Abstracts
1999 Digestive Disease Week

# 2203 NSAIDS ATTENUATE PROLIFERATION OF COLONIC CARCINOMA CELLS BY BLOCKING EPIDERMAL GROWTH FACTOR (EGF) INDUCED CA++ MOBILIZATION
E R Kokoska, G S Smith, T A Miller, Saint Louis Univ Health Sci Ctr, St. Louis, MO

We have previously reported that NSAIDs, in human colonic carcinoma cells (Caco-2), attenuate EGF induced cellular proliferation through a process independent of prostaglandin synthesis inhibition. Furthermore, separate studies have also suggested that NSAIDs inhibit EGF induced store operated Ca++ influx. Thus, we developed the hypothesis that NSAIDs may limit the activity of EGF by altering intracellular Ca++ ([Ca++]i) mobilization. [Ca++]i was measured with Fluo-3 (5 mM) and extracellular Ca++ influx was monitored by quenching Fluo-3 fluorescence with Mn++. Proliferation was quantitated (relative fluorescence) with two assays: CyQuant (nucleic acid; NA) and NanoOrange (protein; PROT). Cells exposed to EGF (10 ng/ml) for < 18 mins demonstrated no increased proliferation while longer incubations (20 min - 48 hrs) achieved increased but similar growth rates. Caco-2 cells exposed to EGF demonstrated an initial increase in [Ca++]i (0.5 - 3 min) which was blocked by neomycin (Neo; 100 mM), an inhibitor of IP3 generation, and the phospholipase C (PLC) inhibitor U73122 (U731; 1 mM) but not U73343 (U733, inactive control; 1 mM). This was followed by sustained extracellular Ca++ influx (3 - 18 min) which was attenuated with calcium free buffer (-Ca++), the store operated Ca++ channel (SOCC) blocker lanthanum (La+++; 25 mM), indomethacin (Indo; 5 mM), ibuprofen (Ibu; 10 mM), and aspirin (ASA; 20 mM),. In subsequent studies, cells were serum starved (48 hrs), treated (30 min) with either serum-free media (MEM) or EGF ± the aforementioned inhibitors, and again serum-starved (48 hrs). Data are below (mean ± SEM; n = 8-12/group). Cells exposed to EGF ± the inactive PLC inhibitor U733 demonstrated a significant increase in nucleic acid (*p<0.01) and protein (*p<0.01). However, proliferation induced by EGF was not observed when [Ca++]i elevation was prevented by blocking either internal Ca++ store release via PLC/IP3 (†p<0.01) or sustained Ca++ influx through SOCCs (p<0.01). Thus, sustained [Ca++]i elevation, as induced by EGF, appears to be required for mitogenesis. These data support our premise that one mechanism whereby NSAIDs may attenuate colonic neoplasia is by blocking EGF induced Ca++ mobilization.

	NA	PROT		NA	PROT
MEM	1387±52	3487±153	-Ca+++EGF	1621±132	3721±95
EGF	2501±66*	4621±167*	La++++EGF	1481±128	3642±113
Neo+EGF	1561±83†	3680±112†	Indo+EGF	1532±102	3498±167
U731+EGF	1485±107†	3549±104†	Ibu+EGF	1589±162	3641±139
U733+EGF	2470±117*	4533±149*	ASA+EGF	1504±99	3683±143

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