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[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 adenocarcinoma 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

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. Inhibitors. Tranylcypromine, a PGIS inhibitor (13), was obtained from EIA. The activities of TXAS or PGIS were indirectly estimated by meas- Aldrich. Tranylcypromine (0.7 mg/kg/day) was dissolved in water and admin- uring their stable metabolites, TXB2 or 6-keto PGF1␣, by EIA, respectively. istered to animals daily through gavage tubes.

Fig. 2. Transduction of TXAS gene and PGIS gene to C26 cells. a, schematic presentation of the retroviral constructs. The aminoglycoside phospho- transferase gene (neoR) that confers neomycin re- sistance to the cells is transcribed from the LTR of the Moloney murine leukemia virus (Mo-MuLV). TXAS or PGIS cDNAs are transcribed from an internal cytomegalovirus (CMV) immediate early gene promoter (pLNCX-TXAS; pLNCX-PGIS). ⌿ϩ, Mo-MuLV retroviral packaging signal. b, Northern blots. Total RNAs from C26-TXAS or C26-PGIS as well as C26-neo and C26 (wild type) were subjected to Northern blot analyses to monitor the expression levels of TXAS (left panel) and PGIS (right panel). Expression of the GAPDH gene is shown to confirm equal loading of RNA. Arrow, position of each transcript. c, production of TXB2 and 6-keto PGF1␣. Quantitative analyses of TXB2 (left panel) and 6-keto PGF1␣ (right panel) produced by C26-TXAS and C26-PGIS, respectively, were determined as described under “Materials and Methods.” As controls, prostanoids from C26-neo and C26 (wild type) cells under the same growth conditions were quantitatively determined. Data, the mean Ϯ SD of four independent experiments. d, growth rate of the cell lines C26-TXAS, C26-PGIS, C26-neo, and C26 (wild type). Cell number of each clone was determined at the indicated growth time. Results were presented in a semilog plot. No significant differences were observed in the cell growth rates.

Seratrodast, a TXA2 receptor inhibitor (14), was from Takeda Pharmaceu- tical, Japan. Seratrodast (3 mg/kg/day) was suspended in 5% arabic gum solution and administered daily through gavage tubes. Statistical Analysis. Significant differences in the means were examined by Student’s unpaired, two-tailed t test. Survival curves were analyzed by the method of Kaplan and Meier (15).

Results and Discussion

The retroviral constructs used in this study are schematically shown in Fig. 2a. The constructs, pLNCX-TXAS, pLNCX-PGIS, and pLXIN, gave comparable numbers of colonies after the corresponding retroviruses were transduced into C26 cells. All of these colonies were collected and were grown as mass population cultures to reduce the artifacts caused by the differences in growth rates among colonies attributable to the position effects of random insertion of retroviral constructs. Both C26-TXAS, and C26-PGIS express high amounts of mRNAs derived from the transduced cDNAs (Fig. 2b).

TXAS converts PGH2 to TXA2, and PGIS converts PGH2 to PGI2

(Fig. 1). Both TXA2 and PGI2 have extremely short half-lives and cannot easily be measured quantitatively. Instead, we measured the amounts of TXB2 and 6-keto PGF1␣, stable compounds derived from

TXA2 and PGI2, respectively. As expected, C26-TXAS and C26-

PGIS produced significantly higher amounts of TXB2 and 6-keto

PGF1␣, respectively (Fig. 2c). Thus, transduced TXAS and PGIS cDNAs both produced functioning enzymes in the cells. We next studied the effect of TXAS and PGIS expression on the cell growth in vitro. C26-TXAS and C26-PGIS did not show any significant differences in their growth rates (Fig. 2d). These results indicate that exogenous expression of TXAS and PGIS did not affect the cell growth in vitro. The cell growth rate in vitro does not predict the tumor growth rate in vivo. The latter is affected by host factors, such as migration of endothelial cells into tumors to generate blood vessels, involvement of fibroblasts to form tumor interstitium, or reactions of the immune cells against tumors. Mice carrying C26-TXAS and C26-PGIS transformants exhibited contrasting effects in tumor characteristics (Fig. 3, a–d). Tumors established from C26-TXAS grew more rapidly than C26-neo or C26 (wild type; P Ͻ 0.05 at days 7, 8, and 9), and resulted in the death of all of the mice at day 13 (P Ͻ 0.01). On the other hand, tumors from C26-PGIS grew more slowly (P Ͻ 0.05 after day 12), and resulted in longer survivals (P Ͻ 0.05) in these mice (Fig. 3, a and b). These results indicate that TXA2 and PGI2 exerted antithetical effects on tumor growth through their actions on the host cells. To identify the TXAS or PGIS target(s) that modified the tumor Fig. 3. Characterization of the tumors of BALB/c mice inoculated with C26-TXAS, growth, we conducted the immunohistological analyses on the estab- C26-PGIS, C26-neo, and C26 (wild type). a, growth of the tumors established from the the ,ء .individual cell line. Tumor volumes are presented as the mean Ϯ SE of five tumors lished tumors. H&E staining or immunostaining using lineage- statistical significance, analyzed using Student’s unpaired, two-tailed t test. b, survival specific antibodies did not show any differences in the numbers or rates of the BALB/c mice bearing tumors established from the individual cell line (n ϭ 5). The statistical significance was analyzed by the method of Kaplan and Meier. c, immu- subsets of the invading immune cells (data not shown). Therefore, the nohistochemical staining of the tumors established from individual cell line (ϫ100). immune system was probably not the main target. In contrast, the Brown stain, vascular endothelial cells. C26-TXAS tumor has denser and C26-PGIS staining of these sections using vascular endothelial cell-specific tumor has sparser blood vessels than C26-neo and C26 (wild type) tumors. d, numerical comparison of the blood vessels formed in individual tumor. Blood vessels were visual- antibody (antimurine CD31) revealed a marked difference in the ized by immunohistochemical staining against antimouse CD31. Data, the mean Ϯ SD of density of the tumor vasculature. Tumors established from C26-TXAS four tumors. The statistical significance was analyzed using Student’s unpaired, two-tailed had significantly richer vasculature (204%, versus C26-neo; t test. P Ͻ 0.01), and tumors from C26-PGIS had much poorer vasculature Ͻ (52%, versus C26-neo; P 0.01), than tumors from C26-neo or C26 of PGH2, might be involved in the angiogenic activity observed in (wild type). The density of the tumor vasculature was correlated with those studies (17, 18). the tumor growth rate: the denser the tumor vasculature, the faster the We next tested whether the effects of TXA2 and PGI2 on tumor tumors grew (compare Fig. 3a with Fig. 3, c and d). This indicates that angiogenesis and tumor growth could be reversed by the specific

TXA2 and PGI2 modified tumor growth through tumor angiogenesis. inhibitors. Administration of seratrodast, a TXA2 receptor inhibitor, A recent study showed that COX-2 overexpression in colon cancer reduced the vasculature and tumor growth in C26-TXAS-derived cells stimulated angiogenesis and that the stimulation was inhibited by tumors. Administration of tranylcypromine, a PGIS inhibitor, in-

NSAIDS or COX-2 inhibitors (16). TXA2, a downstream metabolite creased the vasculature and tumor growth in C26-PGIS-derived tu- 65

investigated in this study, PGE2 is of special interest, because in- creased PGE2 levels were reported in intestinal adenoma and colon cancer (8), and a recent study in which the EP2 gene (a PGE2 ⌬716 receptor) was disrupted in APC mice showed that PGE2 is involved in tumor angiogenesis (19). Studies on prostanoid profiles will enable us to select candidate prostanoids that can be molecular targets for future cancer treatment. Acknowledgments

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Prasenohadi Pradono, Ryushi Tazawa, Makoto Maemondo, et al.

Cancer Res 2002;62:63-66.

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