Department of Medicine, University of Illinois at Chicago; and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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The purpose of this study was to determine whether exposure of cultured chemically transformed hamster oral keratinocytes (HCPC-1) to an aqueous extract of smokeless tobacco (STE) potentiates DNA synthesis elicited by vasoactive intestinal peptide (VIP), an autocrine neuropeptide, and, if so, whether this response is associated with inactivation of neutral endopeptidase 24.11 (NEP 24.11), an ectoenzyme that cleaves and inactivates VIP very effectively, in these cells. I found that STE and VIP each elicited a modest, albeit significant, increase in DNA synthesis in cultured HCPC-1 cells (P < 0.05). However, incubation of HCPC-1 cells with STE together with VIP evoked a significant, concentration- dependent increase in DNA synthesis that was mediated by VIP receptors. The effects of STE and VIP were synergistic. Maximal response was observed after a 48-h incubation. STE significantly attenuated NEP 24.11 activity in HCPC-1 cells at a time when VIP-induced DNA synthesis was maximal. Collectively, these data indicate that STE potentiates VIP-induced DNA synthesis in cultured oral keratinocytes, and that this response is temporally related to STE-induced inactivation of NEP 24.11 in these cells. I suggest that NEP 24.11 modulates the mitogenic effects of smokeless tobacco in the oral epithelium, in part, by inactivating VIP.
oral mucosa; epithelium; leukoplakia; oral cancer; snuff; 5-bromo-2'-deoxyuridine; peptidase; hamster
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INTRODUCTION |
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IT IS ESTIMATED THAT EVERY YEAR more than 300,000 adolescents become regular users of smokeless tobacco in the United States (8, 47). A growing scientific evidence suggests that regular use of smokeless tobacco is associated with oral mucosa injury and inflammation (12, 14, 18). Importantly, smokeless tobacco may predispose susceptible individuals to oral epithelial cell dysplasia and cancer (14, 18, 28). However, Müns et al. (27) showed that an aqueous extract of smokeless tobacco (STE) has no significant effects on proliferation of cultured chemically transformed golden Syrian hamster oral keratinocytes (HCPC-1) (51). These disparate results suggest that host factors elaborated in the oral mucosa could modulate the deleterious effects of smokeless tobacco.
To this end, the oral epithelium, which is exposed directly to smokeless tobacco, is densely innervated by nerve fibers containing neuropeptides, most notably vasoactive intestinal peptide (VIP), an autocrine neuropeptide (10, 17, 36, 49). Nicotine, a major constituent of smokeless tobacco (18, 33, 34), has been shown to stimulate VIP release from nerves in the rat gastric fundus (5, 25). It is well established that VIP promotes growth of cultured skin keratinocytes and enterocytes (15, 31, 32, 41, 44, 50). However, under normal physiological conditions the biological effects of VIP are short-lived, due, most likely, to proteolytic inactivation and spontaneous hydrolysis of the peptide (26, 30, 35). Hence, for VIP to play a significant role as a growth factor in the oral epithelium, local peptide catabolism has to be slowed.
Previous work from my laboratory showed that neutral endopeptidase 24.11 (NEP 24.11; EC 3.4.24.11), an ectoenzyme widely distributed in the oral epithelium that cleaves and inactivates VIP very effectively in various organs (6, 19, 22, 35, 48, 52), modulates the vasorelaxant effects of VIP in the in situ hamster oral mucosa (43). In addition, Gao et al. (12) showed that short-term suffusion of an aqueous STE on the in situ hamster cheek pouch decreases NEP 24.11 activity in tissue homogenates. Importantly, expression of NEP 24.11 is reduced in stomach, colon, and lung cancer relative to normal tissues and regulates the growth of certain malignant tumors (2-4, 10, 38, 40). For instance, Bunn et al. (2) showed that exogenous NEP 24.11 inactivates autocrine peptides involved in lung cancer signal transduction and inhibits the growth of lung cancer and normal lung cell lines in vitro and in vivo in athymic nude mice.
Taken together, these data suggest that smokeless tobacco placed on the oral mucosa could stimulate nerves to release VIP and inactivate NEP 24.11 in the epithelium. This, in turn, will slow local VIP catabolism thereby promoting epithelial cell growth (36, 37, 42). The purpose of this study was to begin to address this issue by determining whether exposure of HCPC-1 to STE potentiates VIP-induced DNA synthesis and, if so, whether this response is associated with inactivation of NEP 24.11.
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METHODS |
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General
Preparation of STE.
The extract was prepared in my laboratory according to the
method of Oh et al. (29) as previously described (11, 12, 27, 37).
Briefly, 10 g of smokeless tobacco (1S3 moist snuff; Tobacco and Health
Research Institute, University of Kentucky, Lexington, KY) were mixed
with 100 ml DMEM and incubated at 37°C for 2 h. The mixture was
then centrifuged at 450 g for 10 min. The supernatant was
collected and centrifuged at 13,000 g for 1 h. After adjusting
the pH to 7.4 using 0.1 N HCl, the resulting supernatant, designated
arbitrarily as 1:10 aqueous dilution of raw smokeless tobacco (11, 12,
27, 28, 37), was filtered through a Millipore filter (pore size, 0.45 µm), divided into 2-ml samples, snap-frozen in liquid nitrogen, and
stored at 70°C until used.
Culture of oral keratinocytes. 7,12-Dimethylbenz[a]anthracene-transformed golden Syrian HCPC-1 were kindly provided by Dr. D. T. Wong (51). They have been previously used in my laboratory (37). Cells were seeded in microtiter, flat-bottom 96-well cell culture cluster (Costar, Cambridge, MA) at a density 30,000 cells/well in 100 µl DMEM supplemented with 10% FCS (GIBCO, Grand Island, NY) and antibiotics (50 U penicillin/ml, 50 µg streptomycin/ml, and 2 µg fungizone/ml; GIBCO). The cells were maintained in 95% air-5% CO2 at 37°C for 24-72 h as outlined below. Cell viability was always >95% as determined by morphological examination using phase-contrast microscopy and 0.1% trypan blue dye exclusion test.
Experimental Protocols
Effects of STE and VIP on DNA synthesis in HCPC-1 cells.
HCPC-1 cells (30,000 cells/well) were incubated in the absence and
presence of increasing concentrations of STE (1:1,000, 1:500, and 1:100
aqueous dilutions) or human VIP (107 to
10
5 M) for 24-72 h. In a second series of
experiments, cells were incubated in the absence and presence of STE
(1:1,000, 1:500, and 1:100 aqueous dilutions) together with VIP
(10
6 M) or VIP (10
7 to
10
5 M) together with STE (1:100 aqueous dilution)
for 24-72 h. In another series of experiments, cells were
incubated with VIP10-28 (10
5 M), a
selective VIP receptor antagonist in the in situ hamster cheek pouch
(39), alone and in the presence of VIP (10
6 M), STE
(1:100 aqueous dilution), and VIP (10
6 M) together
with STE (1:100 aqueous dilution) for 24-72 h. Incubation of cells
with VIP10-28 alone at concentrations higher than 10
5 M was associated with a significant increase in
DNA synthesis (data not shown). In some experiments, HCPC-1 cells were
incubated with VIP1-12 (10
5 M), an
inactive peptide fragment, alone and in the presence of VIP
(10
6 M) and VIP (10
6 M) together
with STE (1:100 aqueous dilution) for 24-72 h. Each experiment was
conducted in triplicate. The concentrations of STE, VIP,
VIP10-28, and VIP1-12 used in these
experiments are based on preliminary and previous studies in my
laboratory and reports in the literature (11, 12, 27, 28, 37, 39, 43).
Effects of STE on NEP 24.11 activity in HCPC-1 cells. HCPC-1 cells were seeded in 100-mm tissue culture dishes (Costar) at a density of 1 × 105/ml for 24-72 h in the absence and presence of STE (1:100 aqueous dilution). At the end of the incubation period, the cells were scraped from the dish using a rubber policeman and PBS (GIBCO), and cell extracts were prepared as previously described (36). Protein concentration in cell lysates was determined by the method of Bradford (1). NEP 24.11 activity in cell lysates was determined in duplicate by a sensitive two-stage enzymatic reaction using the synthetic substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4 nitroanilide supplemented with bacterial (Streptomyces griseus) aminopeptidase I (SGAPI) kindly provided by Dr. S. Blumberg as previously described in my laboratory (21, 48). The assay was performed with 0.4 mM substrate in 50 mM Tris · HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5 and 6.7 pg/ml SGAPI in a volume of 200 µl. Incubation was performed in flat-bottom 96-microtiter plates at 23°C for 30 min. NEP 24.11 activity was measured by following the increase in absorbency at 405 nm that is due to the release of p-nitroaniline from the substrate using an ELISA microplate reader. The amount of SGAPI used is optimal for the assay conditions, and its activity is not significantly augmented by the comparatively small amount of endogenous aminopeptidase I present in tissue extracts. Tissue NEP 24.11 activity was expressed as picomoles per minute per milligram protein.
Drugs and chemicals. DMEM was obtained from GIBCO. Human VIP was obtained from American Peptide (Sunnyvale, CA). VIP10-28 and VIP1-12 were obtained from Sigma Chemical (St. Louis, MO). STE and drugs were diluted in DMEM to the desired concentrations on the day of the experiment.
Data and statistical analyses. Data are expressed as means ± SE. Statistical analysis was performed by two-way ANOVA and the Newman-Keuls test. P < 0.05 was considered significant.
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RESULTS |
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Effects of STE and VIP on DNA Synthesis in HCPC-1 Cells
Incubation of HCPC-1 cells with increasing concentrations of STE alone elicited a small but significant increase in DNA synthesis in HCPC-1 cells relative to media at the highest concentration used (1:100 aqueous dilution; Fig. 1A; each group, n = 4; P < 0.05). Likewise, incubation of HCPC-1 cells with VIP (10
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Effects of STE on NEP 24.11 Activity in HCPC-1 Cells
Incubation of HCPC-1 cells in media alone for 24-72 h was associated with a significant, time-dependent decrease in NEP 24.11 activity that was maximal after 48 h (Table 1; each group, n = 4; P < 0.05). Incubation of cells with STE (1:100 aqueous dilution) for 24-72 h was associated with a significant decrease in NEP 24.11 activity relative to media that was maximal after 48 h (Table 1; each group, n = 4; P < 0.05). Enzyme activity began to recover after 72-h incubation of HCPC-1 cells with STE (1:100 aqueous dilution; Table 1; each group, n = 4; P < 0.05 in comparison to 48 h).
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DISCUSSION |
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There are two new findings from this study. First, we found that exposure of HCPC-1 to aqueous STE, at noncytotoxic concentrations that have been previously shown to evoke inflammation in the in situ hamster cheek pouch (11, 12), together with VIP, a ubiquitous autocrine neuropeptide in the oral epithelium (17), is associated with significant, concentration-dependent, synergistic increase in DNA synthesis as assessed by BrdU incorporation. These effects were mediated by VIP receptors because VIP10-28, a VIP receptor antagonist in the in situ hamster cheek pouch (43), but not VIP1-12, an inactive peptide fragment (43), significantly attenuated VIP-induced increase in DNA synthesis in the absence or presence of STE. Second, STE inactivated NEP 24.11, an ectoenzyme widely distributed in oral epithelium that cleaves and inactivates VIP very effectively (12, 35, 43, 52), in HCPC-1 cells after 48-h incubation, a time when VIP-induced DNA synthesis was maximal. This process appears to be reversible after 72 h.
These data suggest that inactivation of NEP 24.11 amplifies VIP-induced DNA synthesis in HCPC-1 cells. This contention is supported, in part, by the studies of Suzuki et al. (43) and Gao et al. (11) who showed that selective inhibitors of NEP 24.11 amplify VIP-induced vasodilation and STE-induced increase in clearance of macromolecules from the in situ hamster cheek pouch, respectively. Moreover, Burns et al. (3) showed recently that pharmacologic inhibition of NEP 24.11 in cultured breast cancer cells amplifies bombesin-induced cell growth. Importantly, Ganju et al. (10) showed that cultured airway epithelial cells grow more rapidly when NEP 24.11 is inhibited. Conversely, exogenous NEP 24.11 inhibits growth of lung cancer and normal lung cell lines in vitro and in vivo in athymic nude mice (2, 4). On balance, these data suggest that NEP 24.11 modulates the mitogenic effects of smokeless tobacco and VIP in oral keratinocytes.
It is not feasible to expose HCPC-1 cells to raw smokeless tobacco because of cytotoxicity (18, 27-29, 37). Hence, cells are exposed to a diluted aqueous extract of raw smokeless tobacco. This approach has been previously used to determine the effects of smokeless tobacco on oral keratinocytes (27-29, 37). In addition, several toxic and carcinogenic constituents of aqueous STE used in this study are qualitatively similar to those of raw smokeless tobacco (18, 34). People place smokeless tobacco on the oral mucosa where it is continuously being mixed with and diluted in saliva, thereby producing, in essence, an aqueous extract in the vicinity of oral keratinocytes. Collectively, these data indicate that the use of an aqueous extract of raw smokeless tobacco in this study is appropriate.
The chemical composition of smokeless tobacco is complex (34). Consequently, it is difficult to identify constituents of STE that could stimulate DNA synthesis and inactivate NEP 24.11 in cultured HCPC-1 cells. Nonetheless, the role of nicotine, a major constituent of smokeless tobacco that has been shown to stimulate VIP release from nerves in the rat gastric fundus (5, 18, 25, 34), should be considered. Ringdahl et al. (33) showed that daily application of nicotine on the mandibular lip of rats for 6 wk is not associated with oral keratinocyte proliferation. Similar observations were reported by Theilig et al. (45) using cultured human skin keratinocytes. Whether nicotine inactivates NEP 24.11 has not been determined in these studies. Clearly, additional studies are warranted to characterize constituents of STE that amplify VIP-induced DNA synthesis and inactivate NEP 24.11 in oral keratinocytes.
The intracellular signal transduction mechanism(s) underlying STE potentiation of VIP-induced DNA synthesis and inactivation of NEP 24.11 in HCPC-1 cells was not elucidated in this study. Conceivably, membrane-bound NEP 24.11 could downregulate VIP-induced calcium flux and activation of protein kinase C, an important intracellular signal transduction pathway involved in cell growth (36, 42), and/or decrease apoptosis of oral keratinocytes (9, 42). For instance, Cohen et al. (4) showed that exogenous NEP 24.11 attenuates intracellular calcium flux elicited by gastrin-releasing peptide and bradykinin, two autocrine peptides, in H345 lung cancer cell line. In addition, pharmacologic inhibition of NEP 24.11 augmented gastrin-releasing peptide- and bradykinin-induced responses in these cells.
Certain constituents of STE may stimulate HCPC-1 cells to elaborate reactive oxygen species that inactivate NEP 24.11 (28, 37). This contention is supported, in part, by the study of Dusser et al. (7) who showed that cigarette smoke inactivates airway NEP 24.11 due, most likely, to the effect of reactive oxygen species. Exposure to STE may also increase the number and/or affinity of VIP receptors on oral keratinocytes thereby potentiating VIP-induced responses (39).
Irrespective of NEP 24.11 downregulation, the cell growth-promoting effects of VIP are tissue and species specific because VIP has been shown to inhibit human small-cell lung cancer and airway smooth muscle proliferation and to have no significant effects on human lung fibroblast proliferation (16, 23, 24). Additional studies using molecular, biochemical, and cell biology techniques are indicated to elucidate the mechanisms mediating the disparate effects of VIP on cell growth.
In summary, we found that STE potentiates VIP-induced DNA synthesis in cultured oral keratinocytes and that this response is temporally related to STE-induced inactivation of NEP 24.11 in these cells. We suggest that NEP 24.11 modulates the mitogenic effects of smokeless tobacco in the oral epithelium, in part, by inactivating VIP.
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ACKNOWLEDGEMENTS |
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I thank Sergei Pakhlevaniants for expert technical assistance and Drs. Wong and Blumberg for providing HCPC-1 cells and bacterial aminopeptidase I, respectively.
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FOOTNOTES |
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This study was supported, in part, by National Institutes of Health Grant DE-10347. Dr. Rubinstein is a recipient of a Research Career Development Award from the National Institutes of Health (DE-00386) and a University of Illinois Scholar Award.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Rubinstein, Dept. of Medicine (M/C 787), University of Illinois at Chicago, 840 South Wood St., Chicago, IL 60612-7323 (E-mail: IRubinst{at}uic.edu).
Received 23 December 1998; accepted in final form 7 September 1999.
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