0066101. Our results lend further support to the hypothesis that PKD family members provide a novel target for PDAC therapy. J. Cell. Physiol. 226: 1074–1085, 2011.

Zyflamend suppresses growth and sensitizes human pancreatic tumors to gemcitabine in an orthotopic mouse model through modulation of multiple targets

Agents that can potentiate the efficacy of standard chemotherapy against pancreatic cancer are of great interest. Because of their low cost and safety, patients commonly use a variety of dietary supplements, although evidence of their efficacy is often lacking. One such commonly used food supplement is Zyflamend, a polyherbal preparation with potent anti‐inflammatory activities and preclinical efficacy against prostate and oral cancer. Whether Zyflamend has any efficacy against human pancreatic cancer alone or in combination with gemcitibine, a commonly used agent, was examined in cell cultures and in an orthotopic mouse model. In vitro, Zyflamend inhibited the proliferation of pancreatic cancer cell lines regardless of p53 status and also enhanced gemcitabine‐induced apoptosis. This finding correlated with inhibition of NF‐κB activation by Zyflamend and suppression of cyclin D1, c‐myc, COX‐2, Bcl‐2, IAP, survivin, VEGF, ICAM‐1 and CXCR4. In nude mice, oral administration of Zyflamend alone significantly inhibited the growth of orthotopically transplanted human pancreatic tumors, and when combined with gemcitabine, further enhanced the antitumor effects. Immunohistochemical and Western blot analyses of tumor tissue showed that the suppression of pancreatic cancer growth correlated with inhibition of proliferation index marker (Ki‐67), COX‐2, MMP‐9, NF‐κB and VEGF. Overall, these results suggest that the concentrated multiherb product Zyflamend alone can inhibit the growth of human pancreatic tumors and, in addition, can sensitize pancreatic cancers to gemcitabine through the suppression of multiple targets linked to tumorigenesis.

Epidermal growth factor down-regulates the expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells

The authors previously reported that neutrophil gelatinase‐associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)‐EGF receptor (EGFR) axis is up‐regulated significantly in PDAC, they examined EGF‐mediated NGAL regulation in these cells.

Target-specific agents imaging ectopic and orthotopic human pancreatic cancer xenografts

Objectives: This study aimed to develop target-specific binding agents for in vitro and in vivo imaging of human pancreatic cancer. Methods: A monoclonal neutrophil gelatinase-associated lipocalin (NGAL)-specific antibody and a peptide specific for matrix metalloproteinase (MMP) were labeled with a near-infrared dye for in vitro and in vivo imaging studies. Fluorescence or confocal microscopy was used to determine antibody or peptide binding and internalization of agents into human AsPC-1, Panc-1, and MiaPaCa pancreatic cancer cell lines and in mice bearing ectopic or orthotopic pancreatic tumor transplants. Results: Both the NGAL-specific antibody and MMP peptide bound to pancreatic cancer cells with high specificity; most NGAL-specific antibody localized to the cytosol. In vivo imaging results demonstrated high signal intensity of both agents bound to the tumor. The average tumortr-to-background ratio of antibody and peptide was 1.29 and 2.86, respectively. Signal was also detectable in the liver, kidneys, and bladder. Conclusions: Both NGAL-specific antibody and MMP peptide bound to cancer cells, and the labeled antibody was internalized. These results demonstrate that both agents can be used to enhance detection of human pancreatic cancer xenografts. However, the biodistribution patterns of these agents might limit their use in research and clinical practice.

Neutrophil Gelatinase Associated Lipocalin: Structure, Function and Role in Human Pathogenesis

Glycoproteins have a unique position in the pathogenesis of human diseases. Most of the commonly employed protein biomarkers are glycoproteins. Examples include CA19-9 (carbohydrate antigen 19-9) to follow-up patients with pancreatic cancer, CEA (carcinoembryonic antigen) for multiple solid tumors and CA125 (carbohydrate antigen 125) used in the diagnosis, follow-up and therapy of patients with ovarian cancer. Most of these glycoproteins are large molecules. However, there is a family of smaller, secreted glycoproteins (called lipocalins) that are important in the maintenance of health and in combating diseases effectively. A prototype of this family is a small, secreted glycoprotein called Neutrophil gelatinase associated lipocalin or NGAL. In recent years, NGAL has emerged as a novel biomarker in several benign and malignant diseases. Further, studies in cultured cells and in murine models have revealed a pivotal role for this molecule in several physiological processes and pathological conditions. In this Chapter, we review the biology of lipocalins, focusing specifically on NGAL and its role in human health and disease.