VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis.

Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR-2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR-2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR-2 expression and (iii) to assess the effect of VEGFR-2 on BON cell invasion, migration and proliferation. We found that, although VEGFR-2 is expressed in BON cells, reduction in VEGFR-2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR-2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR-2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR-2 (sVEGFR-2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR-2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR-2, in addition to its well-defined role in angiogenesis. The expression of sVEGFR-2 may explain the inverse relationship between VEGFR-2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR-2 has on BON cell proliferation, migration and invasion.

Development and characterization of a novel in vivo model of carcinoid syndrome.

PURPOSE: Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome. EXPERIMENTAL DESIGN: Here, we have injected BON cells, a novel human carcinoid cell line established in our laboratory, into the spleens of athymic nude mice to establish liver metastases. RESULTS: The majority of mice injected intrasplenically with BON cells developed significant increases in plasma serotonin and urine 5-hydroxyindoleacetic acid, and several mice exhibited mesenteric fibrosis, diarrhea, and fibrotic cardiac valvular disease reminiscent of carcinoid syndrome by both echocardiographic and histopathologic evaluation. Mice pretreated with octreotide, a long-acting somatostatin analogue, or bevacizumab, a vascular endothelial growth factor inhibitor, developed fewer liver metastases and manifestations of carcinoid syndrome, including valvular heart disease. CONCLUSION: We have provided an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome that will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease (e.g., mesenteric fibrosis and valvular heart disease).

Alternative medicine products as a novel treatment strategy for inflammatory bowel disease.

Inflammatory bowel disease (IBD) affects the mucosal lining of the gastrointestinal tract; the etiology is unknown and treatment is directed at systemic immunosuppression. Natural products, including medicinal herbs, have provided approximately half of the drugs developed for clinical use over the past 20 years. The purpose of our current study was to determine the effects of a novel combination of herbal extracts on intestinal inflammation using a murine model of IBD. Female Swiss-Webster mice were randomized to receive normal water or 5% dextran sulfate sodium (DSS) drinking water to induce colitis. Mice were treated with either a novel combination of herbal aqueous extracts or vehicle control per os (po) or per rectum (pr) every 24 hours for 7-8 days. Disease activity index score (DAI) was determined daily; mice were sacrificed and colons were analyzed by H & E staining, MPO assay, and cytokine (TNF-alpha, IL-6) ELISAs. Mice treated with the combination of herbal extracts, either po or pr, had significantly less rectal bleeding and lower DAI scores compared to the vehicle-treated group. Moreover, colonic ulceration, leukocytic infiltration, and cytokine levels (TNF-alpha and IL-6) were also decreased in the colons of herbal-treated mice, reflected by H & E staining, MPO assay, and cytokine ELISA. Treatment with the combination of medicinal herbs decreases leukocyte infiltration and mucosal ulceration, ameliorating the course of acute colonic inflammation. This herbal remedy may prove to be a novel and safe therapeutic alternative in the treatment of IBD.

Effectiveness of siRNA uptake in target tissues by various delivery methods.

BACKGROUND: RNA interference offers clinical potential as a therapeutic modality for a variety of diseases; the efficacy of in vivo delivery remains poorly understood. The purpose of our study was to compare and contrast short interfering RNA (siRNA) uptake in vivo using various delivery techniques. METHODS: DY547- and rhodamine-labeled siRNA was administered to mice by one of four delivery methods: (1) hydrodynamic intravenous tail vein injection, (2) standard intravenous tail vein injection, (3) intraperitoneal injection, and (4) rectal administration. Mice were killed over a time course; representative tissue samples were collected and analyzed using fluorescent microscopy to determine siRNA uptake. RESULTS: siRNA uptake was noted in the liver, kidney, pancreas, spleen, and bone marrow by both hydrodynamic and standard IV injection. siRNA uptake was detected in the spleen, liver, and bone marrow after intraperitoneal administration. Rectal administration resulted in siRNA uptake in the spleen, bone marrow, and colon. CONCLUSION: Our results demonstrate differential siRNA uptake depending on delivery technique. Importantly, our results demonstrate the potential of siRNA as a systemic therapeutic option in vivo for selected disease processes.

Increased incidence of well-differentiated thyroid cancer associated with Hashimoto thyroiditis and the role of the PI3k/Akt pathway.

BACKGROUND: The link between inflammation and cancer is well-established, but the link between Hashimoto thyroiditis (HT) and thyroid cancer remains controversial. The purpose of our study was to determine the incidence of patients with thyroid cancer and associated HT at our institution, to correlate our patient population demographics with the Surveillance, Epidemiology and End Results (SEER) database, and to assess the expression of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in patients with HT. STUDY DESIGN: Demographic and histologic data were collected from patients undergoing thyroid resection at the University of Texas Medical Branch from 1987 to 2002 and compared with the SEER database. Immunohistochemistry for phosphorylated Akt (a marker of PI3K activity), Akt isoforms and PTEN (an inhibitor of PI3K) was performed on paraffin-embedded blocks of resected thyroid tissue. RESULTS: Our patient population demographics and thyroid cancer incidence by histologic type were similar to patients in the SEER database. Ninety-eight (37.7%) resected specimens had pathologic changes consistent with HT; 43 (43.8%) had an associated well-differentiated thyroid cancer. Increased phosphorylated Akt, Akt1, and Akt2 expression was noted in regions of HT and thyroid cancer compared with regions of normal surrounding thyroid tissue. CONCLUSIONS: Patients with HT were three times more likely to have thyroid cancer, suggesting a strong link between chronic inflammation and cancer development. PI3K/Akt expression was increased in both HT and well-differentiated thyroid cancer, suggesting a possible molecular mechanism for thyroid carcinogenesis.

Nuclear factor of activated T cells (NFAT) signaling regulates PTEN expression and intestinal cell differentiation.

The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3α/ß. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27(kip1) expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27(kip1) expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation.

The effect of PTEN on serotonin synthesis and secretion from the carcinoid cell line BON.

BACKGROUND: Carcinoid tumors are associated with the carcinoid syndrome, a set of symptoms resulting from the peptide and amine products, including serotonin, secreted from the cancer cells. The purpose of this study was to investigate the relationship between the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) inhibitor PTEN (phosphatase and tensin homolog deleted on chromosome ten) and serotonin synthesis and secretion in the carcinoid cancer cell line BON. MATERIALS AND METHODS: PTEN was inhibited by pharmacological and molecular approaches, and the resultant secretion of serotonin and expression of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin synthesis, was assessed. RESULTS: Inhibition of PTEN in vitro, with concomitant increased Akt signaling, resulted in decreased secretion of serotonin, as well as decreased serotonin synthesis, as confirmed by reduced expression of TPH1. Inhibition of PTEN in BON cells in an animal model resulted in decreased serum serotonin. CONCLUSION: By inhibiting signaling through Akt, PTEN indirectly promotes serotonin synthesis and secretion.

Suppression of neurotensin receptor type 1 expression and function by histone deacetylase inhibitors in human colorectal cancers.

Neurotensin, a gut peptide, stimulates the growth of colorectal cancers that possess the high-affinity neurotensin receptor (NTR1). Sodium butyrate (NaBT) is a potent histone deacetylase inhibitor (HDACi) that induces growth arrest, differentiation, and apoptosis of colorectal cancers. Previously, we had shown that NaBT increases nuclear GSK-3beta expression and kinase activity; GSK-3beta functions as a negative regulator of extracellular signal-regulated kinase (ERK) signaling. The purpose of our current study was to determine: (a) whether HDACi alters NTR1 expression and function, and (b) the role of GSK-3beta/ERK in NTR1 regulation. Human colorectal cancers with NTR1 were treated with various HDACi, and NTR1 expression and function were assessed. Treatment with HDACi dramatically decreased endogenous NTR1 mRNA, protein, and promoter activity. Overexpression of GSK-3beta decreased NTR1 promoter activity (> 30%); inhibition of GSK-3beta increased NTR1 expression in colorectal cancer cells, indicating that GSK-3beta is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACi significantly decreased phosphorylated ERK while increasing GSK-3beta. Selective MAP/ERK kinase/ERK inhibitors suppressed NTR1 mRNA expression in a time- and dose-dependent fashion, and reduced NTR1 promoter activity by approximately 70%. Finally, pretreatment with NaBT prevented neurotensin-mediated cyclooxygenase-2 and c-myc expression and attenuated neurotensin-induced interleukin-8 expression. HDACi suppresses endogenous NTR1 expression and function in colorectal cancer cell lines; this effect is mediated, at least in part, through the GSK-3beta/ERK pathway. The downregulation of NTR1 in colorectal cancers may represent an important mechanism for the anticancer effects of HDACi.

Regulation of proliferation, apoptosis and cell cycle in gastrointestinal disorders.

Carcinogenesis is a multifactorial process representing the accumulation of acquired and genetic defects, and it has become apparent that many gastrointestinal cancers originate from a state of chronic inflammation. Advances in the field of inflammation-induced carcinogenesis over the past several years have focused on the creation of agonists or antagonists of cytokines and pathways regulating proliferation and apoptosis, and include advances such as the discovery of pharmacological inhibitors of the histone deacetylase (HDAC) family of transcriptional co-repressors, which induce apoptosis of neoplastic cells; discovery of natural products, such as curcumin, which have been shown to regulate cytokine expression; and further investigation into the role of annexin A1, a downstream mediator of glucocorticoid action, in the regulation of inflammation, to name a few.

PKD3 is the predominant protein kinase D isoform in mouse exocrine pancreas and promotes hormone-induced amylase secretion.

The protein kinase D (PKD) family of serine/threonine kinases, which can be activated by gastrointestinal hormones, consists of three distinct isoforms that modulate a variety of cellular processes including intracellular protein transport as well as constitutive and regulated secretion. Although isoform-specific functions have been identified in a variety of cell lines, the expression and function of PKD isoforms in normal, differentiated secretory tissues is unknown. Here, we demonstrate that PKD isoforms are differentially expressed in the exocrine and endocrine cells of the pancreas. Specifically, PKD3 is the predominant isoform expressed in exocrine cells of the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly expressed in the pancreatic islets. Within isolated mouse pancreatic acinar cells, PKD3 undergoes rapid membrane translocation, trans-activating phosphorylation, and kinase activation after gastrointestinal hormone or cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs viaaCa2+-independent, diacylglycerol- and protein kinase C-dependent mechanism. PKD phosphorylation can also be induced by physiologic concentrations of secretagogues and by in vivo stimulation of the pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK, ribosomal S6 kinase) signaling and significantly enhances cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a novel distinction between the exocrine and endocrine cells of the pancreas and further identify PKD3 as a signaling molecule that promotes hormone-stimulated amylase secretion.

An analysis of trends and growth factor receptor expression of GI carcinoid tumors.

INTRODUCTION: The purpose of our study was twofold: (1) to determine the incidence, patient and tumor characteristics, and outcome of patients with gastrointestinal carcinoid tumors using the Surveillance, Epidemiology and End Results (SEER) database, and (2) to delineate the expression pattern of growth factor receptors (GFRs) in carcinoid tumors. MATERIALS AND METHODS: The SEER database search provided information on patients diagnosed with carcinoid tumors from 1990 to 2002. Carcinoid tumor sections (n = 46) were stained for the GFRs: epidermal growth factor receptor, insulin-like growth factor receptor (IGFR), vascular endothelial growth factor receptor (VEGFR), and HER-2/neu. RESULTS: Over the 12-year analysis period, 18,180 patients were identified with carcinoid tumors of the foregut, midgut, and hindgut; the incidence of carcinoid tumors increased approximately 2-fold during this time period. Of the patients with carcinoid tumors, there was a trend of increased expression of VEGFR and IGFR, particularly in the foregut and midgut carcinoids. Analysis of the SEER database confirms that the incidence of carcinoid tumors is increasing with an approximate doubling in the number of carcinoid cases from 1990 to 2002. Furthermore, an increase in VEGFR and IGFR expression suggests that GFR inhibitors may be effective adjuvant therapy for carcinoid cancer.

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy.

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85alpha regulatory and a p110alpha catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85alpha and p110alpha subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85alpha or p110alpha with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85alpha or p110alpha, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.

Targeted molecular therapy of the PI3K pathway: therapeutic significance of PI3K subunit targeting in colorectal carcinoma.

OBJECTIVE: The phosphatidylinositol 3-kinase (PI3K) pathway promotes cancer cell proliferation and survival. The authors determined the pattern of distribution of PI3K pathway components (ie, the p85alpha regulatory subunit, p110alpha catalytic subunit, Akt1, Akt2, and the tumor suppressor PTEN) in human colorectal cancer. In addition, inhibition of in vitro proliferation and in vivo liver metastasis by p85alpha or p110alpha siRNA treatment was analyzed. SUMMARY BACKGROUND DATA: Small interfering RNA (siRNA) molecules suppress expression of target genes and may have therapeutic applications as target-specific therapies for cancer. Therefore, the purpose of this study was 2-fold: 1) to analyze the distribution pattern of PI3K pathway components in human normal colorectal cancers, and 2) to determine whether targeted inhibition of PI3K inhibits colon cancer growth in vitro and suppresses metastatic growth in vivo. METHODS: Immunohistochemical analysis was performed on colorectal adenocarcinomas and adjacent normal mucosa for PI3K pathway components, including p85alpha, p110alpha, Akt1, Akt2, and the tumor suppressor PTEN, which inhibits PI3K. HT29 and KM20 human colon cancer cells were treated with siRNA directed to p85alpha or p110alpha, and cell viability and apoptosis assessed. HT29 cells, transfected with a plasmid containing green fluorescent protein (GFP), were injected into the spleen of athymic nude mice to establish liver metastases; mice were randomized to receive either nontargeting control (NTC), p85alpha or p110alpha siRNA. RESULTS: PI3K pathway components p85alpha and Akt2 were highly expressed in glandular elements of colon cancers, with a correlation between staining intensity and clinical stage; PTEN expression was decreased in the colon cancers of all stages. PI3K-specific siRNA treatment decreased cell viability in vitro and suppressed metastatic tumor growth in vivo. CONCLUSIONS: Selective targeting of PI3K pathway components may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment of selected colorectal cancers.

Overexpression of wild-type PKD2 leads to increased proliferation and invasion of BON endocrine cells.

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2WT) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.