Identification of a novel bombesin bioactive peptide QUB2995 and its insulin secreting activity / 中华内分泌代谢杂志

Objective:To identify a novel bombesin bioactive peptide from the skin secretion of Hylarana Latouchii, and to explore its effect on insulin secretion in islet cells.Methods:The skin secretion from Hylarana Latouchii was extracted by electrical stimulation, and the single chain of bombesin peptide was cloned and sequenced. The peptide QUB2995 was synthesized via solid-phase synthesis, then purified using reversed-phase high performance liquid chromatography (HPLC). Matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was applied to validate. QPCR and ELISA were used to probe the effect of QUB2995 on insulin secretion in MIN6 and INS-1 cells.Results:A novel bombesin peptide named QUB2995 (GAFGDFLKGAAKA GALKILSIAQCKLSGTC) was found in the skin secretion of Hylarana Latouchii through molecular cloning. The bioactive peptide could significantly promote the proliferation and insulin secretion from mouse islet MIN6 cells and rat islet INS-1 cells. The effect reached a climax at the concentration of 10 -5 mol/L. Conclusion:A novel bombesin bioactive peptide named QUB2995 was found from Hylarana Latouchii. It could significantly promote insulin secretion in MIN6 cells of mouse islets and INS-1 cells of rat islets, indicating its potential in the treatment of diabetes.

Discovery of exogenous ligands for orphan receptor BRS-3 from Chinese herbs / 中国中药杂志

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.

Evaluation of 99mTc-HYNIC-βAla-Bombesin(7-14) as an agent for pancreas tumor detection in mice

Pancreatic adenocarcinoma is important in oncology because of its high mortality rate. Deaths may be avoided if an early diagnosis could be achieved. Several types of tumors overexpress gastrin-releasing peptide receptors (GRPr), including pancreatic cancer cells. Thus, a radiolabeled peptide derivative of gastrin-releasing peptide (GRP) may be useful as a specific imaging probe. The purpose of the present study was to evaluate the feasibility of using99mTc-HYNIC-βAla-Bombesin(7-14)as an imaging probe for Capan-1 pancreatic adenocarcinoma. Xenographic pancreatic tumor was developed in nude mice and characterized by histopathological analysis. Biodistribution studies and scintigraphic images were carried out in tumor-bearing nude mice. The two methods showed higher uptake by pancreatic tumor when compared to muscle (used as control), and the tumor-to-muscle ratio indicated that99mTc-HYNIC-βAla-Bombesin(7-14)uptake was four-fold higher in tumor cells than in other tissues. Scintigraphic images also showed a clear signal at the tumor site. The present data indicate that99mTc-HYNIC-βAla-Bombesin(7-14)may be useful for the detection of pancreatic adenocarcinoma.

Comparison of two peptide radiotracers for prostate carcinoma targeting

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95 percent. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32 percent of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

Preparation and evaluation of 99Tcm-(HYNIC-Lys3 -bombesin) (tricine) (TPPTS) for imaging the Balb/c nude mice bearing human pancreatic cancer / 中华核医学杂志

Objective To synthesize 99Tcm- (hydrazinonictinamide- [Lys3] -bombesin) (tricine)(trisodium triphenylphosphine-3,3',3"-trisulfonate) ((HYNIC-[Lys3]-BBS) (tricine) (TPPTS)) and evaluate its biodistribution and binding capability with tumor tissue in Balb/c nude mice bearing human pancreatic cancer xenografts. Methods HYNIC was conjugated to the [Lys3] -BBS at pH = 9.0 with SnCl2 as reducing agent and both tricine and TPPTS as coligands for 99Tcm-labeling. 99Tcm-HYNIC-[Lys3]-BBS)(tricine) (TPPTS) was purified by Sep-Pak C18 cartridge and was analysed by HPLC. The radiochemical purity and radiolabeling yield were measured. The stability of 99Tcm-(HYNIC-[Lys3]-BBS) (tricine)(TPPTS) in serum, biodistribution (% ID/g) in the normal mice and imaging of the Balb/c nude mice bearing human pancreatic cancer xenografts in vivo were studied. Results The radiolabeling yield was (90 ±2)% and the radiochemical purity was over 95%. The radiochemical purity after 4 h in serum was over 85%. The distribution in normal mice showed rapid clearance from blood (the uptake was (0.07 ±0.01) %ID/g at 2 h postinjection). 99Tcm-(HYNIC-[Lys3]-BBS) (tricine) (TPPTS) was excreted mainly via the kidney with little radioactivity accumulation in the liver and gastrointestinal tract (the uptake of liver, stomach, intestine was (0.27 ±0.03), (0.06 ±0.03), (0.04 ±0.00) %ID/g at 2 h postinjection). Marked uptake of radioactivity was found in tumor tissue of the Balb/c nude mice bearing human pancreatic cancer with maximum T/NT ratio of 3.71 ± 0.57 at 2 h postinjection. Conclusions 99Tcm-(HYNIC-[Lys3]-BBS)(tricine) (TPPTS) can be easily prepared with high radiolabeling yield and radiochemical purity. The stability in serum and good biodistribution charateristics make it useful for the diagnosis of human pancreatic cancer with over-expression of the gastric-releasing peptide(GRP) receptor.

Function of bombesin in reducing body temperature of febrile rats and its relativity with PGE_2 / 中国药理学通报

Aim To study the effect of bombesin on body temperature thermothreshold.Methods With IL-1?-induced febrile rats as research subject,the content of PGE2 in hypothalamus and plasma was detected with radioimmunoassay.Results(1) Induced by i.c.v.injection of IL-1?(0.1 ?g),the content of PGE2 in hypothalamus and plasma of the febrile rats significantly increased(P

Inhibition of BN IL-1?-induced fever and effects on cAMP in POA and plasma / 中国药理学通报

Aim To study the effects of Bombesin(BN) on hypothermia of rats and its relation with cAMP.Methods SD male rats were dealed with intracerebroventricular administration of bombesin and IL -1?. Change in body temperature was measured and content of cAMP in hypothalam us and plasma was detected. Results ①In the febrile rats induced by icv. injection of IL-1?, cAMP level in POA and plasma was significantly higher (P

Expression of Bombesin Family Ligands and Receptors in Human Gastric Cancer Tissues and Cell Lines

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.

Study on the Regulatory Effects of Bombesin on the Gastrointestinal Morphology and Proliferation of Mucosa Cells in Neonatal Rabbits / 中华围产医学杂志

Objective To investigate the regulatory effects of bombesin on the gastrointestinal morphology and proliferation of mucosa cells in neonatal rabbits. Methods Twenty four neonatal rabbits were divided into big,small dose experimental group and control group. The gastrointestinal morphology in neonatal rabbits was observed by using Video Image Digtal Analysis System and electron microscopy, and the proliferative rate of gastrointestinal epithelium cells was detected by using immunohistochemical assay. Results The villous height of duodenum were (520?76),(513?31),(379?44) ?m in three groups respectively. That in experimental group with big or small dose were significantly higher than that in control group( P

Further characterization of bombesin like immunoreactivities from the skin of Korean fire-bellied toad, Bombina orientalis

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.

The effect of bombesin on the growth of human immortalized gastric epithelial cell line and its mechanisms / 中华消化杂志

Objective To investigate the regulatory effects of bombesin on the growth of human immortalized gastric epithelial cell line(GES-1), and its mechanisms. Methods ① The expression of gastrin releasing peptide receptor(GRP-R) mRNA in GES-1 was detected. ② The expression of GRP-R protein was tested by cross-linking experiment and the location of the receptors in the cells were investigated by cytochemistry. ③GES-1 cell line was incubated with varying concentrations of bombesin with or without its antagonist and growth of the cell line was determined. ④The effect of protein kinase C (PKC) inhibitor on cell growth induced by bombesin was studied. ⑤After treated with bombesin, the intracellular IP 3 and translocation of PKC activity were measured in GES-1. ⑥Semiquantification of GRP-R mRNA in this cell line treated with bombesin was performed. Results ①Expression of mRNA of GRP-R was demonstrated in GES-1 cells. ②The GRP-R protein was about 75?103 as revealed by cross-linking study, and the receptors were identified on the cell membranes by cytochemistry. ③Bombesin stimulated the growth of GES-1 significantly, which could be inhibited by specific antagonist of bombesin. ④Bombesin-induced growth of GES-1 was also inhibited by PKC inhibitor. ⑤Bombesin induced an increase of IP 3 generation in GES-1 as well as remarkable translocation of PKC activity from cytoplasm to the cell membranes. ⑥An increase in GRP-R mRNA was induced by treatment of cell line with bombesin. Conclusions Bombesin stimulates the growth of this GES-1 via its receptor GRP-R and through IP 3, PKC signal pathway. The increase in expression of GRP-R mRNA in GES-1 induced by bombesin indicates that bombesin might upregulate the GRP-R in the GES-1 cells.

Reinforcement of anticancer effect of combined mitomycin and bombesin on human gastric cancer cell in vitro / 重庆医科大学学报

Objective: To study the effects of combined mitomycin(MMC) and bombesin(BOM) on apoptosis of human gastric cancer cell in vitro.Methods: Four groups were formed: control group,MMC group,BOM group and MMC+BOM group.The effects of different concentrations of MMC and BOM,on the rate of cell existence,and cell apoptosis in human gastric cancer line SGC 7901 were studied in four groups by using cell culture,MTT assay and flow cytometry.Results : Within the scope of certain density,the multiplication effect of BOM,and the inhibitory action of MMC on stomach cancer cell line SGC 7901 were in a time and dose dependent manner.The rates of cell existence in four groups were respectively 90.00% in BOM group?82.96% in control group?65.07% in MMC group and 60.07% in MMC+BOM group,lowered one by one in order.There was significant difference in statistics about the rate of apoptosis between MMC+BOM group(29.26%) and control group(4.72%) or BOM group(5.13%)(P