Kirk L. Ives

Curcumin Inhibits Neurotensin-Mediated Interleukin-8 Production and Migration of HCT116 Human Colon Cancer Cells

Purpose: Neurotensin, a gut tridecapeptide, acts as a potent cellular mitogen for various colorectal and pancreatic cancers that possess high-affinity neurotensin receptors. Cytokine/chemokine proteins are increasingly recognized as important local factors that play a role in the metastasis and invasion of multiple cancers. The purpose of this study was to (a) determine the effect of neurotensin on cytokine/chemokine gene expression and cell migration in human cancer cells and (b) assess the effect of curcumin, a natural dietary product, on neurotensin-mediated processes. Experimental Design: The human colorectal cancer, HCT116, was treated with neurotensin, with or without curcumin, and interleukin (IL)-8 expression and protein secretion was measured. Signaling pathways, which contribute to the effects of neurotensin, were assessed. Finally, the effect of curcumin on neurotensin-mediated HCT116 cell migration was analyzed. Results: We show that neurotensin, acting through the native high-affinity neurotensin receptor, induced IL-8 expression in human colorectal cancer cells in a time- and dose-dependent fashion. This stimulation involves Ca2+-dependent protein kinase C, extracellular signal-regulated kinase–dependent activator protein-1, and extracellular signal-regulated kinase–independent nuclear factor-κB pathways. Curcumin inhibited neurotensin-mediated activator protein-1 and nuclear factor-κB activation and Ca2+ mobilization. Moreover, curcumin blocked neurotensin-stimulated IL-8 gene induction and protein secretion and, at a low concentration (i.e., 10 μmol/L), blocked neurotensin-stimulated colon cancer cell migration. Conclusions: Neurotensin-mediated induction of tumor cell IL-8 expression and secretion may contribute to the procarcinogenic effects of neurotensin on gastrointestinal cancers. Furthermore, a potential mechanism for the chemopreventive and chemotherapeutic effects of curcumin on colon cancers may be through the inhibition of gastrointestinal hormone (e.g., neurotensin)–induced chemokine expression and cell migration.

Multiple protein kinase pathways are involved in gastrin-releasing peptide receptor-regulated secretion.

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca2+([Ca2+] i ), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca2+] i oscillations or a biphasic elevation in [Ca2+] i . The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca2+, by chelation of [Ca2+] i , and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca2+] i were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca2+] i . Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca2+] i ; however, elevated [Ca2+] i is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca2+] i levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca2+-sensitive PKC.

The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to G(q), but not G(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores

As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both Gq/11 and Gi/o, and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca2+] i . However, oxytocin-stimulated ERK-2 phosphorylation and prostaglandin E2 synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from Gq-mediated pathways. The Δ51 receptor is coupled to Gi, as oxytocin-stimulated Ca2+ transients were inhibited by pertussis toxin, and a Gβγ sequestrant. Preincubation of Δ51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A Δ39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with Gq/11, but not Gi/o. Furthermore, an increase in intracellular calcium was generated via a Giβγ-tyrosine kinase pathway from intracellular stores that are distinct from Gq-mediated inositol trisphosphate-regulated stores.

Gastrin-Releasing Peptide Receptor in Breast Cancer Mediates Cellular Migration and Interleukin-8 Expression

BACKGROUND Breast cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor, gastrin-releasing peptide receptor (GRP-R). Experimental evidence suggests that bombesin (BBS), the pharmacological homologue of GRP, promotes breast cancer growth and progression. The contribution of GRP-R to other poor prognostic indicators in breast cancer, such as the expression of the EGF-R family of growth factors and hormone insensitivity, is unknown. MATERIALS AND METHODS Two estrogen receptor (ER)-negative breast cancer cell lines were used. MDA-MB-231 overexpress both EGFR and GRPR, whereas SK-BR-3 cells express EGF-R but lack GRP-R. Cellular proliferation was assessed by Coulter counter. Chemotactic migration was performed using Transwell chambers, and the migrated cells were quantified. Northern blot and real-time PCR were used to evaluate proangiogenic factor interleukin-8 (IL-8) mRNA expression. RESULTS In MDA-MB-231 cells, GRP-R and EGF-R synergize to regulate cell migration, IL-8 expression, but not cell proliferation. In SK-BR-3 cells, ectopic expression of GRP-R was sufficient to increase migration and IL-8 mRNA. CONCLUSIONS These data suggest relevant roles for GRP-R in ER-negative breast cancer progression. Future mechanistic studies to define the molecular role of GRP-R in breast cancer metastasis provide novel targets for the treatment of ER-negative breast cancers.

CD133+ colon cancer cells are more interactive with the tumor microenvironment than CD133− cells

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133−) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133− cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133− cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs – cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133−, cells is due to their increased ability to interact with their neighboring CAF.Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca2+]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.

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.

Oxytocin Receptor Regulation and Action in a Human Granulosa-Lutein Cell Line

Abstract Although oxytocin and its receptor have been identified in human ovary, its regulatory role in granulosa cell or corpus luteum function has not been clearly defined. To better understand oxytocin action in the human ovary, we have characterized the expression and function of oxytocin receptors in an immortalized human granulosa-lutein cell line, HGL5. Expression of oxytocin receptor mRNA was demonstrated by reverse transcriptase-polymerase chain reaction analysis, and by specific binding of an iodinated oxytocin antagonist (apparent dissociation constant of 131 ± 0.15 pM, and a Bmax of 12 ± 0.5 fmol/μg DNA). Receptor levels were down-regulated by serum starvation, and rapidly up-regulated by serum restoration. Stimulation of protein kinase C activity increased oxytocin receptor levels in a concentration-dependent manner. Conversely, protein kinase C inhibition blocked up-regulation of oxytocin receptors. Treatment of cells with 10 nM oxytocin resulted in a rapid, transient increase in intracellular Ca2+, and the response was blocked by an oxytocin antagonist. Because HGL5 cells secrete progesterone and estradiol in response to agents that elevate intracellular cAMP concentrations, we studied the effect of oxytocin on steroid production. Oxytocin enhanced the effects of forskolin on progesterone production. These results suggest that oxytocin augments the activity of luteotropins in vivo. Our studies are the first to show an ovarian cell line that expresses functional oxytocin receptors. These cells can serve as a useful model for studying oxytocin signal pathways and their cross-talk with respect to progesterone synthesis. These cells also will be useful in the analysis of mechanisms of oxytocin receptor regulation, including regulation of its gene.

THE CD133-POSITIVE COLON CANCER CELL PHENOTYPE IS MORE INTERACTIVE WITH THE TUMOR MICROENVIRONMENT COMPARED TO CD133-NEGATIVE CELL

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133−) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133− cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133− cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs – cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133−, cells is due to their increased ability to interact with their neighboring CAF.Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca2+]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.

CCK2 RECEPTOR EXPRESSION TRANSFORMS NON-TUMORIGENIC HUMAN NCM356 COLONIC EPITHELIAL CELLS INTO TUMOR FORMING CELLS

Expression of gastrin and cholecystokinin 2 (CCK2) receptor splice variants (CCK2R and CCK2i4svR) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK2 receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non‐tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK2i4svR or CCK2R. RT‐PCR and intracellular Ca2+ ([Ca2+]i) imaging of RIE/CCK2R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK2R or CCK2i4svR (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK2 receptor selective antagonist, YM022, partially inhibited the growth of both receptor‐expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor‐expressing cells. Vector‐NCM356 cells did not form tumors in nude mice, whereas, either CCK2 receptor‐expressing cells formed large tumors. Autocrine activation CCK2 receptor variants are sufficient to increase in vitro growth and tumorigenicity of non‐transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.

Differential Response to Gamma Radiation of Human Stomach Cancer Cells in Vitro

In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0.5 to 10 Gy of 60Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2.0 and 10 Gy were 1.22 and 17.8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D0) were 1.08 and 1.45 Gy for AGS and SII parent lines, respectively. The D0 values for AGS parent cells and sublines were similar (1.01 to 1.08 Gy), but SII parent cells and sublines had D0 values of 1.45, 1.36, 1.37 and 1.12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2.0 and 10 Gy that were 1.3 and 11.3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.