Bombesin and Analogs

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What is bombesin?

Bombesin (Bn), a biologically active tetradecapeptide, was first extracted from the skin of two European aquatic toads belonging to the family Bombinatoridae, namely Bombina bombina and Bombina variegata. Bn is a diminutive regulatory peptide characterized by significant cellular permeability and biocompatibility. Consequently, Bn is a more appealing targeting ligand compared to alternatives such as folic acid, β-hydroxybutyric acid, biomolecules, or galactose. Owing to its diminutive dimensions, Bn does not influence the overall size of the nanocarrier and may be integrated at a high surface density (quantity of peptides per nanoparticle). Peptides are mostly hydrophilic and are unable to traverse the blood-brain barrier, with less than 0.1% of the total injected peptide achieving this passage. Consequently, the characteristics of Bn are advantageous when peripheral cancers are the intended targets. There are four known BN receptor subtypes, including three mammalian: BB1 (neuromedin B receptor), BB2 (GRPR), and BB3 (orphan BN receptor), and one amphibian: BB4. BN has a strong affinity for the human GRPR, a G-protein-coupled receptor that is overexpressed in several malignancies, including prostate, breast, pancreatic, gastrointestinal, and small cell lung cancer.

Bombesin sequence

A 14-amino acid peptide found in amphibians called bombesin is structurally similar to the 27-amino acid gastrin-releasing peptide (GRP) found in mammals. For immunogenicity and high-affinity binding to the bombesin/GRP-preferring receptor, bombesin and GRP share a 7-amino acid C-terminal sequence (Trp-Ala-Val-Gly-His-Leu-Met-NH2). A number of malignancies, such as small cell lung, prostate, breast, and gastrointestinal cancers, exhibit overexpression of the human GRP receptor, which both bombesin and GRP bind with high affinity.

Peptide sequences. (Fani M., et al., 2012)

Bombesin-like peptides

Profs. Espamer and Nakajima and colleagues extracted a plethora of other Bn-related peptides from various amphibian skins; these peptides were then categorized into three broad categories. One set of peptides belonged to the Bn family and ended in Gly-His-Leu-Met-NH2. Another set of peptides belonged to the litorin-ranatensin family and ended in Gly-His-Phe-Met-NH2. Finally, a third set of peptides belonged to the phyllolitorin family and ended in Gly-Ser-Phe (Leu)-Met-NH2. The Bn peptide subgroup's mammalian member, the 27-amino acid peptide known as GRP, was not identified from swine stomachs until 1980 by McDonald and colleagues. Since it shares the same seven COOH-terminal amino acids as Bn, the peptide's physiologically active end, it was determined to have a high degree of similarity with Bn. Minamino discovered in 1983 that neuromedin B (NMB), a decapeptide, occurs in bigger forms of 30 and 32 amino acids in pig spinal cord. Identicalities were found between ranatensin and six of NMB's seven COOH terminal amino acids. Afterwards, in 1984, Minamino discovered neuromedin C, also known as the COOH terminal decapeptide of GRP, from pig spinal cord [GRP18-27]. There is currently no known mammalian analogue of phyllolitorin.

Research at the molecular level has shown that different Bn peptides originate from different prohormones. Studies on amphibians have shown that several frog species have skin peptides that include different kinds of Bn peptides, each obtained from a different gene. These peptides have penultimate residues that are either Phe or Leu. Mammalian GRP is not the same as frog Bn, which was previously thought to be the mammalian equivalent of GRP. This changed when researchers discovered that the cDNA encoding GRP and Bn were different in the same frog, with the frog GRP being significantly more similar to mammalian GRP than frog Bn. Consequently, no Bn peptide that is structurally similar to frog Bn has been identified in mammals as of yet.

The mammalian bombesin-like peptides are GRP and neuromedin B (NMB). Phylogenetic analysis of the prohormones encoding the bombesin-like peptides suggests there are three distinct branches of bombesin-like peptides: the GRP branch, the NMB branch, and a branch that contains the multiple bombesin-like peptides found in amphibian skin.

Similar to other peptides, Bombesin-like peptides are extensively dispersed and exert various effects contingent upon their location and the specific conditions of their action. While injection into the central nervous system of cats enhances tidal volume and similarly elevates respiratory rate in rats, these effects are unlikely to be significant for Bombesin-like peptides generated from pulmonary epithelium. Of more relevance is the evidence that they induce bronchoconstriction in guinea pigs, but their impact on vasomotor tone remains unknown. Due to their capacity to induce the release of various peptides, they may control other secretory products of PECs. Pulmonary Bombesin-like peptides may have a function in the inflammatory response. Inflammatory lung illness significantly stimulates the proliferation of PECs, with calcitonin-containing cells predominating, but the number of Bombesin-like peptide-containing cells also rises. The evidence that Bombesin-like peptides serve as chemoattractants for monocytes has significant ramifications.

However, their trophic function is likely to be of paramount relevance. This is seen not only in the lungs but also in the gastrointestinal tract and pancreas, where oral or parenteral treatment induces hyperplasia of gastrin-secreting cells in the stomach and secretory acini in the duodenum and pancreas. Rozengurt and Sinnett-Smith (1983) and Willey, Lechner, and Harris (1984) have shown that, in addition to promoting the proliferation of small cell carcinoma, they are also significantly trophic to murine fibroblasts and normal human bronchial epithelial cells in vitro. Sunday et al. (1990) demonstrated the enhancement of tritiated thymidine incorporation into the developing bronchial epithelium of fetal mouse lungs.

GRP/NMB/bombesin family peptides. (Hirooka A., et al., 2021)

Similarities between amino acid sequences of bombesin and bombesin-like peptides. (Serafin P., et al., 2023)

Bombesin function

(1) Neuroprotective effect

The cognitive and synaptic plasticity deficits, as measured by the Morris water maze and long-term potentiation tests, were considerably mitigated by the injection of bombesin (30 μg/kg/day, for 14 consecutive days). Based on the results of the mechanistic investigations, bombesin greatly reduced the reduced activity of total superoxide dismutase (T-SOD) and catalase (CAT), and it changed the elevated malondialdehyde (MDA) content. Drug treatment also boosted the downregulation of synaptic plasticity-related proteins, such as synaptophysin (SYP) and calcium-calmodulin-dependent protein kinase II (CaMKII), in the hippocampus. Finally, bombesin might mitigate the effects of chronic cerebral ischemia on synaptic plasticity and protein expression, two factors crucial to cognitive function. Memory loss and impaired long-term synaptic plasticity caused by chronic cerebral ischemia may be ameliorated by administering Bombesin, which has promise as a treatment for diseases with cognitive impairments.

Administration of bombesin (BB) improved cognitive deficits in rats. (Yao Y., et al., 2018)

(2) Regulate diet, exercise and endocrine function

The powerful appetite suppressant effects of Bombesin (BN) are well-known. Also, ethanol consumption was shown to be reduced as a result of BN. Consistent with this, Deschodt-Lanckman's group found that BN inspired the mammalian pancreas to release amylase in an in vitro setting. Additionally, the peptide was shown to be responsible for the release of cholecystokinin CCK and other substances, such as vasoactive intestinal peptide or insulin, from the small intestine's I cells. Mitogenic activity of BN was also shown, notably in Swiss 3T3 cells, and it may function as an autocrine growth factor for small-cell lung cancer (SCLC). Additionally, it was noted that BN might have antioxidant properties. In a study conducted by Assimakopoulos and colleagues, they demonstrated that BN reduced intestine oxidative stress after partial hepatectomy and in an animal model of experimental obstructive jaundice by lowering intestinal lipid peroxidation. After being administered centrally and systemically, BN is shown in several articles to cause a hypertensive response. There is evidence from other sources that BN may, in a dose-and time-dependent manner, greatly enhance locomotor, rearing, and grooming behaviors. The fact that neuroleptics like haloperidol were able to prevent these behavioral effects in research provides further evidence that BN may interact with the dopaminergic system. Also discovered in 2008 was the fact that BN promotes the expression of factors that are involved in wound healing.

The beneficial and deleterious biological effects of bombesins in human. (Serafin P., et al., 2023)

(3) Antibacterial action

Studies suggesting the potential antibacterial action of the BN family have also shown these positive benefits. As a matter of fact, frogs may be able to ward against bacterial infections and terrestrial predators by secreting bombesins, which may serve as either poisons or antimicrobial peptides. Given the alarming rise of bacterial resistance to many antimicrobial medications, it is clear that such an endeavor is very beneficial for humans. Here, it was discovered that BN, via its own effects on mucosal immunity, helps keep respiratory defenses up against germs and viruses. For GRP analogues, other research has had positive outcomes. One such example is the selective BB2 receptor antagonist RC-3096, which has shown protective effects against a locally delivered Escherichia coli endotoxin in the lungs.

Bombesin cancer

Bombesin peptides and their messenger RNAs are present in a wide variety of tumor types; they serve as autocrine growth factors that promote tumor proliferation by binding to certain receptors. New blood vessel formation has been linked to these peptides in several cancer models. The results indicate that the matching receptor is functionally relevant and is expressed in tumor cells and tumor vascular beds in a systematic manner. Recent research has shown that transactivation of tumor human epidermal growth factor receptors (EGFR) is a common mechanism for activating bombesin-like receptors 3, 4, or 3 (BRS-3), as well as GRPR and NMBR. Protein kinase C (PKC) activity, proto-oncogene tyrosine protein kinase (Src) activation, ROS stimulation, matrix metalloproteinase activation, and EGFR ligand production are common components of this signaling pathway. A new method of treating tumors may emerge from this interaction, which involves combining an EGFR inhibitor with a bombesin receptor antagonist.

In PC-3 cells (human prostate cancer cell line), Bombesin activation of GRP-R led to the upregulation of IL-8 and VEGF expression via an NFκB-dependent pathway. Bombesin resulted in the degradation of the NFκB inhibitor, translocation of NFκB to the cell nucleus, enhanced binding of NFκB to its DNA consensus sequence, and increased mRNA expression and protein secretion of IL-8 and VEGF. Treatment with the proteasome inhibitor MG-132 inhibited Bombesin-induced NFκB DNA binding, as well as the production and secretion of IL-8 and VEGF. Ultimately, media obtained from PC-3 cell cultures post- Bombesin treatment induced NFκB-dependent migration of human umbilical vascular endothelial cells in vitro. Bombesin and GRP-R in the NFκB-mediated upregulation of proangiogenic gene expression, suggesting a potential molecular mechanism that connects neuroendocrine differentiation with the heightened metastatic potential of androgen-insensitive prostate tumors.

Sonic hedgehog (Shh) and bombesin-cognate receptor (GRPR) are co-expressed in small cell lung cancer (SCLC) samples. We further show that Bombesin-mediated SCLC proliferation is dependent on Bombesin activating Gli in SCLC cells; this is supported by the fact that the effects of Bombesin were inhibited by cyclopamine, an inhibitor of the Shh pathway. In a similar manner, Gli was activated by additional Gαq and Gα13 linked receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants, as well as by bombesin binding to GRPR via its downstream Gαq and Gα12/13 GTPases. Using Gαq and Gα12/13 null cells, we show that these G proteins are absolutely required for Bombesin to activate Gli. In addition, we demonstrate that Rho coordinates Gli activation that is reliant on G-protein-coupled receptors (GPCRs), Gαq, and Gα12/13 by using constitutively active Rho small G-protein (Rho QL) and its inhibitor, C3 toxin. Molecularly, Bombesin triggered an upregulation of Shh gene transcription and protein secretion, which was reliant on the activation of nuclear factor κB (NFκB) by Bombesin-induced GPCR/Gαq-Gα12/13/Rho, which in turn might activate an NF-κB response element within the Shh gene promoter. It has been suggested that Shh inhibitors might be a new way to treat aggressive SCLC, according to a new molecular network that links autocrine Bombesin and Shh circuitries.

Schematic representation of the mechanism of action of bombesin on the Shh pathway. (Castellone M D., et al., 2015)

References

1. Okarvi S M., et al., Radiometallo-labeled peptides in tumor diagnosis and targeted radionuclide therapy[M]//Advances in Inorganic Chemistry. Academic Press, 2016, 68: 341-396.
2. Fani M., et al., Radiolabeled peptides: valuable tools for the detection and treatment of cancer, Theranostics, 2012, 2(5): 481.
3. Hirooka A., et al., The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus, Scientific reports, 2021, 11(1): 13315.
4. Serafin P., et al., Bombesins: A new frontier in hybrid compound development, Pharmaceutics, 2023, 15(11): 2597.
5. Yao Y., et al., Bombesin attenuated ischemia-induced spatial cognitive and synaptic plasticity impairment associated with oxidative damage, Biomedicine & Pharmacotherapy, 2018, 103: 87-93.
6. Castellone M D., et al., Cross talk between the bombesin neuropeptide receptor and Sonic hedgehog pathways in small cell lung carcinoma, Oncogene, 2015, 34(13): 1679-1687.