-
Molecular Biology of the Cell Sep 2003The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the...
The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the tyrosine phosphatase SHP-1. We now identified Src and SHP-2 as sst2-associated molecules and demonstrated their role in sst2 signaling. Surface plasmon resonance and mutation analyses revealed that SHP-2 directly associated with phosphorylated tyrosine 228 and 312, which are located in sst2 ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This interaction was required for somatostatin-induced SHP-1 recruitment and activation and consequent inhibition of cell proliferation. Src interacted with sst2 and somatostatin promoted a transient Gbetagamma-dependent Src activation concomitant with sst2 tyrosine hyperphosphorylation and SHP-2 activation. These steps were abrogated with catalytically inactive Src. Both catalytically inactive Src and SHP-2 mutants abolished somatostatin-induced SHP-1 activation and cell growth inhibition. Sst2-Src-SHP-2 complex formation was dynamic. Somatostatin further induced sst2 tyrosine dephosphorylation and complex dissociation accompanied by Src and SHP-2 inhibition. These steps were defective in cells expressing a catalytically inactive Src mutant. All these data suggest that Src acts upstream of SHP-2 in sst2 signaling and provide evidence for a functional role for Src and SHP-2 downstream of an inhibitory G protein-coupled receptor.
Topics: Amino Acid Motifs; Animals; Antineoplastic Combined Chemotherapy Protocols; COS Cells; Cell Division; Chlorocebus aethiops; Cricetinae; Cyclophosphamide; DNA Mutational Analysis; Doxorubicin; Intracellular Signaling Peptides and Proteins; Models, Molecular; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Rats; Receptors, Somatostatin; Somatostatin; Surface Plasmon Resonance; Vincristine; src-Family Kinases
PubMed: 12972574
DOI: 10.1091/mbc.e03-02-0069 -
Oncotarget Apr 2016Malignant brain glioma is the most lethal and aggressive type of cancer. Surgery and radiotherapy cannot eliminate all glioma stem cells (GSCs) and blood-brain barrier...
Malignant brain glioma is the most lethal and aggressive type of cancer. Surgery and radiotherapy cannot eliminate all glioma stem cells (GSCs) and blood-brain barrier (BBB) restricts the movement of antitumor drugs from blood to brain, thus leading to the poor prognosis with high recurrence rate. In the present study, the targeting conjugates of cholesterol polyethylene glycol polyethylenimine (CHOL-PEG2000-PEI) and D-a-tocopheryl polyethylene glycol 1000 succinate vapreotide (TPGS1000-VAP) were newly synthesized for transporting drugs across the BBB and targeting glioma cells and GSCs. The multifunctional targeting vinorelbine plus tetrandrine liposomes were constructed by modifying the targeting conjugates. The studies were undertaken on BBB model, glioma cells, GSCs, and glioma-bearing mice. In vitro results showed that multifunctional targeting drugs-loaded liposomes with suitable physicochemical property could enhance the transport drugs across the BBB, increase the intracellular uptake, inhibit glioma cells and GSCs, penetrate and destruct the GSCs spheroids, and induce apoptosis via activating related apoptotic proteins. In vivo results demonstrated that multifunctional targeting drugs-loaded liposomes could significantly accumulate into brain tumor location, show the specificity to tumor sites, and result in a robust overall antitumor efficacy in glioma-bearing mice. These data suggested that the multifunctional targeting vinorelbine plus tetrandrine liposomes could offer a promising strategy for treating brain glioma.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzylisoquinolines; Brain Neoplasms; Cell Line, Tumor; Glioma; Liposomes; Mice; Mice, Inbred ICR; Molecular Targeted Therapy; Neoplastic Stem Cells; Random Allocation; Vinblastine; Vinorelbine
PubMed: 27029055
DOI: 10.18632/oncotarget.8360 -
Proceedings of the National Academy of... Mar 1991Analogues of somatostatin (SS) and luteinizing hormone-releasing hormone (LH-RH) activate tyrosine phosphatases in MIA PaCa-2 human pancreatic cancer cell line membranes... (Comparative Study)
Comparative Study
Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors.
Analogues of somatostatin (SS) and luteinizing hormone-releasing hormone (LH-RH) activate tyrosine phosphatases in MIA PaCa-2 human pancreatic cancer cell line membranes and inhibit growth. We compared the substrates phosphorylated by epidermal growth factor (EGF) to those dephosphorylated by the SS analogue RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) and [D-Trp6]LH-RH in cancer cell lines such as MIA PaCa-2 (human pancreatic cancer), HCPC (hamster cheek pouch carcinoma), A-549 (human lung cancer), HT-29 (human colon cancer), and R3230AC (breast cancer). EGF phosphorylated proteins of 170, 65, and 60 kDa and analogues of SS and LH-RH promoted the dephosphorylation of these proteins in MIA PaCa-2 and HCPC cell lines. The EGF receptor is 170 kDa. pp60src (60 kDa) is known to be a substrate for EGF receptor. The LH-RH receptor is also 60 kDa. The effects of RC-160 and [D-Trp6]LH-RH were quantitatively different. Examinations of HT-29, A-549, and R3230AC cancer cell lines revealed no phosphorylation by EGF or dephosphorylation by RC-160 and [D-Trp6]LH-RH. In addition to the 170-, 65-, and 60-kDa proteins, 35-kDa proteins were also phosphorylated in some cancer cell lines. This work demonstrates that analogues of SS and LH-RH can reverse the effects of EGF biochemically as well as functionally.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Autoradiography; Breast Neoplasms; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Kinetics; Lung Neoplasms; Membrane Proteins; Molecular Sequence Data; Pancreatic Neoplasms; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Somatostatin; Triptorelin Pamoate; Tyrosine
PubMed: 1672042
DOI: 10.1073/pnas.88.5.1656 -
Journal of Neurochemistry Jan 1997Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant...
Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA-induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7-10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5-10 microM), simultaneously applied with excitatory amino acid, significantly reduced in a dose-dependent manner the neurotoxic effect of NMDA but not that of KA (0.25-0.5 mM). GABA (10 microM) partially protected neurons to a similar extent from NMDA- or KA-induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201-995; 10 microM) and vapreotide (RC 160; 10 microM), did not influence the NMDA-dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl-cyclic GMP (10 microM), a cyclic GMP analogue, whereas 8-(4-chlorphenylthio)-cyclic AMP (10 microM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose-dependent manner by SRIF (2.5-10 microM). Finally, both specific (Rp-8-bromoguanosine 3',5'-monophosphate, 10 microM) and nonspecific [1-(5 isoquinolinylsulfonyl)-2-methylpiperazine (H7), 10 microM] cyclic GMP-dependent protein kinase (cGMP-PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA-induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP-PK presumably by regulation of the intracellular Ca2+ level.
Topics: Animals; Cell Death; Cyclic GMP; Excitatory Amino Acid Agonists; N-Methylaspartate; Neurons; Neuroprotective Agents; Rats; Somatostatin
PubMed: 8978741
DOI: 10.1046/j.1471-4159.1997.68010319.x -
Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5.Proceedings of the National Academy of... Aug 1997We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably...
We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.
Topics: Alkaloids; Aminoquinolines; Animals; CHO Cells; Carbazoles; Cell Division; Cholecystokinin; Cricetinae; Gene Transfer Techniques; Humans; Indoles; Receptors, Somatostatin; Signal Transduction; Somatostatin
PubMed: 9256484
DOI: 10.1073/pnas.94.17.9343 -
Proceedings of the National Academy of... Sep 2009Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant...
Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.
Topics: 2-Hydroxyphenethylamine; Adrenal Gland Neoplasms; Adrenal Glands; Aniline Compounds; Animals; Cell Line, Tumor; Cell Proliferation; Cytostatic Agents; Doxorubicin; Gene Expression; Humans; Neuropeptides; Oligonucleotide Array Sequence Analysis; PC12 Cells; Pyrroles; RNA, Messenger; Rats; Receptors, LHRH; Receptors, Neuropeptide; Receptors, Somatostatin; Somatostatin
PubMed: 19717419
DOI: 10.1073/pnas.0907843106 -
Biochemical Pharmacology Jul 1995
Review
Topics: Amino Acid Sequence; Antineoplastic Agents; Humans; Molecular Sequence Data; Neoplasms; Octreotide; Peptides, Cyclic; Receptors, Somatostatin; Somatostatin
PubMed: 7646531
DOI: 10.1016/0006-2952(95)00066-9 -
British Journal of Cancer Nov 1994We investigated the effects of our synthetic bombesin/gastrin-releasing peptide (GRP) antagonists and somatostatin analogue RC-160 on the growth of human small-cell lung...
Effects of somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonists on the growth of human small-cell and non-small-cell lung carcinomas in nude mice.
We investigated the effects of our synthetic bombesin/gastrin-releasing peptide (GRP) antagonists and somatostatin analogue RC-160 on the growth of human small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (non-SCLC) lines in nude mice. Athymic nude mice bearing xenografts of the SCLC NCl-H69 line or non-SCLC NCl-H157 line were treated for 5 and 4 weeks, respectively, with somatostatin analogue RC-160 or various bombesin/GRP antagonists. RC-160, administered s.c. peritumorally at a dose of 100 micrograms per animal per day, inhibited the growth of H69 SCLC xenografts as shown by more than 70% reduction in tumour volumes and weights, as compared with the control group. Bombesin/GRP antagonists, RC-3440, RC-3095 and RC-3950-II, given s.c. peritumorally at a dose of 20 micrograms per animal per day, also inhibited the growth of H69 SCLC tumours. RC-3950-II had the greatest inhibitory effect and decreased tumour volume and weights by more than 80%. The growth of H-157 non-SCLC xenografts was significantly reduced by treatment with RC-160, but not with bombesin/GRP antagonist RC-3095. In mice bearing either tumour model, administration of RC-160 significantly decreased serum growth hormone and gastrin levels. Specific high-affinity receptors for bombesin and somatostatin were found on membranes of SCLC H69 tumours, but not on non-SCLC H157 tumours. Receptor analyses demonstrated high-affinity binding sites for epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the membranes of H69 and H157 tumours. EGF receptors were down-regulated on H69 tumours after treatment with RC-160 and bombesin/GRP antagonists. The concentration of binding sites for EGF and IGF-I on the H157 tumours was decreased after treatment with RC-160, but bombesin/GRP antagonist RC-3095 had no effect. These results demonstrate that bombesin/GRP antagonists inhibit the growth of H-69 SCLC, but not of H-157 non-SCLC xenografts in nude mice, whereas somatostatin analogue RC-160 is effective in both tumour models. This raises the possibility that these peptide analogues could be used selectively in the treatment of various subclasses of lung cancer.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Body Weight; Bombesin; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Gastrin-Releasing Peptide; Gastrins; Gonadotropin-Releasing Hormone; Growth Hormone; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Peptide Fragments; Peptides; Receptors, Somatotropin; Somatostatin; Substrate Specificity; Transplantation, Heterologous
PubMed: 7947094
DOI: 10.1038/bjc.1994.415 -
Proceedings of the National Academy of... Mar 1993Somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 were infused at 2 micrograms per day via miniosmotic pumps implanted s.c. in...
Somatostatin analogue RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 were infused at 2 micrograms per day via miniosmotic pumps implanted s.c. in hamsters with premalignant disease to examine the effect of these peptides on cancer promotion and progression. These analogues have been shown to inhibit growth of certain tumors, especially those that overexpress tyrosine kinase activity. Progression of premalignant lesions initiated by applying 0.5% 9,10-dimethyl-1,2-benzanthracene (DMBA) to the hamster buccal cheek pouch was measured by Photofrin-induced fluorescence 24 hr after injecting the porphyrin (1.0 mg/kg) by using in vivo fluorescence photometry. This method of monitoring progression was reaffirmed by the observations that fluorescence increased significantly as compared with controls in lesions receiving 4 additional weeks of continuous promotion by DMBA application (P < 0.01 in two independent trials) and in lesions receiving transient promotion by laser incision (P < 0.01 and < 0.05 at the same time in the two trials). Twelve weeks after treatment, fluorescence had decreased significantly among animals treated for 2 weeks with RC-3095 (control, 0.53 +/- 0.03 V vs. RC-3095, 0.28 +/- 0.03 V; P < 0.0005) or with RC-160 (control, 0.85 +/- 0.03 V vs. RC-160, 0.24 +/- 0.03 V; P < 0.0001). These data were obtained 20 weeks after DMBA initiation. Thus, treatment with RC-160 and RC-3095 decreased the progression, measured by fluorescence, compared with control animals. In addition, there was also an absolute continuous decrease in fluorescence for the 22 weeks after the cessation of RC-160 treatment. That the changes in tumor progression produced by RC-160 extended beyond the treatment period supports the hypothesis that the changes were irreversible. Histopathological analysis revealed normal tissue and/or mild-moderate dysplasia in hamster buccal mucosa treated with the RC-160 (an improvement compared to pretreatment), whereas 40% of the animals receiving no treatment after DMBA initiation developed invasive squamous cell carcinomas after 20 weeks. These results show that the antagonists of bombesin/gastrin-releasing peptide can delay the development of malignancies and the agonists of somatostatin can potentially reverse this development.
Topics: Animals; Bombesin; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Cricetinae; Fluorescent Dyes; Hematoporphyrin Derivative; Microscopy, Fluorescence; Mouth Neoplasms; Peptide Fragments; Peptides; Somatostatin
PubMed: 8095335
DOI: 10.1073/pnas.90.5.1897 -
Proceedings of the National Academy of... Feb 1995Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO...
Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.
Topics: Amino Acid Sequence; Animals; CHO Cells; Calcium; Carbachol; Cholecystokinin; Cricetinae; Cyclic AMP; Growth Inhibitors; Insulin; Molecular Sequence Data; Phosphoprotein Phosphatases; Receptors, Somatostatin; Recombinant Proteins; Signal Transduction; Somatostatin; Thrombin; Vanadates
PubMed: 7878022
DOI: 10.1073/pnas.92.5.1580