Effect of platelet-derived growth factor receptor-beta inhibition with STI571 on radioimmunotherapy

Whereas radioimmunotherapy of hematologic malignancies has evolved into a viable treatment option, the responses of solid tumors to radioimmunotherapy are discouraging. The likely cause of this problem is the interstitial hypertension inherent to all solid tumors. Remarkable improvements in tumor responses to radioimmunotherapy were discovered after the inclusion of STI571 in the therapy regimen. A combination of the tumor stroma-reactive STI571, a potent platelet-derived growth factor receptor-beta (PDGFr-beta) antagonist, and the tumor-seeking radiolabeled antibody B72.3 yielded long-lasting growth arrest of the human colorectal adenocarcinoma LS174T grown as s.c. xenografts in athymic mice. The interaction of STI571 with the stromal PDGFr-beta reduced tumor interstitial fluid pressure (P(IF)) by >50% and in so doing improved the uptake of B72.3. The attenuation of P(IF) also had a positive effect on the homogeneity of antibody distribution. These effects were dose-dependent and under optimized dosing conditions allowed for a 2.45 times increase in the tumor uptake of B72.3 as determined in the biodistribution studies. Single-photon emission computed tomography imaging studies substantiated these results and indicated that the homogeneity of the radioisotope distribution was also much improved when compared with the control mice. The increased uptake of radioimmunotherapy into the tumor resulted in >400% increase in the tumor absorbed radiation doses in STI571 + radioimmunotherapy-treated mice compared with PBS + radioimmunotherapy-treated mice. The improved antibody uptake in response to the attenuation of tumor P(IF) was identified as the primary reason for the growth arrest of the STI571 + radioimmunotherapy-treated tumors. Two related causes were also identified: (a) the improved homogeneity of monoclonal antibody distribution in tumor and (b) the increased tumor radiosensitivity resulting from the improved tumor oxygenation.     

Chemosensitization by STI571 targeting the platelet-derived growth factor/platelet-derived growth factor receptor-signaling pathway in the tumor progression and angiogenesis of gastric carcinoma

BACKGROUND: Autocrine and paracrine growth mediated by the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR)-signaling pathway plays an important role in the progression of solid tumors. The authors assessed the effect of STI571 on the tumor growth of gastric carcinoma in combination with 5-fluorouracil (5-FU) or paclitaxel targeting the PDGF/PDGFR-signaling pathway. METHODS: In MKN-45 gastric carcinoma cells, the cytotoxic effect was evaluated by 3-(4,5 dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the in vivo antitumor effect was evaluated in a nude mouse xenograft. Both STI571 and an antitumor drug were administered intraperitoneally. Gene expression was assessed by Western blot analysis and immunohistochemical staining. Apoptotic cell death was evaluated by the terminal deoxyuridine triphosphate-biotin nick-end labeling assay, and tumor angiogenesis was evaluated by microvessel density analysis. RESULTS: Treatment with STI571 alone was not effective in vitro, as assessed by a 50% inhibitory concentration value of 24.3 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the cytotoxic effect somewhat when the concentration of STI571 was increased to 10 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the antitumor effect of the antitumor drug significantly in vivo. The enhanced antitumor effect was associated with increased apoptotic cell death and inhibition of tumor angiogenesis. Treatment with STI571 down-regulated the expression of PDGF-BB and PDGFR-beta in tumor cells and decreased the production of phosphorylated PDGFR-beta and phosphorylated Akt. Furthermore, treatment with STI571 inhibited the expression of PDGFR-beta in stromal cells. CONCLUSIONS: STI571 was an effective chemosensitizer of antitumor drugs, such as 5-FU and paclitaxel for gastric carcinoma, targeting the PDGF/PDGFR-signaling pathway of tumor cells and stromal cells in disease progression and angiogenesis.     

Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans.

PURPOSE: Dermatofibrosarcoma protuberans is caused by activation of the platelet-derived growth factor B (PDGFB) receptor, a transmembrane tyrosine kinase. We investigated the response of dermatofibrosarcoma protuberans to the tyrosine kinase inhibitor imatinib mesylate. PATIENTS AND METHODS: A patient with unresectable, metastatic dermatofibrosarcoma protuberans received imatinib mesylate (400 mg bid). Response to therapy was assessed by [18F]fluorodeoxyglucose (FDG) positron emission tomography, magnetic resonance imaging, and histopathologic and immunohistochemical evaluation. RESULTS: The patient was treated for 4 months with imatinib mesylate. The hypermetabolic uptake of FDG fell to background levels within 2 weeks of treatment, and the tumor volume shrank by over 75% during the 4 months of therapy, allowing for resection of the mass. There was no residual viable tumor in the resected specimen, indicating a complete histologic response to treatment with imatinib mesylate. CONCLUSION: Imatinib mesylate is highly active in dermatofibrosarcoma protuberans. The dramatic response seen in this patient demonstrates that inhibition of PDGFB receptor tyrosine kinase activity can significantly impact viability of at least one type of solid tumor.     

Chronic myeloproliferative disorders with rearrangement of the platelet-derived growth factor alpha receptor: a new clinical target for STI571/Glivec

Two cases of atypical chronic myeloid leukaemia (CML) carrying the t(4;22)(q12;q11) translocation involving the breakpoint cluster region (BCR) and platelet-derived growth factor alpha receptor (PDGFRA) genes have been recently characterized. We report a third case of atypical CML with the same translocation but with a distinct breakpoint fusing BCR exon 1 with PDGFRA exon 13. The patient had a clinical presentation of CML with progressive transformation in B-cell acute lymphoblastic leukaemia. The involvement of PDGFRA led us to treat the patient with the small organic compound imatinib mesylate/STI571 (Glivec) that blocks the ATP binding site of tyrosine kinases such as Abelson, KIT and platelet-derived growth factor receptors. The patient subsequently achieved a rapid clinical and molecular response clearly demonstrating, for the first time, that Glivec is active against PDGFRA in vivo. Therefore, our study expands the list of Glivec targets and has direct biological and also clinical implications.     

Inhibition of platelet-derived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model.

PURPOSE: We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice. EXPERIMENTAL DESIGN: Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine. RESULTS: In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were >70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P < 0.0002 and P < 0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells. CONCLUSIONS: Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.     

Inhibition of platelet-derived growth factor-mediated proliferation of osteosarcoma cells by the novel tyrosine kinase inhibitor STI571.

PURPOSE: Osteosarcoma is an aggressive primary bone cancer characterized by expression of platelet-derived growth factor (PDGF) and its cognate receptor. Coexpression of the growth factor and receptor suggests their role in autocrine or paracrine growth mechanisms. It has been reported previously that STI571 has specific activity in inhibiting select tyrosine kinase receptors, including PDGF and c-Kit. Osteosarcomas express low levels of c-Kit but abundant levels of PDGF receptor (PDGFR). EXPERIMENTAL DESIGN: To investigate the potential of STI571 as therapy for osteosarcoma, we studied its effects on PDGF-mediated cell growth in vitro and in an in vivo mouse model. RESULTS: PDGF acted as a potent mitogen in a dose-dependent manner in two osteosarcoma cell lines. STI571 (1.0 micro M) inhibited both PDGFR-alpha and PDGFR-beta phosphorylation and the downstream phosphorylation targets extracellular signal-regulated kinase and Akt. STI571 also inhibited PDGF-mediated growth and induced apoptosis in vitro as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. To study the effect of STI571 alone or in combination with Taxol in an in vivo model, an osteosarcoma cell line (KRIB) was transplanted into the tibia of athymic nude mice. Mice were treated with STI571 (50 mg/kg p.o. q M-F), Taxol (8 mg/kg i.p. weekly), or STI571 plus Taxol for 6 weeks. There was no significant difference in tumor size between treatment and control mice. Aberrant signaling pathways downstream of the PDGFR in the v-Ki-ras oncogene-transformed KRIB cell line may in part explain this finding. CONCLUSIONS: Our data demonstrate that STI571 inhibits PDGF-mediated growth and leads to apoptosis of osteosarcoma cells in vitro by selective inhibition of the PDGFR tyrosine kinase. The effectiveness of STI571 in our studies suggests targeting of PDGFRs as a novel treatment for osteosarcoma.     

Development of hygromas or severe edema during treatment with the tyrosine kinase inhibitor STI571 is not associated with platelet-derived growth factor receptor (PDGFR) gene polymorphisms

STI571 is active against Bcr/Abl-, c-kit- and platelet-derived growth factor receptor (PDGFR)-driven malignancies. Mild to moderate edema is common, whereas severe edema, body cavity effusions and subdural hygromas are rarely observed. These effects have been suggested to involve inhibition of PDGFR signaling, but predisposing factors are unknown. We examined SNPs in the PDGFR alpha and beta gene regions in STI571-treated patients with and without life-threatening edema or cerebral hygromas, and in healthy volunteers. By RFLP analysis of 15 SNPs, the frequencies of genotypes did not differ between the three groups. SNPs of PDGFR genes do not appear to play a role in patient's susceptibility to clinically severe edema formation during treatment with STI571.     

Arsenic exposure followed by the development of dermatofibrosarcoma protuberans

A case of documented significant arsenic exposure followed by the development of dermatofibrosarcoma protuberans is reported. Exposure to arsenic is associated with an increased chance of the subsequent development of a variety of neoplasms. It is possible that dermatofibrosarcoma protuberans may be one such tumor.     

Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors.

Analysis of genomic organization and expression of platelet-derived growth factor receptors (PDGFR) and epidermal growth factor receptor (EGFR) in human malignant gliomas showed amplification and overexpression of both receptors in distinct subsets of tumors. Amplification of the alpha PDGFR was detected in 4 of 50 glioblastomas (8%). EGFR was amplified in 9 of the 50 tumors (18%). Western blot analysis showed elevated expression of alpha PDGFR and EGFR proteins in 4 (24%) and 3 (18%), respectively, of 17 tumor specimens analyzed. Increased production of alpha PDGFR as well as EGFR proteins was observed in the presence or absence of gene amplification. Three of the 4 tumors with elevated levels of alpha PDGFR also overexpressed the beta PDGFR, which was present as a single copy gene in all 50 tumors analyzed. Our findings suggest that the amplification and/or overexpression either of EGFR or of the alpha PDGFR along with the coordinate overexpression of the beta PDGFR can contribute to the malignant phenotype of distinct subsets of human glioblastoma.     

Growth inhibition of A549 human lung carcinoma cells by beta-lapachone through induction of apoptosis and inhibition of telomerase activity

The objective of the present study was to investigate the effect of beta-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae), on the cell growth and apoptosis in human lung carcinoma cell line A549. Exposure of A549 cells to beta-lapachone resulted in growth inhibition and induction of apoptosis in a time- and dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy and flow cytometry analysis. This increase in apoptosis was associated with a decrease in Bcl-2 and expression, an increase of Bax, and an activation of caspase-3 and caspase-9. beta-lapachone treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the levels of human telomerase RNA (hTR) and c-myc expression were progressively down-regulated by beta-lapachone treatment. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of beta-lapachone.     

Growth inhibition of U937 leukemia cells by aqueous extract of Cordyceps militaris through induction of apoptosis

Cordyceps militaris is a traditional herbal ingredient, which has been used for patients suffering from cancer in Oriental medicine. In the present study, we investigated the biochemical mechanisms of anti-proliferative effects by aqueous extract of C. militaris (AECM) in human leukemia U937 cells. It was found that AECM could inhibit cell growth of U937 cells in a dose-dependent manner, which was associated with morphological change and apoptotic cell death such as formation of apoptotic bodies and DNA fragmentation. We observed the down-regulation of anti-apoptotic Bcl-2 expression and proteolytic activation of caspase-3 in AECM-treated U937 cells. However, AECM did not affect the pro-apoptotic Bax expression and activity of caspase-9. Furthermore, Western blotting and RT-PCR revealed that AECM treatment caused a dose-dependent inhibition of cyclooxygenase-2 and prostaglandin E2 accumulation. Taken together, these results indicated that the anti-proliferative effects of AECM were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products such as Bcl-2 family expression and caspase protease activity, and AECM may have therapeutic potential in human leukemia treatment.     

Growth inhibition and modulation of kinase pathways of small cell lung cancer cell lines by the novel tyrosine kinase inhibitor STI 571

Small cell lung cancer (SCLC) is an aggressive cancer characterized by several autocrine growth mechanisms including stem cell factor and its receptor c-Kit. In order to arrive at potentially new and novel therapy for SCLC, we have investigated the effects of the tyrosine kinase inhibitor, STI 571, on SCLC cell lines. It has been previously reported that STI 571 does not only inhibit cellular Abl tyrosine kinase activity but also the PDGF receptor and c-Kit tyrosine kinases at similar concentrations (approximately 0.1 microM). There is no expression of the PDGF-receptor, and the Abl kinase is not activated by SCLC, but over 70% of SCLC contain the c-Kit receptor. Utilizing this preliminary data, we have determined that three (NCI-H69, NCI-H146 and NCI-H209) of five (including NCI-H82 and NCI-H249) SCLC cell lines had detectable c-Kit receptors and were inhibited in growth and viability at concentrations 1 - 5 microM of STI 571 after 48 h of treatment. The SCLC cell lines, NCI-H69, NCI-H146 and NCI-H209, showed a dose-response (tested between 0.1 - 10 microM) inhibition of tyrosine phosphorylation of c-Kit as well as in vitro kinase activity (at 5 microM) of c-Kit in response to STI 571. STI 571 inhibited cell motility, as assessed by time-lapsed video microscopy, within 6 h of STI 571 treatment (5 microM). STI 571 also decreased intracellular levels of reactive oxygen species (ROS) by at least 60%, at a concentration (5 microM) that also inhibited cell growth. Cell cycle analysis of STI 571 responsive cells showed that cells were generally slowed in G2/M phase, but there was no arrest at G1/S. A downstream phosphorylation target of c-Kit, Akt, was not phosphorylated in response to stem cell factor in the presence of STI 571. These data imply that STI 571 inhibits growth of SCLC cells through a mechanism that involves inactivation of the tyrosine kinase c-Kit. The effectiveness of STI 571 in this study suggests this drug may be useful in a clinical trial, for patients with SCLC. Oncogene (2000) 19, 3521 - 3528     

Disruption of the inhibitor of apoptosis protein survivin sensitizes Bcr-abl-positive cells to STI571-induced apoptosis

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides -1,194 and -1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl-transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl-transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.     

STI571 sensitizes nasopharyngeal carcinoma cells to cisplatin: sustained activation of ERK with improved growth inhibition

Previously, we demonstrated that c-kit and stem cell factors (SCF) commonly co-expressed in primary and metastatic nasopharyngeal carcinomas (NPC), and in HONE-1 NPC cells with tyrosine autophosphorylation of c-kit. These findings suggest that the SCF/c-kit signaling may contribute to pathogenesis of NPC. Therefore, the efficacy of STI571 treatment alone and when combined with cisplatin on HONE-1 cells were evaluated. STI571 induced growth inhibition at the IC50 concentration (14.9 microM). When the concentration was at or higher than 30 microM, the induction of cell apoptosis was observed. The effects of STI571 were shown to be mediated by the sustained activation of ERK but did not involve the inhibition of c-kit signal activity. When the STI571 (5 microM) and cisplatin (5 microg/ml) treatments were combined, there were further inductions of ERK activation resulting in obviously enhanced growth inhibition and induction of cell apoptosis. In a xenograft model, STI571 (50 mg/kg/day) showed only a limited ability to inhibit HONE-1 cell growth, but when combined with cisplatin (3 mg/kg/twice a week) treatment, there was a significant improvement in growth inhibition compared with STI571 or cisplatin treatment alone. Our results provide experimental support for the advanced NPC therapeutic trials using the combined STI571 and cisplatin treatment.     

Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist

A lipophilic farnesyl moiety attached to the carboxyl terminal cysteine of ras proteins structurally supports their membrane anchorage, required for ras-dependent growth-factor signaling and for transforming activity of ras oncoproteins. It has been shown that inhibition of ras farnesylation can block tumor growth in nude mice but that some ras-dependent tumors escape such blockage as a result of prenylation of ras. S-trans-transfarnesylthiosalicylic acid (FTS) is a potent ras-dislodging antagonist that does not affect ras prenylation but rather acts on the mature, membrane-bound ras and facilitates its degradation. Here we demonstrate that FTS induces reappearance of stress fibers in H-ras-transformed rat-1 cells (EJ cells) in vitro, inhibits their anchorage-independent growth in vitro, and blocks EJ-tumor growth in nude mice. The anchorage-independent growth of cells expressing ErbB2 (B104), but not that of v-raf-transformed cells, is also inhibited by FTS, suggesting specificity towards activated ras. FTS treatment (5 mg/kg i.p. daily) caused inhibition (75-80%) of tumor growth in nude mice implanted with EJ, but not in mice implanted with v-raf-transformed cells, with no evidence of systemic toxicity. Moreover, FTS treatment increased the survival rate of EJ-tumor-bearing mice from 48 to 68 days. Here we demonstrate anti-tumor potency in a synthetic, non-toxic, ras-dislodging antagonist acting independently of farnesyltransferases.     

In situ distribution of the beta-subunit of platelet-derived growth factor receptor in nonneoplastic tissue and in soft tissue tumors

The distribution of the beta-subunit of platelet-derived growth factor receptor (PDGFR-beta) was assessed by a sensitive immunoalkaline phosphatase technique using the monoclonal antibody PR7212. Frozen tissue sections of several nonneoplastic human tissues were stained along with 42 soft tissue sarcomas, 16 benign soft tissue proliferations, and 7 epithelial tumors. In all nonneoplastic tissue, there was intense labeling of cell processes of perivascular fibroblasts or pericytes in and about the walls of muscular blood vessels and of fibroblast cell processes around some glandular and ductal epithelia. No PDGFR-beta was found in the endothelial cells of muscular arteries and veins, but cells of uncertain identity within some capillaries were immunoreactive and the possibility that endothelial cells of some small capillaries express PDGFR-beta could not be excluded. In kidney there was strong labeling of glomerular mesangial cells and interstitial fibroblasts. Some histological types of soft tissue sarcomas were uniformly and strongly labeled with anti-PDGFR-beta, but other types were infrequently labeled or unreactive. The order of decreasing frequency and strength of labeling of the various types of benign and malignant soft tissue proliferations was as follows: benign fibromatosis and neurofibroma greater than malignant fibrous histiocytoma greater than liposarcoma greater than leiomyosarcoma greater than rhabdomyosarcoma. No tumor cell labeling was detected in epithelioid, synovial or clear cell sarcomas, leiomyomas, or carcinomas, but there was usually strong labeling of fibroblast and/or pericyte cell processes within tumor, especially around blood vessels. We conclude that PDGFR-beta is strongly expressed by vascular and stromal tissues of most tumors and normal organs and by tumor cells of several types of soft tissue tumors and proliferations, most notably those of fibroblastic origin.     

Growth inhibition and induction of apoptosis in colorectal tumor cells by cyclooxygenase inhibitors

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal carcinogenesis and prevent or revert the growth of premalignant colonic polyps. They inhibit cyclooxygenase (COX) but recent data indicate that this is not the only or even the most important mechanism of inhibition in colorectal tumor cells. We have used colonic carcinoma and adenoma cell lines to study the effects of the NSAID sulindac sulfide, its COX-inactive metabolite, sulindac sulfone, and the isoenzyme-specific inhibitors SC58125, SC236 and SC58560 on tumor cell growth in relation to COX-2 expression and prostaglandin production. To establish the role of COX-2 in NSAID action, we constructed clones expressing different levels of COX-2 from SW480 cells. All five compounds inhibited DNA synthesis and/or induced apoptosis, each with a characteristic pattern. ID(50)s were very similar in all the cell lines and were independent of COX expression, except for the COX-1 inhibitor SC58560, which was least effective in HT29/HI1, the cell line expressing the highest level of COX-1 (ID(50) 70 microM; in other cells lines the ID(50) was 15 microM). For all other compounds ID(50) concentrations varied less than two-fold: 25-40, 40-90 and 150 microM for SC236, sulindac sulfide and sulindac sulfone, respectively. SC58125 was the weakest inhibitor, never causing >50% cell loss. All compounds modulated expression of Bcl-2 and Bak and activated caspase 3. Overexpression of COX-2 in SW480 cells protected them against induction of apoptosis by sulindac sulfide. The effect was restricted to clones producing high levels of prostaglandin E(2). In summary, our data indicate that both COX-dependent and COX-independent mechanisms are involved in NSAID-induced growth in colorectal tumor cells. The concentrations necessary to inhibit growth were higher than serum concentrations that can be obtained in vivo, indicating that the therapeutic effect of NSAIDs cannot be explained by a direct effect of NSAIDs on the epithelial cells alone. For therapeutic purposes, compounds using different targets could be used to minimize side effects while optimizing therapeutic effect.