Hirozumi Sawai

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.

Loss of PTEN expression is associated with colorectal cancer liver metastasis and poor patient survival

BackgroundThe tumour suppressor phosphatase and tensin homolog (PTEN) is an important negative regulator of cell-survival signaling. To evaluate the correlation between PTEN expression and clinicopathological characteristics of colorectal cancer patients with and without liver metastases, we investigated PTEN expression in primary colorectal cancer and colorectal cancer liver metastases.MethodsSixty-nine pairs of primary colorectal cancer and corresponding liver metastasis specimens were analyzed immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. Seventy primary colorectal cancer specimens from patients without liver metastases were used as controls.ResultsPTEN was strongly expressed in 44 (62.9%) colorectal cancer specimens from patients without liver metastases. In contrast, PTEN was weakly expressed in 52 (75.4%) primary colorectal cancer specimens from patients with liver metastases, and was absent in liver metastases. Weak PTEN expression in colorectal cancer tissues was significantly associated with advanced TNM stage (p < 0.01) and lymph node metastasis (p < 0.05). PTEN expression was significantly stronger in primary colorectal cancer specimens from patients without liver metastases. Furthermore, among colorectal cancer patients with liver metastases, the 5-year survival rate was significantly higher in patients with positive PTEN expression compared to those with negative PTEN expression (p = 0.012).ConclusionOur results suggest that loss of PTEN expression is involved with colorectal cancer aggressive capacity and that diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for colorectal cancer patients.

Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation

BackgroundInteraction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1α and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.ResultsIn immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and β1 integrin. Enhancement of FAK protein association with β1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1α. Preincubation with anti-β1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with β1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1α of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1α stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1α-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, β1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1α-induced enhancement of adhesion and invasion in pancreatic cancer cells.ConclusionOur results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.

PTEN regulate angiogenesis through PI3K/Akt/VEGF signaling pathway in human pancreatic cancer cells

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.

IGF-1 Mediates PTEN Suppression and Enhances Cell Invasion and Proliferation via Activation of the IGF-1/PI3K/Akt Signaling Pathway in Pancreatic Cancer Cells

BACKGROUND Type-1 insulin-like growth factor (IGF-1) up-regulates cell proliferation and invasiveness through activation of PI3K/Akt signaling pathway. IGF-1 also down-regulates the tumor suppressor chromosome 10 (PTEN). We investigated the mechanism by which IGF-1 affects cell proliferation and invasion by suppression of PTEN phosphorylation and interaction with PI3K/PTEN/Akt/NF-small ka, CyrillicB signaling pathway in pancreatic cancer. MATERIALS AND METHODS The expression of IGF-1 receptor (IGF-1R) and PTEN in five pancreatic cancer cell lines was determined by RT-PCR and Western blot. Proliferation and invasion were investigated by WST-1 assay and Matrigel-double chamber assay. Pancreatic cancer cells were transfected with PTEN siRNA to investigate which signaling pathway correlates in regulation of cancer cell proliferation and invasion. RESULTS Five pancreatic cancer cell lines expressed PTEN and IGF-1R in mRNA and protein levels. Suppression of PTEN phosphorylation strongly enhanced cell proliferation and invasion stimulated with IGF-1 via activation of PI3K/Akt/NF-small ka, CyrillicB signaling pathway. In addition, knockdown of PTEN by siRNA transfection also enhanced activation of PI3K/Akt/NF-small ka, CyrillicB pathway, subsequently up-regulating cell invasiveness and proliferation. CONCLUSIONS The IGF-1/PI3K/PTEN/Akt/NF-small ka, CyrillicB cascade may be a key pathway stimulating metastasis of pancreatic cancer cells. We suggest that interfering with the functions of IGF-1/PI3K/Akt/NF-small ka, CyrillicB might be a novel therapeutic approach to inhibit aggressive spread of pancreatic cancer.

The G691S RET Polymorphism Increases Glial Cell Line–Derived Neurotrophic Factor–Induced Pancreatic Cancer Cell Invasion by Amplifying Mitogen-Activated Protein Kinase Signaling

Mutations of the RET proto-oncogene are responsible for several inherited human diseases and may function as genetic modifiers of the disease. However, the role of RET mutations in pancreatic cancer has not been studied. Expression of the glial cell line-derived neurotrophic factor (GDNF) receptors RET and GDNF family receptor alpha1 (GFRalpha1) in human pancreatic cancer cells was determined by Western blot, immunofluorescence, and flow cytometry. The effect of GDNF on cell proliferation and invasion was assessed. Small interfering RNA and antibodies were used to evaluate the involvement of RET. The G691S RET polymorphism was analyzed by sequencing and restriction analysis. The modifying effect of G691S RET on GDNF-induced invasion and mitogen-activated protein kinase (MAPK) signaling was evaluated. Transfection studies with wild-type and mutated RET determined the functional role of the G691S polymorphism. Pancreatic cancer specimens and matched tissues were analyzed for the presence of the G691S RET polymorphism. GDNF receptors were found on all cell lines. GDNF increased pancreatic cancer cell proliferation and invasion, which was mediated by RET. The effect of GDNF was more profound in cells with the G691S RET polymorphism (P < 0.01). G691S RET correlated with an enhanced activation of the downstream extracellular signal-regulated kinase pathway. Overexpression of G691S RET increased pancreatic cancer cell invasion. The G691S RET polymorphism was also detected in human pancreatic tumors and represented a somatic mutation in some patients. These findings indicate that the G691S RET single nucleotide polymorphism may directly correlate with the aggressive growth of pancreatic cancers and may function as a genetic modifier or even low-penetrance gene.

The stem cell factor/c- kit receptor pathway enhances proliferation and invasion of pancreatic cancer cells

BackgroundThe transmembrane protein c-kit is a receptor tyrosine kinase (KIT) and KIT is expressed in solid tumors and hematological malignancies such as gastrointestinal stromal tumor (GIST), small-cell lung cancer and chronic myelogenous leukemia (CML). KIT plays a critical role in cell proliferation and differentiation and represents a logical therapeutic target in GIST and CML. In pancreatic cancer, c-kit expression has been observed by immunohistochemical techniques. In this study, we examined the influence of c-kit expression on proliferation and invasion using five pancreatic cancer cell lines. In addition, the inhibitory effect of imatinib mesylate on stem cell factor (SCF)-induced proliferation and invasion was evaluated. Finally, we also analyzed KIT and SCF expression in pancreatic cancer tissues using immunohistochemistry and correlated the results with clinical features.ResultsRT-PCR revealed that two pancreatic cancer cell lines, PANC-1 and SW1990, expressed c-kit mRNA. By Western blot analysis, c-kit protein was also present in those lines. In KIT-positive pancreatic cancer cell lines, proliferation and invasion were significantly enhanced by addition of SCF. In contrast, SCF did not enhance proliferation and invasion in the three KIT-negative lines (BxPC-3, Capan-2 and MIA PaCa-2). 5 μM imatinib mesylate significantly inhibited SCF-enhanced proliferation to the same extent compared with the control. Similarly, SCF-enhanced invasive ability was significantly inhibited by 5 μM imatinib mesylate. KIT was expressed in 16 of 42 clinical specimens by immunohistochemistry, and KIT expression was significantly related to venous system invasion. Furthermore, patients expressing both KIT and SCF had a somewhat lower survival.ConclusionOur results demonstrated that the SCF-KIT pathway enhanced the proliferation and invasiveness in KIT-positive pancreatic cancer cell lines and that the enhanced proliferation and invasion were inhibited by imatinib mesylate. We propose that inhibitors of c-kit tyrosine kinase receptor have the potential to slow the progression of KIT-positive pancreatic cancers.

Opposing effects of n-6 and n-3 polyunsaturated fatty acids on pancreatic cancer growth.

Objectives: Epidemiologic studies suggest that fish oil, rich in n-3 polyunsaturated fatty acids (PUFA), possesses antitumor activity, whereas n-6 PUFAs may stimulate the development of cancers. The aim of this study was to evaluate the effects of n-6 and n-3 PUFAs on the growth of pancreatic cancer. Methods: The n-6 PUFA arachidonic acid (AA) stimulated the growth of cyclooxygenase (COX) 2 positive human pancreatic cancer (PaCa) cells, which was mediated by COX-2 generated prostaglandin E2 (PGE2) binding to EP2 and EP4 receptors. In contrast, the n-3 PUFA eicosapentaenoic acid decreased the growth of COX-2-positive and COX-2-negative PaCa cells. The COX-2-dependent mechanism of eicosapentaenoic acid was mediated by binding of PGE3 to EP2 and EP4 receptors. Dietary intake of n-3 PUFAs decreased the growth of pancreatic cancers in a xenograft model, which was accompanied by a decrease of PGE2 and an increase of PGE3 in the tumors. Conclusions Our studies provide evidence that n-3 PUFAs possess antitumor activities, whereas n-6 PUFAs stimulate pancreatic tumor growth. The opposite effects of n-3 and n-6 PUFAs are mediated by the formation of different prostaglandin species. n-3 PUFAs may prove beneficial as monotherapy or combination therapy with standard chemotherapeutic agents in pancreatic cancer patients.

Stem Cell Factor/c-kit Receptor Signaling Enhances the Proliferation and Invasion of Colorectal Cancer Cells Through the PI3K/Akt Pathway

In this study, we examined the role of c-kit receptor (KIT) signal transduction on the proliferation and invasion of colorectal cancer cells. We found that c-kit was expressed in 2 colorectal cancer cell lines as determined by RT-PCR, Western blot, and flow cytometry. In KIT-positive lines, KIT was activated by stem cell factor (SCF). SCF enhanced cellular proliferation of positive lines as demonstrated by the WST-1 proliferation assay. Furthermore, SCF enhanced the invasive ability of KIT-positive cell lines. SCF stimulation upregulated p44/42 mitogen-activated protein kinase (MAPK) and Akt as shown by Western blot. We examined the roles played by p44/42 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways in proliferation and invasion. PI3K/Akt activity strongly correlated with proliferation and invasion and p44/42 MAPK was correlated with only invasion. In conclusion, the SCF-enhanced proliferation and invasion of KIT-positive colorectal cancer cells is achieved mainly through the PI3K/Akt pathway.

Integrin-linked kinase activity is associated with interleukin-1α-induced progressive behavior of pancreatic cancer and poor patient survival

Cancer cell adhesion and invasion into extracellular matrix are regulated by integrin-linked kinase (ILK) activity in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. In this study, we demonstrated that ILK and β1-integrin play important roles in interleukin (IL)-1α-induced enhancement of adhesion and invasion of pancreatic cancer cells through p38 mitogen-activated protein kinase (MAPK) signaling pathway and activator protein-1 (AP-1) activation. Alteration of ILK kinase activity controlled IL-1α-induced p38 MAPK phosphorylation and its downstream AP-1 activation with subsequent regulation of pancreatic cancer cell adhesion and invasion. Overexpressed ILK enhances the IL-1α-induced p38 MAPK phosphorylation more strongly through glycogen synthase kinase 3 (GSK-3) activation, and subsequently induces AP-1 activation, which promotes aggressive capabilities of pancreatic cancer cells. In contrast, knockdown of ILK kinase activity inhibits the IL-1α-induced activation of MAPK/AP-1 pathway via inhibition of GSK-3 phosphorylation. In immunohistochemical analysis, statistically significant association between strong expression of ILK and poor prognosis of pancreatic cancer patients were observed, and strong expression of ILK in cancerous tissues can be a significant prognostic indicator of pancreatic cancer patients. Our results suggest that ILK is involved with aggressive capability in pancreatic cancer and that these regulations can be helpful to understand biological processes for a better translational treatment for pancreatic cancer patients.