Aberrant signal transduction pathways involved in the development of metastatic disease are poorly defined in both small cell lung carcinoma(SCLC) and non-small cell lung carcinoma (NSCLC). Neuropeptide-driven positive feedback loops stimulating cell proliferation are characteristic of SCLC. The activation of phospholipase C (PLC)-β is an early and common response to stimulation of G protein-coupled receptors by these neuroendocrine growth factors. The importance of PLC-β in neuropeptide signaling prompted us to compare PLC-βisoform expression and activity in four independent SCLC cell lines and four independent NSCLC cell lines. We found that PLC-β1 is more highly expressed in SCLC than in NSCLC, as indicated by Western blotting of cell lysates. All SCLC lines studied express PLC-β1; only one of the NSCLC lines investigated showed detectable levels of the enzyme. NSCLC lines are significantly more sensitive to the antiproliferative effects of ET-18-OCH3 (edelfosine)compared with the SCLC lines, as indicated by[3H]thymidine uptake. The only SCLC cell line (NCI-H345)that is as sensitive as the NSCLC cell lines to ET-18-OCH3also expresses uniquely low levels of PLC-β1. The participation of PLC-β1 in signaling by SCLC growth factor receptors is indicated by our finding that PLC-β1 (but not PLC-β3) coimmunoprecipitates with Gαq/11 upon activation of neurotensin receptors; this association is inhibited by ET-18-OCH3. Ca2+mobilization mediated by neurotensin receptors is also inhibited by ET-18-OCH3. The binding of GTPγS to Gαq/11upon treatment of SCLC cells with neurotensin is not inhibited by ET-18-OCH3. These findings indicate that ET-18-OCH3 does not interfere with Gαq/11activation but rather inhibits the association of Gαq/11with PLC-β1. Our data suggest that PLC-β is an important mediator of both SCLC and NSCLC proliferation. Differences in PLC-β1 expression may be exploitable in the development of effective diagnostic and therapeutic tools.

Lung cancer is currently the leading cause of cancer mortality in the United States, resulting in >150,000 deaths/year (1). Malignant lung tumors are generally divided into two major categories,SCLC4and NSCLC (reviewed in Ref. 2). SCLC accounts for 20–25%of all bronchogenic malignancies and follows the most aggressive clinical course. Less than 5% of patients with SCLC currently survive 5 years past the initial diagnosis (3). The prognosis for patients diagnosed with NSCLC is almost as poor; ∼15% of these patients survive 5 years after diagnosis (4). SCLC is initially highly responsive to radiation and chemotherapeutic regimes;in contrast, NSCLC is unresponsive to such therapies (reviewed in Ref.5). The inadequacy of current treatment protocols reinforces the need for a greater knowledge of the basic biology of the several forms of this disease and a greater understanding of the differences between SCLC and NSCLC. Novel therapeutic strategies could thus be designed based on this information.

SCLC is distinguished by its neuroendocrine phenotype. SCLC cells can secrete >30 regulatory peptides and hormones and may express receptors for almost half of these factors (reviewed in Ref. 6). Several of these peptides act as autocrine growth factors that activate GPCRs, establishing positive feedback loops that stimulate cell proliferation (reviewed in Refs. 5, 6, 7, 8) and growth of SCLC xenografts in rodents (9, 10). Potent inhibitors, which block the interaction of these neuropeptides with their receptors, have been demonstrated to affect cell proliferation (10, 11, 12, 13). However, the expression of multiple types of neuropeptide GPCRs limits the successful therapeutic application of specific neuropeptide antagonists for the treatment of SCLC (reviewed in Ref.14). Efforts have been directed toward the development of broad spectrum antagonists, such as substance P analogues, which block a number of receptors (15). Investigations have also focused on increasing the activity of endopeptidases to remove neuropeptide activity (16, 17). Other means of inhibiting neuroendocrine receptor function may present a more efficient treatment modality.

A number of SCLC neuroendocrine growth factors, such as gastrin-releasing peptide, neurotensin, vasopressin, and cholecystokinin, act by stimulating GPCRs, initiating multiple signaling cascades and activating different phospholipid enzymes. The activation of PLC-β appears to be one of the earliest and most common responses after binding of these agonists to their GPCR(reviewed in Refs. 7 and 8). Agonist binding to these receptors causes α subunits of the Gqfamily of G proteins (Gq/11/14/16) to associate with PLC-β, resulting in activation of the enzyme. Many subsequent events are dependent on PLC-β activation, including the generation of inositol 1,4,5-triphosphate and 1,2-sn-diacylglycerol second messengers (reviewed in Refs. 7 and 8). Because PLC-β activation may be a crucial event in autocrine growth factor stimulation of SCLC proliferation, blocking PLC-β activity could prove to be a profitable therapeutic objective.

The PLC-β class of phospholipases is composed of four isozymes(β1–β4) encoded by different genes (18). PLC-β3 is expressed in diverse tissue types (reviewed in Refs. 19and 20); the other PLC-β isozymes appear to have a more restricted tissue distribution (20). PLC-β1 is highly expressed in neuronal tissue (21), PLC-β2 is expressed in hematopoetic cells (22), and PLC-β4 occurs in certain brain regions, notably those involved in visual perception(23). The levels of PLC-β isozymes expressed by different types of lung cancer have not been reported previously.

The roles of PLC-β in cell viability and proliferation have been investigated using the ether-linked phospholipid analogue ET-18-OCH3(24, 25, 26). ET-18-OCH3 selectively inhibits the in vitro activity of phosphatidylinositol-specific PLCs(27), which include members of the PLC-β class(28). Previous reports indicate that ET-18-OCH3 inhibits the proliferation of a variety of neoplastically transformed cells, including some human lung cancer cell lines (29, 30). However, the molecular targets of ET-18-OCH3 in these cells, and the differential effects of ET-18-OCH3 on SCLC versus NSCLC, have not been characterized completely.

The potential involvement of PLC-β in the autocrine-stimulated proliferation of SCLC prompted us to compare the expression and activity of PLC-β in SCLC and NSCLC cell lines. We report that significantly greater levels of PLC-β1 are expressed by SCLC compared with NSCLC. In contrast, SCLC and NSCLC express similar levels of PLC-β3. ET-18-OCH3 inhibits the proliferation of all lung cancer cell lines tested. However, greater concentrations of ET-18-OCH3 are required to inhibit the proliferation of most SCLC cell lines compared with the NSCLC lines. This result may be related to the greater reserves of PLC-β1 expressed by SCLC compared with NSCLC. We found that neurotensin induces the association of Gαq/11 with PLC-β1, but not with PLC-β3, in serum-starved SCLC cells. In contrast, Gαq/11 associates with both PLC-β1 and PLC-β3 when these cells are exposed to serum. These findings indicate that signaling by neurotensin receptors relies more upon PLC-β1 than PLC-β3 in these SCLC cells. The neurotensin-mediated association of Gαq/11 with PLC-β1 is inhibited by ET-18-OCH3, consistent with ET-18-OCH3 blocking PLC-β function and inhibiting SCLC proliferation.

This is the first demonstration of a difference in PLC-β levels between SCLC and NSCLC cells. Our data indicate that PLC-β is a critical mediator of both SCLC and NSCLC proliferation. The increased expression of PLC-β1 by SCLC cells and the specific activation of PLC-β1 by neuropeptide agonists in SCLC cells indicate that PLC-β1 may play an important role in the neuroendocrine growth factor stimulation of SCLC proliferation. Blocking signaling by PLC-β may,therefore, prove to be a valuable approach to inhibit tumor growth and metastasis.

Reagents.

Rabbit polyclonal antibodies to Gαq/11,PLC-β1, or PLC-β3 were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). Horseradish peroxidase-labeled antirabbit antibodies,ECL reagents, [3H]thymidine (specific activity,74 Ci/mmol), and 35S-labeled GTPγS (specific activity, 1030 Ci/mmol) were purchased from Amersham (Arlington Heights, IL). ET-18-OCH3 (edelfosine), D609, and thapsigargin were obtained from Calbiochem (La Jolla, CA). The fluorescent dye Fura-2 AM and pluronic were purchased from Molecular Probes (Eugene, OR). DSP was obtained from Pierce Chemical Co.(Rockford, IL). All other reagents were purchased from the Sigma Chemical Co. (St. Louis, MO) unless otherwise noted in the text.

Cell Culture.

The SCLC cell line SCC-9, established from a biopsy specimen of an SCLC skin metastasis, has been characterized extensively(31, 32, 33). The SCLC cell lines NCI-H345, NCI-H69, and NCI-H146 were obtained from the American Type Culture Collection(Rockville, MD). The SCLC cells were cultured in complete SCLC medium consisting of RPMI 1640 (Cellgro Mediatech, Herndon, VA) containing 10% calf bovine serum (Hyclone Laboratories, Logan, UT), 0.3 mg/ml glutamine, 20 units/ml penicillin, and 20 μg/ml streptomycin sulfate. The NSCLC lines NCI-H520, NCI-H522, NCI-H23, and NCI-H157 were obtained from the American Type Culture Collection and cultured in complete NSCLC medium consisting of RPMI 1640 containing 10% FCS (Hyclone Laboratories), 0.3 mg/ml glutamine, 20 units/ml penicillin, and 20μg/ml streptomycin sulfate. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air at densities that promoted exponential proliferation.

ECL-Western Blotting.

Cells were lysed by periodic agitation for 15 min in ice-cold lysis buffer (50 mm Tris-HCl, 120 mm NaCl, 2.5 mm EDTA, 1 mm DTT, and 0.5% NP40, pH 7.4)containing protease inhibitors (400 μmphenylmethylsulfonyl fluoride and 20 μg/ml leupeptin) and phosphatase inhibitors (10 mm sodium fluoride, 1 mm sodium orthovanadate, 0.2 mm sodium PPi, and 10 mm β-glycerophosphate). After centrifugation(16,000 × g for 10 min at 4°C), the supernatants were diluted with lysis buffer to equal protein concentrations and boiled for 5 min with sample buffer (75 mm Tris, 2% SDS, 10% glycerol, 5%β-mercaptoethanol, and 0.005% bromphenol blue, pH 6.8). Aliquots containing equal protein concentrations were subjected to SDS-PAGE using a 5% stacking gel and a 10% separating gel and electrophoretically transferred to PVDF membranes. The blocked PVDF membranes were incubated with antibodies to PLC-β1 or PLC-β3,followed by incubation with horseradish peroxidase-labeled antirabbit antibodies, as described previously (33). Bound antibody was visualized by ECL and quantified by densitometry.

[3H]Thymidine Uptake.

Uptake of [3H]thymidine by cells was used as an indicator of DNA synthesis, as described previously (31). SCLC cells were plated in 96-well microtiter plates at a density of 104 cells/well in complete SCLC medium. NSCLC cells were plated in 96-well microtiter plates at a density of 103 cells/well in complete NSCLC medium. The cells were incubated for 2 days at 37°C in a humidified atmosphere of 5% CO2/95% air before the addition of drugs. Cells were exposed to drugs for 3 days, and[3H]thymidine was added for the final 3 h of the incubation period. The SCLC cells were washed and lysed with distilled water and collected on filters using an automatic cell harvester (Skatron, Sterling, VA). The NSCLC cells were incubated (10 min at 37°C) with PBS containing 5 mm EDTA and 5 mm EGTA to induce detachment of the cells from the microtiter plates before collecting the cells as described above. The filters were placed in Ultima-Gold scintillation fluid (Packard Bioscience, Downers Grove, IL) and counted with an LS-6000 β-counter(Beckman Instruments, Fullerton CA).

Viability Studies.

The viability of the cells after incubation with or without ET-18-OCH3 was assayed by measuring the uptake of the vital dye neutral red (34, 35). NSCLC cells were plated in 24-well plates at a density of 104cells/ml in complete NSCLC medium. SCLC cells were plated in 24-well plates at a density of 105 cells/ml in complete SCLC medium. Cells were incubated for 2 days at 37°C in a humidified atmosphere of 5% CO2/95% air before the addition of ET-18-OCH3. After addition of the drug, cells were incubated for an additional 3 days, and neutral red vital dye was added for the final 2 h at a final concentration of 0.033%. The SCLC cells were collected and washed with PBS, and the cell pellet was solubilized in 1% acetic acid in 50% ethanol. NSCLC cells suspended in culture supernatants were pooled with NSCLC cells,which were detached from the plates by incubation with 5 mmEDTA and 5 mm EGTA in PBS. The pooled NSCLC cells were washed with PBS and solubilized in 1% acetic acid in 50% ethanol. The absorbance at 560 nm (A560) of each sample was determined spectrophotometrically using a Dynatech MR 5000 96-well plate reader (Dynex Technologies, Chantilly, VA).

Intracellular Ca2+ mobilization.

Cells attached to glass coverslips were incubated for 45 min at 37°C in DMEM containing 10 mm HEPES (pH 7.4), 0.28 mg/ml probenecid, 0.03% pluronic F127, and 2 μg/ml Fura-2 AM at 37°C in a humidified atmosphere of 5%CO2/95% air. The cells were washed three times in DMEM containing 10 mmHEPES (pH 7.4) and 0.28 mg/ml probenecid. The ratio of intracellular Fura-2 AM fluorescence at 340 and 380 nm was measured with a CMX Scanning Cation MicroIlluminator (Spex Industries, Edison,NJ). Ratios were converted to intracellular calcium concentrations as described previously (36).

Coprecipitation of PLC-β with Gαq/11.

Coimmunoprecipitation of PLC-β1 or PLC-β3 with Gαq/11 was performed using a modification of a method described by Luttrell et al.(37). NCI-H345 cells were incubated for 2 h in serum-free RPMI and exposed to 50 μmET-18-OCH3 or no drug for 15 min at 37°C. Serum(15%), neurotensin (1 μm), or no growth factors were added to the cultures for 1 min. The cells were then treated with DSP to induce protein cross-linking (37) and subsequently lysed in lysis buffer [1% Triton-X100, 0.5% NP40, 50 mm HEPES (pH 7.4), 0.2% BSA, 130 mm NaCl, 50 mm NaF, 1 mm MgCl2, 1 mm CaCl2, 15% glycerol, 40 mmKH2PO4, and 1 mm orthovanadate]. The lysate was centrifuged at 13,000 × g for 10 min, and the resulting supernatant was rotated (90 min at 4°C) with Gαq/ll antibody and protein A-agarose beads(Life Technologies, Inc., Grand Island, NY). After washing three times in lysis buffer, the immunoprecipitates were eluted with sample buffer(30 min at 40°C), subjected to SDS-PAGE, and electrophoretically transferred to PVDF membranes. The PVDF membranes were blocked and probed by ECL-Western blotting, as described above using antibodies to Gαq/ll, PLC-β1, or PLC-β3.

Binding of [35S]GTPγS to Gαq/ll.

Binding of [35S]GTPγS to Gαq/11 was determined by modification of a method described by Barr and Manning (38). NCI-H345 cells were washed in PBS, suspended in reaction buffer (50 mmHEPES, 100 mm NaCl, 6 mmMgCl2, 2 mm EDTA, 10 μmGDP, and 150 nm GTPγS, pH 7.4), and subjected to a−70°C freeze/thaw cycle to disrupt cell membranes. The freeze/thawed cells were incubated in the absence or presence of 50 μmET-18-OCH3 for 15 min (37°C), followed by incubation with 30 nm [35S]GTPγS in the absence or presence of neurotensin (200 nm) for 10 min (37°C). The samples were solubilized by rocking in lysis buffer(50 mm HEPES, 150 mm NaCl, 20 mmMgCl2, 100 μm GDP, 100μ m GTP, 0.5% NP40, 1 mm phenylmethylsulfonyl fluoride, and 200 μg/ml leupeptin, pH 7.4). The samples were centrifuged (13,000 × g for 10 min at 4°C), and the resulting supernatants were incubated (1.5 h at 4°C)with Gαq/ll antibody and protein A-agarose. The immunoprecipitates bound to protein A-agarose were washed two times in lysis buffer, resuspended in distilled water, and transferred to scintillation vials containing Ultima-Gold scintillation fluid (Packard Bioscience). The amounts of [35S]GTPγS bound to the immunoprecipitated Gαq/ll were determined by liquid scintillation counting using an LS-6000β-counter.

Higher PLC-β1 Levels Are Expressed by SCLC Compared with NSCLC.

The expression of PLC-β enzymes by different lung carcinoma lines was examined by Western blotting. Detectable expression of PLC-β1 occurs in 100% of SCLC lines tested but occurs in only 25% of NSCLC lines tested (Fig. 1). Levels of PLC-β1 expression are generally higher in SCLC cells compared with NSCLC cells. However, PLC-β1 expression is significantly reduced in the NCI-H345 SCLC cell line, similar to the level of PLC-β1 expressed by the NCI-H23 NSCLC cell line. In contrast to the cell type-specific expression of PLC-β1, other forms of PLC-β are expressed similarly by the SCLC and NSCLC lines. Expression of PLC-β3 occurs in all SCLC and NSCLC lines tested (Fig. 1), whereas PLC-β2 and PLC-β4 expression is undetectable in all of the cell lines (data not shown).

Greater Concentrations of ET-18-OCH3 Are Required to Inhibit the Proliferation of SCLC Compared with NSCLC.

The phosphatidylinositol-specific PLC inhibitor ET-18-OCH3 was used to investigate the role of PLC-β in SCLC and NSCLC cell proliferation. Although ET-18-OCH3 inhibits DNA synthesis in all SCLC and NSCLC lines tested, most of the SCLC cell lines exhibit increased resistance to this drug (Fig. 2,A). Greater concentrations of ET-18-OCH3 are generally needed to inhibit DNA synthesis in the SCLC lines compared with the NSCLC lines (Fig. 2,A and Table 1). However, the NCI-H345 cell line is uniquely sensitive to ET-18-OCH3, exhibiting ET-18-OCH3 ICmax and IC50 values comparable with those exhibited by the NSCLC cell lines (Table 1). The sensitivities of the cell lines to ET-18-OCH3 are specific for this class of inhibitor, because none of the cell lines respond to the phosphatidylcholine-specific PLC inhibitor D609 (Fig. 2 B).

It is possible that ET-18-OCH3 inhibits S-phase progression, thereby reducing the uptake of[3H]thymidine during the 3-h pulse-labeling,without significantly affecting cell number. To determine whether ET-18-OCH3 decreases the number of viable cells,we measured the cellular uptake of the vital dye neutral red. Neutral red passes through the intact plasma membrane and becomes concentrated in the lysosomes of viable cells (34). We found that ET-18-OCH3 diminishes neutral red uptake by all cell lines tested (Fig. 3). The ability of ET-18-OCH3 to inhibit neutral red uptake positively correlates with its ability to inhibit thymidine uptake. Concentrations of ET-18-OCH3 that half-maximally inhibit thymidine uptake cause a small but significant reduction in the number of viable cells. A greater reduction in the number of viable cells occurs when cells are treated with concentrations of ET-18-OCH3 that maximally inhibit thymidine uptake (Fig. 3). These findings indicate that treatment of both SCLC and NSCLC cells with ET-18-OCH3 reduces the number of viable cells in culture.

ET-18-OCH3 Diminishes Agonist-induced Ca2+Mobilization in SCLC Cells.

ET-18-OCH3 is a known inhibitor of PLC activity;however, the molecular mechanism by which it acts remains uncertain(reviewed in Ref. 26). To investigate the effects of ET-18-OCH3 on PLC-β activity, we measured the ability of the drug to alter intracellular Ca2+mobilization induced by PLC-β activation. Mobilization of intracellular Ca2+ is induced by activating M3 mAChRs on SCC-9 cells (Fig. 4,A) and by activating neurotensin receptors on NCI-H345 cells(Fig. 4,D). Treatment with ET-18-OCH3diminishes Ca2+ mobilization induced by activating both types of receptors in these cells (Fig. 4). However,ET-18-OCH3 more effectively inhibits Ca2+ mobilization induced by neurotensin receptors in NCI-H345 cells, compared with mAChR in SCC-9 cells. Neurotensin-mediated Ca2+ mobilization is significantly inhibited by 5 μmET-18-OCH3 and completely inhibited by 50μ m ET-18-OCH3. In contrast, mAChR-mediated Ca2+ mobilization is only partially inhibited by 50 μmET-18-OCH3 and still occurs in the presence of 200 μm ET-18-OCH3 (Fig. 4).

In addition to altering agonist-stimulated Ca2+mobilization, ET-18-OCH3 also increases basal intracellular Ca2+ concentrations in SCC-9 and NCI-H345 cells (Fig. 4). This increase in intracellular Ca2+ may occur because ET-18-OCH3 alters Ca2+mobilization from intracellular stores. However, we found that thapsigargin elicits similar increases in intracellular Ca2+ in ET-18-OCH3-treated and untreated cells(Fig. 5). This finding indicates that intracellular Ca2+stores are not grossly altered by ET-18-OCH3treatment.

ET-18-OCH3 Blocks the Association of PLC-β with Gαq/11.

To confirm that ET-18-OCH3 interferes with PLC-β activation, we investigated the association of PLC-β1 and PLC-β3 with the G proteins Gαq/11. The ability of Gαq/11 to bind to PLC-β1 and PLC-β3 was assessed by immunoprecipitating Gαq/11 from cells activated with the appropriate ligands and assaying the immunoprecipitates for the presence of PLC-β1 and PLC-β3 by Western blotting (Fig. 6). Both PLC-β1 and PLC-β3 coprecipitate with Gαq/11 when NCI-H345 cells are exposed to serum(Fig. 6, Lanes 3). In contrast, only PLC-β1 coprecipitates with Gαq/11 when these cells are exposed to neurotensin (Fig. 6,A, Lane 2). Treatment with 50μ m ET-18-OCH3 prior to the addition of serum or neurotensin abolishes the interaction of Gαq/11 with both PLC-β1 and PLC-β3 (Fig. 6, Lanes 4 and 5). These results indicate that ET-18-OCH3 interferes with the agonist-induced activation of PLC-β1 or PLC-β3 by affecting the association of the enzyme with its appropriate G protein.

ET-18-OCH3 Does Not Inhibit the Agonist-induced Binding of GTPγS to Gαq/11.

To further determine the site of ET-18-OCH3interference with PLC signaling by GPCR, we investigated the effect of the drug on the interaction of Gαq/11 with GPCR. The interaction of GPCR with Gαq/11 was determined by quantitatively measuring the ability of neurotensin to activate Gαq/11. Treatment of NCI-H345 cells with neurotensin results in a 2-fold increase in the binding of GTPγS to Gαq/11 (Fig. 7). This response is not inhibited by incubation with 5 μmor 50 μm ET-18-OCH3 (Fig. 7), which are concentrations of ET-18-OCH3 that significantly inhibit neurotensin-induced Ca2+mobilization (Fig. 4). These results indicate that ET-18-OCH3 does not interfere with Gαq/11 activation induced by neurotensin receptor stimulation.

This study demonstrates that PLC-β1 is more highly expressed in SCLC than in NSCLC. Increased PLC-β1 expression may arise as part of the neuroendocrine phenotype exhibited by SCLC cells, because PLC-β1 is most commonly expressed by neuronal tissues (21). Genetic alterations in the PLC-β1 locus, which maps to human chromosome 20q12–13 (18, 39, 40), may also contribute to greater expression of PLC-β1 by SCLC. However, we are not aware of any genetic abnormalities in this region that could readily explain increased PLC-β1 expression by SCLC. Although gains in 20q genetic material occur in SCLC (41), 20q gains also occur in NSCLC(41, 42) and several other types of cancer(43) with equal or greater frequency. We are also not aware of 20q abnormalities in NCI-H345 cells that might explain why these SCLC cells express less PLC-β1 than the other SCLC cell lines. Additional testing will be required to determine whether there are other SCLC cell lines with PLC-β1 levels as low as those expressed by NCI-H345 cells. We are currently investigating whether the generally higher PLC-β1 levels in SCLC cells compared with NSCLC cells are attributable to increased mRNA transcription or stability or attributable to increased PLC-β1 protein stability.

Our findings also demonstrate that PLC-β1 participates in signaling by SCLC growth factor receptors. Although both PLC-β1 and PLC-β3 are expressed by SCLC, only PLC-β1 associates with Gαq/11 upon activation of neurotensin receptors. Inhibiting PLC activity with ET-18-OCH3 inhibits the association of Gαq/11 with PLC-β1 and diminishes Ca2+ mobilization mediated by neurotensin receptors. These results are consistent with the participation of PLC-β1 in signaling by these receptors.

The importance of PLC-β in SCLC and NSCLC cell proliferation is indicated by the ability of ET-18-OCH3 to inhibit the proliferation of these cells. Although previous studies found that ET-18-OCH3 inactivates phosphatidylinositol-specific PLCs in vitro(27), the effects of ET-18-OCH3 on different PLC-β isoforms in vivo are not completely characterized. Our results show that ET-18-OCH3inactivates both PLC-β1 and PLC-β3 in vivo, as indicated by the inability of Gαq/11 to associate with these phospholipases in ET-18-OCH3-treated SCLC cells. The inactivation of PLC-β1 and PLC-β3 by ET-18-OCH3 probably contributes to the antiproliferative effects of the drug, because altering PLC-βactivity by other means significantly affects the proliferation and neoplastic transformation of various cell types (reviewed in Ref.24). The importance of PLC-β1 inactivation in the antiproliferative effects of ET-18-OCH3 is further supported by a previous demonstration that expressing catalytically inactive PLC-β1 inhibits the clonogenic growth of SCLC cells (44).

We found that greater concentrations of ET-18-OCH3 are generally required to inhibit the proliferation of SCLC cells, compared with NSCLC cells. It is reasonable to assume that increased ET-18-OCH3concentrations are needed to abolish PLC-β1 activity in cells expressing higher levels of PLC-β1. Thus, increased PLC-β1 expression may contribute to the resistance of the SCLC cell lines to ET-18-OCH3. This possibility is supported by our finding that the three SCLC cell lines (SCC-9, NCI-H69, and NCI-H146)that express significantly greater PLC-β1 levels also exhibit significantly higher ET-18-OCH3IC50s. The NCI-H345 SCLC cell line uniquely exhibits reduced PLC-β1 expression and a low ET-18-OCH3 IC50; these characteristics also indicate a positive correlation between expression of PLC-β1 and resistance to ET-18-OCH3. Previous studies similarly found that cells with increased phosphatidylinositol-specific PLC activity exhibit increased resistance to the antiproliferative effects of ET-18-OCH3(27).

The molecular targets of ET-18-OCH3 have important bearing on our study. ET-18-OCH3 is unquestionably an inhibitor of PLC, but the bases of its antineoplastic effects are uncertain (reviewed in Refs. 24, 25, 26). The drug may inhibit many other membrane-delimited signaling events, and some of these may be very important in determining the antiproliferative effects of the drug. ET-18-OCH3 has been reported to inhibit a number of transmembrane signaling processes, including the receptor-mediated activation of pathways dependent upon protein kinase C or mitogen-activated protein kinases (reviewed in Ref.26). Many of these effects of ET-18-OCH3 may result from PLC inactivation,because PLC activation is a proximal receptor-mediated event that initiates these pathways. Thus, some of the confusion over the points at which ET-18-OCH3 acts may be attributable to the secondary wide-ranging effects resulting from the ability of the drug to inhibit PLC.

Treatment with ET-18-OCH3 has a cytotoxic effect on several cell types, in addition to its cytostatic activity(45, 46, 47, 48, 49, 50). The cytotoxicity of ET-18-OCH3 is believed to occur independently and by a different mechanism from the cytostatic effects of the drug(45, 46). Some cytotoxicity may be attributable to the reported increased permeability of ET-18-OCH3-treated cells to Ca2+(46, 51). The increased basal intracellular Ca2+ concentrations in SCC-9 and NCI-H345 cells treated with ET-18-OCH3 that we observed may reflect the ability of the drug to alter membrane Ca2+ permeability. Several reports indicate that ET-18-OCH3 also induces apoptosis in different cell types (45, 48, 49, 50). We are currently investigating whether apoptosis contributes to the observed reduction in viable SCLC and NSCLC cells cultured for prolonged periods with ET-18-OCH3.

The expression of PLC-β1 by SCLC may have potential clinical relevance in addition to its role as a regulator of growth factor-mediated signaling. Many features of the SCLC neuroendocrine phenotype are used as diagnostic and prognostic aids, in addition to being targets for therapeutic intervention (reviewed in Ref.6). PLC-β1 expression may be another unique feature of SCLC cells, which can be similarly exploited. For example, PLC-β1 expression might be useful for differentiating SCLC from NSCLC since significantly higher levels of PLC-β1 are expressed by SCLC compared with NSCLC. Further studies are needed to clarify the role of PLC-β1 in lung cancer biology and to assess the potential uses of PLC-β1 in treating this disease.

We are grateful to Rebecca Porter for excellent technical assistance.

Fig. 1.

Expression of PLC-β1 and PLC-β3 by SCLC and NSCLC cell lines. A, the indicated SCLC and NSCLC cell lines were probed by ECL-Western blotting using antibodies to PLC-β1(Lanes 1–3) or PLC-β3 (Lanes 4 and 5). The concentration of antibodies used were 200 ng/ml(Lane 1), 100 ng/ml (Lane 2), 50 ng/ml(Lane 3), 2 μg/ml (Lane 4), or 1μg/ml (Lane 5). Results shown are representative of three independent experiments. B, densitometry of the ECL-Western blots was performed to determine the levels of PLC-β1 and PLC-β3 identified in the indicated SCLC and NSCLC cell lines. Results shown are the means of three independent experiments; bars, ±1 SE.

Fig. 1.

Expression of PLC-β1 and PLC-β3 by SCLC and NSCLC cell lines. A, the indicated SCLC and NSCLC cell lines were probed by ECL-Western blotting using antibodies to PLC-β1(Lanes 1–3) or PLC-β3 (Lanes 4 and 5). The concentration of antibodies used were 200 ng/ml(Lane 1), 100 ng/ml (Lane 2), 50 ng/ml(Lane 3), 2 μg/ml (Lane 4), or 1μg/ml (Lane 5). Results shown are representative of three independent experiments. B, densitometry of the ECL-Western blots was performed to determine the levels of PLC-β1 and PLC-β3 identified in the indicated SCLC and NSCLC cell lines. Results shown are the means of three independent experiments; bars, ±1 SE.

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Fig. 2.

Inhibition of [3H]thymidine incorporation induced by ET-18-OCH3 and D609. [3H]Thymidine uptake by SCLC and NSCLC cells was measured for the final 3 h of a 3-day exposure to ET-18-OCH3 (A) or D609(B). Cells incubated in the absence of drugs served as controls. Results are the means of nine determinations from at least three independent experiments; bars, SE.

Fig. 2.

Inhibition of [3H]thymidine incorporation induced by ET-18-OCH3 and D609. [3H]Thymidine uptake by SCLC and NSCLC cells was measured for the final 3 h of a 3-day exposure to ET-18-OCH3 (A) or D609(B). Cells incubated in the absence of drugs served as controls. Results are the means of nine determinations from at least three independent experiments; bars, SE.

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Fig. 3.

Reduction in cell viability induced by ET-18-OCH3. Neutral red uptake by SCLC and NSCLC cells was measured after a 3-day incubation of cells with or without ET-18-OCH3. The concentrations of ET-18-OCH3that were used corresponded to the ET-18-OCH3IC50 and ICmax values for each cell line, as indicated in Table 1. Cells incubated in the absence of ET-18-OCH3 served as controls. Results are the means of 15 determinations from three independent experiments; bars,±1 SE.

Fig. 3.

Reduction in cell viability induced by ET-18-OCH3. Neutral red uptake by SCLC and NSCLC cells was measured after a 3-day incubation of cells with or without ET-18-OCH3. The concentrations of ET-18-OCH3that were used corresponded to the ET-18-OCH3IC50 and ICmax values for each cell line, as indicated in Table 1. Cells incubated in the absence of ET-18-OCH3 served as controls. Results are the means of 15 determinations from three independent experiments; bars,±1 SE.

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Fig. 4.

Inhibition of agonist-induced Ca2+mobilization by ET-18-OCH3. SCC9 cells loaded with Fura-2 AM were pretreated for 15 min with no drug (A), 50μ m ET-18-OCH3 (B), or 200μ m ET-18-OCH3 (C) prior to challenge with the mAChR agonist carbachol (10 μm). Fura-2 AM-loaded NCI-H345 cells were pretreated for 15 min with no drug(D), 5 μm ET-18-OCH3(E), or 50 μm ET-18-OCH3(F) prior to challenge with the agonist neurotensin (200 nm). Results shown are representative of three independent experiments.

Fig. 4.

Inhibition of agonist-induced Ca2+mobilization by ET-18-OCH3. SCC9 cells loaded with Fura-2 AM were pretreated for 15 min with no drug (A), 50μ m ET-18-OCH3 (B), or 200μ m ET-18-OCH3 (C) prior to challenge with the mAChR agonist carbachol (10 μm). Fura-2 AM-loaded NCI-H345 cells were pretreated for 15 min with no drug(D), 5 μm ET-18-OCH3(E), or 50 μm ET-18-OCH3(F) prior to challenge with the agonist neurotensin (200 nm). Results shown are representative of three independent experiments.

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Fig. 5.

Mobilization of Ca2+ by thapsigargin. NCI-H345 cells loaded with Fura-2 AM were pretreated for 15 min with no drug or 50 μm ET-18-OCH3 before exposure to thapsigargin (100 nm). Results shown are representative of three independent experiments.

Fig. 5.

Mobilization of Ca2+ by thapsigargin. NCI-H345 cells loaded with Fura-2 AM were pretreated for 15 min with no drug or 50 μm ET-18-OCH3 before exposure to thapsigargin (100 nm). Results shown are representative of three independent experiments.

Close modal
Fig. 6.

Association of Gαq/11 with PLC-β1, but not with PLC-β3, upon activation of neurotensin receptors. The agonist-induced association of Gαq/11 with PLC-β1(A) or PLC-β3 (B) was determined using NCI-H345 cells. The cells were incubated in the absence (Lanes 1–3) or presence (Lanes 4 and 5)of 50 μm ET-18-OCH3 for 15 min and then exposed to 1 μm neurotensin (Lanes 2 and 4) or 15% serum (Lanes 3 and 5) for 1 min. Gαq/11 was immunoprecipitated from the cells after protein cross-linking with DSP and subjected to ECL-Western blotting using antibodies to PLC-β1(A), PLC-β3 (B), or Gαq/11 (A and B). Similar results were obtained in two other independent experiments.

Fig. 6.

Association of Gαq/11 with PLC-β1, but not with PLC-β3, upon activation of neurotensin receptors. The agonist-induced association of Gαq/11 with PLC-β1(A) or PLC-β3 (B) was determined using NCI-H345 cells. The cells were incubated in the absence (Lanes 1–3) or presence (Lanes 4 and 5)of 50 μm ET-18-OCH3 for 15 min and then exposed to 1 μm neurotensin (Lanes 2 and 4) or 15% serum (Lanes 3 and 5) for 1 min. Gαq/11 was immunoprecipitated from the cells after protein cross-linking with DSP and subjected to ECL-Western blotting using antibodies to PLC-β1(A), PLC-β3 (B), or Gαq/11 (A and B). Similar results were obtained in two other independent experiments.

Close modal
Fig. 7.

Neurotensin-mediated activation of Gαq/11 in the presence of ET-18-OCH3. NCI-H345 cells were freeze/thawed and pretreated with ET-18-OCH3 (5 or 50μ m) or no drug for 15 min at 30°C. The preparations were immediately assayed for neurotensin-stimulated binding of[35S]GTPγS to Gαq/11. Binding of[35S]GTPγS was determined by liquid scintillation counting of the [35S]GTPγS radioactivity associated with immunoprecipitated Gαq/11. Cells incubated in the absence of neurotensin and ET-18-OCH3 served as controls. Results are the means of nine determinations from three independent experiments; bars, ±1 SE.

Fig. 7.

Neurotensin-mediated activation of Gαq/11 in the presence of ET-18-OCH3. NCI-H345 cells were freeze/thawed and pretreated with ET-18-OCH3 (5 or 50μ m) or no drug for 15 min at 30°C. The preparations were immediately assayed for neurotensin-stimulated binding of[35S]GTPγS to Gαq/11. Binding of[35S]GTPγS was determined by liquid scintillation counting of the [35S]GTPγS radioactivity associated with immunoprecipitated Gαq/11. Cells incubated in the absence of neurotensin and ET-18-OCH3 served as controls. Results are the means of nine determinations from three independent experiments; bars, ±1 SE.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This research was funded by the Arthur T. Cantwell Charitable Foundation.

4 The abbreviations used are: SCLC, small cell lung carcinoma; NSCLC, non-small cell lung carcinoma; PLC,phospholipase C; GPCR, G-protein coupled receptor;ET-18-OCH3,1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine,edelfosine; PVDF, polyvinylidene difluoride; mAChR, muscarinic acetylcholine receptor; DSP, dithiobis[succinimidyl propionate]; ECL,enhanced chemiluminescence.

Table 1

Inhibitory effect of ET-18-OCH3 on [3H]thymidine uptake by lung cancer cell lines

Cell lineIC50aICmaxa
NSCLC cell lines   
NCI-H23 4.1 ± 1.1 23.5 ± 5.8 
NCI-H157 5.1 ± 0.7 12.0 ± 1.4 
NCI-H520 1.2 ± 0.5 10.5 ± 0.5 
NCI-H522 4.8 ± 0.8 51.5 ± 6.6 
SCLC cell lines   
SCC9 44.8 ± 3.2 83.5 ± 4.6 
NCI-H69 66.5 ± 5.3 >100b 
NCI-H146 12.2 ± 3.7 85.3 ± 4.4 
NCI-H345 6.4 ± 0.9 37.7 ± 6.6 
Cell lineIC50aICmaxa
NSCLC cell lines   
NCI-H23 4.1 ± 1.1 23.5 ± 5.8 
NCI-H157 5.1 ± 0.7 12.0 ± 1.4 
NCI-H520 1.2 ± 0.5 10.5 ± 0.5 
NCI-H522 4.8 ± 0.8 51.5 ± 6.6 
SCLC cell lines   
SCC9 44.8 ± 3.2 83.5 ± 4.6 
NCI-H69 66.5 ± 5.3 >100b 
NCI-H146 12.2 ± 3.7 85.3 ± 4.4 
NCI-H345 6.4 ± 0.9 37.7 ± 6.6 
a

The IC50 and ICmax values represent the mean ± 1 SE of at least three independent experiments, which were conducted as described in the legend to Fig. 2.

b

The maximum concentration of drug tested was 100μ m.

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