The Pattern of mRNA Expression Is Changed in Sinoatrial Node from Goto-Kakizaki Type 2 Diabetic Rat Heart.

BACKGROUND: In vivo experiments in Goto-Kakizaki (GK) type 2 diabetic rats have demonstrated reductions in heart rate from a young age. The expression of genes encoding more than 70 proteins that are associated with the generation and conduction of electrical activity in the GK sinoatrial node (SAN) have been evaluated to further clarify the molecular basis of the low heart rate. MATERIALS AND METHODS: Heart rate and expression of genes were evaluated with an extracellular electrode and real-time RT-PCR, respectively. Rats aged 12-13 months were employed in these experiments. RESULTS: Isolated spontaneous heart rate was reduced in GK heart (161 ± 12 bpm) compared to controls (229 ± 11 bpm). There were many differences in expression of mRNA, and some of these differences were of particular interest. Compared to control SAN, expression of some genes were downregulated in GK-SAN: gap junction, Gja1 (Cx43), Gja5 (Cx40), Gjc1 (Cx45), and Gjd3 (Cx31.9); cell membrane transport, Trpc1 (TRPC1) and Trpc6 (TRPC6); hyperpolarization-activated cyclic nucleotide-gated channels, Hcn1 (HCN1) and Hcn4 (HCN4); calcium channels, Cacna1d (Cav1.3), Cacna1g (Cav3.1), Cacna1h (Cav3.2), Cacna2d1 (Cavα2δ1), Cacna2d3 (Cavα2δ3), and Cacng4 (Cav γ 4); and potassium channels, Kcna2 (Kv1.2), Kcna4 (Kv1.4), Kcna5 (Kv1.5), Kcnb1 (Kv2.1), Kcnd3 (Kv4.3), Kcnj2 (Kir2.1), Kcnk1 (TWIK1), Kcnk5 (K2P5.1), Kcnk6 (TWIK2), and Kcnn2 (SK2) whilst others were upregulated in GK-SAN: Ryr2 (RYR2) and Nppb (BNP). CONCLUSIONS: This study provides new insight into the changing expression of genes in the sinoatrial node of diabetic heart.

Frondoside A potentiates the effects of conventional therapeutic agents in acute leukemia.

Acute leukemia is the major cause of mortality in hematological malignancies. Despite improvement of survival with current chemotherapies, patients die from the disease or side-effects of treatment. Thus, new therapeutic agents are needed. Frondoside A is a triterpenoid glycoside originally isolated from the sea cucumber, Cucumaria frondosa that has potent antitumor effects in various cancers. The current study investigated the effects of frondoside A in acute leukemia cell lines alone and in combination with drugs used for this malignancy. This study is the first comparing the efficacy of frondoside A to available conventional drugs. The acute leukemia cell lines used were CCRF-CEM, HL-60 and THP-1. Cells were cultured and treated with different concentrations of vincristine sulphate, asparaginase and prednisolone alone and in combination with frondoside A. The inhibitory concentration 50 (IC50) for each compound was determined for the cell lines. CCRF-CEM cells were very sensitive to frondoside A treatment while HL-60 and THP1 were less sensitive. Frondoside A markedly enhanced the anticancer effects of all of the conventional drugs. Synergistic effects were seen with most of the combinations. Frondoside A may be valuable in the treatment of acute leukemia, particularly when used in combination with current therapeutic drugs.

Effects of a sucrose-enriched diet on the pattern of gene expression, contraction and Ca(2+) transport in Goto-Kakizaki type 2 diabetic rat heart.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM), and cardiovascular disease is the major cause of morbidity and mortality in diabetic patients. A variety of diastolic and systolic dysfunctions have been demonstrated in type 2 diabetic heart. The consumption of sugar-sweetened beverages has been linked to rising rates of obesity, which in turn is a risk factor for development of T2DM. In this study, the effects of a sucrose-enriched diet on the pattern of gene expression, contraction and Ca(2+) transport in the Goto-Kakizaki T2DM rat heart were investigated. Genes encoding cardiac muscle proteins (Myh7, Mybpc3, Myl1, Myl3 and Mylpf), intercellular proteins (Gja4), cell membrane transport (Atp1b1), calcium channels (Cacna1c, Cacna1g and Cacnb1) and potassium channels (Kcnj11) were upregulated and genes encoding potassium channels (Kcnb1) were downregulated in GK compared with control rats. Genes encoding cardiac muscle proteins (Myh6, Mybpc3 and Tnn2), intercellular proteins (Gja1 and Gja4), intracellular Ca(2+) transport (Atp2a1 and Ryr2), cell membrane transport (Atp1a2 and Atp1b1) and potassium channel proteins (Kcnj2 and Kcnj8) were upregulated and genes encoding cardiac muscle proteins (Myh7) were downregulated in control rats fed sucrose compared with control rats. Genes encoding cardiac muscle proteins (Myh7) and potassium channel proteins (Kcnj11) were downregulated in control and GK rats fed sucrose compared with control and GK rats, respectively. The amplitude of shortening was reduced in myocytes from the control-sucrose group compared with control rats and in the GK-sucrose group compared with GK rats. The amplitude of the Ca(2+) transient was increased in myocytes from control-sucrose compared with control rats and decreased in GK-sucrose compared with GK rats. Subtle alterations in the pattern of expression of genes encoding a variety of cardiac muscle proteins are associated with changes in shortening and intracellular Ca(2+) transport in ventricular myocytes from GK T2DM and control rats fed a sucrose-enriched diet.

Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer.

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 µg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

Effects of exercise training on excitation-contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats.

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation-contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation-contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10-11 months following 2-3 months of treadmill exercise training. Shortening, [Ca(2+)]i and L-type Ca(2+) current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca(2+) transients and L-type Ca(2+) current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca(2+) transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.

Rectal taurocholate increases L cell and insulin secretion, and decreases blood glucose and food intake in obese type 2 diabetic volunteers.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are secreted from enteroendocrine L cells in response to numerous stimuli, including bile salts. Both have multiple effects that are potentially useful in treating diabetes and obesity. L cell number and hormone content in the intestine are highest in the rectum in humans. We investigated the effects of intrarectal sodium taurocholate on plasma GLP-1, PYY, insulin and glucose concentrations, and on food intake of a subsequent meal. METHODS: Ten obese type 2 diabetic volunteers were each studied on five separate occasions after an overnight fast and oral administration of 100 mg sitagliptin 10 h before the study. They then received an intrarectal infusion of either one of four doses of taurocholate (0.66, 2, 6.66 or 20 mmol, each in 20 ml of vehicle) or vehicle alone (1% carboxymethyl cellulose) single-blind over 1 min. Hormone and glucose measurements were made prior to, and for 1 h following, the infusion. The consumption of a previously selected favourite meal eaten to satiety was measured 75 min after the infusion. RESULTS: Taurocholate dose-dependently increased GLP-1, PYY and insulin, with 20 mmol doses resulting in peak concentrations 7.2-, 4.2- and 2.6-fold higher, respectively, than those achieved with placebo (p < 0.0001 for each). Plasma glucose decreased by up to 3.8 mmol/l (p < 0.001). Energy intake was decreased dose-dependently by up to 47% (p < 0.0001). The ED(50) values for effects on integrated GLP-1, insulin, PYY, food intake and glucose-lowering responses were 8.1, 10.5, 18.5, 24.2 and 25.1 mmol, respectively. CONCLUSIONS/INTERPRETATION: Therapies that increase bile salts (or their mimics) in the distal bowel may be valuable in the treatment of type 2 diabetes and obesity.

Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca(2+) transient were unaltered; however, the time-to-peak shortening was prolonged and time-to-half decay of the Ca(2+) transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation-contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca(2+) transport.

Contractility of ventricular myocytes is well preserved despite altered mechanisms of Ca2+ transport and a changing pattern of mRNA in aged type 2 Zucker diabetic fatty rat heart.

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The objective of the study was to investigate ventricular myocyte shortening, intracellular Ca(2+) signalling and expression of genes encoding cardiac muscle proteins in the aged Zucker diabetic fatty (ZDF) rat. There was a fourfold elevation in non-fasting blood glucose in ZDF rats (478.43 ± 29.22 mg/dl) compared to controls (108.22 ± 2.52 mg/dl). Amplitude of shortening, time to peak (TPK) and time to half (THALF) relaxation of shortening were unaltered in ZDF myocytes compared to age-matched controls. Amplitude and THALF decay of the Ca(2+) transient were unaltered; however, TPK Ca(2+) transient was prolonged in ZDF myocytes (70.0 ± 3.2 ms) compared to controls (58.4 ± 2.3 ms). Amplitude of the L-type Ca(2+) current was reduced across a wide range of test potentials (-30 to +40 mV) in ZDF myocytes compared to controls. Sarcoplasmic reticulum Ca(2+) content was unaltered in ZDF myocytes compared to controls. Expression of genes encoding cardiac muscle proteins, membrane Ca(2+) channels, and cell membrane ion transport and intracellular Ca(2+) transport proteins were variously altered. Myh6, Tnnt2, Cacna2d3, Slc9a1, and Atp2a2 were downregulated while Myl2, Cacna1g, Cacna1h, and Atp2a1 were upregulated in ZDF ventricle compared to controls. The results of this study have demonstrated that preserved ventricular myocyte shortening is associated with altered mechanisms of Ca(2+) transport and a changing pattern of genes encoding a variety of Ca(2+) signalling and cardiac muscle proteins in aged ZDF rat.

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+ signalling in young type 2 Zucker diabetic fatty rat heart.

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca(2+) transport in young (9- to 13-week-old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time-to-peak shortening and time to half-relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age-matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca(2+) transient was unaltered; however, time-to-peak Ca(2+) transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L-type Ca(2+) current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L-type Ca(2+) current was 1.19 ± 0.28 pA pF(-1) in ZDF myocytes compared with 2.42 ± 0.40 pA pF(-1) in control myocytes. Sarcoplasmic reticulum Ca(2+) content, release and uptake and myofilament sensitivity to Ca(2+) were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L-type Ca(2+) channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca(2+) transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L-type Ca(2+) channel proteins might partly underlie alterations in the time course of contraction and Ca(2+) transients in ventricular myocytes from ZDF rats.

Review of the apoptosis pathways in pancreatic cancer and the anti-apoptotic effects of the novel sea cucumber compound, Frondoside A.

Pancreatic cancer cells are resistant to the growth-inhibitory and apoptosis-inducing effects of conventional chemotherapeutic agents. There are multiple genetic and epigenetic events during the process of carcinogenesis that enable the cancer cells to avoid normal growth constraints and apoptosis. Investigation of the mechanisms involved has led to multiple strategies that encourage cell death and apoptosis to occur. The pathways involved are summarized in this review, together with some recently developed strategies to promote cell death in this cancer and with a particular focus on the frondoside A, a novel triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A inhibited proliferation of AsPC-1 human pancreatic cancer cells in a concentration- and time-dependent manner, as measured by (3)H-thymidine incorporation and cell counting. In concert with inhibition of cell growth, frondoside A induced significant morphological changes consistent with apoptosis. Propidium iodide DNA staining showed an increase of sub-G0/G1 cell population of apoptotic cells induced by frondoside A. Frondoside A-induced apoptosis was confirmed by annexin V binding and TUNEL assay. Furthermore, western blotting showed a decrease in expression of Bcl-2 and Mcl-1, an increase in Bax expression, activation of caspases 3, 7, and 9, and an increase in the expression of the cyclin-dependent kinase inhibitor, p21. These findings show that frondoside A induced apoptosis in human pancreatic cancer cells through the mitochondrial pathway and activation of the caspase cascade. Finally, a very low concentration of frondoside A (10 mug/kg/day) inhibited growth of AsPC-1 xenografts in athymic mice. In conclusion, new chemotherapeutic agents are desperately needed for pancreatic cancer because of the poor responsiveness to currently available treatment options. Frondoside A has potent growth inhibitory effects on human pancreatic cancer cells, and the inhibition of proliferation is accompanied by marked apoptosis. Frondoside A may be valuable for the treatment or chemoprevention of this devastating disease.

BLT2 is expressed in PanINs, IPMNs, pancreatic cancer and stimulates tumour cell proliferation.

Pancreatic cancer has an abysmal prognosis. Targets for early detection, prevention and therapy are desperately needed. Inflammatory pathways have an important impact on tumour growth and progression. Expression of BLT2 (the second leukotriene B(4) receptor) was investigated by real-time RT-PCR and immunohistochemistry. Cell proliferation was studied after stable transfection with BLT2, after treatment with siRNA and Compound A as an agonist. BLT2 is expressed in all pancreatic cancer cell lines. Results from real-time RT-PCR revealed significant overexpression of BLT2 in malignant intraductal papillary mucinous neoplasias (IPMNs) and pancreatic adenocarcinoma. Intense staining was evident in IPMNs, infiltrating tumour cells and advanced pancreatic intraepithelial neoplasias (PanINs) but not in normal ductal cells. Overexpression of BLT2 as well as stimulation of Colo357, Panc-1 and AsPC1 cells with Compound A caused a significant increase in tumour cell proliferation, an effect reversed after siRNA treatment. This study demonstrates for the first time the expression of BLT2 in the pancreas and overexpression in pancreatic cancers and malignant IPMNs in particular. Upregulation of BLT2 is already evident in precursor lesions (PanINs, IPMNs). Overexpression of this receptor leads to significant growth stimulation. Therefore, we suggest BLT2 as a new target for chemoprevention and therapy for pancreatic cancer.

Inhibition of pancreatic cancer cell growth.

Pancreatic cancer cells are resistant to the growth-inhibitory and apoptosis-inducing effects of conventional chemotherapeutic agents. There are multiple genetic and epigenetic events during the process of carcinogenesis that enable the cancer cells to avoid normal growth constraints and apoptosis. Investigation of the mechanisms involved has led to multiple strategies that encourage cell death and apoptosis to occur. The pathways involved are summarized in this review, together with some recently developed strategies to promote cell death in this cancer.

Mechanisms of skeletal muscle degradation and its therapy in cancer cachexia.

Severe or chronic disease can lead to cachexia which involves weight loss and muscle wasting. Cancer cachexia contributes significantly to disease morbidity and mortality. Multiple studies have shown that the metabolic changes that occur with cancer cachexia are unique compared to that of starvation. Specifically, cancer patients seem to lose a larger proportion of skeletal muscle mass. There are three pathways that contribute to muscle protein degradation: the lysosomal system, cytosolic proteases and the ubiquitin (Ub)-proteasome pathway. The Ub-proteasome pathway seems to account for the majority of skeletal muscle degradation in cancer cachexia and is stimulated by several cytokines including tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, interferon-gamma and proteolysis-inducing factor. Cachexia is particularly severe in pancreatic cancer and contributes significantly to the quality of life and mortality of these patients. Several factors contribute to weight loss in these patients, including alimentary obstruction, pain, depression, side effects of therapy and a high catabolic state. Although no single agent has proven to halt cachexia in these patients there has been some progress in the areas of nutrition with supplementation and pharmacological agents such as megesterol acetate, steroids and experimental trials targeting cytokines that stimulate the Ub-proteasome pathway.

On the role of the islets of Langerhans in pancreatic cancer.

Pancreatic cancer is a devastating disease characterized by a dismal prognosis with most patients dying within six months after diagnosis. Surgery is an option in less than one in five of these patients, and even with tumor resection the majority of patients succumb to the disease. Other effective treatment options are not available. Common features of pancreatic cancer are severe cachexia, marked insulin resistance and diabetes mellitus. Several studies have demonstrated connections between pancreatic cancers and the endocrine pancreas and this has raised questions regarding the role of the islets of Langerhans in pancreatic adenocarcinoma. This manuscript reviews the recent literature in this field and addresses several questions regarding the interaction between the islets of Langerhans and pancreatic cancer. This review considers the histological findings in pancreatic cancer, cell culture and animal experiments, the four islet cell types and the hormones they secrete, as well as the influence of the arachidonic acid pathways on islet cell function and pancreatic cancer. While pancreatic adenocarcinomas are ductal in nature, the cell of origin has not been identified and there is even some evidence that the islets may harbor the precursor cell. Considerable evidence suggests that the diabetes is caused by the tumor, while other studies have identified diabetes as a risk factor. Clearly, the islets are important in many aspects of this disease. However, even though progress has been made, some questions regarding the interaction of pancreatic cancer and the endocrine pancreas remain unanswered.

12-lipoxygenase metabolite 12(S)-HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation.

We previously reported that inhibition of the 12-lipoxygenase pathway abolished proliferation and induced apoptosis in several pancreatic cancer cell lines. Furthermore, the 12-lipoxygenase product 12(S)-HETE stimulated pancreatic cancer cell proliferation and reversed 12-lipoxygenase inhibitor-induced growth inhibition. We investigated the underlying mechanism for 12(S)-HETE-induced pancreatic cancer cell proliferation, using 2 human pancreatic cancer cell lines, PANC-1 and HPAF. Cell proliferation was monitored by both thymidine incorporation and cell number. Western blotting was used to investigate the effect of 12(S)-HETE on cellular protein tyrosine phosphorylation as well as ERK, P38 MAPK and JNK/SAPK phosphorylation. 12(S)-HETE markedly stimulated proliferation of pancreatic cancer cells in a time- and concentration-dependent manner. In parallel, 12(S)-HETE induced tyrosine phosphorylation of multiple cellular proteins, while inhibition of tyrosine kinase by genestein abolished 12(S)-HETE-induced proliferation, indicating that intracellular protein tyrosine kinase activation is involved in the mitogenic effects of 12(S)-HETE. Following treatment with 12(S)-HETE, both ERK and P38 MAPK, but not JNK/SAPK, were phosphorylated. The specific MEK inhibitors PD098059 and U0126, which in turn suppress ERK, abolished 12(S)-HETE-stimulated proliferation. In contrast, inhibition of P38 MAPK with SB203580 did not affect 12(S)-HETE-stimulated pancreatic cancer cell proliferation. Furthermore, 12(S)-HETE-stimulated ERK phosphorylation was inhibited by genestein, indicating that tyrosine phosphorylation is essential for ERK activation. These findings suggest that both ERK and cellular protein tyrosine kinase activation are involved in 12(S)-HETE-induced pancreatic cancer cell proliferation but P38 and JNK/SAPK are not involved in this mitogenic effect.

Responses of python gastrointestinal regulatory peptides to feeding.

In the Burmese python (Python molurus), the rapid up-regulation of gastrointestinal (GI) function and morphology after feeding, and subsequent down-regulation on completing digestion, are expected to be mediated by GI hormones and neuropeptides. Hence, we examined postfeeding changes in plasma and tissue concentrations of 11 GI hormones and neuropeptides in the python. Circulating levels of cholecystokinin (CCK), glucose-dependent insulinotropic peptide (GIP), glucagon, and neurotensin increase by respective factors of 25-, 6-, 6-, and 3.3-fold within 24 h after feeding. In digesting pythons, the regulatory peptides neurotensin, somatostatin, motilin, and vasoactive intestinal peptide occur largely in the stomach, GIP and glucagon in the pancreas, and CCK and substance P in the small intestine. Tissue concentrations of CCK, GIP, and neurotensin decline with feeding. Tissue distributions and molecular forms (as determined by gel-permeation chromatography) of many python GI peptides are similar or identical to those of their mammalian counterparts. The postfeeding release of GI peptides from tissues, and their concurrent rise in plasma concentrations, suggests that they play a role in regulating python-digestive responses. These large postfeeding responses, and similarities of peptide structure with mammals, make pythons an attractive model for studying GI peptides.

The role of eicosanoids in the process of adaptation following massive bowel resection in the rat.

BACKGROUND: Long chain polyunsaturated fatty acids (LCPUFAs) such as arachidonic acid (AA) and eicosapentaenoic acid (EPA) stimulate intestinal adaptation. Prostaglandins also enhance intestinal adaptation. The purpose of this study was to determine by which eicosanoid pathway dietary arachidonic acid enhances intestinal adaptation. Cyclo-oxygenase or lipoxygenase were selectively inhibited to determine whether either of them enhanced or inhibited adaptation. METHODS: Sixty Sprague-Dawley rats were divided into 2 groups, one receiving an 80% small bowel resection and the other receiving a sham operation. Rats were further divided into groups receiving either a placebo, a general lipoxygenase inhibitor (nordihydroguaiaretic acid [NDGA] at 40 mg/kg per day), or a cyclo-oxygenase-2 inhibitor (Etodolac at 3 mg/kg per day). Rats were pair-fed a diet containing 30% kcal from fat, primarily consisting of AA. RESULTS: After 14 days, mucosal mass, protein, DNA, and disaccharidase activity were measured in the remaining small intestine. There was a significant decrease in ileal mucosal mass in rats receiving Etodolac and a significant increase in mucosal mass in the duodenum in rats receiving NDGA (both p < .001). Mucosal DNA, protein, and disaccharidase data showed similar trends. CONCLUSIONS: These findings suggest that after small bowel resection, dietary arachidonic acid may facilitate the adaptation process by acting as a substrate for the synthesis of prostaglandins, and not through the derivatives of lipoxygenase such as leukotrienes or thromboxanes.

Transdifferentiation of human islet cells in a long-term culture.

It has been established that ductal cells or precursor cells within the ductal tree of the pancreas can differentiate into islet cells. Although islet cells can also form exocrine cells, it is unclear whether they arise from precursor (stem) cells or from mature endocrine cells by transdifferentiation. Using a defined culture medium and technique for islet purification, for the first time we were able to maintain human islets in culture for more than a year. Multilabeling immunohistochemical and immunoelectron microscopic examination of the islets at different days of culture using islet cell markers (antibodies to hormones, neuron-specific enolase, chromogranin A) and ductal cell markers (cytokeratins 7 and 19, carbonic anhydrase II, DU-PAN2, CA 19-9, and MUC1) revealed that endocrine cells gradually transdifferentiate to ductal, acinar, and intermediary cells. Although islet hormone secretion ceased after day 28 in culture, endocrine cells were still detectable at day 60. However, later, all endocrine and exocrine cells were replaced by undifferentiated cells that expressed neuron-specific enolase, chromogranin A, laminin, vimentin, cytokeratin 7 and 19, alpha-1-antitrypsin, transforming growth factor-alpha, and epidermal growth factor receptor. Our data thus show that, under proper conditions, human islets can be maintained in vitro over a long period and that, in the culture condition, islet cells seem to transdifferentiate to exocrine cells and undifferentiated cells, which may be considered pancreatic precursor (stem) cells.

MEK/ERK-mediated proliferation is negatively regulated by P38 map kinase in the human pancreatic cancer cell line, PANC-1.

Pancreatic carcinoma is characterized by a poor prognosis and lack of response to conventional therapy. The regulatory mechanisms for the rapid proliferation of pancreatic cancer cells and the particular aggressiveness of this cancer are still not fully understood. In mammalian cells, three MAPK families including ERK, JNK, and P38 MAPK have been characterized. ERK is known to play an important role in regulating pancreatic cancer cell proliferation. However, the role of P38 kinase in pancreatic cancer cell proliferation and its relationship with ERK are unclear. Using the specific P38 inhibitor, SB203580 we found that blockade of P38 MAP kinase significantly enhanced proliferation of the pancreatic cancer cell line, PANC-1 cell, in a concentration-dependent manner. In parallel with the stimulation of proliferation, blockade of P38 MAP kinase markedly induced MEK and ERK1/2 phosphorylation, indicating an interaction between MEK/ERK and P38 MAP kinase signaling. Clearly, the interaction between these kinase pathways does not involve transcription and translation because MEK/ERK was activated immediately upon SB203580 treatment. Furthermore, inhibition of the MEK/ERK cascade using the MEK inhibitor, PD098059 abolished SB203580-induced PANC-1 cell proliferation. From these results, we conclude that a MEK/ERK and P38 MAP kinase interaction is important for pancreatic cancer cell proliferation. Breaking the balance between these two signaling pathways will modify pancreatic cancer cell proliferation.