Previously, we have isolated authentic bombesin and another bombesin like peptide named bombesin like immunoreactivity (BLI)-K2 from the skin of Korean fire-bellied toad, Bombina orientalis. In the present study, we have newly purified three heterogeneous forms of BLI named BLI-K3, BLI-K4, and BLI-K5 from side fractions obtained in previous isolation of bombesin like peptide. The BLIs were separated into five peaks on a column of C18 preparative HPLC. Among them, three minor peaks containing BLI-K3, K4, and K5 were purified by means of sequential chromatography on the columns of SP cation exchange HPLC and C18 reverse phase HPLC. The purified BLI-K3 and K4 showed high binding affinity to an anti-bombesin serum (LBE 2G-2) with binding potency of 72 and 95%, respectively, relative to that of bombesin. However, they did not possess any distinctive biological activity of bombesin like peptide. On the contrary, the biological activity of BLI-K5 was similar to that of bombesin but its binding affinity to an anti-bombesin serum was low. The results indicate that three heterogeneous forms of BLI were coexpressed with bombesin and BLI-K2 in the skin of B. orientalis. All forms of the purified BLI in the present study were immunologically active but only BLI-K5 possessed the distinctive biological activity of bombesin like peptide.
Anura* ; Bombesin* ; Chromatography ; Chromatography, High Pressure Liquid ; Population Characteristics ; Skin*

Bombesin receptor subtype-3(BRS-3) is an orphan receptor in the bombesin receptor family. Its signal transduction mechanism and biological function have attracted much attention. Seeking the ligand for BRS-3 is of great significance for exploring its function. Considering the fact that the activation of BRS-3 receptor can induce the change in intracellular Ca~(2+) concentration, the fluo-rometric imaging plate reader(FLIPR) was utilized for ligand screening at the cellular level. Among more than 400 monomeric compounds isolated from Chinese herbs, yuanhunine from Corydalis Rhizoma and sophoraisoflavanone A and licoriphenone from Glycyrrhizae Radix et Rhizoma antagonized BRS-3 to varying degrees. It was confirmed in HEK293 cells expressing BRS-3 that yuanhunine, sophoraisoflavanone A, and licoriphenone inhibited the calcium current response after the activation of BRS-3 by [D-Phe~6,β-Ala~(11),Phe~(13),Nle~(14)]bombesin-(6-14) in a dose-dependent manner with the IC_(50) values being 8.58, 4.10, and 2.04 μmol·L~(-1), respectively. Further study indicated that yuanhunine and sophoraisoflavanone A exhibited good selectivity for BRS-3. In this study, it was found for the first time that monomers derived from Chinese herbs had antagonistic activity against orphan receptor BRS-3, which has provided a tool for further study of BRS-3 and also the potential lead compounds for new drug discovery. At the same time, it provides reference for the research and development of innovative drugs based on the active ingredients of Chinese herbs.
Drugs, Chinese Herbal/chemistry* ; HEK293 Cells ; Humans ; Ligands ; Receptors, Bombesin

Neuromedin B (NMB) acts as a growth factor or a morphogen and plays a role in cancer progression. Indeed, the NMB receptor (NMB-R) is overexpressed in different types of tumors. In our current study, we investigated the involvement of NMB-R in the proliferation of oral cancer cells. Human oral squamous cell carcinoma (SCC) and human oral cancer cells, SCC-25 cells were found to be NMB-R-positive. The NMB-R antagonist PD168368 inhibited the proliferation of SCC-25 cells and reduced their colony formation capacity. We also found that PD168368 induced the cell cycle arrest and apoptosis of SCC-25 cells in a dose-/time-dependent manner. Overall, this antitumor activity of PD168368 in human oral cancer cells suggests that NMB-R is a potential target for the future prevention and treatment of human cancers.
Apoptosis ; Carcinoma, Squamous Cell ; Cell Cycle Checkpoints ; Humans ; Mouth Neoplasms* ; Receptors, Bombesin*

PURPOSE: Bombesin-like peptides are known to be important in the autocrine growth of a number of small cell lung cancer cell lines. The aim of this study was to investigate the extent of bombesin family ligands/receptors expression in human gastric cancer tissues and cell lines, and to evaluate the relationship between the expression of bombesin family ligands/receptor and clinicopathologic parameters. METHODS: We measured the expression of gastrin releasing peptide (GRP), neuromedin B (NMB), and their receptors, in human gastric cancer tissues and cell lines. Ligand and receptor mRNA studies were carried out on; 20 tumor and matched normal samples, and 9 gastric cell lines. The expression of mRNA of GRP/NMB, and their receptors, was examined by the reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Expression of GRP, NMB and GRPR, NMBR mRNA was found in 55%, 100%, 40%, and 100% of gastric cancer tissue, respectively. GRP/GRPR co-expression was observed in 30% of gastric cancer tissues and expression of gastric cancer was higher than that of normal mucosa. GRP and GRPR were highly expressed in the differentiated type of gastric cancer. In gastric cancer cell lines, these peptides and receptors were expressed equally. CONCLUSION: The result demonstrate that GRP, NMB, GRPR, and NMBR were expressed in gastric cancer tissues and cell lines. This result suggests that these may have a role as growth factors in gastric cancer growth, and these peptides may act in an autocrine fashion as a morphogen in gastric cancer.
Bombesin* ; Cell Line* ; Gastrin-Releasing Peptide ; Humans ; Humans* ; Intercellular Signaling Peptides and Proteins ; Ligands* ; Mucous Membrane ; Peptides ; RNA, Messenger ; Small Cell Lung Carcinoma ; Stomach Neoplasms*

OBJECTIVETo observe the effect of bombesin noncytoskeleton form and intracellular free calcium ([Ca2+]i) concentration in PC-3 prostate cancer cell line.

METHODSImmunofluorescent histochemistry (IH) combined with laser scanning confocal microscopy (LSCM) was used to examine the expression of cytokeratin (CK) in PC-3 cells treated with definite concentrations of BBS and observe its effect on cytoskeleton form. Fluo-3/AM fluorescence technique and LSCM were adopted to measure the [Ca2+]i concentration after different concentrations (10(-9), 10(-7) and 10(-5) mol/L) of BBS were added in PC-3 cells.

RESULTSBBS (10(-5) mol/L) stimulated the expression of CK in PC-3 cells and the formation of lamellipodium, and increased the [Ca2+]i concentration, with concentration dependence.

CONCLUSIONDefinite concentrations of BBS could obviously enhance the [Ca2+] i concentration, CK expression and cytoskeleton morphology of PC-3 cells. The results provide a basis for further studies on the role of BBS in tumour researches as well as in intracellular signal transmission.

Bombesin ; pharmacology ; Calcium ; analysis ; Cytoskeleton ; drug effects ; metabolism ; Fluoroimmunoassay ; Humans ; Keratins ; biosynthesis ; Male ; Microscopy, Confocal ; Prostatic Neoplasms ; metabolism ; Serum Albumin, Bovine ; Tumor Cells, Cultured

AIMTo determine whether bombesin prevents IFN-alpha-induced fever and it's possible mechanism.

METHODSEffects of BN on changes in body temperature and arginine vasopressin(AVP) content in the ventral septal area(VSA) and hypothalamus were measured in the rats following intracerebroventricular (ICV) injection of IFN-alpha.

RESULTS(1) IFN-alpha produced a dose-dependent rise in colonic temperature simultaneously with increase in AVP content in the VSA in the rats. (2) BN produced a dose-dependent hypothermia and significantly elevated AVP content in the VSA in rats. (3) BN injected intracerebroventricularly at 30 min after IFN-alpha prevented the increase in colonic temperature which recovered to the control level as well as AVP content in the VSA in rats at 150 min.

CONCLUSIONAVP in the VSA may play a role in IFN-alpha-induced fever. AVP in the VSA may play a partial role in the BN antipyretic action and hypothermic action.

Animals ; Arginine Vasopressin ; metabolism ; Body Temperature Regulation ; drug effects ; Bombesin ; pharmacology ; Brain ; drug effects ; metabolism ; Fever ; chemically induced ; physiopathology ; Interferon-alpha ; adverse effects ; Male ; Rats ; Rats, Wistar

BACKGROUNDTumor cells can reduce the number of dendritic cells (DCs) in the tumor environment and cause DC dysfunction through autocrine or paracrine pathways. We sought to measure cyclooxygenase-2 (COX-2) expression in bombesin-inhibited DCs treated with theanine in vitro and to explore the protection and activation effects of theanine on DCs.

METHODSEnzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were used to analyze the effects of theanine on COX-2 expression and interleukin (IL)-12/IL-10 secretion of bombesin-treated DCs.

RESULTSDCs acquired an impaired phenotype as a result of bombesin treatment. Theanine increased the expression of mature DC surface molecules. The number of cell apoptosis with the treatment of bombesin and theanine significantly decreased, accounting for 15.9%, compared with 26.1% of cell apoptosis with bombesin. COX-2 expression in bombesin-treated DCs was inhibited by theanine in a dose-dependent manner. Theanine promoted DC secretion of IL-12. IL-12 levels reached (137.4 ± 4.9) pg/ml with theanine at 200 µmol/L. However, theanine inhibited the secretion of IL-10 in a dose-dependent manner. IL-10 levels were only (58.4 ± 6.9) pg/ml with theanine at 200 µmol/L.

CONCLUSIONTheanine inhibits the transcription and translation of COX-2 and regulates the balance of IL-10/IL-12 secretion in bombesin-inhibited DCs, leading to the recovery of a state of activation in DCs.

Bombesin ; pharmacology ; Cells, Cultured ; Cyclooxygenase 2 ; metabolism ; Dendritic Cells ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Glutamates ; pharmacology ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism

Recently various peptide receptors which displayed the highest binding affinity and specificity with their peptide ligands by ligand-receptor have been exploited to develop drug delivery system which can directionally deliver drug to targeted cell. It is significant to study and applicate, including targeted drug delivery system mediated by bombesin receptor, somatostatin receptor, SynB3 receptor, LH-RH receptor and other peptide receptor, et al. Several small peptide fragments were selected as carriers radicals combining doxorubicin, 2-pyrrolino-DOX, methotrexate, cis-platinum, and camptothecin to form hybrid cytotoxic analogs. These highly potent cytotoxic analogs have been designed as targeted anti-tumor agents for the treatment and study of various cancers that possess receptors for the carrier peptide.
Animals ; Antineoplastic Agents ; therapeutic use ; Doxorubicin ; analogs & derivatives ; therapeutic use ; Drug Delivery Systems ; Humans ; Neoplasms ; drug therapy ; metabolism ; Oligopeptides ; metabolism ; Pyrroles ; therapeutic use ; Receptors, Bombesin ; metabolism ; Receptors, LHRH ; metabolism ; Receptors, Somatostatin ; metabolism

OBJECTIVE: To investigate the role and mechanism of bombesin receptor subtype 3 (BRS-3) in the proliferation and protection against injury of human brochial epithelial cells (HBECs). METHODS: Effect of P3513 (a specific agonist of BRS-3) on the proliferation of HBECs was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; the release rate of 3H-Udr, and LDH activity, catalase activity, and the expression of cadherin and integrin beta1 were also analyzed under O3 stress with or without P3513 treatment. RESULTS: The proliferation of HBECs was accelerated by P3513 in a concentration-dependent manner (10(-9) approximately 10(-7) mol/L). Ozone stress could promote the release rate of 3H-Udr, and LDH activity, which could be inhibited by P3513. P3513 could promote the activity of catalase. The effect of proliferation and protection against injury caused by P3513 could be inhibited by W7 (calmodulin inhibitor), PD98059 (tyrosin kinase inhibitor) and H89 (PKA inhibitor). P3513 could stimulate the expression of caderin and integrinbeta1 of ozone-stressed HBECs. CONCLUSION Activation of BRS-3 caused by P3513 may play an important role in protecting HBECs from oxidant injury, and the signal pathway is possibly relevant to Ca2+, MEK and PKA.
Bronchi ; cytology ; Cadherins ; metabolism ; Catalase ; metabolism ; Cell Line ; Cell Proliferation ; Epithelial Cells ; cytology ; Humans ; Integrin beta1 ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Protective Agents ; Reactive Oxygen Species ; adverse effects ; Receptors, Bombesin ; physiology

OBJECTIVE: To screen the differentially expressed gene profile from the smooth muscles in the fundus uterus at the active stage of labor, and to provide candidate genes for picking out the drug targets related to uterine contraction. METHODS: Differentially expressed genes of uterine smooth muscles in the corpus from pro and post spontaneous parturition and those induced by oxytocin,as well as those from the corpus and the lower portion spontaneous parturition,were scanned respectively by human full-length genetic cDNA microarray with 8064 probe sets. Semi-quantitative RT-PCR was applied to testify the expression of voltage dependent calcium channel-L subtype (CACNA). The differentially expressed genes in the structure and function of the drug targets were picked out by bio-informatics to serve as candidate drug targets related to uterine contraction. RESULTS: The expressions of 29 genes were upregulated in fundus smooth muscles from the pro and post natural parturition, the pro and post inductive parturition of oxytocin, and the natural parturition. The expression of CACNA gene in RT-PCR was in accordance with that in the microarray. Among the 29 genes, neuromedin B receptor (NMBR) gene and neuropeptide Y (NPY) gene were the genes which not only had the targets of uterine contracted medicine, but also could contract the uterine. The differential expression ratios of NMBR in the above 3 types of uterine myometrium were 6.9,11.3, and 9.0, respectively while those of NPY were 6.0,29.8, and 2.9 respectively. CONCLUSION NMBR, whose expression in the uterine smooth muscles is always up-regulated at different parturition conditions, is likely to be an ideal candidate target of uterotonic drugs.
Calcium Channels ; genetics ; Drug Evaluation, Preclinical ; Female ; Gene Expression ; Gene Expression Profiling ; Humans ; Myometrium ; drug effects ; Neuropeptide Y ; genetics ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Receptors, Bombesin ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Uterine Contraction ; drug effects