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Experimental Therapeutics |
Lung Cancer Program and Department of Medicine, University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262 [A. F. S., B. H., D. C. C., L. E. H., P. A. B.], and Laboratory of Biochemical Pharmacology, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021 [T-C. C.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Understanding lung cancer biology opened the opportunity for new treatment strategies. Increased levels of eicosanoids have been reported in both SCLCs3 and NSCLCs, which indicates that they may play a role in tumorigenesis (4 , 5) . In SCLC cells, neuropeptide autocrine growth factors stimulate arachidonic acid release through activation of phospholipase A2 (5) . In NSCLC cells, especially those with ras mutations, there is constitutively high expression of the Mr 85,000 cytosolic phospholipase A2 and COX-2 (6) . The arachidonic acid metabolic pathway generates bioactive lipids that modulate physiological and pathological responses involved in tumor growth and promotion (5) . LOXs generate various hydroperoxides (such as HETES), which tend to promote invasion and metastases (5 , 7) . Several LOX inhibitors inhibit the growth of human lung cancer cell lines and inhibit lung tumor carcinogenesis (7 , 8) . COX-1 and COX-2 generate prostaglandins and thromboxanes. COX-1 is a constitutive enzyme present in most cells, whereas COX-2 is inducible and is often up-regulated in tumors (5 , 9 , 10) .
Classical NSAIDs (such as sulindac) inhibit both of the COX enzymes and were shown to inhibit tumor formation in animal models (11, 12, 13) . However, the inhibition of COX-1 has serious side effects such as peptic ulcer formation and renal dysfunction. Selective COX-2 inhibitors and exisulind were developed to avoid these toxicities (14, 15, 16) . Exisulind lacks the inhibitory effects on COX-1, COX-2, and LOX but induces apoptosis and inhibits tumor growth in studies involving rodent models of chemically induced mammary and colon carcinogenesis (14, 15, 16) . Exisulind was also reported to inhibit lung tumor carcinogenesis in mice (17) . A recent study reported that exisulind inhibits cGMP phosphodiesterases, which leads to apoptosis (18) .
The combination of COX and LOX inhibitors produced synergistic growth inhibition against Lewis lung carcinomas when used together and in combination with cisplatin (4) . In another study, both exisulind and sulindac sulfide produced synergistic cytotoxicity with doxorubicin and VP-16 against the lung cancer cell line A549, which overexpresses MRP. These synergistic cytotoxicities did not occur with cell lines overexpressing MDR-1p170(19) . On the basis of these results, we chose to evaluate the effects of a COX-1 and -2 inhibitor (sulindac sulfide), a non-COX inhibitor (exisulind), a LOX inhibitor (NDGA), 13-cis-retinoic acid, cisplatin and paclitaxel on the growth of human lung cancer cells (both SCLC and NSCLC) alone and in combination. Our goal was to determine whether combinations of these agents would produce synergistic activity which would open the potential for combination-chemoprevention and therapeutic strategies.
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MATERIALS AND METHODS |
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-estradiol, and sodium selenium) medium. All of the cell lines were grown in 5% CO2 at 37°C, 100% humidity.
Immunofluorescent Staining for MDR-1, MRP, and LRP.
For MDR-1 cell surface staining, 5 x 105 cells were stained with the murine monoclonal antibody 4E3 (Signet Laboratories Inc., Dedham, MA) or the isotype matched control UPC-10 (IgG2a, Sigma Immunochemicals, St. Louis, MO). The cells were counterstained with a goat antimouse IgG2a-FITC (Southern Biotechnology Associates, Inc., Birmingham, AL). All of the staining was done at 4°C for 45 min followed by washing three times with HEPES/balanced salt solution + 5% Newton calf serum. The samples were analyzed by flow cytometry for the percentage of positive cells (see Table 1
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LRP cytoplasmic staining was performed as above using the LRP murine monoclonal antibody (LRP-56, IgGb) which reacts with an internal epitope of the Mr 110,000 LRP, MDR-related antigen (kindly provided by Dr. R. J. Scheper, Free University Hospital, Amsterdam, the Netherlands). The isotype-matched control was MOPC-141 (IgG2b, Sigma). The counterstaining antibody was goat antimouse IgG2b-FITC (Southern Biotechnology).
Agents.
NDGA, 13-cis-retinoic acid and cisplatin were obtained from Sigma Chemical Co. (St. Louis, MO). Paclitaxel was kindly provided by Terry Dugan (Bristol Myers-Squibb Co., Princeton, NJ). Sulindac sulfide and exisulind were kindly provided by Cell Pathways Inc. (Horsham, PA).
MTT Growth Assay.
Cell viability was assayed in triplicate using a MTT assay (22)
. Briefly, 10,000 NSCLC or 20,000 SCLC cells were plated in each well of 96-well flat-bottomed microtiter plates (Corning Glass Works, Corning NY). Cell populations were harvested and plated when 75–80% confluent. Adherent cell lines were trypsinized. The cells were allowed to recover overnight. The following day, the various agents to be tested were added alone or in combination at various concentrations to each triplicate well. The drug solvent (DMSO and H2O) concentrations in all of the samples were 
1%. Appropriate solvent controls were run with the assays and produced no cytotoxic effects. After a 5 day incubation, 50 µl of a 2 mg/ml solution of the tetrazolium salt MTT (Sigma Chemical Company), dissolved in RPMI 1640, was added to each well. The microtiter plates were incubated for 4 h at 37°C. At the end of the 4-h incubation, the medium was aspirated off leaving the dark blue formazan product in the bottom of the wells. The reduced MTT product was solubilized by adding 100 µl of 0.2 N HCL in 75% isopropanol, 25% milliQ water to each well. Thorough mixing was done using a Titertek multichannel pipetter. The absorbency of each well was measured using an automated plate reader. The data were analyzed using a SlideWrite program to determine the IC50 of each drug alone. The CI-isobologram by Chou and Talalay (23)
was used to analyze the drug combination assays. Variable ratios of drug concentrations were used in the studies, and mutually exclusive equations were used to determine the CIs. Each CI was calculated from the mean affected fraction at each drug ratio concentration (triplicate). CI > 1, CI = 1, and CI < 1 indicate antagonism, additive effect, or synergy, respectively.
Cell Cycle Analysis.
SHP77 cells were added to each well of a 6-well plate (Corning Glass Works, Corning NY) at a concentration of 1 x 105 cells/well and incubated overnight. Drugs, at various concentrations, were added to each well the following day. Cells were harvested at several time points. EDTA at a concentration of 0.6 mM was used to separate the cells from the plate. Cells were spun and resuspended in Krishan’s reagent (24)
. After an overnight incubation, the cells were analyzed by flow cytometry at a wavelength of 488 nm on an EPICS XL-MCL (Coulter Electronics, Hialeah, FL) for cell cycle distribution. Data were analyzed by Modfit LT Software (Verity House software, Topsham, Maine).
Apoptosis Assay.
A549 and SHP77 cell lines at a concentration of 1 x 105 cells were added to each well of 6-well plates and incubated overnight. Drugs at various concentrations that were found to inhibit growth in an MTT assay were added the following day. The plates were then incubated for various times–24, 48,72, 96 and 120 h. On the scheduled day, 15 µl of a dye made of propridium iodine (1 mg/ml) and Hoechst (1 mg/ml) at a ratio of 6:1 were added to each well and incubated for 1 h. Cells were then removed from the plates using EDTA (0.6 mM) and spun at 1000 RPM. The supernatant was removed, and the precipitate containing the cells was placed on a slide. Cells were viewed on a fluorescent microscope (Zeiss Axioskop) under x40 with oil immersion, and 200 cells were scored for the percent of apoptotic cells.
Data Analysis.
Statistical analysis was done using the SAS PROC general linear models procedure (SAS PKG 6.14 manual; SAS Institute, Cary, NC). The significant results in Table 3
indicate contrasts comparing each drug regimen with the control. The overall ANOVA was significant at the 0.05 level for each column, and the individual contrasts indicated in bold are significant at the 0.05 level.
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RESULTS |
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and Fig. 1
). Of the two chemotherapeutic agents tested, paclitaxel was the most potent with an IC50 ranging from 3 to 50 nM in all of the cell lines with the exception of the MDR-1-positive SHP77 cell line, which, as expected, required a higher concentration (0.15 µM) to inhibit 50% of control cell growth. NDGA was the next most potent drug with an average IC50 of 28 µM in the SCLC cell lines and 52 µM in the NSCLC cell lines. Sulindac sulfide had cytotoxicities similar to those of NDGA in five of the cell lines tested. The SCLC cell line H69 was more sensitive to NDGA. The SCLC cell line H345 was the cell line most sensitive to four of the drugs (excepting exisulind and cisplatin) and was particularly sensitive to NDGA (IC50 = 10 µM), sulindac sulfide (IC50 = 8 µM), and 13-cis-retinoic acid (IC50 = 4 µM). Like sulindac sulfide and NDGA, 13-cis-retinoic acid had a similar effect on all of the cell lines with the exception of H345, which was the most sensitive (IC50 = 4 µM). Exisulind, was the least potent of the NSAID-related compounds (IC50 = 175–225 µM). The inhibitory effects of exisulind were similar in both NSCLC and SCLC cell lines. Cisplatin was found to be the least potent drug tested (IC50 = 200–500 µM). Like exisulind, cisplatin produced similar effects on NSCLC and SCLC cell lines. Overall, no relationship between p53 status, the presence of MDR-1, MRP, or LRP, and drug cytotoxicity were observed with the exception of paclitaxel resistance in the MDR-1-positive SHP77 cell line.
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and Table 3
, each of the six drugs produced strikingly different effects in the SCLC line SHP77 on cell cycle distribution and apoptosis. These differences are consistent with different mechanisms of action. As previously reported, paclitaxel resulted in a block in G2-M with a shift in cell population from G0-G1 to G2-M (see Table 3
). The peak effect was evident after 72 h of drug exposure, but increases in G2-M were seen as early as after 24 h, the first time point evaluated. Paclitaxel’s cell cycle effects lasted through 120 h, the last time point evaluated. The only other drug that produced an increase in the G2-M fraction was cisplatin which led to a maximum of 52% of cells in G2-M at 96 h (in contrast to 23% of control cells). A striking finding was the increase in G1 and the decrease in the S phase fraction produced by exisulind. This effect was seen as early as 24 h, peaked at 96 h, and was still present at 120 h. The reasons for this previously unreported accumulation in G1 and decrease in S phase are not completely clear. Piazza et al. and others have shown that the drug causes G1 arrest in colon cancer cells (14)
. None of the other drugs led to either an increase in G1 or a decrease in the S phase fraction. In fact, NDGA and 13-cis-retinoic acid produced an S-phase block with a higher percentage of cells in the S phase. Sulindac sulfide produced little change in the cell cycle distribution. These results imply that sulindac sulfide, exisulind, and NDGA work by completely different mechanisms of action. Similar results were seen in the other five cell lines (data not shown).
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. A greater than 10-fold increase in apoptotic cells was seen at the highest paclitaxel concentration. This effect was evident after 48 h of drug exposure with early evidence of apoptosis seen on fluorescent microscopy. Although cisplatin was the least potent drug for growth inhibition, it produced the second highest precentage of apoptotic cells (44% in SHP77 cells), a 9-fold increase compared with controls. This effect was observed as early as 72 h, peaked at 96 h and persisted through 120 h. Exisulind, sulindac sulfide, and NDGA produced similar degrees of apoptosis in the SHP77 line. A maximum of 25% of cells was apoptotic at 96 h with NDGA, 22% with sulindac sulfide, and 19% with exisulind. 13-cis-retinoic acid, produced the lowest maximal percentage (17%) of apoptotic cells.
Growth Inhibitory Effects of Drug Combinations.
Paclitaxel and cisplatin are frequently used in combination to treat both SCLC and NSCLC patients. Modest synergy (CI ± to 2+) was noted between these agents. Peak synergy (2+) with these two cytotoxics occurred in the two NSCLC cell lines at moderate concentrations. Greater synergy was achieved when several of the inhibitors of arachidonic acid metabolism were combined with the cytotoxic agents. For example, exisulind at the highest concentration produced synergy with paclitaxel and additive or synergistic effects with cisplatin in all three of the cell lines tested. (Fig. 3
and Table 4
). Exisulind combined with NDGA or 13-cis-retinoic acid also produced 2+ or greater synergy in each of the three cell lines tested. The combination of exisulind and sulindac sulfide produced synergy in the two NSCLC cell lines but antagonism in the SCLC cell line SHP77. The above synergies occurred most often at the highest concentration of exisulind tested. The synergy between these agents is consistent with the differing mechanism of actions of these agents.
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The synergy of the noncytotoxic agents suggests that combination-chemoprevention strategies are warranted, and the synergy with cytotoxic agents suggests that preclinical and clinical trials of these combinations in advanced stages are indicated. The SCLC cell line SHP77 has strong expression of MDR-1 and is more resistant to cytotoxic agents including paclitaxel compared with other lung cancer cell lines. In this cell line, these inhibitors of arachidonic acid metabolism produced greater synergy than the combination of cytotoxic agents.
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DISCUSSION |
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Abnormalities in arachidonic acid metabolism are present in both NSCLC and SCLC (4 , 5) . The generation of bioactive lipids, from this metabolic pathway modulates physiological and pathological responses involved in both tumor growth and promotion. LOX and COX inhibitors have been shown to inhibit the growth of human lung cancer cell lines in vitro and in animal models (7 , 8) . LOX inhibitors have been shown to prevent lung carcinogenesis in a xenograft model as well as in A/J mice given the tobacco-specific carcinogen 4-(methlynitrosamino)-1-(3-pyridyl)-1-butanone (NNK; Ref. (7) . Similarly, sulindac, a potent COX inhibitor, has also been shown to inhibit tumor formation in animal models (11, 12, 13) . The mechanism of growth inhibition of sulindac sulfide has been attributed to the inhibition of COX. One study noted the markedly up-regulated COX-2 expression in about one-third of atypical adenomatous hyperplasia and carcinoma in situ specimens and a significant increase in COX-2 expression in 70% of invasive adenocarcinomas (9) . In contrast, small cell carcinomas showed virtually negligible expression, and squamous cell carcinomas showed infrequent and low expression (9) . In the present study, sulindac sulfide showed similar growth inhibitory effects on all of the lung cancer cell lines except for the SCLC line H345, which was more sensitive.
In a study involving A549 NSCLC cells, Huang et al. (26)
demonstrated increased PGE2 production in response to IL-1. Tumor-derived PGE2 promotes lymphocyte and macrophage IL-10 induction while simultaneously inhibiting macrophage IL-12 production, thus, altering the cytokine balance in the lung cancer microenvironment. They further demonstrated that specific inhibition of COX-2 abrogated the capacity of IL-1B-stimulated A549 cells to induce IL-10 in lymphocytes and macrophages as well as reversing the tumor-derived PGE2-dependent inhibition of macrophage IL-12 production. An effect on cell cycle regulation may be involved in tumor growth. Aspirin, a COX inhibitor, was shown to induce cell cycle arrest with a decrease in the proportion of cells in G0-G1 and a relative increase in the percentage of cells in S phase and G2-M when given in high concentrations (27)
. In the present study, however, no effect on the cell cycle was seen with the addition of the COX inhibitor sulindac sulfide. Higher concentrations may, thus, be needed to see this effect. In a study involving colon cancer cells, Tsujii et al. (28)
demonstrated an inhibitory effect of NS-398 (a selective COX-2 inhibitor) on endothelial migration and tube formation, which suggested a possible role of COX in endothelial cell migration and angiogenesis. Thus, it remains unclear whether the inhibition of COX is necessary for growth inhibition.
The sulindac metabolite, exisulind, which is devoid of inhibiting prostaglandin synthesis activity, was the least potent of the NSAIDs but produced a similar apoptotic rate to NDGA and sulindac sulfide. Unlike NDGA or sulindac sulfide, exisulind seemed to cause cell cycle arrest in G0-G1 with a significant proportion of the cell population remaining in G1. This effect was evident by 48 h of drug exposure and lasted up to 120 h. Cell cycle regulation involves multiple factors known as cyclins and CDKs. The phosphorylation of Rb by CDKs results in the release of E2F, which drives the cell into completing the cell cycle. The activity of the CDKs requires cyclin binding, and this activity is suppressed by the inhibitors p16ink4A and p15ink4B. Exisulind may affect the cell cycle by either indirectly or directly inhibiting cyclin D1 or stimulating p16 and thus regulating the effect of Rb on the cell cycle in NSCLC. A similar effect was seen in the SCLC lines that lack Rb. Thus, the mechanism by which exisulind blocks cell cycle in G0-G1 remains to be elucidated. Other possible targets for the effects of exisulind may be the reduction of B-catenins, which are also known to regulate the levels of cyclin D1.
Piazza et al (14) . also studied the mechanism of growth inhibition of both exisulind and sulindac sulfide and showed that it was produced by the induction of apoptosis, independent of p53 status in comparison to 5-fluorouracil, an agent that requires p53 to induce apoptosis. Apoptosis by exisulind has been shown to occur via a COX-independent pathway in multiple tumor cell lines (29) . Recently, Thompson et al. (18) reported that exisulind induced apoptosis in cultured tumor cells via the inhibition of cyclic GMP phosphodiesterase. Phosphodiesterases are responsible for the hydrolysis of cyclic AMP and cyclic GMP, which act as intracellular second messengers in a variety of cellular functions.
The cure rate for lung cancer is a low 14%. More than 85% of cases are found in current and former smokers. One-half of the newly diagnosed cases develop in former smokers, which indicates a need for chemopreventive agents. 13-cis-retinoic acid is a vitamin-A derivative that has been shown as an effective chemopreventive agent. Two small studies suggested a benefit of 13-cis-retinoic acid in the prevention of secondary malignancies in high-risk individuals (30
, 31)
. A clinical trial of 13-cis-retinoic acid as a chemopreventive agent for second malignancies in patients with completely resected NSCLC has been completed, and the final results are eagerly awaited. A smaller trial evaluating 13-cis-retinoic acid in combination with 
-tocopherol is currently in progress for patients with longstanding tobacco abuse and evidence of sputum dysplasia. The present study showed that the addition of sulindac sulfide, exisulind, or NDGA to 13-cis-retinoic acid produced synergistic growth-inhibitory effects. These data suggest that clinical combination-chemoprevention trials be warranted.
Their side effects and the development of drug resistance limit the use of conventional cytotoxic chemotherapeutic agents for treating patients with lung cancers. Continuous usage of cisplatin and paclitaxel often results in severe peripheral neuropathy, myelosuppression, and resistance to multiple cytotoxic chemotherapeutic agents. In the present study, the addition of exisulind or sulindac sulfide to cisplatin and paclitaxel resulted in synergistic growth inhibition, especially at high concentrations, in all of the cell lines including SHP77, which overexpresses MDR-1 and is resistant to many chemotherapeutic agents. The addition of sulindac sulfide, exisulind, or NDGA to paclitaxel and cisplatin may increase effectiveness, limit toxicity, and overcome drug resistance. Sulindac sulfide at high concentrations can result in serious gastrointestinal and renal toxicity because of its effect on prostaglandins. Exisulind, being devoid of any antiprostaglandin activity, would be a logical candidate for combination studies with present chemotherapeutic agents. The combination of the sulfide metabolites with either cisplatin or paclitaxel is presently being evaluated in animal models and should be evaluated also in a clinical setting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a grant from the Cancer Research Foundation of America (to A. F. S. and P. A. B.) and by NIH Grants CA P50 CA58187 and CA46934. 
2 To whom requests for reprints should be addressed, at University of Colorado Cancer Center Box B188, 4200 East 9th Avenue, Denver, CO 80262. Phone: (303) 315-3007; Fax: (303) 315-3304; E-mail: Paul.Bunn{at}UCHSC.edu
3 The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-SCLC; NDGA, nordihydroguaiaretic acid; COX, cyclooxygenase; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; MDR, multidrug-resistant/multidrug-resistance; MDR-1, multidrug resistant gene 1; MRP, multidrug resistance-associated protein; LRP, lung cancer resistance-associated protein; CI, combination index; MTT, modified tetrazolium salt; NSAID, nonsteroidal anti-inflammatory drug; PGE, prostaglandin expression; IL, interleukin; CDK, cyclin-dependent kinase; Rb, retinoblastoma.
Received 6/ 1/99. Accepted 10/19/99.
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T. Hida, K.-i. Kozaki, H. Muramatsu, A. Masuda, S. Shimizu, T. Mitsudomi, T. Sugiura, M. Ogawa, and T. Takahashi Cyclooxygenase-2 Inhibitor Induces Apoptosis and Enhances Cytotoxicity of Various Anticancer Agents in Non-Small Cell Lung Cancer Cell Lines Clin. Cancer Res., May 1, 2000; 6(5): 2006 - 2011. [Abstract] [Full Text] [PDF]
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IC50 concentrations of cisplatin, paclitaxel, NDGA, sulindac sulfide, exisulind, and 13-cis-retinoic acid. The cells were harvested after drug exposure, resuspended in Krishan’s reagent, and analyzed by flow cytometry. The data were analyzed using Modift LT software to determine the percent cells in each phase of the cell cycle. 
, cells alone; 
, exisulind (400 µM); 
, cisplatin (200 µM); 
, paclitaxel (150 nM); 
, NDGA (80 µM); •, sulindac sulfide (100 µM); 
, 13-cis-retinoic acid (100 µM).