[CANCER RESEARCH 62, 63–66, January 1, 2002] Advances in Brief Gene Transfer of Thromboxane A2 Synthase and Prostaglandin I2 Synthase Antithetically Altered Tumor Angiogenesis and Tumor Growth1 Prasenohadi Pradono, Ryushi Tazawa,2 Makoto Maemondo, Masashi Tanaka, Kazuhiro Usui, Yasuo Saijo, Koichi Hagiwara, and Toshihiro Nukiwa Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan Abstract inhibitors of the procancer prostanoids retard cancer growth and vice versa. Cyclooxygenase, involved in tumor growth and angiogenesis, converts Prostanoids exert a wide range of biological functions on a variety arachidonic acid to prostaglandin (PG)H , which is immediately con- 2 of cells. Some of these actions are in opposition to each other, for verted to bioactive prostanoids including PGE2, PGD2, thromboxane example, TXA2 promotes platelet aggregation and vasoconstriction, (TX)A2 and PGI2. To test the hypothesis that changes in the prostanoid profile alter cancer growth, we transduced the retroviral vectors carrying whereas PGIS inhibits platelet aggregation and promotes vasodilata- tion (10). Because of the fact that a variety of the host cells are TXA2 synthase cDNA or PGI2 synthase cDNA to colon-26 adenocarci- noma cells and subsequently inoculated each transformant to syngeneic involved in tumor growth, the effects of an individual prostanoid on BALB/c mice. Tumors derived from TXA2 synthase transformants grew tumor growth are hard to predict and must be studied in vivo. There- faster (280%, day 8, versus null-vector control; P < 0.05) and showed fore, to test our hypotheses, we have investigated the effects of TXA2 more abundant vasculature (204%, versus null-vector control; P < 0.01), and PGI2 on tumor growth using a mouse model system. We chose whereas tumors from PGI2 synthase transformants presented opposite these prostanoids because they have biological functions opposing effects. These effects by the transgenes were reversed by administration of each other and may provide us with a clear view of the relationship specific inhibitors. These results suggest that the profile of downstream between prostanoid function and tumor growth. In this study, TXAS metabolites of cyclooxygenase in cancer cells can be a determinant for tumor development. and PGIS were introduced into murine colon-26 adenocarcinoma cell line (C26) to alter the prostanoid balance. The resulting cells were Introduction then inoculated into syngeneic BALB/c mice, tumor growth and animal survival were monitored, and tumor histology was examined. 3 NSAIDs inhibit both COX-1, a constitutively expressed isozyme Comparing the results of PGIS and TXAS gene transduction, we implicated in maintaining normal cellular functions, and COX-2, an could test our hypotheses and infer the underlying mechanisms that inducible isozyme expressed in inflammatory lesions and in many resulted from differential prostanoid profiles. types of cancers including colon, stomach, esophagus, lung, breast, prostate, skin, and melanoma (1, 2). Epidemiological, animal, and Materials and Methods clinical studies have established that NSAIDs are effective for the prevention and size-reduction of colon cancers and have suggested Cell Lines and Animals. Murine colon-26 adenocarcinoma cell line (C26) that they may also be effective for other types of cancers (3–5). was maintained in RPMI 1640 (Life Technologies) with 10% FCS. The ␺CRIP Studies using COX-2-specific inhibitors have demonstrated that the cells, a packaging cell line that produces replication-incompetent retrovirus anticancer effect of NSAIDs is likely attributable to the inhibition of (11), were maintained in DMEM (LifeTechnologies) with 10% calf serum (CS). Female BALB/c mice at 6–8 weeks of age were obtained from Charles COX-2 activity (6), although the contribution of a COX-independent River Japan. mechanism has also been suggested (7). Retroviral Vector Construction and Transduction into the C26 Cells. COXs convert arachidonic acid to PGH2, a common precursor to a The human TXAS cDNA and the human PGIS cDNA were gifts from Dr. variety of prostanoids (summarized in Fig. 1). PGH2, which by itself Lee-Ho Wang. A 1.8-kb BamHI fragment that contained the entire coding has no known physiological functions, is immediately catalyzed to sequence of TXAS and a 1.6-kb BamHI fragment that contained the entire bioactive prostanoids PGE2, PGD2, TXA2, PGF2␣, and PGI2. The coding sequence of PGIS were each blunt-ended and inserted into the HpaI site effects of COX expression in cancer cells are considered to be related of the retroviral vector pLNCX (Clontech) to generate pLNCX-TXAS and to the fractional amounts of these prostanoids (i.e., the prostanoid pLNCX-PGIS, respectively (see Fig. 2a). The three retroviral vector con- profile; Refs. 8, 9). However, little information is available for the structs, pLNCX-TXAS, pLNCX-PGIS, and pLXIN (a retroviral vector carry- R Ј relationship of prostanoid profile and cancer growth. We hypothesize ing the neo gene driven by 5 Moloney murine leukemia virus LTR, used as a control null vector; Clontech) were individually transfected into ␺CRIP. that: (a) changes in the prostanoid profile alter cancer growth; and (b) Neo-resistant, retrovirus-producing cells were selected with 400 ␮g/ml G418 (Life Technologies, Inc.), and named ␺CRIP-TXAS, ␺CRIP-PGIS, and Received 7/25/01; accepted 11/8/01. ␺CRIP-LXIN. C26 cells were incubated with each viral supernatant in the The costs of publication of this article were defrayed in part by the payment of page presence of 8 ␮g/ml Polybrene (Aldrich Chemical), and the transduced cells charges. This article must therefore be hereby marked advertisement in accordance with ␮ Ͼ 18 U.S.C. Section 1734 solely to indicate this fact. were selected with 600 g/ml of G418. The resultant colonies ( 200 colonies) 1 Supported in part by a grant for cancer research from The Sagawa Foundation for were collected as a mass population culture and designated as C26-TXAS, Promotion of Cancer Research and Grant-in-Aid 10770261 for scientific research from the C26-PGIS, and C26-neo. Ministry of Education, Science, Sports, Culture and Technology of Japan. 2 Northern Blot. Total cellular RNA was extracted using the Isogen kit To whom requests for reprints should be addressed, at Department of Respiratory ␮ Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku (Nippon Gene). RNA (10 g) from each cell line was electrophoresed on a University 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8539; 1.2% agarose gel containing 2.2 M formaldehyde and transferred to Hybond Fax: 81-22-717--8549; E-mail: [email protected]. nylon membrane (Amersham Pharmacia Biotech). TXAS, PGIS, and GAPDH 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, cDNAs, labeled with [␣-32P]dCTP (DuPont) using Prime-It kit (Stratagene), cyclooxygenase; TX, thromboxane; PG, prostaglandin; PGIS, PGI synthase; TXAS, 2 were used as probes. Hybridization was performed in Quikhyb solution (Strat- TXA2 synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EIA, enzyme immunoabsorbent assay; LTR, long terminal repeat. agene) at 68°C overnight. Filters were washed three times in 2ϫ SSC, 0.1% 63 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. GENE TRANSDUCTION OF TXAS AND PGIS TO COLON CANCER CELLS The TXB2 EIA kit and the 6-keto PGF1␣ EIA kit were purchased from Cayman ϫ 5 Chemical. For TXB2,3 10 cells were plated in 2 ml of RPMI 1640 2 h prior ϫ 5 to the assay. For 6-keto PGF1␣, 3 10 cells were plated in 2 ml of growth medium 24 h prior to the assay. The media were collected and subjected to EIA. Cell Growth Assay. C26-TXAS, C26-PGIS, C26-neo, and wild-type C26 cells were plated in 35-mm dishes (1 ϫ 105 cells/well, in 2 ml of RPMI medium containing 10% FCS). The number of cells was counted after 24, 48, and 72 h of seeding. Tumor Growth Assay. C26-TXAS, C26-PGIS, C26-neo, and wild-type C26 cells (5 ϫ 105 cells) were s.c. inoculated into the left flanks of BALB/c mice that are syngeneic with C26 cells. Two perpendicular diameters of the resultant tumors were measured daily using calipers. Tumor volumes were calculated as described previously (12). Immunohistochemical Staining of the Tumor Tissue. When tumors reached 1 cm in the longer diameter, they were resected, embedded in Tissue-Tek OCT embedding medium (Sakura Finetechnical) and stored at Ϫ80°C until use. Thin sections of the tumor tissues were prepared by cryostat and placed on glass slides. Sections were then fixed in 1% paraformaldehyde at room temperature for 30 min, washed three times with PBS, and incubated overnight with a 1:100 dilution of biotin-conjugated rat antimouse CD31 (platelet endothelial cell adhe- Fig. 1. Schematic presentation of the metabolic pathways of prostanoids. Phospho- sion molecule-1; PharMingen) to detect the vascular endothelial cells. The bound lipase A2 (PLA2) converts phospholipid localized in cell membrane to arachinonic acid, which in turn is converted to PGH2 by COXs. PGH2 is immediately catalyzed to various antibody was coupled with streptavidin-peroxidase complex (Histofine; Nichirei prostanoids. In ellipses, enzymes. Black arrows, catalytic pathways. Opposite effects of Corporation) and visualized by 3,3Ј-diaminobenzidine tetrahydrochloride (DAB). TXA2 and PGI2 on platelet aggregation and vasoconstriction are shown: gray arrow, The sections were then counterstained with methylgreen for 1 min and observed stimulation; gray T-bar, inhibition. under a microscope. Four high-power fields (ϫ400) from the tumor region were arbitrarily selected, and two pathologists (M. M. and M. T.) independently counted SDS at 68°C, three times in 0.2ϫ SSC, 0.1% SDS at 68°C, and then exposed the number of the vessels stained. to XA-R film (Kodak) at Ϫ70°C overnight.