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Objectives To investigate the effects and mechanism of Frondanol-A5P, a polar extract from Cucumaria frondosa, on growth inhibition and apoptosis in S2013 and AsPC-1 human pancreatic cancer cells. Methods The effects of Frondanol-A5P on proliferation, cell cycle, expression of cell cycle proteins and p21, phosphorylation of MAP kinases, annexin V binding, and caspase-3 activation were examined. Results Frondanol-A5P inhibited proliferation and induced G2/M phase cell cycle arrest in both cell lines with decreased expression of cyclin A, cyclin B, and cdc25c. Frondanol-A5P induced phosphorylation of stress-activated protein kinase and Janus kinase (SAPK/JAK) and p38 mitogen-activated protein kinase (MAP) within 5 minutes. Frondanol-A5P markedly increased expression of p21 messenger RNA and protein at 3 hours in both cell lines. This effect was reduced by the p38 kinase inhibitor, SB203580. Frondanol-A5P markedly increased annexin V binding and activated caspase-3. Conclusions Frondanol-A5 causes cell cycle arrest and apoptosis in human pancreatic cancer cells. These changes are associated with decreased expression of cyclin A, cyclin B, and cdc25c and increased expression of p21 that, at least in part, is mediated by a p38 kinase-dependent mechanism. Because Frondanol-A5P is derived from an edible, nontoxic, sea cucumber, it may be valuable for nutritional therapy or prevention of pancreatic cancer.

OBJECTIVES Five distinct somatostatin receptors (SSTRs) have been cloned, characterized, and designated SSTRs 1-5. The role of these receptors in B-cell signaling has not been well characterized. METHODS In the current study, the isolated perfused human pancreas model was used to determine the specific effect of 4 different somatostatin receptor agonists on insulin secretion. CONCLUSION We demonstrated that the SSTR 2 agonist and octreotide significantly suppressed insulin secretion. Furthermore, even during the immunoneutralization of endogenous intrapancreatic somatostatin, the SSTR 2 agonist was able to reverse the effect of somatostatin immunoneutralization by suppressing insulin secretion. These results demonstrate that activation of SSTR 2 suppresses insulin secretion in the isolated perfused human pancreas.

We have previously shown that arsenic trioxide blocks proliferation and induces apoptosis in human pancreatic cancer cells at low, non-toxic concentrations. The mechanisms of the apoptosis was investigated in MiaPaCa2 and PANC-1 cells that have been previously shown to be responsive to arsenic trioxide. The results show the caspase-3, caspase-7, and caspase-9 are all activated by arsenic trioxide, together with cleavage of the downstream caspase-3 target poly ADP ribose polymerase (PARP). Expression of the anti-apoptosis proteins, Bcl-2 and Mcl-1 expression decreased time-dependently while Bax expression increased. These findings indicate that the Bcl family of proteins, the mitochondrial pathway and activation of the caspase cascade are responsible for arsenic-induced apoptosis. Flow cytometric analysis revealed changes of cell cycle distribution from a G0/G1 phase arrest at 24 hours to G2/M phase arrest at 72 hours following arsenic treatment. The sub-G0/G1 cell population of apoptotic cells was increased at these times. Arsenic increased expression of the P21 protein and decreased levels of cyclin A, cyclin B1 and cyclin D1, but expression of CDK2, CDK4, CDK6, and cyclin E were not affected. Arsenic trioxide markedly enhanced the expression of GADD45 and GADD153 in a time-dependent manner. In summary, arsenic trioxide induced apoptosis in pancreatic cancer cells through activating the caspase cascade via the mitochondrial pathway, GADD expression and by modifying cell cycle progress and changes in several cycle-regulating proteins. This old drug may be valuable for treatment of pancreatic cancer.

Because of lack of early diagnosis and poor therapeutic responsiveness, median survival in patients with pancreatic cancer is6 months, and survival beyond 5 years is rare. Thus, another dimension in chemotherapeutic agents for pancreatic cancer would be beneficial to control metastatic and unresectable disease. Resveratrol, a natural product from grapes, has been shown to be chemopreventive for carcinogen-induced skin cancer and also to inhibit proliferation of oral squamous, breast, colonic, and prostate cancer cells.To investigate the effect of resveratrol in pancreatic cancer.To evaluate the potential role of resveratrol on pancreatic cancer cell proliferation, two human pancreatic cancer cell lines, PANC-1 and AsPC-1, were used.Resveratrol inhibited proliferation of both PANC-1 and AsPC-1 in a concentration- and time-dependent manner as measured by [ H]thymidine incorporation. Cell number of both PANC-1 and AsPC-1 was also significantly decreased following 48 and 72 hours of treatment with 100 micromol/L resveratrol. The growth inhibition induced by resveratrol was accompanied by apoptotic morphologic changes, characterized by cell rounding and cell membrane blebbing suggesting apoptosis. Propidium iodide staining of DNA, measured by flow cytometry, showed a dramatic increase in the fraction of sub-G0/G1 cells following resveratrol treatment in both PANC-1 and AsPC-1. The substantial apoptosis inducted by resveratrol on these two cell lines was confirmed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay.These findings suggest that the natural product resveratrol may have a potent antiproliferative effect on human pancreatic cancer with induction of apoptosis. Resveratrol is likely to be valuable for the management and prevention of human pancreatic cancer.

INTRODUCTION Pancreatic cancer cells are susceptible to antiproliferative effects of cytokines such as tumor necrosis factor-alpha (TNF-alpha). However, little is known about the mechanisms involved. AIM To determine the mechanisms of the antiproliferative effects of TNF-alpha on pancreatic cancer cells. METHODOLOGY AND RESULTS In the current study, four of five pancreatic cancer cell lines tested were responsive to the antiproliferative effects of TNF-alpha. In two cell lines, the effects of TNF-alpha were completely abolished by superoxide dismutase, suggesting that superoxide anion mediates the effects. Further, inhibition of nitric oxide (NO) synthase by L-NAME potentiated the TNF-alpha response, suggesting a protective role for endogenously produced NO in these two cell lines. MiaPaCa-2 cells, which were unresponsive to the antiproliferative effects of TNF-alpha, produced five times more nitrite than the other cell lines. Treatment of MiaPaCa-2 cells with interferon-gamma (IFN-gamma) increased inducible nitric oxide synthase mRNA as shown by reverse transcription-polymerase chain reaction. This induction was potentiated by TNF-alpha and further enhanced by a combination of three cytokines (INF-gamma, TNF-alpha, and interleukin 1-beta (IL-1beta). This combination of cytokines increased nitrite accumulation fourfold and inhibited the proliferation of this resistant cell line. These effects were prevented by L-NAME. CONCLUSIONS These studies suggest that TNF-alpha inhibits proliferation of pancreatic cancer cells by increasing the production of superoxide anion and that endogenously produced NO protects against this effect. In addition, there is a direct correlation between the amount of NO produced and resistance to TNF-alpha in the only resistant cell line. In contrast, treatment with a combination of IFN-gamma, TNF-alpha and IL-1beta upregulates inducible nitric oxide synthase, and the resulting markedly enhanced NO production inhibits pancreatic cancer cell growth. This pathway may provide a valuable target for therapy of this devastating disease.

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy for reasons that are not clear. Because of the structural relationship between the exocrine and endocrine pancreas and high concentrations of islet hormones bathing pancreatic tissue, we hypothesized that pancreatic cancer cell proliferation and glucose utilization are regulated by pancreatic islet hormones, particularly insulin. Based on this, the effect of islet hormones on pancreatic cancer cells in vitro was investigated. Five pancreatic cancer cell lines, CD11, CD18, HPAF, PANC-1, and MiaPaCa2 were used to investigate the effect of islet hormones on cell proliferation, glucose utilization, and GLUT-1 expression. Insulin, but not somatostatin and glucagon, induced pancreatic cancer cell growth in a concentration- and time-dependent manner. At concentrations within the range of those in the intrapancreatic vasculature, insulin (10(-10)-10(-8) mol/L) markedly increased [3H]-thymidine incorporation. Insulin significantly enhanced glucose utilization of pancreatic cancer cells before it enhanced cell proliferation. The MAPK kinase inhibitor PD 098059 abolished insulin-stimulated DNA synthesis and partially reduced insulin-stimulated glucose uptake. In contrast, the PI3 kinase inhibitor wortmannin substantially inhibited insulin-induced glucose uptake and partially blocked thymidine incorporation. Furthermore, after 24-hour treatment with insulin, GLUT-I expression in pancreatic cancer cells was markedly increased, indicating that insulin enhances glucose utilization partly through increasing glucose transport. These findings suggest that insulin stimulates proliferation and glucose utilization in pancreatic cancer cells by two distinct pathways. Insulin augments DNA synthesis mainly by MAP kinase activation and glucose uptake mainly by PI3 kinase activation and enhancement of GLUT-I expression. High intrapancreatic concentrations of insulin are likely to play an important role in stimulating pancreatic cancer growth indirectly by increasing substrate availability as well as by direct action as a trophic factor.

The diabetes or impaired glucose tolerance that occurs in most patients with pancreatic cancer is characterized by profound insulin resistance. Recent evidence suggests that the diabetes may result from the presence of the tumor rather than being a predisposing factor to development of the malignancy. Some islet hormones have been shown to exhibit diabetogenic effects. To investigate the potential role of these hormones in the diabetic state associated with pancreatic cancer, we measured islet hormones during fasting in pancreatic cancer patients (n = 30), patients with other malignancies (n = 43), and healthy controls (n = 25). Preoperative pancreatic cancer patients were classified as normal glucose tolerance (NGTT), impaired glucose tolerance (IGTT), non-insulin-requiring diabetes (NIRD), and insulin-requiring diabetes (IRD). Nine pancreatic cancer patients were studied after tumor removal by subtotal pancreatectomy. Some preoperative pancreatic cancer patients (n = 19), postoperative patients (n = 9), and controls (n = 8) were also studied during hyperglycemia and following glucagon injection. Fasting plasma C-peptide was elevated in NIRD pancreatic cancer patients compared to controls. Fasting levels of islet amyloid polypeptide (IAPP), glucagon, and somatostatin were elevated in NIRD and IRD patients. IAPP and glucagon, but not somatostatin, normalized following subtotal pancreatectomy. During hyperglycemia, increases in C-peptide and IAPP were seen only in controls and in NGTT and postoperative pancreatic cancer patients. After glucagon infusion, IAPP levels increased in controls and nondiabetic cancer patients; C-peptide levels increased in controls, nondiabetic patients, and NIRD. Responses of C-peptide and IAPP to glucagon normalized after pancreatectomy. During hyperglycemia, glucagon levels fell in all groups except IGTT patients and a decrease in somatostatin concentrations was seen in controls.

OBJECTIVES Protein kinase C (PKC) is involved in cell growth, differentiation, and apoptosis. We investigated the effects of the PKC activator, the tetradecanylphorbol acetate (TPA), in human pancreatic cancer cells. METHODS Cell proliferation was measured by thymidine incorporation. Expression of cell cycle proteins was investigated by Western blot. Real-time reverse transcriptase-polymerase chain reaction was used to measure p21 messenger RNA expression, whereas knockdown of its expression was accomplished with a specific small interferring RNA. Cell cycle phases were determined by flow cytometry. RESULTS TPA time and concentration dependently inhibited thymidine incorporation in Panc-1 and CD18 cells and induced G2/M cell cycle arrest. The TPA decreased cyclin A and B expression, increased cyclin E, and markedly increased the expression of p21 at both the messenger RNA and protein levels. TPA-induced p21 expression and growth inhibition were blocked by the PKC inhibitor, bisindoylmaleimide. TPA induced extracellular signal-regulated kinase1/2 phosphorylation, whereas the MEK inhibitor, PD98059, blocked the TPA-induced p21 expression. Small interferring RNA targeted to p21 blocked TPA-induced p21 protein expression but not TPA-induced cell growth arrest. CONCLUSIONS TPA-induced p21 expression is mediated by the MEK/ERK pathway but is not involved in TPA-induced growth inhibition. In contrast, cyclin A and cyclin B are likely involved in TPA-induced G2/M arrest because both proteins are involved in S phase and G2/M transition during cell proliferation.

Overexpression of autocrine growth factors and their receptors has been reported in many human cancers. The study of autocrine-regulated pathways using in vitro culture systems can be hindered by the presence of fetal bovine serum in culture medium. A human pancreatic cancer cell line (HPAF) was slowly weaned from its dependence on fetal bovine serum and subsequently maintained in serum-free conditions. Growth factor secretion studies showed that production of autocrine growth factors such as transforming growth factor alpha, gastrin-releasing peptide, and insulin-like growth factor I from weaned cells increased three times compared with nonweaned cells (p < 0.01). The epidermal growth factor and gastrin-releasing peptide receptor densities were also increased in weaned cells (2 times and 2.5 times, respectively, p < 0.05). The proliferation of weaned cells cultured continuously in the same medium was significantly greater than of nonweaned cells (p < 0.05). Collectively, these data indicate that weaned pancreatic cancer cells can proliferate in the absence of serum by up-regulating autocrine pathways.

Islet amyloid polypeptide (IAPP) and insulin are co-stored and generally secreted in parallel; however, studies have demonstrated that the IAPP/insulin molar secretory ratio may be altered in response to certain stimuli. Because we previously demonstrated that intraislet somatostatin is an inhibitory regulator of basal insulin secretion in the isolated perfused human pancreas, this study was designed to determine the relative influence on the regulation of IAPP versus insulin secretion. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors with continuous perfusion of a modified Krebs media with the glucose level maintained at constant 3.9 mM. Intraislet somatostatin was immunoneutralized by the infusion of either a highly sensitive monoclonal somatostatin antibody (SAb) or its FAb fragment (SFAb). Sequential test periods separated by basal periods were performed by infusion of either of the following: glucose, SAb, SFAb, or appropriate controls. IAPP/insulin molar secretory ratio decreased by 33% in response to infusion of either SAb or the SFAb, respectively (p < 0.01), and decreased by 67% in response to glucose infusion (p < 0.01). An alteration of the IAPP/insulin secretory ratio is seen in response to infusion of exogenous glucose or in response to the neutralization of intraislet somatostatin.

Abnormal insulin and islet amyloid polypeptide (IAPP) secretion are usually seen in patients with exocrine pancreatic cancer. The beta-cell dysfunction is a characteristic of the glucose intolerance found in pancreatic cancer patients. The effects of pancreatic cancer cells on insulin and IAPP secretion from beta cells are unclear. In this study, isolated rat pancreatic islets were cocultured with two human pancreatic adenocarcinoma cell lines (Panc-1 and HPAF) and a human colonic adenocarcinoma cell line (HT-29). As a control, islets were incubated in the absence of malignant cells. The accumulation of insulin and IAPP in culture media was measured by radioimmunoassay. Output of insulin and IAPP was decreased in islets cocultured with each malignant cell line. Molar ratio of secreted IAPP and insulin (IAPP/insulin) was increased in the islets cocultured with Panc-1 or HPAF cells, but not HT-29 cells. The decreased insulin and IAPP secretion were partly recovered after Panc-1, HPAF, or HT-29 cells were removed. The IAPP/insulin ratio was normalized after the removal of Panc-1 or HPAF cells. This study indicates that insulin and IAPP secretion are altered by the human adenocarcinoma cells investigated. The impairment induced by pancreatic adenocarcinoma cells is associated with a hypersecretion of IAPP relative to insulin on a molar basis.

Diabetes associated with pancreatic cancer is characterized by profound peripheral insulin resistance. The intracellular mechanism of insulin resistance was investigated in skeletal muscles from N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. Effects of high-fat diet and exercise also were studied. BOP (20 mg/kg body weight) was administrated weekly for 2 weeks. Hyperinsulinemia was found in BOP-treated hamsters at 20 weeks after BOP treatment, suggesting the peripheral insulin resistance is an early feature in pancreatic cancer. Hamsters were killed at 42 weeks, and soleus muscles were taken for the analysis. Skeletal muscle insulin-receptor binding and insulin receptor tyrosine kinase activities were similar between the control and BOP-treated hamsters. However, maximal muscle glycogen synthase activity was significantly reduced in BOP-treated hamsters compared with the control group. Muscle glycogen phosphorylase activity was increased in the BOP-treated group fed with high-fat diet as well as in BOP-treated groups with exercise. These findings indicate that insulin resistance in the hamster pancreatic cancer model is caused by a postreceptor defect, which led to significant decrease of muscle glycogen synthase activity. Whereas a high-fat diet causes more severe insulin resistance in BOP-treated hamsters, high-fat diet and exercising had no significant effects on skeletal muscle insulin-receptor function and glycogen synthase activity. Furthermore, both high-fat diet and exercise enhanced glycogen phosphorylase activity in BOP-treated hamsters.

The role of cholecystokinin (CCK) in pancreatic growth and secretory development in fetal and neonatal guinea pigs was investigated by CCK receptor blockade. For the last 20 days of gestation, sows received the CCK A receptor antagonist, MK329 (25 nmol/kg/h) by continuous subcutaneous infusion. Alternatively, neonates from untreated females received an MK329 infusion for the first 4 or 15 days following birth. Pancreatic weight, DNA, RNA, protein, and amylase content per 100 g body weight and secretory responses to CCK, carbamylcholine, and phorbol ester were determined at birth and 4 days in animals receiving MK329 in utero and were measured at 4 and 15 days in neonatally infused animals. No significant changes in pancreatic growth parameters were seen in MK329-treated animals compared to controls, except for a small (14% ; p < 0.02) decrease in weight after 15 days of neonatal exposure. Enhanced amylase secretion in response to CCK and carbamylcholine was seen in all groups receiving MK329 (all p values < 0.00001). Pancreatic growth and secretion were not inhibited by CCK A receptor blockade, which suggests that the effects of CCK mediated by the CCK A receptor are not essential for growth or development of the pancreatic amylase secretory response in the neonatal guinea pig.

The effect of native prostaglandins and their methylated analogues on pancreatic enzyme secretion remains unclear, with previous studies reporting inconsistent results. To determine whether the E series prostaglandins directly influence pancreatic secretion, we studied the effect of rioprostil, a prostaglandin E1 analogue, and 16,16-dimethyl prostaglandin E2 (DMPGE2), a prostaglandin E2 analogue, on enzyme release from dispersed guinea pig pancreatic acini. Basal amylase release (4.3 +/- 0.6% of total acinar content) was not altered by either analogue (10(-10)-10(-5) M). A 50% inhibition of maximal cholecystokinin stimulation (10(-9) M; 28.8 +/- 1.2%) was seen with rioprostil (10(-7) M; 14.6 +/- 1.3%) and DMPGE2 (10(-6) M; 15.9 +/- 0.7%) (both p less than 0.005). Prostaglandin inhibition of carbachol-stimulated amylase was less pronounced. The most effective inhibitory dosage with maximal carbachol (10(-5) M; 30.2 +/- 1.9%) was 10(-6) M for both rioprostil (19.2 +/- 1.6%) and DMPGE2 (22.4 +/- 1.7%) (both p less than 0.005). Incubation of acini with A23187, phorbol ester, and 1-oleoyl-2-acetyl-glycerol resulted in a dose-dependent increase in amylase release that was not altered by maximal concentrations of either prostaglandin analogue. Our results indicate that rioprostil and DMPGE2 can directly inhibit pancreatic acinar secretion. This effect appears to occur before activation of the inositol phospholipid system.