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Experimental Therapeutics, Preclinical Pharmacology |
Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica [G. T., R. C., V. D., D. M., Ro. Bianco, S. D. P., Ra. Bianco, F. C.] e Dipartimento di Biologia e Patologia Cellulare e Molecolare [B. M. V.], Università di Napoli Federico II, 80131 Napoli, and Istituto di Anatomia Patologica, Università di Pisa, 56100 Pisa [G. F.], Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: The COX-2 inhibitor SC-236 was combined with the selective EGFR tyrosine kinase inhibitor ZD1839 (Iressa) and the DNA/RNA-mixed backbone oligonucleotide AS-PKAI to study their effect on human cancer growth and angiogenesis, measuring vascular endothelial growth factor (VEGF) and basic fibroblast growth factor expression and vessel formation, in vitro and after oral administration of these agents in mice.
Results: A cooperative effect was observed with SC-236 in combination with either ZD1839 or AS-PKAI, as well as with all three agents together, on the proliferation of human colon and breast cancer cells in soft agar at doses that were ineffective for each agent alone. The antiproliferative effect was accompanied by inhibition of COX-2 expression. Moreover, combination of SC-236 with either agent or the triple combination markedly reduced VEGF secretion in the conditioned medium and completely suppressed VEGF and basic fibroblast growth factor expression. In nude mice bearing human colon cancer xenografts, a low, noninhibitory dose of SC-236 with ZD1839 and AS-PKAI, all given p.o., caused a dramatic cooperative antitumor effect, with no histological evidence of tumor in 60% of mice 5 weeks after treatment withdrawal, at which time all mice were alive. Moreover, analysis of tumor specimens revealed inhibition of vessel formation and expression of COX-2 and VEGF.
Conclusions: This is the first demonstration that three novel agents blocking multiple signaling pathways, in absence of cytotoxic drugs, may have a potent antitumor and antiangiogenic activity after oral administration. Because all agents are under clinical evaluation, our results provide a rationale to translate this feasible therapeutic strategy into a clinical setting.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGFR is a major transducer of mitogenic signals involved in cancer pathogenesis and progression and is considered an important target for anticancer therapy (1) . ZD1839 (Iressa, AstraZeneca) is a p.o. active, selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth and is under clinical development in cancer patients (1 , 3) . ZD1839 inhibits the growth of a variety of human cancer types, and additive/cooperative effects have been observed when combined with a variety of cytotoxic drugs in vitro and in vivo in nude mice (4) . EGFR activation has been directly related to induction of angiogenic growth factor expression and secretion. We and others have shown that ZD1839 is able to inhibit VEGF expression and neoangiogenesis in vitro and in nude mice xenografts (5) .
Overexpression of PKA isoform type I (PKAI), as compared with the type II isoform (PKAII), is a hallmark of the great majority of human tumors, correlating with worse clinicopathological features in several tumor types (reviewed in Ref. 2
). PKAI overexpression is associated with cell proliferation and neoplastic transformation, with G1
S cell cycle transition and with transduction of mitogenic signals from different growth factors, including TGF-
and epidermal growth factor. We have demonstrated that PKAI, through its RI subunit, has a structural interaction with the ligand-activated EGFR and cooperates in the propagation of mitogenic signals originated by different growth factors and hormones (6
, 7)
. For these reasons, PKAI is considered a relevant target for therapeutic intervention and different pharmacological tools able to inhibit PKAI expression and function have been developed (2
, 6
, 8)
. The most recent one is a DNA/RNA hybrid MBO targeting its RI subunit (AS-PKAI), which causes growth arrest and differentiation in a variety of cancer cell lines in vitro and in nude mice, synergizing with several class of cytotoxic drugs, and is active after oral administration (9, 10, 11, 12)
. Phase II clinical trials in cancer patients are now ongoing with this MBO AS-PKAI (defined GEM 231) in combination with different cytotoxic agents.
On the basis of the molecular interactions between PKAI and EGFR, we have developed a therapeutic strategy achieving the combined blockade of these two signaling pathways by different selective inhibitors and demonstrating their ability to target the respective kinases. This results in a cooperative antitumor and antiangiogenic activity both in vitro and in vivo in nude mice models (6 , 13) .
COX-2 is an enzyme known to be involved in inflammation but increasing evidence now supports its role in promoting tumor cell growth and angiogenesis, probably through the activity of COX-2-derived prostaglandins (14) . COX-2 overexpression has been shown in the biopsies of a variety of human cancers, including colon, cervix, lung, prostate, and breast, and has been associated with poor prognosis in patients affected by some of these cancers (14, 15, 16, 17) . It has been reported that newly formed blood vessels in tumors express COX-2, whereas quiescent vasculature expresses only the COX-1 enzyme (14 , 18) . Moreover, COX-2-dependent promotion of neoangiogenesis has been associated with induction of bFGF and VEGF (14 , 18) . Recent studies have demonstrated that activation of receptor tyrosine kinases such as EGFR may induce COX-2 expression and prostaglandin production (14 , 16) and that cAMP may regulate COX-2 transcription through the CRE present in its promoter (14 , 19) .
An earlier study has shown that sulindac, a nonsteroid anti-inflammatory drug that inhibits both COX-1 and COX-2 in combination with an irreversible inhibitor of EGFR, is able to confer protection from development of intestinal neoplasia in a murine model of familial adenomatous polyposis (20) .
On the basis of these functional interactions between EGFR, PKAI, and COX-2, we have evaluated whether a combined blockade of these signaling pathways by the respective inhibitors may result in significant antitumor effects. For this purpose, we have used ZD1839, AS-PKAI, and SC-236, a COX-2 inhibitor that has shown antiproliferative and antiangiogenic activity after oral administration (21) . We have studied the effect of these inhibitors on growth and angiogenesis of human cancer cells in vitro and in nude mice after oral administration.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunit of PKA (9)
. The control is a scramble MBO obtained by mixing all four nucleosides at each position. Sequence, structure, and purification methods were as published previously (12
, 22)
.
Cell Lines.
GEO colon and ZR-75-1 breast human cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). GEO and ZR-75-1 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES (pH 7.4), penicillin (100 UI/ml), streptomycin (100 µg/ml), and 4 mM glutamine (ICN, Irvine, United Kingdom) in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Growth in Soft Agar.
On day 0, cells (104 cells/well) were suspended in 0.5 ml of 0.3% Difco Noble agar (Difco, Detroit, MI) supplemented with complete culture medium. This suspension was layered over 0.5 ml of 0.8% agar-medium base layer in 24-multiwell cluster dishes (Becton Dickinson, Lincoln Park, NJ) and treated on days 0, 2, and 4 with the following concentrations of drugs: SC-236, from 0.01 to 2.5 µM; ZD1839 (0.1 µM); and AS-PKAI (0.1 µM). After 10–14 days, cells were stained with nitro blue tetrazolium (Sigma), and colonies > 0.05 mm were counted.
Western Blot Analysis.
Total cell lysates were obtained from either cells cultured in vitro or from homogenized tumor specimens. The protein extracts were resolved by 4–15% SDS-PAGE and probed with antihuman polyclonal COX-2, monoclonal VEGF, and monoclonal bFGF antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham International, London, United Kingdom), as described previously (7)
.
Evaluation of VEGF Secretion.
The concentration of VEGF in the CM obtained from cultured cells was measured using commercially available sandwich ELISA kits and according to manufacturers’ instructions. The ELISA kit for VEGF was purchased from R&D Systems, Inc. (Minneapolis, MN). Cells were plated in 60-mm dishes (Becton Dickinson) and treated for 4 days with SC-236 (0.1 µM), ZD1839 (0.1 µM), and AS-PKAI (0.1 µM), alone and in combination. Assays were performed using 24-h collected serum-free CM.
GEO Xenografts in Nude Mice.
Five to 6-week-old female Balb/cAnNCrlBR athymic (nu+/nu+) mice were purchased from Charles River Laboratories (Milan, Italy). The research protocol was approved, and mice were maintained in accordance with institutional guidelines of the University of Naples Animal Care and Use Committee. Mice were acclimatized to the University of Naples Medical School Animal Facility for 1 week before they received injections of cancer cells: 107 GEO human colon cancer cells were resuspended in 200 µl of Matrigel (Collaborative Biomedical Products, Bedford, MA) and injected s.c. in mice. After 7 days, when well-established tumors of 
0.2 cm3 were detected, mice were randomized to receive different treatments. Groups of 10 mice were treated with oral administration of SC-236 (6 mg/kg) and/or AS-PKAI (10 mg/kg) and/or ZD1839 (150 mg/kg) daily on days 7–11, 14–18, 21–25, and 28–32. Tumor volume was measured using the formula 
/6 x larger diameter x (smaller diameter)2, as reported previously (13)
. Two mice were sacrificed at day 32 to perform biochemical and immunohistochemical analysis.
Immunohistochemical Analysis.
New blood vessels were detected as previously described (23)
, using a monoclonal antibody against the human factor VIII-related antigen (Dako, Milan, Italy) at 1:50 dilution and stained with a standard immunoperoxidase method (Vectastain ABC kit; Vector). Each slide was firstly scanned at low power (x10–100 magnification), and the area with the higher number of new vessels was identified (hot spot) and then scanned at x250 microscope magnification (0.37 mm2). Stained blood vessels were counted in each of five different fields. For individual tumors, the microvessel count was scored by averaging the counts from five fields. All analyses were performed in a blind fashion.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cooperative Effect of the Different Agents in Combination.
Combination of either AS-PKAI or ZD1839 with different doses of SC-236 caused a supra-additive effect in both cell lines, particularly with lower doses (Fig. 1, A and B)
. A similar supra-additive effect was also obtained with the combination of AS-PKAI and ZD1839 (Fig. 1, A and B)
. When we combined the three agents together, a clear cooperative inhibitory effect was observed on the soft agar growth of GEO and ZR-75-1 cancer cells. In fact, low doses that alone induce up to 15–20% inhibition of colony formation caused an almost complete suppression of growth (Fig. 1, A and B)
. No cooperative effect was obtained when either SC-236 or ZD1839 were combined with the scramble sequence oligo (data not shown).
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shows that SC-236 and ZD1839 inhibit COX-2, whereas AS-PKAI is unable to do so. Combination of any two agents markedly reduced COX-2 expression, which was completely suppressed by the three agents in combination. Protein expression was compared with that of actin (Fig. 2)
. Interestingly, Western blot analysis of RI, EGFR, and phosphorylated EGFR tyrosine expression showed that SC-236 is unable to affect their expression (data not shown).
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18 ng of VEGF for 106 cells in 24 h. Treatment with either AS-PKAI or ZD1839 alone caused 20% inhibition of VEGF secretion, respectively, whereas SC-236 was almost ineffective (Fig. 2A)
. When SC-236 was combined with AS-PKAI, an additive inhibitory effect was observed, whereas combination of SC-236 with ZD1839 caused a supra-additive effect with 50% inhibition of VEGF secretion. An additional cooperative inhibitory effect was achieved by the three agents used together, reducing the VEGF secretion to 7 ng/106 cells (Fig. 2A)
.
Western blot analysis of VEGF and bFGF expression on the same cells demonstrated that each single agent is able to reduce the amount of both angiogenic factors, as compared with the expression of the control protein actin (Fig. 2B)
. Combination of SC-236 with either AS-PKAI or ZD1839 caused >80% inhibition of both VEGF and bFGF expression, whereas the three agents together completely suppressed the expression of these two angiogenic factors (Fig. 2B)
.
Effect on the Growth of Tumor Xenografts.
We investigated the antitumor activity of SC-236, AS-PKAI, and ZD1839 administered p.o., alone and in combination, in nude mice bearing GEO colon cancer xenografts. When established GEO tumors of 0.2 cm3 were detectable, groups of 10 mice were treated with SC-236, AS-PKAI, and ZD1839, alone and in combination. Two mice were sacrificed on day 32 to perform biochemical and histochemical analysis; therefore, tumor growth studies were performed on the remaining 8 mice. As shown in Fig. 3A
, within 5 weeks, GEO tumors reached a size not compatible with normal life. Treatment with the SC-236 alone at the low dose of 6 mg/kg was almost ineffective. AS-PKAI and ZD1839 alone each caused a similar significant inhibition of tumor growth, delaying the death of mice by 2 weeks. When SC-236 was combined with either AS-PKAI or ZD1839, an increase in the growth inhibitory effect and a delay of 1 week in the death of mice were observed, as compared with PKAI or EGFR inhibitors alone. An additional inhibitory effect was obtained when AS-PKAI and ZD1839 were used in combination. A dramatic and sustained inhibitory effect was obtained by the combination of three drugs altogether. In fact, no tumor growth was observed for 5 weeks after treatment withdrawal, and a modest increase in tumor size was recorded at the end of the experiment, 10 weeks after tumor cell injection (Fig. 3A)
. Moreover, at this time point, pathologic evaluation showed that 5 of 8 mice were still tumor free. The combined treatment with two drugs or with three drugs altogether was well tolerated; no weight loss or other signs of acute or delayed toxicity were observed.
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. Combination of SC-236 with AS-PKAI caused 90% inhibition of COX-2 expression, whereas protein was no longer detectable in the specimens from animals treated with SC-236 in combination with ZD1839 or with the three agents together (Fig. 3B)
.
Effect of Treatment on Angiogenesis in Vivo.
We have quantified by immunohistochemistry the tumor-induced vascularization as microvessel count in the most intense areas of neovascularization, using an antifactor VIII-related antigen monoclonal antibody. At day 32, two tumor samples were analyzed for each group of mice. SC-236 or AS-PKAI used as single drug caused 20% inhibition of vessel formation, whereas ZD1839 caused 30% inhibition, as compared with samples from untreated mice (Fig. 4A)
. A supra-additive effect was achieved by treatment with SC-236 combined with ZD1839 or AS-PKAI or with the three agents together, which inhibited vessel formation by 50–60 and 80%, respectively (Fig. 4A)
.
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70% by combination of SC-236 and AS-PKAI and completely suppressed in the animals treated with SC-236 and ZD1839 in combination or with the three agents altogether (Fig. 4B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several recent studies have correlated EGFR ligands such as TGF- and EGFR activation with the ability to stimulate normal endothelial cell growth through paracrine mechanisms and to up-regulate VEGF production in human cancer cells at the transcriptional level (24
, 25)
. In this regard, we have demonstrated that ZD1839 inhibits the expression of TGF-, bFGF, and VEGF in a large variety of human cancer cell lines (5)
. We have also shown that PKAI is involved in the angiogenesis and that PKAI-selective inhibitors, including oral AS-PKAI, are able to down-regulate the production of angiogenic growth factors (12
, 26)
.
Many studies have shown that COX-2 is involved in cancer cell proliferation, induction of neoangiogenesis, and increased invasiveness (14 , 27, 28, 29) . These events may be responsible for the worse prognosis observed in patients with different types of cancer overexpressing COX-2 (14, 15, 16, 17) . For such reasons, different selective inhibitors have been developed and proposed as novel agents for cancer treatment and for chemoprevention (14) . It has been demonstrated that COX-2 expression is transcriptionally induced by the signaling cascade triggered by EGFR activation and is controlled by the cAMP-dependent pathway through a CRE-regulated AP-1 transcription factor (14 , 19) .
In this study, we have evaluated the possibility of obtaining a control of tumor growth without using cytotoxic drugs, by the combined blockade of EGFR, PKAI, and COX-2, three molecules that interact in nodal points of distinct yet related signaling pathways. To translate this hypothesis in an experimental setting, we have used three novel agents with specific properties, including oral activity: the selective EGFR tyrosine kinase inhibitor ZD1839; a hybrid DNA/RNA MBO AS-PKAI; and the COX-2 inhibitor SC-236. All these agents have demonstrated antiproliferative and antiangiogenic properties in different tumor models, alone and in combination with cytotoxic drugs.
We have demonstrated, in human colon and breast cancer cell types, that these agents in combination have a cooperative growth inhibitory effect, achieving maximal activity when the three agents are used together, even at very low doses. The antitumor effect is accompanied by down-regulation of the expression of COX-2 as well as of VEGF and bFGF angiogenic proteins.
Moreover, secretion of VEGF in the CM was inhibited by combined treatments. In all cases, the combination of SC-236 and ZD1839 was the most effective.
Taken together, these results support the evidence of a functional interaction among EGFR, PKAI, and COX-2 in promoting cell proliferation and angiogenesis.
We translated these results in vivo in nude mice bearing GEO xenografts using an oral administration for all of the agents tested. We used a suboptimal dose of SC-236 and effective doses of AS-PKAI and ZD1839 to determine any potential for cooperative effect among the drugs used in combination. Addition of SC-236 to either ZD1839 or AS-PKAI clearly increased the effect of the latter two agents alone. An effective cooperative activity was also obtained when AS-PKAI and ZD1839 were used in combination, as demonstrated previously (30)
. A dramatic cooperative effect was obtained when the three agents were used together, resulting in no tumor growth at the end of the experiment on day 70 after tumor injection and 6 weeks after treatment withdrawal. At this time point, 60% of mice were still tumor free. Analysis of tumor samples removed at the end of treatment demonstrated a clear inhibition of COX-2 expression by two drugs in combination and a complete suppression when the three agents were used together. Moreover, we have demonstrated that two drugs in combination are able to reduce the VEGF expression in the specimen from treated nude mice, whereas a complete suppression was caused by the three drugs together. An inhibitory effect was also obtained on the vessels count, suggesting a direct interference of the respective drugs on the angiogenic signaling pathways.
The dramatic in vivo activity of the three agents together, as compared with even two agents in combination, may be explained only by a potent cooperativity. Because each pathway blocked by the respective inhibitors has different downstream branches, involving other signaling molecules, it may be hypothesized that alternative signaling pathways may lead to proliferation. In this context, combination of the three agents together would provide an effective multisignaling blockade. The previously reported ability of SC-236 to prevent the DNA synthesis and proliferative response to other growth factors such as platelet-derived growth factor (31) and the prostaglandin-dependent activation of CREB, which stimulates cell proliferation in certain murine models (19) , may greatly enhance the cooperative effect previously demonstrated for AS-PKAI and ZD1839 in combination.
There is growing consensus that the blockade of key mitogenic and angiogenic molecules may be a successful strategy for both chemoprevention and long-term control of cancer. Agents suitable for these purposes should have mild and differing toxicity patterns and simple administration routes. In agreement with this approach, we have shown that using oral administration of different noncytotoxic selective agents targeting molecules involved in mitogenic signaling and angiogenesis, it is possible to achieve an antitumor and antiangiogenic effect. Because all of the agents used in our study are under clinical development, we believe that this study provides a strong rationale to translate this feasible therapeutic strategy into a clinical setting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and the CNR-MIUR 449/97-99. 
2 To whom requests for reprints should be addressed, at Cattedra di Oncologia Medica, Dipartimento Endocrinologia e Oncologia Molecolare e Clinica, Università di Napoli Federico II Via S. Pansini 5 80131 Napoli, Italy. Phone: 39-081-7462061; Fax: 39-081-7462066; E-mail: gtortora{at}unina.it
3 The abbreviations used are: EGFR, epidermal growth factor receptor; PKAI, protein kinase A type I; VEGF, vascular endothelial growth factor; TGF-, transforming growth factor ; MBO, mixed backbone oligodeoxynucleotide; COX, cyclooxygenase; bFGF, basic fibroblast growth factor; CRE, cAMP-responsive element; CM, conditioned medium.
Received 5/21/02; revised 9/17/02; accepted 9/17/02.
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subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor. Oncogene, 14: 923-928, 1997.[CrossRef][Medline]
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C. F Singer, G. Hudelist, W. Lamm, R. Mueller, K. Czerwenka, and E. Kubista Expression of tyrosine kinases in human malignancies as potential targets for kinase-specific inhibitors Endocr. Relat. Cancer, December 1, 2004; 11(4): 861 - 869. [Abstract] [Full Text] [PDF]
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M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]
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Z. Chen, X. Zhang, M. Li, Z. Wang, H. S. Wieand, J. R. Grandis, and D. M. Shin Simultaneously Targeting Epidermal Growth Factor Receptor Tyrosine Kinase and Cyclooxygenase-2, an Efficient Approach to Inhibition of Squamous Cell Carcinoma of the Head and Neck Clin. Cancer Res., September 1, 2004; 10(17): 5930 - 5939. [Abstract] [Full Text] [PDF]
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 L. Castillo, M. C. Etienne-Grimaldi, J. L. Fischel, P. Formento, N. Magne, and G. Milano Pharmacological background of EGFR targeting Ann. Onc., July 1, 2004; 15(7): 1007 - 1012. [Abstract] [Full Text] [PDF]
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F. Ciardiello, R. Bianco, R. Caputo, R. Caputo, V. Damiano, T. Troiani, D. Melisi, F. De Vita, S. De Placido, A. R. Bianco, et al. Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy Clin. Cancer Res., January 15, 2004; 10(2): 784 - 793. [Abstract] [Full Text] [PDF]
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C. L. Arteaga Molecular Therapeutics: Is One Promiscuous Drug against Multiple Targets Better than Combinations of Molecule-specific Drugs? Clin. Cancer Res., April 1, 2003; 9(4): 1231 - 1232. [Full Text] [PDF]
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) of each couple shows the effect obtained when the drugs were actually used in combination. Therefore, the comparison between the height of the first bar and that of the second bar of each couple shows whether a supra-additive effect is obtained and the magnitude of such effect. The data represent means and SEs of triplicate determination of at least two experiments. C, Western blotting analysis of COX-2 expression in ZR-75-1 cells. Cells were plated in 60-mm dishes (Becton Dickinson) and treated for 4 days with SC-236 (1 µM), AS-PKAI (0.5 µM), and ZD1839 (0.5 µM), alone and in combination. Cell lysates were processed as described in "Materials and Methods."
