Autocrine inhibitory influences of -melanocyte-stimulating hormone in malignant pleural mesothelioma

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1 Autocrine inhibitory influences of -melanocyte-stimulating hormone in malignant pleural mesothelioma Anna Catania,*,1 Gualtiero Colombo,* Andrea Carlin,* Letizia Garofalo,* Stefano Gatti, Roberto Buffa, Nadia Carboni,* Lorenzo Rosso, Luigi Santambrogio, Luigi Cantalamessa,* and James M. Lipton Departments of *Internal Medicine and Surgery, Ospedale Maggiore di Milano IRCCS, Italy; Institute of Pathology, University of Milano Bicocca, Monza, Italy; and Zengen, Inc., Woodlands Hills, California Abstract: Malignant pleural mesothelioma is a highly aggressive tumor arising from the mesothelial cells that line the pleural cavities. This tumor is resistant to most conventional anticancer treatments and appears to be very sensitive to growthpromoting influences of cytokines and growth factors. Identification of natural inhibitory pathways that control growth should aid discovery of novel therapeutic approaches. We hypothesized that -melanocyte-stimulating hormone ( -MSH), which is produced by many cell types and antagonizes cytokines and growth factors, could be an endogenous inhibitory molecule in mesothelioma. Twelve mesothelioma cell lines were established from pleural effusions of patients with malignant mesothelioma. Mesothelioma cells were found to express mrna for proopiomelanocortin and its processing enzymes; release -MSH peptide into supernatants; and express melanocortin 1 receptor (MC1R), the high-affinity receptor for -MSH. Immunoneutralization of MC1R in the cell lines enhanced expression of interleukin-8 (IL-8), IL-6, and transforming growth factor-. These molecules promote mesothelioma proliferation and are considered therapeutic targets in this tumor. Coincubation of mesothelioma cells with synthetic -MSH significantly reduced cell proliferation. The present research shows an autocrine-inhibitory circuit based on -MSH and its receptor MC1R. Activation of MC1R by selective peptides or peptidomimetics might provide a novel strategy to reduce mesothelioma cell proliferation by taking advantage of this endogenous inhibitory circuit. J. Leukoc. Biol. 75: ; Key Words: melanocortin receptors (MCR) proopiomelanocortin (POMC) malignant mesothelioma growth factors INTRODUCTION Malignant pleural mesothelioma is a relatively rare but highly aggressive tumor arising from the mesothelial cells that line the pleural cavities. This tumor appears to have a complex etiology in which environmental carcinogens (asbestos and erionite), ionizing radiation, viruses, and genetic factors act alone or in concert to cause or promote malignancy [1]. Malignant mesothelioma is resistant to most conventional anticancer treatments and appears to be very sensitive to endogenous factors that promote growth. Indeed, several autocrine and paracrine substances including cytokines, growth factors, and their receptors enhance mesothelioma proliferation and contribute to invasiveness of the tumor [2 7]. Therefore, identification of natural inhibitory pathways should promote discovery of novel, therapeutic approaches. We hypothesized that -melanocytestimulating hormone ( -MSH), which is produced by many cell types and antagonizes cytokines and growth factors, could be an endogenous inhibitory molecule in mesothelioma. Adrenocorticotropic hormone (ACTH) and -, -, and -MSH derive from the precursor molecule proopiomelanocortin (POMC) [8]. These POMC-derived molecules are collectively called melanocortin peptides or melanocortins. Recent research indicates that melanocortin peptides and mainly -MSH have multiple anti-inflammatory and anticytokine effects on host cells [9, 10]. Recognition of these effects has been hastened by identification and cloning of five melanocortin receptors (MC1R MC5R) [11 16]. The anti-inflammatory effects of -MSH are exerted, in some part, through reduced translocation to the nucleus of the transcription factor nuclear factor (NF)- B [17, 18]. -MSH limits degradation of the I B -inhibitory protein and consequently, reduces activation of the NF- B signaling pathway [17, 18]. NF- B is an essential nuclear factor that induces transcription of many cell mediators; its inhibition has, therefore, broad consequences for mediator production and cell functions [19]. Administration of synthetic -MSH inhibits production of proinflammatory cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor (TNF- ) [20], chemokines [21, 22], platelet-derived growth factor (PDGF), transforming growth factor- (TGF- ), endothelin-1 [23], and adhesion molecules [24]. Therefore, -MSH, which reduces production of cytokines and growth factors in vivo and in vitro [25], could be an endogenous inhibitory molecule in cancer cells. 1 Correspondence: Department of Internal Medicine, Ospedale Maggiore di Milano IRCCS, Via F. Sforza 35, Milano, Italy. Received June 11, 2003; revised September 30, 2003; accepted October 4, 2003; doi: /jlb Journal of Leukocyte Biology Volume 75, February

2 The specific aim of the present research was to determine whether mesothelioma cells produce -MSH and express MCR. We established multiple mesothelioma cell lines from pleural effusions of patients with ascertained, previously untreated malignant mesothelioma and determined expression of mrna for POMC and its processing enzymes; presence of -MSH immunoreactivity in cultured cells and in the original tissue biopsy; and release of immunoreactive -MSH in supernatants of mesothelioma cells maintained in culture. As any autocrine effect of the peptide would be mediated by one or more MCR, we explored expression of MC1R and MC3R, the receptors involved in the anti-inflammatory and anticytokine influences of -MSH [26, 27]. When we did find production of -MSH and expression of the MC1R, we determined influences of immunoneutralization of this receptor on the expression of molecules such as IL-8, IL-6, TGF-, and PDGF-B, agents that are known to promote mesothelioma proliferation. To confirm the idea that -MSH has inhibitory influences on mesothelioma cell growth, proliferation of the cell lines was evaluated in the presence of a synthetic peptide. MATERIALS AND METHODS Mesothelioma cell lines Specimens were obtained from pleural effusions of 12 patients with histologically demonstrated, previously untreated, pleural, malignant mesothelioma. Ten were monophasic epithelioid and two, biphasic malignant mesotheliomas. Pleural effusions obtained at the time of surgery were centrifuged (200 g at 20 C for 10 min), and cell pellets were treated with erythrocyte lysis buffer (ammonium chloride 0.829%, potassium bicarbonate 0.1%, EDTA %) and seeded at high density into 25 cm 2 tissue-culture flasks. Cell cultures were performed according to standard methods. The medium consisted of RPMI 1640 supplemented with 15% fetal bovine serum, 2 mm glutamine, 10 mm HEPES buffer, 100 U/mL penicillin, 100 g/ml streptomycin, and 0.25 g amphotericin. Cultures were maintained in a humidified atmosphere of 5% CO 2 at 37 C and examined daily. When cells were confluent, a 1-min treatment with a trypsin EDTA mixture in phosphate-buffered saline (PBS) was used to detach the cells, which were then reseeded at high density. Cell lines were considered established after 2 months (passage, 15 30). All the cell lines had the immunocytological characteristic of mesothelial cells: coexpression of low molecular weight cytokeratins, vimentin, and calretinin and negative reactivity with antibody to carcinoembryonic antigen (CEA) and thyroid transcription factor (TTF)-1. Most specimens were also examined by electron microscopy for characteristic microvilli and intermediate filaments. Three cell lines were obtained from the pleural effusion of patients with nonsmall cell lung cancer (NSCLC). These CEA, TTF-1-positive, and vimentin-negative cell lines were established and maintained in culture using the same procedures described for mesothelioma. Gene expression of POMC, processing enzymes, and MCR Mesothelioma cell pellets were used for RNA extraction. Total RNA was isolated using acid guanidinium thiocyanate-phenol-chloroform extraction [28]. As MCR genes lack introns, to prevent misinterpretation as a result of genomic contamination, total RNA was treated with amplification grade RNase-free DNase I (Life Technologies, Gaithersburg, MD) at room temperature for 15 min. DNase I was then inactivated by adding 2.5 mm EDTA and heating at 65 C for 10 min. RNA was checked for integrity by agarose gel electrophoresis and quantitated by optical density (OD) measurement (260 nm). First-strand cdna (cdna) synthesis was performed using 1 g each RNA sample, 20 pmoles oligo (dt) 18 primer, and 200 U Moloney murine leukaemia virus reverse transcriptase (RT; Clontech Laboratories, Palo Alto, CA) in a 20- l reaction volume. Portions (4 10%) of each diluted (1:5) cdna were used as templates and were polymerase chain reaction (PCR)-amplified with gene-specific primers. The primer pairs were synthesized according to published mrna sequences. PCR amplifications were performed in a 25- l reaction volume containing 20 pmoles each of upstream and downstream primers, 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), 200 M dntps, and mm MgCl 2 using a GeneAmp 9700 thermal cycler (Applied Biosystems). PCR reaction mixture and temperature profile conditions were tested against positive controls for each specific mrna (mrna) in preliminary experiments. The PCR products were single bands of the expected size, and their identity was confirmed by sequencing. The number of amplification cycles was selected to obtain a linear correlation between the amount of cdna and the yield of PCR product. The POMC primer pair (upstream 5 -GAGGGCAAGCGCTCCTACTCC-3, downstream 5 -GGGGCCCTCGTCCTTCTTCTC-3 ) generated a 260-base pair (bp) product; the MC1R primer pair (upstream 5 -GCCACCATCGCCAA- GAACC-3, downstream 5 -ATAGCCAGGAAGAAGACCA-3 ), a 416-bp product; the MC3R pair (upstream 5 -CGGTGGCCGACATGCTGGTAAGTG- 3, downstream 5 -TGAGGAGCATCATGGCGAAGAACA-3 ), a 461-bp product; the paired basic amino acid cleaving enzyme 4 PACE4 pair (upstream 5 -AGTGCCCATCAGTCCCGCTC-3, downstream 5 -GCAAGACAGGCACT- GCGTCG-3 ), a 380-bp product; the furin pair (upstream 5 -GTGGGCCG- GAAAGTGAGCCA-3, downstream 5 -GCTGCCCTAAAGGTTTCACTCC-3 ), a 1393-bp product; the 7B2 protein pair (upstream 5 -CCTCGGTTGACAAT- GGTCTC-3, downstream 5 -TTGCCCAAGCCTGGATAGT-3 ), a 494-bp product; the precursor convertase (PC)2 pair (upstream 5 -GATCCTCTTTT- TACAAAGCAGTGG-3, downstream 5 -GGTGAGCACAGTCAGATGCTG- CAT-3 ), a 880-bp product; and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pair (upstream 5 -TGAAGGTCGGAGTCAACGGATTTGGT- 3, downstream 5 -CATGTGGGCCATGAGGTCCACCAC-3 ), a 980-bp product. Amplified products were resolved on 1.5% agarose gels loaded with ethidium bromide and were visualized by UV transillumination. Real-time RT-PCR analysis Expression of IL-6, IL-8, TGF- 1, and PDGF-B mrnas after immunoneutralization of MC1R was evaluated by real-time RT-PCR based on TaqMan methodology. In brief, total RNA and cdnas were prepared as stated above. PCR was performed in an ABI PRISM 7000 sequence detection system (Applied Biosystems) using 2.5 l of each diluted (1:5) cdna in a final volume of 25 l. The PCR mixture contained 1 TaqMan Universal PCR Master Mix with AmpErase UNG enzyme and 1 Assays-on-Demand predesigned primers and probe mix (900 nm primer/250 nm probe final concentrations; Applied Biosystems). Hs _m1 for IL-6, Hs _m1 for IL-8, Hs _m1 for TGF- 1, and Hs _m1 for PDGF-B were the assay IDs. Based on the 5 nuclease chemistry, each set consists of two unlabeled PCR primers and a 6-carboxyfluorescein dye-labeled TaqMan minor groove binder (MGB) probe, which spans an exon junction and will not detect genomic DNA. Heating for 2 min at 50 C activated the AmpErase UNG enzyme; then, heating for 10 min at 95 C activated the AmpliTaq Gold enzyme. All genes were amplified by a first step of 15 s at 95 C followed by 1 min at 60 C for 50 cycles. To normalize for differences in the amount of total RNA added to each reaction mixture, GAPDH was selected as an endogenous RNA control, using a VIC-labeled TaqMan MGB probe. The relative amount of each inducible mrna was calculated by reference to the GAPDH mrna in the sample using the comparative C T method and then compared with the amount in cells incubated with medium alone (calibrator). C T is the point at which the exponential increase in signal (fluorescence) crosses a somewhat arbitrary signal level (usually 10 background); the amount of target, normalized to the endogenous reference and relative to the calibrator, is given by 2 CT. Immunohistochemistry Mesothelioma tissue specimens were fixed in 10% buffered formalin and were paraffin-embedded. Tissue sections (3 m) were cut from the paraffin-embedded blocks. The sections were dewaxed and hydrated, and endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide in distilled water. After antigen retrieval using microwave irradiation, 3 5 min, 780 W cycles at 90 C in ph 6.0, 0.01 M citrate buffer for 15 min, the slides 254 Journal of Leukocyte Biology Volume 75, February 2004

3 were allowed to cool at room temperature for 20 min and incubated at 4 C overnight with a rabbit polyclonal antibody against -MSH (Eurodiagnostica, Malmö, Sweden) using a 1:600 dilution or with goat polyclonal antibodies against MC1R (1:400; Santa Cruz Biotechnology, Santa Cruz, CA). Sections were subsequently rinsed in Tris-NaCl buffer, ph 7.6, 0.1 M, and incubated at room temperature for 30 min with biotin-conjugated donkey anti-goat immunoglobulin G or donkey anti-rabbit secondary antibodies (Santa Cruz Biotechnology). Negative-control samples were incubated only with secondary antibodies. For MC1R, preincubating the antibody with the competitor peptide provided by the manufacturer also performed a specificity control. After rinsing in Tris-NaCl buffer, sections were incubated for 30 min with avidin-biotinylated horseradish peroxidase complex. The final reaction was run by incubating the sections in 0.002% 3 3 -diaminobenzidine tetrahydro-chloride chromogen solution, added with 0.01% hydrogen peroxide for 5 min. The sections were then washed in deionized H 2 O and counterstained with Harry s hematoxylin. Immunocytochemistry for -MSH and MC1R in mesothelioma cell lines was performed in cells cultured in two-well glass chamber slides (Nalgene Nunc International, Naperville, IL). Chambers were filled with cell suspension ( ) and incubated in complete medium in a humified atmosphere of 5% CO 2 at 37 C for 24 h. At the end of the incubation period, supernatants were removed, and cells were washed in Tris-NaCl buffer, ph 7.6, and fixed in 50% methanol and acetone solution. -MSH release by mesothelioma cells in culture Mesothelioma cells ( ) were seeded in six-well culture plates and incubated in complete medium (see above) in a humified atmosphere of 5% CO 2 at 37 C for 24 h. At the end of the incubation period, supernatants were removed and stored at 80 C. -MSH was measured using a double-antibody radioimmunoassay (RIA) method (Eurodiagnostica); the sensitivity, calculated from a decrease in binding of 2 SD in the 0 standard, is 4.8 pg/ml, and the crossreactivity with other POMC peptides [ACTH (1 24), ACTH (1 39), -MSH, -MSH] is 0.002%. ACTH was also measured in supernatants using an immunoradiometric assay (DiaSorin, Stillwater, MN). Immunoneutralization of MC1R Mesothelioma cells ( ) were seeded in six-well culture plates in complete medium (see above) and allowed to adhere overnight in a humified atmosphere of 5% CO 2 at 37 C. Cells were then incubated for 24 h in the presence of anti-mc1r goat polyclonal antibody diluted 1:250 in medium (Santa Cruz Biotechnology); control goat polyclonal antibody (antisheep red cells) at the same dilution; or an equal volume of medium alone. At the end of the incubation period, cells were detached using a trypsin EDTA mixture in PBS, and pellets were used for RNA extraction (see above). Cell-growth inhibition assay Mesothelioma cells were counted and dispensed into 96-well tissue-culture plates (Costar, Cambridge, MA) at the concentration of 2000 cells/well in 100 l culture medium. After 24 h incubation at 37 C, 5% CO 2, to allow cells to adhere, 100 l culture medium, containing -MSH (Sigma Chemical Co., St. Louis, MO) at the final concentrations of 10 5 and 10 4 M, was dispensed into wells (six replicates for each concentration). Control wells received an equal volume of medium alone. Medium alone or containing the same concentrations of -MSH was aspirated and renewed every 48 h of incubation. Cell proliferation was determined at 72 h and 96 h using 3(4,5-dimethylthiazol-2yl)2,5- diphenyltetrazolium bromide (MTT), a tetrazolium salt that is reduced to a colored formazan product by reducing enzymes present only in metabolically active cells [29]. MTT solution (25 l) was added to culture wells. Plates were incubated at 37 C for 3 h. After incubation, the culture medium was removed by careful aspiration and replaced with 200 l dimethyl sulfoxide to solubilize formazan. Using a plate shaker for 10 min completed formazan solubilization. An automated reader (Titertek Multiskan MCC/340) at 540 nm measured absorption of each well. Cell proliferation assays were repeated in at least three separate experiments on the same cell line. Statistical analysis Statistical analysis was performed using one-way repeated measures of ANOVA followed by Bonferroni t-test for specific comparisons. A probability value 0.05 was considered significant. RESULTS The mesothelioma cell lines expressed mrna for POMC (Fig. 1). POMC expression varied considerably across samples. However, repeated experiments showed that expression level was peculiar for each individual patient and not related to experimental conditions or cell passage (not shown). Further, POMC expression was observed during the exponential growth phase or cell confluence. The cell lines expressed a set of POMC-processing enzymes belonging to the subtilisin family of PCs (Fig. 1). mrnas for PACE4, furin, and the PC2-specific binding protein known as 7B2 were found in all subjects. Most of the cells also clearly expressed the PC2 (Fig. 1). As mesothelioma cells expressed mrna for POMC and its processing enzymes, it was reasonable to believe that these cells had the capacity to produce the -MSH peptide. Therefore, using a specific RIA, we measured -MSH in the supernatants of mesothelioma cells maintained in culture for 24 h. All the supernatants contained variable concentrations of Fig. 1. Gene expression of POMC, PACE4, furin, 7B2, PC2, and MC1R in cell lines derived from two biphasic, MM8 and MM12, and ten monophasic epithelioid malignant mesotheliomas and from three NSCLCs. GAPDH amplification was used as RT-PCR control. Catania et al. Autocrine -MSH in malignant mesothelioma 255

4 Fig. 2. (Left) Production of -MSH in supernatants of 12 mesothelioma cell lines maintained in culture for 24 h. Each point represents the mean of three determinations. (Right) -MSH immunoreactivity in a mesothelioma cell line in culture. (Original magnification, 40.) -MSH (Fig. 2). The precursor molecule ACTH was not detected in any supernatant. Immunocytochemistry techniques confirmed the presence of -MSH immunoreactivity in mesothelioma cells in culture (Fig. 2). To exclude the possibility that -MSH was induced in cells as a consequence of cell manipulations or cell passage, we analyzed -MSH expression in the original tissue biopsies obtained at surgery. Immunohistochemistry revealed the presence of -MSH in mesothelioma specimens as much as in cell culture (Fig. 3). As any effect of circulating/locally produced -MSH would require expression of one or more MCR, we determined whether mesothelioma cells express MC1R and/or MC3R, the melanocortin receptors that are known to mediate the antiinflammatory and anticytokine effects of -MSH. mrna for MC1R was expressed in all mesothelioma cell lines (Fig. 1). No expression of MC3R was detected in any sample of mesothelioma cells. Tissue immunohistochemistry/immunocytochemistry confirmed the presence of MC1R immunoreactivity in mesothelioma cells in culture and in mesothelioma tissue (Fig. 4). Presence of MC1R immunoreactivity in stroma of the mesothelioma tumor biopsy confirmed previous observations that this receptor subtype occurs in macrophages, fibroblasts, and endothelial cells [30 32]. Expression of POMC, its processign enzymes, and MC1R was also found in cells obtained from three NSCLCs (Fig. 1). Therefore, it appears that -MSH production and MC1R expression are not restricted to malignant mesothelioma but rather occur in other cancer cells. To explore the functional significance of -MSH production and its high-affinity receptor expression in mesothelioma cells, we performed immunoneutralization studies in which cells were incubated with an affinity-purified anti-mc1r polyclonal antibody or a control antibody. Immunoneutralization of the -MSH receptor MC1R caused a significant enhancement of mrnas for IL-6, IL-8, and TGF- (Fig. 5; P 0.05). This enhancement of gene expression was present in epithelioid and biphasic mesotheliomas. A small increase in PDGF-B was also observed, but the difference was not statistically significant. Incubation of cells with the control antibody did not cause any significant change in expression of any mrna. Immunoneutralization of MC1R did not cause any significant change in POMC and MC1R mrna steady-state levels (data not shown). Therefore, up-regulation of cytokines and growth factors induced by the anti-mc1r antibody was not associated with a modulation of POMC or MC1R gene expression. To confirm the idea that -MSH is an inhibitory factor in mesothelioma, cell proliferation was measured in the presence or absence of a synthetic peptide. Coincubation of cells with concentrations of -MSH caused a significant reduction in mesothelioma cell proliferation, which was more pronounced at 96h(Fig. 6). DISCUSSION Although several stimulatory pathways have been identified and targeted in mesothelioma cells [3, 7, 33, 34], endogenous inhibitory circuits have not been described. The present research on malignant mesothelioma cells suggests the existence of an autocrine, inhibitory circuit based on -MSH and its high-affinity receptor MC1R. Indeed, disruption of this circuit Fig. 3. Immunohistochemistry for -MSH in tissue specimens of five monophasic epithelioid (A E) and one biphasic (F) malignant mesotheliomas. All specimens show clear cytoplasmic immunoreactivity for -MSH. (Original magnification, 40 ; hematoxylin counterstain.) Fig. 4. Immunohistochemistry for MC1R in a tissue biopsy obtained from a patient with biphasic malignant mesothelioma (A) and in the cell line from the same subject (B). (Original magnification, 40.). 256 Journal of Leukocyte Biology Volume 75, February 2004

5 Fig. 5. Influence of immunoneutralization of MC1R on gene expression of IL-6, IL-8, TGF-, and PDGF-B in two biphasic and four monophasic epithelioid mesotheliomas in culture. Cells were incubated with medium alone, a control antibody, or an anti-mc1r antibody, and steady-state levels of mrna were measured using quantitative real-time RT-PCR. Data are expressed as fold change of the targeted gene relative to medium alone. Bars denote mean SE. *,P 0.05; **, P through immunoneutralization of MC1R enhanced expression of endogenous factors that are known to promote mesothelioma cell proliferation and are potential therapeutic targets for novel anticancer therapies [35]. A direct evidence of inhibitory influences of -MSH was found when addition of synthetic -MSH to culture medium significantly reduced mesothelioma cell proliferation. Activation of MC1R by selective peptides or peptidomimetics might provide a novel strategy to reduce mesothelioma cell proliferation by taking advantage of an endogenous inhibitory circuit based on this melanocortin receptor. Secretion of POMC products by cancer cells has been known for decades and regarded as an aberrant hormone production by a disregulated cell [36]. Although POMC expression is common in tumors such as small cell carcinoma of the lung or bronchial carcinoid tumors, clinical Cushing s syndrome is rare. The reason for this discrepancy is that tumor-associated POMC processing differs substantially from that occurring in the anterior pituitary. Indeed, whereas pituitary corticotrophs express predominantly the serine protease PC1, which gives rise to ACTH (1 39), neuroendocrine tumor cells predominantly express the PC2/7B2 enzyme complex, responsible for further POMC processing into -MSH (ACTH 1 13) and corticotropin-like intermediate lobe peptide (ACTH 18 39) [37]. These smaller melanocortin peptides lack the adrenal-stimulatory capacity of ACTH, and therefore, their production is not associated with enhanced cortisol secretion. It is now clear that POMC derivatives such as -MSH and other melanocortins have regulatory effects on host cells including lymphocytes, monocytes, keratinocytes, and melanocytes [38 42]. The present data on malignant mesothelioma indicate that these tumor cells express the POMC gene and its processing enzymes PCs. Therefore, it is clear that mesothelioma cells have the capacity to process the POMC precursor to yield specific peptides. Indeed, -MSH production by mesothelioma was confirmed by two independent techniques. The peptide was released in the supernatants of cultured cells, where it was measured using a radioimmunoassay. Further, mesothelioma cell lines stained for the peptide at immunocytochemistry. -MSH immunoreactivity was also present in the mesothelioma tissue obtained at surgery. Therefore, peptide production was not induced by culture conditions, but it occurred in the tumor before any cell manipulation. With regard to biological significance of the amount of peptide produced by cultured cells, it is worth mentioning that -MSH occurs in picogram amounts in the circulation, and it was found that other cell types such as macrophages, microglia, and endothelial cells produced similar concentrations [25]. Picomolar concentrations of -MSH inhibited NF- B activation in several cell systems [18]. Any effect of -MSH on cell functions would require the presence of one or more of its receptors. The receptors that mediate the effects of -MSH and related melanocortins have been identified and cloned [12 16, 43]. Five MCR (MC1R MC5R) are currently recognized. They are small, seven-transmembrane, G protein-linked receptors, which differ in their tissue distribution and affinity for the various melanocortins [44]. The transmembrane signaling involves stimulation of adenylyl cyclase followed by cyclic adenosine monophosphateinduced activation of protein kinase(s) and protein phosphorylation. MC1R is the receptor with the highest affinity for -MSH and is expressed by virtually all cells involved in the inflammatory response including monocytes [40, 45], neutro- Fig. 6. Inhibitory effects of concentrations of synthetic -MSH on proliferation of 12 mesothelioma cell lines. Measurements of in vitro growth in microculture wells were performed through evaluation of cell-mediated reduction of tetrazolium. Data are expressed as the mean OD ( SE) measured at the indicated time intervals. *, P 0.05; **, P 0.01, versus control. Catania et al. Autocrine -MSH in malignant mesothelioma 257

6 phils [46], microglia [47], astrocytes [48], and endothelial cells [31]. The present research indicates that mesothelioma cells express mrna for the MC1R as well as the receptor protein, which was demonstrated by immunohistochemistry. It appears, therefore, that mesothelioma cells express -MSH and its high-affinity receptor MC1R. Previous research showed that immunoneutralization of MC1R in monocytes increases TNF- production by these cells [32]. The present data indicate that disruption of the circuit based on -MSH and MC1R enhances expression of cytokines and growth factors in mesothelioma cells. Indeed, several growth factors are all known to promote growth of mesothelioma cells and are targets for novel therapies [3]. One of the most promising new therapies for mesothelioma is based on the highly selective inhibitor of the PDGF receptor tyrosine kinase STI-571 (Gleevec; Novartis, Basel, Switzerland); this molecule is being tested in mesothelioma patients [3]. Antisense oligonucleotides specific for TGF- inhibited the growth of malignant mesothelioma in vitro and in vivo [49]. IL-8, a proinflammatory and angiogenic chemokine, likewise appears to contribute to mesothelioma progression [5, 50, 51]. Antibody treatment against IL-8 decreased mesothelioma progression in athymic nude mice injected intrapleurally with human malignant mesothelioma cells [50]. IL-6 occurs in high concentrations in patients with malignant pleural mesothelioma [52]. This proinflammatory cytokine is believed to contribute to systemic manifestations of malignant mesothelioma, including fever, cachexia, and thrombocytosis [53]. Antagonists for these growth-promoting molecules are actively sought. However, it is important to mention that all the new potential treatments are based on inhibition of a single mediator. -MSH or MC1Rspecific agonists [54] would cause a collective inhibition of growth factors, providing, therefore, a more complete control of molecules that promote tumor proliferation and systemic host reactions. As three pulmonary adenocarcinoma cell lines also showed expression of POMC, PCs, and MC1R, it appears that the autocrine circuit, based on -MSH and its receptor, is not peculiar to mesothelioma, but it may be present in other tumor cells. Therefore, inhibitory influences of -MSH are potentially beneficial in other tumors, especially those more sensitive to stimulatory effects of endogenous growth factors. Blocking these stimulatory pathways can increase sensitivity of tumor cells to more specific, antiproliferative treatments. In conclusion, there is general agreement that no single treatment modality will be effective by itself in mesothelioma. The development of more active drug combinations might arise from recognition of endogenous inhibitory pathways. ACKNOWLEDGMENT Progetto a Concorso, Ospedale Maggiore di Milano, Italy, supported this work. REFERENCES 1. Carbone, M., Kratzke, R. A., Testa, J. R. (2002) The pathogenesis of mesothelioma. Semin. Oncol. 29, Gerwin, B. I., Lechner, J. F., Reddel, R. R., Roberts, A. B., Robbins, K. C., Gabrielson, E. W., Harris, C. C. (1987) Comparison of production of transforming growth factor- and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines. Cancer Res. 47, Nowak, A. K., Lake, R. A., Kindler, H. L., Robinson, B. W. (2002) New approaches for mesothelioma: biologics, vaccines, gene therapy, and other novel agents. Semin. Oncol. 29, Demetri, G. D., Zenzie, B. W., Rheinwald, J. G., Griffin, J. D. (1989) Expression of colony-stimulating factor genes by normal human mesothelial cells and human malignant mesothelioma cells lines in vitro. 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