Elsevier

Neuropeptides

Volume 42, Issue 3, June 2008, Pages 255-265
Neuropeptides

Cholecystokinin (CCK) and CCK receptor expression by human gliomas: Evidence for an autocrine/paracrine stimulatory loop

https://doi.org/10.1016/j.npep.2008.02.005Get rights and content

Abstract

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Introduction

Gliomas are tumours originating from supporting glial cells and are the most frequent intraxial brain neoplasm. Incidence of gliomas is around 1% in the general population (Behin et al., 2003). Gliomas are very serious brain tumours with a lethality approaching 80% within the first year of diagnosis. Induction of angiogenesis in glioblastomas (GBM) suggests that it is a necessary part of malignant progression. However, the precise molecular mechanisms underlying the regulation of brain tumour growth and angiogenesis remain unresolved and only recently it was reported that tumour suppressor gene ING4 has an important role in brain tumour pathogenesis (Garkavtsev et al., 2004). Other studies have demonstrated that gliomas commonly express molecular or genetic abnormalities that influence the signal pathways that regulate cell proliferation. For instance, a glioma cell may overexpress oncogenes such as epidermal growth factor receptor (EGFR) overexpression of EGFR, a tyrosine kinase receptor with downstream effects resulting in cell proliferation and invasion, is the most common oncogenic alteration in gliomas (Watanabe et al., 1996). Those gliomas that do not overexpress EGFR, instead, they commonly contain mutations of tumour suppressor genes such as phosphatase and tensin homolog (PTEN) and p53 (Frederick et al., 2000, Kleihues and Ohgaki, 1999). These gains or losses may promote cancerous behavior but also may be targets for new treatments. In a different approach, recent studies have revealed involvement of a number of growth factors and cytokines in regulating invasion of gliomas in surrounding structures. Gastrin has been described as one, and found to act as an endogenous modulator of glioma progression and invasion to surrounding structures (Lefranc et al., 2002). Cholecystokinin (CCK), a peptide of the same family as gastrin is able to powerfully stimulate growth of rat glioma cells in vitro (Kaufmann et al., 1998), whereas older studies remained inconclusive as to the action of gastrin on human gliomas, suggesting a possible role in the growth of a substantial proportion of human astrocytic tumours (Camby et al., 1996).

Gastrin and CCK are both members of the amidated peptide hormone family and are characterized by an identical carboxyl-terminal pentapeptide sequence (-Gly-Trp-Met-Asp-Phe-NH2). Both are widely distributed throughout the central nervous system and the digestive tract and regulate growth of normal tissues as well as of gastrointestinal cancers (Noble et al., 1999, Townsend et al., 1986). On the basis of ligand binding and molecular biology studies, two main classes of receptors have been identified, namely, the CCK-A and CCK-B/ gastrin receptors (Moran et al., 1986, Jensen et al., 1994). Both CCK-A and CCK-B receptors are members of the seven transmembrane G protein-coupled receptors and despite sharing 50% sequence identity they can be clearly identified by using a number of selective CCK agonists and antagonists (Noble et al., 1999). CCK-A receptors predominate in the periphery and mediate actions such as pancreatic enzyme secretion, gall bladder contraction, and gut motility whereas, CCK-B receptors are predominant in the central nervous system (Shulkes and Baldwin, 1997). Since CCK and gastrin are able to regulate growth of normal tissues as well as of gastrointestinal cancers and still remains inconclusive the precise role of CCK on human gliomas (Camby et al., 1996). CCK-A receptors bind sulphated CCK far more efficiently than non-sulphated forms as compared to CCK-B receptors that binds both forms as well as gastrin (Hughes et al., 1990) therefore, it is logical to expect that the CCK receptors might not have similar effects on glioma cell growth upon CCK stimulation. For instance the cAMP-mediated regulation of growth (either stimulatory or inhibitory) by gastrin on two different human colon cancer cell lines was determined by the predominant isoform of dependent protein kinase A (PKA) (Bold et al., 1994). It is the interest of this study to determine the actual effect of CCK on human glioma cell growth and whether blockade of CCK receptors will have an inhibitory effect on human glioma cell growth.

To further investigate the potential that such a system may be involved in human glioma growth, reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence of CCK receptor gene expression CCK peptide itself, and a neutralizing antibody against CCK was incorporated into the growth experiments to investigate possible endogenous CCK activity on glioma cells growth in an autocrine manner. Two forms of CCK peptides were used in the analysis the CCK-33 and the sulfated form of CCK-8, since differences in biological activities between large and small forms of CCK exist, while none of these comparative studies was performed on brain tissue (Liddle et al., 1986, Solomon et al., 1984, Doi et al., 1988). In parallel the effect of exogenously added CCK on glioma cell growth was assessed and antagonized in vitro.

Section snippets

Gene expression analysis

Experiments were performed on 14 human gliomas. Histologically, the tumours consisted of 8 grade III anaplastic astrocytomas (AA), 5 grade IV GBM, 1 gliosacroma grade IV and the U-87 MG cell line. Total RNA was extracted from a portion of tissue using TRIZOL (Gibco-BRL, UK) according to the manufacturer’s instructions. For cells grown in monolayer, 2 ml of Trizol reagent was added directly to the 3.5 cm Petri dish (Greiner). The extracted total RNA (3 μg cell line and tumour samples) was reverse

Determination of mRNA expression for CCK, CCK-A and CCK-B receptor

All 14 human gliomas (8 grade III AA, 5 grade IV GBM and one gliosacroma grade IV) as well as the U-87 MG cells were shown by RT-PCR analysis to express mRNA for CCK itself and CCK-B receptor, whereas the CCK-A receptor was only detected in two GMBs out of the 14 tumours analysed (Fig. 1A, Table 2). Further RT-PCR analysis of the CCK receptor status in six newly established primary cell lines revealed that both cell lines initially expressing CCK-A receptor did retain the expression after

Discussion

Malignant brain tumours such as GBM remain virtually untreatable and inevitably lethal despite extensive surgical excision and adjuvant radio- and chemotherapy (Behin et al., 2003). Their treatment resistance is related to their exceptional migratory nature and ability to insinuate themselves seamlessly and extensively into neuronal tissue. Glioma invasion seems to be dependent on a number of growth factors involved in a paracrine activation of the tumour by surrounding stroma as well as the

Conclusions

In conclusion, in vitro evidence of an autocrine/paracrine growth stimulatory loop by CCK and its receptor(s) in human glioma cells is presented. Even though antagonistic effects exhibited by the CCK receptor antagonists (L-364,718 and L-36,260) on U-87 MG and primary glioma cell growth was encouraging was not proven to be selective. Further development is required for their implementation in the antiglioma/anticancer therapy, whereas, inhibition of CCK will eventually comprise one of many

Acknowledgements

This work was supported by the Association for International Cancer Research Grant Lennox Children’s Fund Trust.

The CCK antagonists were kindly provided by ML Laboratories PLC, London.

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