Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

OBJECTIVES: Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. METHODS: Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. RESULTS: Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. CONCLUSIONS: These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury.

The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10(-7) M) hyperstimulation and bombesin (10(-5) M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

Expression of mRNA for gastrin-releasing peptide receptor by human bronchial epithelial cells. Association with prolonged tobacco exposure and responsiveness to bombesin-like peptides.

Bombesin-like peptides (BLPs) are important regulators of lung development and may also act as autocrine growth factors in lung tumors. We have previously demonstrated expression of mRNA for the three BLP receptor subtypes (neuromedin B [NMB]) receptor, gastrin-releasing peptide [GRP] receptor, and bombesin receptor subtype 3 [BRS-3]) in human non-small cell lung carcinoma (NSCLC) cell lines and bronchial biopsies using the reverse transcription-polymerase chain reaction (RT-PCR; DeMichele, et al. Am. J. Respir. Cell Mol. Biol. 1994; 11:66-74). We have also previously found that growth responses to BLPs could be elicited in some, but not all, cultures of human bronchial epithelial (HBE) cells (Siegfried, et al. Anat. Rec. 1993; 236:241-247). In this report, we utilized RT-PCR to demonstrate mRNA expression of BLP receptor subtypes in cultured HBE cells and also assessed the response of these cultures to BLPs in proliferation assays. The pattern of mRNA expression was correlated with proliferative response, and the results were also analyzed in relation to smoking history and pulmonary function of the subjects studied. Our results suggest that expression of mRNA for the GRP receptor is associated with a long smoking history (> 25 pack-years [PY], p = 0.02). This association was related to past tobacco exposure, regardless of whether the subjects were still active smokers at the time of tissue procurement. Responsiveness to GRP and NMB in proliferation assays was also found only in those HBE cultures with expression of mRNA for at least one of the known receptors for BLPs, and there was a significant association between expression of mRNA for the GRP receptor and proliferative response to both GRP and NMB (p = 0.048). HBE cultures from subjects with a greater than 25 PY smoking history were also more likely to respond to BLPs in the proliferation assays than cells from subjects with less than a 25 PY history (10 of 16 versus 1 of 7, p = 0.06). Cultures of HBE cells from four of the five subjects with severe obstructive lung disease gave a positive response to GRP and NMB in proliferation assays, compared to five of fifteen without severe obstructive lung disease, but this difference was not significant (p = 0.13). These results suggest there is an increased likelihood of expression of the GRP receptor mRNA in the respiratory epithelium of some individuals with a history of prolonged tobacco exposure, and that expression of the GRP receptor mRNA is accompanied by responsiveness to the mitogenic effects of BLPs. These effects appear to persist after smoking cessation.

Bombesin and neuromedin B stimulate the activation of p42(mapk) and p74(raf-1) via a protein kinase C-independent pathway in Rat-1 cells.

The mechanisms by which seven transmembrane receptors activate p42-(mapk)/p44(mapk) are not well defined although p21ras- and protein kinase C (PKC)-dependent pathways have been implicated, typically for Gi- and Gq-coupled receptors, respectively. Here, we demonstrate that in Rat-1 cells transfected with the Gq-coupled bombesin/gastrin releasing peptide receptor, bombesin stimulated activation of p42(mapk) that was not inhibited by the specific PKC inhibitor GF 109203X or by down regulation of phorbol ester-sensitive PKC isoforms. In addition, bombesin rapidly stimulated p74(raf-1) activity that was also independent of PKC activity and insensitive to inhibition by pertussis toxin. Furthermore, addition of neuromedin B to Rat-1 cells transfected with the neuromedin B preferring receptor also activated p42(mapk) and p74(raf-1) in a PKC-independent and pertussis toxin-insensitive manner. Finally we show that addition of bombesin to Rat-1 cells stimulated the GTP loading of p21ras. Our results reveal a novel PKC-independent pathway in the action of Gq-coupled receptors and stress the importance of cell context in defining the signal transduction pathway(s) that link specific receptors to the activation of the mitogen-activated protein kinase cascade.

Different influence of CGRP (8-37), an amylin and CGRP antagonist, on the anorectic effects of cholecystokinin and bombesin in diabetic and normal rats.

Because previous studies had suggested that the anorectic effects of cholecystokinin (CCK) and bombesin (BBS) depend partly on the release of amylin or calcitonin gene-related peptide (CGRP), we investigated the influence of the amylin and CGRP receptor antagonist CGRP (8-37) on the anorectic effects of CCK and BBS in streptozotocin (STZ)-diabetic and nondiabetic rats. STZ-diabetic rats had significantly lower plasma amylin and insulin concentrations than nondiabetic control rats. Amylin (5 micrograms/kg or 2.5 micrograms/rat) injected IP at dark onset after 24-h food deprivation elicited an anorectic effect of similar extent in STZ-diabetic and control rats. Under similar conditions, CCK (0.25 and 2 micrograms/kg) and BBS (5 micrograms/kg) reduced food intake in both STZ-diabetic and nondiabetic rats. These effects were markedly attenuated by CGRP (8-37) (10 micrograms/kg) in non-diabetics but not in STZ-diabetic rats. It is concluded that part of the anorectic effects of CCK and BBS depend on the release of amylin from pancreatic B-cells.

Identification of functional GRP-preferring bombesin receptors on human melanoma cells.

Bombesin was originally isolated from amphibian skin, whereas its mammalian counterpart, gastrin-releasing peptide (GRP), was first identified in the nervous system of the gastrointestinal tract. Whether GRP is present in the human skin is not known. Bombesin-like peptides are also known to modulate growth. We therefore investigated whether human melanoma cell lines express functional GRP-preferring bombesin receptors and whether they alter growth or other specific cellular functions of these tumour cells. GRP receptor mRNA was found in HBL, D-10, Me-28 and A375-6 cell lines, but only A375-6 cells express a large number of high-affinity binding sites for [125I]-[Tyr4] bombesin (K(d) 0.31 +/- 0.04 nmol L(-1), 3880 +/- 429 binding sites per cell). Bombesin dose-dependently increased cytosolic calcium, but did not alter interleukin (IL) 1beta-induced reduction of cell viability or IL-6 secretion, both A375-6-specific cell functions. Growth of A375-6 cells was not altered by bombesin or the specific GRP receptor antagonist BIM26226 as measured by [3H]-thymidine incorporation or methylene blue assay, whereas insulin alone or in combination with other potential growth factors dose-dependently stimulated growth of these cells. The newly characterized GRP-preferring bombesin receptors on highly malignant human melanoma cells could initiate studies of growth effects on solid tumours or in vivo scanning using radiolabelled tracers.