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Keywords:

  • CS-706;
  • COX-2;
  • colorectal adenocarcinoma;
  • combination therapy

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The potent chemopreventive activity of cyclooxygenase-2 (COX-2) inhibitors has been demonstrated in a number of preclinical studies, but their potency in antitumor activity is still in dispute. In this report, we demonstrate the potent antitumor activity of a novel COX-2 inhibitor, CS-706 in mouse colorectal adenocarcinoma colon 26 tumor-bearing mice treated with or without antitumor chemotherapeutic agents. Daily oral administration of CS-706 at doses of 3–100 mg/kg from the day of tumor inoculation (Day 0) inhibited tumor growth dose-dependently, and the maximal inhibition was 67% at a dose of 100 mg/kg. In contrast, celecoxib, a well-known COX-2 inhibitor, did not inhibit tumor growth at doses up to 100 mg/kg. Furthermore, CS-706 at a dose of 1 mg/kg or above markedly prolonged the survival time of tumor-bearing mice. Administration of 30 mg/kg CS-706 from Day 7 combined with a single intravenous treatment of 10 mg/kg cisplatin on Day 7 completely regressed the tumors in all tumor-bearing mice examined, whereas only in 1 of 10 mice tumor was regressed with cisplatin treatment. Similar combination effects were observed with 10 mg/kg CS-706 and 60 mg/kg 5-fluorouracil (5-FU). Moreover, 10 mg/kg CS-706 significantly inhibited angiogenesis induced by implanted chambers with colon 26 cells in a dorsal air sac assay in mice. Collectively, these results suggest that CS-706 is a potent antitumor agent, especially in combination with conventional chemotherapeutic agents, and that the anti-angiogenic activity of CS-706 may contribute at least in part to its marked antitumor activity. © 2007 Wiley-Liss, Inc.

Cyclooxygenase (COX) is a key enzyme of prostaglandin synthesis and plays an important role in a variety of biological responses, including inflammation and pain. There are 3 isoenzymes of COX, namely COX-1, COX-2 and COX-3. COX-1 is constitutively expressed in most tissues including gastric mucosa and platelets to regulate the turnover of epithelial mucosa1 and platelet coagulation.2 Contrary to COX-1, COX-2 expression is regulated by inflammatory stimuli such as TNF-α and IL-1β. COX-3 (also known as COX-1b) is a splicing variant of the COX-1 gene that is reported to be a target molecule of acetaminophen.3

Among them, COX-2 is suggested to be involved in carcinogenesis. COX-2 overexpression is often detected with high frequency in colorectal polyps and in various kinds of tumor tissues, including colorectal, lung,4 pancreatic5 and breast cancers.6 In a genetically engineered animal model, Liu et al. reported a higher frequency of mammary tumors developing when COX-2 expression was forced in mammary epithelial cells under the mouse mammary tumor virus (MMTV) promoter.7 This result suggests that COX-2 may even act as an oncogene. Therefore, numerous experimental and clinical studies have recently been conducted in order to examine the chemopreventive activity of COX-2 inhibitors such as celecoxib and rofecoxib.8–14

Celecoxib, which is a well-known COX-2 selective inhibitor widely used as a nonsteroidal anti-inflammatory agent, showed an inhibitory effect on colorectal polyp formation in a Min mice model8 and in azoxymethane-induced colorectal tumors in F344 rats.9 The chemopreventive activity of COX-2 inhibitors has been demonstrated not only in experimental colorectal carcinogenesis but also in other tumor carcinogenesis models such as prostate,10 skin11 and breast12, 13 carcinogenesis models in mice or rats. In clinical trials, because celecoxib treatment significantly inhibited the formation of colorectal polyps in familial adenomatous polyposis (FAP) patients,14 it was approved as an adjunctive treatment to surgery for FAP in the US. In addition to its effect on FAP, it was reported that a treatment of 200 or 400 mg of celecoxib twice daily reduced the incidence of recurrent adenomas in the Adenoma Prevention with Celecoxib (APC) trial with 2,000 or more patients who had adenomas removed before study entry.15 Moreover, a case–control study demonstrated that both daily 200 mg celecoxib and daily 25 mg rofecoxib significantly reduced the risk of breast cancer in clinical trials.16 In addition to the chemopreventive activity of COX-2 inhibitors, recently, a few reports have shown the significant tumor growth inhibitory activity of COX-2 inhibitors against various tumors in animal models.17–19 With regard to celecoxib, various pilot studies in cancer patients in both monotherapy and combination therapy with antitumor agents have been conducted, but with very limited success.20, 21 In contrast to great number of reports that evaluate the chemopreventive activity of COX-2 inhibitors, there still are few reports that evaluate their tumor growth inhibitory activity in detail.

CS-706 is a novel COX-2 inhibitor with IC50 values of 2.2 μM (95% confidence interval 1.3–3.4) and 0.31 μM (95% confidence interval 0.15–0.63) for COX-1 and COX-2 inhibition, respectively, when measured in human whole blood.22 It has been demonstrated that CS-706 showed potent antiinflammatory activity in various animal models,22 and ongoing Phase II studies in the US have revealed its safety profile and its potent analgesic activity with regard to dental pain.23, 24 Here, we show the detailed examination of the antitumor activity of CS-706 against mouse colorectal adenocarcinoma colon 26 subcutaneously implanted in CDF1 mice in comparison with that of celecoxib. We also investigated the antitumor activities of CS-706 in combination with chemotherapeutic agents such as cisplatin or 5-fluorouracil (5-FU).

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals

CS-706 and celecoxib were synthesized in Sankyo (Tokyo, Japan). Cisplatin and 5-FU were purchased from Nippon Kayaku (Tokyo, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Sodium carboxymethyl cellulose was purchased from Iwai Chemicals Company (Tokyo, Japan). For the inhibition assay of vascular endothelial growth factor (VEGF) production, lipopolysaccharide (LPS) from Escherichia coli Serotype 055:B5 was purchased from Sigma-Aldrich (Tokyo, Japan) and dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemical Industries.

Cell lines and culture

The mouse tumor cell line, colorectal adenocarcinoma colon 26 derived from Balb/c mice, was obtained from the Japanese Foundation for Cancer Research (Tokyo, Japan) as solid tumors. The cell culture line was established by isolating tumor cells from solid tumor tissue by mincing and trypsinizing. Colon 26 tumor cells were maintained in RPMI Medium 1640 with L-glutamine (Invitrogen, Tokyo, Japan) containing 10% fetal bovine serum (FBS, HyClone, Logan, UT). The established colon 26 tumor cell line highly produced prostaglandin E2, and expressed COX-2, of which expression was confirmed by western blot (data not shown). RAW 264.7, a mouse monocyte/macrophage cell line, was purchased from American Type Culture Collection (Manassas, VA). RAW 264.7 cells were maintained in DMEM with L-glutamine (Invitrogen, Tokyo, Japan) containing 10% FBS.

Animals

CDF1 mice, an F1 hybrid of Balb/c and DBA/2 mice, were purchased at the age of 6–8 weeks from Charles River Japan (Yokohama, Japan), and specific pathogen-free female BALB/cA Jcl-nu mice (nude mice) were purchased at the age of 6 weeks from CLEA Japan (Tokyo, Japan). Diet pellets (FR-2, Funabashi Farms, Funabashi, Japan) and water were given to the mice ad libitum. All the animal care and experiments were conducted under the standard operational protocol of the Sankyo Institutional Animal Care and Use Committee.

Evaluation of antitumor activity of CS-706 in tumor-bearing mice

The colon 26 cell suspension of 1 × 106 cells/0.1 ml was inoculated subcutaneously into the right axillary region of the CDF1 mice. CS-706 and celecoxib were suspended in vehicle (0.5% carboxymethyl cellulose solution) at appropriate concentrations and orally administered to the mice. The compounds were daily administered from Day 0 or from Day 7 after tumor inoculation according to the study protocols.

In the experiment where administration was started from Day 0, the mice were inoculated with tumors, divided into experimental groups (n = 10) on the same day, and administered with CS-706 or celecoxib at the doses of 1, 3, 10, 30, and 100 mg/kg. In the experiment where administration was started from Day 7, the mice were divided into experimental groups (n = 10) on Day 7, when the average tumor reached around 300 mm3 (note that over 95% of the tumors were in the range of 200 mm3 and 400 mm3, average volume: 292 ± 54 mm3). Then, CS-706 at doses of 3 and 30 mg/kg was daily administered.

In our preliminary study, the maximum tolerated dose of cisplatin (intravenously (i.v.), 1 shot) and 5-FU (orally, 3 times per week) were determined to be 10 and 60 mg/kg, respectively, in the colon 26 tumor-bearing (TB) mice. We therefore used 10 mg/kg of cisplatin and 60 mg/kg 5-FU in the following experiment. The grouping of the mice was performed on Day 7, as mentioned earlier, and then cisplatin (i.v., 1 shot, on Day 7) alone or in combination with CS-706 or celecoxib (at doses of 3 and 30 mg/kg, from Day 7, everyday) was administered. 5-FU (orally, from Day 1 to Day 13, every 3 days) was administered alone or in combination with CS-706 (at dose of 10 mg/kg, from Day 0).

The tumor volume (mm3) was calculated as a × b2/2, where a and b are the largest and the smallest perpendicular diameters, respectively. The tumor growth inhibition (TGI %) was calculated as (1 − T/C) × 100, where T and C are the mean tumor volume of the compound-treated mice and the mean tumor volume of the vehicle-treated mice, respectively. The tumor growth rates (TGR mm3/day) in each mouse were represented as the slope of the tumor volume in order to analyze the inhibitory effect of the compounds. The slopes of the tumor volume were calculated by linear regression analysis. To evaluate the life-prolonging effect of the compounds, the median survival time (MST days) after tumor inoculation for each group was determined from the survival times of the mice according to a Kaplan-Meier plot using SAS System Release 8.2 (SAS Institute, Cary, NC). The increase in the life span (ILS %) was calculated as (TMST/CMST − 1) × 100, where TMST and CMST are the MST of the compound-treated mice and the MST of the vehicle-treated control mice, respectively. A mouse that exhibited no palpable tumor for more than 2 weeks was regarded as a complete regression (CR).

Measurement of anti-angiogenic activity of CS-706 in vivo

To evaluate the anti-angiogenic activity of CS-706 in vivo, we used a dorsal air sac (DAS) assay.25, 26 Millipore chambers covered with Millipore filters of 0.45 μm pore size were filled with Hank's Balanced Salt Solution (HBSS, Invitrogen, Tokyo, Japan) or colon 26 cell suspension, and implanted subcutaneously into dorsal air sacs created by the injection of an appropriate volume of air under the skin of nude mice (Day 0). In our preliminary experiments, we optimized the cell concentration and suspension volume in the chamber and found that 3 × 106 cells/0.15 ml is appropriate. For a negative control to evaluate the effect of the chamber itself, an air sac was formed on the mice without the implantation of any chambers, and these mice were designated as the sham-operated group. The mice with colon 26 cell-filled chambers were then administered vehicle (0.5% carboxymethyl cellulose solution) or CS-706 at a dose of 10 mg/kg orally for 4 days from Day 0, and the mice with HBSS-filled chambers or the sham-operated mice received neither CS-706 nor vehicle. Each experimental group included 12 mice. The preparation and administration of the compounds was performed in the same way as in the experiment to evaluate antitumor activity. On Day 4, the mice were sacrificed and the area of neovascularization was photographed. The skin of the mice in the sham-operated group was also resected at the region where it was supposed that a chamber would be implanted. An observer who was blinded to the randomized sample numbers evaluated the total lengths of the vessels in each mouse using Win ROOF Ver. 3.1 (Mitani Corp., Fukui, Japan), by tracing the vessels in the neovasculature. The vessel length was represented as pixels. The percent inhibition of the vessel length (VLI %) was calculated as (1 − (TV − HV)/(CV − HV)) × 100, where TV, HV and CV are the mean length of the compound-treated group, the mean length of the HBSS-control group and the mean length of the vehicle-treated group, respectively.

Production of VEGF by RAW 264.7 cells

RAW 264.7 cells were seeded at 1 × 104 cells/well in 96-well plates 24 hr before the compound addition. Then, CS-706 at final concentrations ranging from 0.01 to 300 nM and LPS at a final concentration of 1 μg/mL was added into the appropriate wells. CS-706 and LPS were diluted with DMSO and physiological saline, respectively, and the final concentration of DMSO was 0.1%. DMSO alone at the final concentration of 0.1% was added to the wells as a negative control. The 96-well plates were placed in a CO2 incubator for 24 hr and then the culture supernatant was collected and kept at −80°C until use in the following assay. The number of samples in each group was 4–8. The concentrations of VEGF and prostaglandin E2 (PGE2) in the culture supernatant were measured directly using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's directions. We used AN′ALYZA M mouse VEGF Quantitative Colorimetric Sandwich ELISA (TECHNE Corp., Minneapolis, MN) for mouse VEGF and Prostaglandin E2 EIA Kit-Monoclonal (Cayman Chemical, Ann Arbor, MI) for PGE2.

Statistical analysis

We used a Dunnett's test and a log-rank test based on the joint-ranking method for evaluation of the inhibitory activity on tumor growth and life-prolonging activity, respectively, to compare the vehicle-treated or chemotherapeutic agent (cisplatin or 5-FU) alone groups to the compound-treated group. The time to death of the CS-706-treated mice was compared with that of the celecoxib-treated mice by a Cox proportional hazards model with a continuous variable “Log Dose” as the covariate. Applying this model, the hazard ratio for every 10-fold increase in the dose of each compound was estimated and the hazard ratio of the CS-706-treated mice was compared with that of the celecoxib-treated mice by a Wald's test. A t-test and a Dunnett's test were performed for evaluation of the inhibitory activity on cytokine production and on colon 26-induced angiogenesis in the DAS assay. A p value of less than 0.05 was considered to be statistically significant. We abbreviated the 95% confidential intervals in the Table in order to prevent the Table from becoming too jumbled.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Effect of CS-706 on tumor growth and survival of colon 26 tumor-bearing mice

When CS-706 was daily administered from Day 0 after tumor inoculation, CS-706 dose-dependently inhibited tumor growth at doses above 3 mg/kg, and a 67% maximal tumor growth inhibition (TGI) was observed at the highest dose of 100 mg/kg on Day 14 (p < 0.05, Fig. 1a and Table I). It is noteworthy that 100 mg/kg CS-706 achieved CR in 1 of the 10 mice. On the contrary, celecoxib showed very weak inhibition of tumor growth up to 100 mg/kg, and the TGI by celecoxib at the dose of 100 mg/kg was 8% on Day 14 (Fig. 1b and Table I). CS-706 at doses of 1–100 mg/kg markedly prolonged the MST of tumor-bearing mice, and the ILS were more than 110% at doses examined (Table I). Celecoxib also prolonged the MST at doses above 10 mg/kg, though the ILS at a dose of 10 mg/kg was only 41% (Table I). Because the CS-706 treatment significantly decreased the hazard ratio more than the celecoxib treatment (Cox regression analysis and Wald's test, p < 0.05), CS-706 was demonstrated to have more potent life prolonging activity as compared with that of celecoxib.

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Figure 1. Antitumor activities of CS-706 in the colon 26 tumor-bearing mice. Mouse colorectal adenocarcinoma colon 26 cells were inoculated subcutaneously into female CDF1 mice (Day 0). The COX-2 inhibitors were orally administered daily from Day 0 as described in Material and Methods. The graphs show the results of tumor growth in (a) CS-706 and (b) celecoxib-treated groups. Since experiments (a) and (b) were conducted at the same time, the control curves in (a) and (b) are identical. Each experimental group consisted of 10 mice, respectively. Tumor volumes were determined until one of the compound-treated mice died. Data are shown as the mean ± standard error of the mean (SEM). n.s., not significant. *p < 0.05 compared with Tumor-bearing (TB) control by a parametric Dunnett's test.

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Table I. Anti-Tumor Activities of CS-706 and Celecoxib when Treated Alone from Day 0 After Tumor Inoculation
  Tumor growthSurvial timeCR
  TGR (mm3/day)TGI (%, Day 14)MST (Days)ILS (%) 
  • Tumor volumes were determined until one of the compound-treated mice died. TB, tumor-bearing; TGR, rate of tumor growth from Day 7 to Day 14; TGI, tumor growth inhibition; MST, median survival time; ILS, increase in life span; CR, complete response.

  • 1

    p < 0.05 (versus TB control, parametric Dunnett's test).

  • 2

    p <0.05 (versus TB control, Log rank test).

TB control 22018.50/10
CS-7061 mg/kg1821542.511300/10
 3 mg/kg12723939.011110/10
 10 mg/kg11524546.511510/10
 30 mg/kg11324844.011380/10
 100 mg/kg6826747.011541/10
Celoxib1 mg/kg245−1433.0780/10
 3 mg/kg219−524.5320/10
 10 mg/kg252−1326.01410/10
 30 mg/kg201739.011110/10
 100 mg/kg179835.01890/10

Similar superior antitumor activities of CS-706 were also demonstrated in the experiments where CS-706 was administered from Day 7 when the average tumor volume reached around 300 mm3. CS-706 treatment from Day 7 at a dose of 30 mg/kg significantly inhibited the growth of tumors on Day 14 by 43% (p < 0.05), and prolonged the MST with an ILS of 98% (p < 0.05, supplementary Fig. 1).

Antitumor effects of CS-706 combined with a chemotherapeutic agent

A single treatment of 10 mg/kg cisplatin on Day 7 showed marked tumor growth inhibition (TGI: 79% on Day 25) and significant prolongation in the survival time of the tumor-bearing mice by 107% ILS (Figs. 2a and 2c). However, when cisplatin was given in combination with CS-706 (3, 30 mg/kg), the tumor growth inhibitory activity and life-prolonging activity of cisplatin were markedly potentiated in a dose-dependent manner. Especially when combined with 30 mg/kg CS-706, all the tumors were completely regressed in the cisplatin and CS-706-treated tumor-bearing mice, although the cisplatin alone-treated group showed only 1 CR (Figs. 2a and 2c). Similar results were also observed when 30 mg/kg celecoxib was administered in combination with cisplatin, but with much weaker activity and only 2 CRs (Figs. 2b and 2d). These results clearly demonstrated that CS-706 showed a marked combination antitumor effect with cisplatin. In addition, the antitumor activity of CS-706 with cisplatin seemed to be more potent than the activity of celecoxib with cisplatin.

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Figure 2. Antitumor activities of CS-706 in combination with cisplatin. Antitumor activities of CS-706 in combination with chemotherapeutic agents, cisplatin. Mouse colorectal adenocarcinoma colon 26 cells were inoculated subcutaneously into female CDF1 mice (Day 0) and the COX-2 inhibitors, CS-706 (a and c) and celecoxib (b and d), were administered daily from Day 7 as described in Material and Methods. Cisplatin at a single dose of 10 mg/kg was administered on Day 7. (a and b) tumor growth in volume (mm3) and (c and d) survival rate in percentage. Since experiment of CS-706 (a and c) and that of celecoxib (b and d) were conducted at the same time, the control curves in graphs of CS-706 (a and c) and those of celecoxib (b and d) are identical, respectively. Solid bold line, tumor-bearing (TB) control; gray bold line, cisplatin at a dose of 10 mg/kg; closed triangles, CS-706 at a dose of 3 mg/kg and cisplatin; closed circles, CS-706 at a dose of 30 mg/kg and cisplatin; open triangles, celecoxib at a dose of 3 mg/kg and cisplatin; open circles, celecoxib at a dose of 30 mg/kg and cisplatin. Each experimental group consisted of 10 mice, respectively. Tumor volumes were determined until one of the compound-treated mice died. Since TB control mice started dying too early (from Day 17), the data after day 17 consist of less than 10 mice. Data are shown as the mean ± SEM. The parenthesized italic number in the figure represents the number of complete response in the group. $p < 0.05 compared with cisplatin by a parametric Dunnett's test; #p < 0.05 compared with TB control by a Log-rank test; p < 0.05 compared with cisplatin by a Log-rank test.

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Similar results were also obtained in the combination treatment of CS-706 and 5-FU. When 10 mg/kg CS-706 (from Day 0 after tumor inoculation, everyday) was administered in combination with 60 mg/kg 5-FU (from Day 1 to Day 13, every 3 days), the life prolongation was remarkably enhanced (ILS: 213%, p < 0.05) when compared with 5-FU alone (ILS: 59%, supplementary Fig. 2). Moreover, 2 of 10 mice in combination treatment of CS-706 and 5-FU showed CR, whereas no tumor-free mice were observed under 5-FU treatment alone (supplementary Fig. 2). These results also demonstrated the marked combination effects of CS-706 with 5-FU.

Inhibitory effects of CS-706 on angiogenesis induced by implanted chambers with colon 26 cells

A DAS assay was performed in BALB/cA Jcl-nu (nude) mice to examine the inhibitory activities of CS-706 on neovascularization induced by implanted chambers with colon 26 cells. In the skins of the nude mice in the sham-operated group and in the HBSS-control group, the mean length (±SEM) of the neovascularity were 40 (±40), and 166 (±50) pixels/mouse, respectively (Fig. 3). In contrast, the implantation of chambers with colon 26 cells to the mice remarkably increased the neovascularization, and the mean vessel length of the neovascularity was 1,971 (±276) pixels/mouse. CS-706 treatment at a dose of 10 mg/kg inhibited neovascularization, and the mean vessel length of the CS-706-treated neovascularity was 1,222 (±194) pixels/mouse with 41% inhibition (p < 0.05, Fig. 3).

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Figure 3. Inhibitory effects of CS-706 on angiogenesis induced by implanted chambers with colon 26 cells. BALB/cA Jcl-nu mice were implanted with chambers filled with HBSS or colon 26 tumor cells. The mice with colon 26 cell-filled chambers were administered for four days with vehicle or CS-706 at a dose of 10 mg/kg as described in Material and Methods. The mice with HBSS-filled chambers or the sham-operated mice received neither CS-706 nor vehicle. Vessel length in the abnormal neovasculature in the skin in contact with diffusion membrane on chambers was measured using Win ROOF (Ver. 3.1) software and represented as pixels. Data are shown as the mean ± SEM, n = 12, t-test.

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Inhibitory activity of CS-706 against VEGF production in vitro

We then studied the inhibitory activity of CS-706 against production of VEGF that is one of the important factors for angiogenesis. Mouse monocyte/macrophage cell line RAW 264.7 cells activated with 1 μg/ml lipopolysaccharide (LPS) produced 157.6 ± 11.01 pg/ml of VEGF (mean ± SEM, Fig. 4a), whereas control RAW 264.7 cells produced less than 10 pg/ml of VEGF without LPS stimulation (Fig. 4a). The addition of CS-706 at the concentrations of 0.1–300 nM inhibited production of VEGF in LPS-stimulated RAW 264.7 cells, and the inhibitory activity of CS-706 against VEGF production reached a plateau of approximately 50% at concentrations above 30 nM.

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Figure 4. CS-706 inhibited VEGF and PGE2 production in LPS-stimulated RAW 264.7 cells. The RAW 264.7 cells were plated in a 96-well plate and precultured for 24 hr. The cells were stimulated by LPS at the final concentration of 1 μg/ml. At the same time, CS-706 was added at various concentrations and cultured for 24 hr. Concentrations of VEGF (a) and PGE2 (b) in the culture supernatant were determined by ELISA. The N in the figure represents the number of samples in each treatment group. Data are shown as the mean ± SEM. *p < 0.05 compared with LPS-untreated control by a t-test; #p < 0.05 compared with LPS-treated control by a Dunnett's test.

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In the parallel experiments, we determined PGE2 production in RAW 264.7 cells. CS-706 potently inhibited the LPS-induced PGE2 production in a concentration dependent manner at concentrations above 0.3 nM, and 30 nM CS-706 nearly completely inhibited the PGE2 production (IC50 = 2.8 nM, Fig. 4b). Note that the addition of 1 μM PGE2 increased VEGF production in RAW 264.7 cells, and the increased VEGF production was not inhibited by 300 nM CS-706 (data not shown). On the other hand, it has been shown that LPS induces VEGF production via a Toll-like receptor-NF-κB pathway27 other than the PGE2 pathway. These results suggested that CS-706 partially inhibited the VEGF production through the inhibition of PGE2 production.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this report, we have demonstrated that daily oral treatment with CS-706, a novel COX-2 inhibitor, exhibited potent tumor growth inhibitory activity (Fig. 1a) and life-prolonging activity (Table I) in colon 26 tumor-bearing mice. Furthermore, when administered together with conventional chemotherapeutic agents such as cisplatin or 5-FU, the combination activity of CS-706 was very marked and synergistic (Figs. 2a and 2c and supplementary Fig. 2). It is noteworthy that all the tumors in the tumor-bearing mice, i.e. 10 of 10 tumor-bearing mice, regressed completely by 30 mg/kg CS-706 treatment in combination with a single dosing of 10 mg/kg cisplatin, while 10 mg/kg cisplatin-alone treatment regressed the tumor for only 1 mouse, out of 10 mice (Fig. 2).

CS-706 showed potent life-prolonging activity not only in mice treated from Day 0 but also in mice treated from Day 7 (established tumors about 300 mm3 in average tumor volume, Table I and supplementary Fig. 1b). In addition, CS-706 with cisplatin resulted in increased survival in established tumor bearing mice (Fig. 2c). These results indicate the potent therapeutic activity of CS-706.

Concerning the antitumor mechanisms of the COX-2 inhibitors, there are numerous reports on the possible antitumor mechanisms of celecoxib or rofecoxib both in vitro and in vivo, including the induction of apoptosis of tumor cells,28, 29 the direct inhibition of tumor cell proliferation,30, 31, 32, 33 the inhibition of COX-2-induced increase in tumor invasiveness34 and the inhibition of COX-2-induced tumor-angiogenesis.35 In our preliminary in vitro experiments, we confirmed that CS-706 at concentrations up to 10 μM did not show any cytotoxic effect on colon 26 cells in a 3-day exposure, though it potently inhibited the production of PGE2 in a 24-hr exposure (data not shown). In addition, 10 μM CS-706 induced neither apoptosis nor cell cycle arrest in the cultured tumor cells. Furthermore, CS-706 did not modulate TNF production in LPS-stimulated macrophages and did not activate peroxisome proliferator-activated receptor (PPAR) γ (data not shown), although a great number of reports have demonstrated that other COX-2 inhibitors or NSAIDs have such activities.36, 37 However, as shown in Figures 3 and 4, CS-706 inhibited angiogenesis induced by implanted chambers with colon 26 cells in a dorsal air sac model in vivo, and also attenuated the production of VEGF in LPS-stimulated RAW 264.7 cells, probably through the inhibition of PGE2 production. Consequently, it is anticipated, as also indicated with other COX-2 inhibitors in various tumor models,35 that the inhibition of angiogenesis may be involved at least in part in the significant tumor-growth inhibitory activity demonstrated by CS-706. The synergy in antitumor activity observed in the combination studies with either cisplatin or 5-FU may be explained by the different mechanism of action between CS-706 and these conventional cytotoxic agents.

Since the importance of anti-angiogenic activities is also shown in the growth and progression of metastatic tumors,38, 39 we examined if CS-706 had an anti-metastatic activity in mouse experimental metastasis model. In a mouse Lewis lung carcinoma (LLC) model, in which LLC cells were subcutaneously inoculated, and the resultant tumors were excised to allow metastasis, CS-706 inhibited the number of lung nodules (data not shown). In other preliminary studies, in which mice were subcutaneously inoculated with colon 26 tumor cells, we often found metastatic nodule in lungs. These results may support the marked survival prolongation of the tumor-bearing mice as results of the decreased growth and progression of metastatic colon 26 cells by CS-706. The antimetastatic mechanisms of CS-706 are now under examination.

In the present studies, we found that the antitumor activity of CS-706 was very marked when compared with that of celecoxib. Our preliminary results demonstrated that the COX-2 inhibitory activity of CS-706 was only about 3 times as potent as that of celecoxib in a human whole blood assay. Furthermore, our preliminary pharmacokinetic studies using 10 mg/kg CS-706 and 10 mg/kg celecoxib demonstrated that the Cmax and AUC0–24 hr of CS-706 was approximately half as high as that of celecoxib in mice. From these results, it is indicated that the COX-2 inhibitory activity of 10 mg/kg CS-706 was almost equivalent to that of 10 mg/kg celecoxib in mice in this calculation. Therefore, it is difficult to explain the potent antitumor activity of CS-706 demonstrated with or without chemotherapeutic agents, when compared with that of celecoxib at the same/above dose, solely by differences in COX-2 inhibitory activity. Next, we examined whether there is an alternative mechanism such as COX-1 that might be involved in the differing strengths of the antitumor effects. In our preliminary experiments, we measured the inhibition of human whole blood COX-1 activities in the presence of CS-706 and celecoxib. The COX-1 inhibitory activity of CS-706 was only around 4 times as potent as that of celecoxib. The inhibitory profiles of COX-1 by CS-706 and celecoxib seem to be similar to those of COX-2. We then carried out in vivo experiments to determine the accumulation of each compound. The radioisotope labeled compounds, [14C]CS-706 and [14C]celecoxib were orally administered to rats and the radioisotope activities were counted in various tissues. Importantly, the isotope levels were comparable in all the tissues examined.40 The above results may suggest unique characteristics of CS-706 or the possible involvement of an unknown mechanism in the potent antitumor activity of CS-706, and this mechanism will need to be clarified in the future.

It has been shown that the colon 26 tumor-bearing mice were cachectic41 at the late stage of tumor-bearing. In addition, PGE2, either produced by colon 26 cells or inflammatory macrophages, is known to be one of the well-known cachectic molecules.42 It may be that CS-706 was very effective in prolonging the survival of colon 26 tumor-bearing mice through inhibiting the colon 26-mediated cachexia. Studies on the effect of CS-706 on colon 26 tumor-induced cachexia are now in progress.

Although the precise mode of action of the marked antitumor activity of CS-706 when compared with that of celecoxib in colon 26 tumor-bearing mice is not clear yet and needs further clarification, CS-706 is expected to be a potent antitumor COX-2 inhibitor in clinical situations in combination with conventional chemotherapeutic agents.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We would like to thank Naomi Shimazaki, Fumie Tanzawa and Shigeru Ushiyama for their assistance in the assays and for their constructive criticism.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Cohn SM,Schloemann S,Tessner T,Seibert K,Stenson WF. Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1. J Clin Invest 1997; 99: 136779.
  • 2
    Smith WL,Langenbach R. Why there are two cyclooxygenase isozymes. J Clin Invest 2001; 107: 14915.
  • 3
    Chandrasekharan NV,Dai H,Roos KL,Evanson NK,Tomsik J,Elton TS,Simmons DL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci USA 2002; 99: 1392631.
  • 4
    Wolff H,Saukkonen K,Anttila S,Karjalainen A,Vainio H,Ristimaki A. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res 1998; 58: 49975001.
  • 5
    Tucker ON,Dannenberg AJ,Yang EK,Zhang F,Teng L,Daly JM,Soslow RA,Masferrer JL,Woerner BM,Koki AT,Fahey TJ,III. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res 1999; 59: 98790.
  • 6
    Parrett ML,Harris RE,Joarder FS,Ross MS,Clausen KP,Robertson FM. Cyclooxygenase-2 gene expression in human breast cancer. Int J Oncol 1997; 10: 5037.
  • 7
    Liu CH,Chang SH,Narko K,Trifan OC,Wu MT,Smith E,Haudenschild C,Lane TF,Hla T. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276: 185639.
  • 8
    Jacoby RF,Seibert K,Cole CE,Kelloff G,Lubet RA. The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res 2000; 60: 50404.
  • 9
    Reddy BS,Hirose Y,Lubet R,Steele V,Kelloff G,Paulson S,Seibert K,Rao CV. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res 2000; 60: 2937.
  • 10
    Gupta S,Adhami VM,Subbarayan M,MacLennan GT,Lewin JS,Hafeli UO,Fu P,Mukhtar H. Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res 2004; 64: 333443.
  • 11
    Fischer SM,Lo HH,Gordon GB,Seibert K,Kelloff G,Lubet RA,Conti CJ. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol Carcinog 1999; 25: 23140.
  • 12
    Harris RE,Alshafie GA,Abou-Issa H,Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res 2000; 60: 21013.
  • 13
    Kubatka P,Ahlers I,Ahlersova E,Adamekova E,Luk P,Bojkova B,Markova M. Chemoprevention of mammary carcinogenesis in female rats by rofecoxib. Cancer Lett 2003; 202: 1316.
  • 14
    Steinbach G,Lynch PM,Phillips RK,Wallace MH,Hawk E,Gordon GB,Wakabayashi N,Saunders B,Shen Y,Fujimura T,Su LK,Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 2000; 342: 194652.
  • 15
    Bertagnolli MM,Eagle CJ,Zauber AG,Redston M,Solomon SD,Kim K,Tang J,Rosenstein RB,Wittes J,Corle D,Hess TM,Woloj GM, et al. Celecoxib for the prevention of sporadic colorectal adenomas. N Engl J Med 2006; 355: 87384.
  • 16
    Harris RE,Beebe-Donk J,Alshafie GA. Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors. BMC Cancer 2006; 6: 27.
  • 17
    Zhang X,Chen ZG,Choe MS,Lin Y,Sun SY,Wieand HS,Shin HJ,Chen A,Khuri FR,Shin DM. Tumor growth inhibition by simultaneously blocking epidermal growth factor receptor and cyclooxygenase-2 in a xenograft model. Clin Cancer Res 2005; 11: 62619.
  • 18
    Sawaoka H,Kawano S,Tsuji S,Tsujii M,Gunawan ES,Takei Y,Nagano K,Hori M. Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am J Physiol 1998; 274: G10617.
  • 19
    Fu SL,Wu YL,Zhang YP,Qiao MM,Chen Y. Anti-cancer effects of COX-2 inhibitors and their correlation with angiogenesis and invasion in gastric cancer. World J Gastroenterol 2004; 10: 19714.
  • 20
    Csiki I,Morrow JD,Sandler A,Shyr Y,Oates J,Williams MK,Dang T,Carbone DP,Johnson DH. Targeting cyclooxygenase-2 in recurrent non-small cell lung cancer: a phase II trial of celecoxib and docetaxel. Clin Cancer Res 2005; 11: 663440.
  • 21
    Prince HM,Mileshkin L,Roberts A,Ganju V,Underhill C,Catalano J,Bell R,Seymour JF,Westerman D,Simmons PJ,Lillie K,Milner AD, et al. A multicenter phase II trial of thalidomide and celecoxib for patients with relapsed and refractory multiple myeloma. Clin Cancer Res 2005; 11: 550414.
  • 22
    Ushiyama S,Yamada T,Murakami Y,Kumakura S,Inoue S,Suzuki K,Nakao A,Kawara A,Kimura T. Preclinical pharmacology of CS-706, a novel cyclooxygenase-2 selective inhibitor, with potent antinociceptive and anti-inflammatory effects. Eur J Pharmacol, Sep 11, 2007, (Epub ahead of print).
  • 23
    Moberly JB,Harris SI,Riff DS,Dale JC,Breese T,McLaughlin P,Lawson J,Wan Y,Xu J,Truitt KE. A randomized double-blind one-week study comparing effects of a novel COX-2 inhibitor and naproxen on the gastric mucosa. Dig Dis Sci 2007; 52: 44250.
  • 24
    Rohatagi S,Kastrissios H,Truitt1 K,Moberly J,Wada R,Salazar D. Pain relief model for a Cox-2 inhibitor in patients with post-operative dental pain, Submitted for publication.
  • 25
    Yonekura K,Basaki Y,Chikahisa L,Okabe S,Hashimoto A,Miyadera K,Wierzba K,Yamada Y. UFT and its metabolites inhibit the angiogenesis induced by murine renal cell carcinoma, as determined by a dorsal air sac assay in mice. Clin Cancer Res 1999; 5: 218591.
  • 26
    Goi T,Fujioka M,Satoh Y,Tabata S,Koneri K,Nagano H,Hirono Y,Katayama K,Hirose K,Yamaguchi A. Angiogenesis and tumor proliferation/metastasis of human colorectal cancer cell line SW620 transfected with endocrine glands-derived-vascular endothelial growth factor, as a new angiogenic factor. Cancer Res 2004; 64: 190610.
  • 27
    Kiriakidis S,Andreakos E,Monaco C,Foxwell B,Feldmann M,Paleolog E. VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2. J Cell Sci 2003; 116: 66574.
  • 28
    Hsu AL,Ching TT,Wang DS,Song X,Rangnekar VM,Chen CS. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000; 275: 11397403.
  • 29
    Wu GS,Zou SQ,Liu ZR,Tang ZH,Wang JH. Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines. World J Gastroenterol 2003; 9: 13026.
  • 30
    Kaur BS,Khamnehei N,Iravani M,Namburu SS,Lin O,Triadafilopoulos G. Rofecoxib inhibits cyclooxygenase 2 expression and activity and reduces cell proliferation in Barrett's esophagus. Gastroenterology 2002; 123: 607.
  • 31
    Cui W,Yu CH,Hu KQ. In vitro and in vivo effects and mechanisms of celecoxib-induced growth inhibition of human hepatocellular carcinoma cells. Clin Cancer Res 2005; 11: 821314.
  • 32
    Patel MI,Subbaramaiah K,Du B,Chang M,Yang P,Newman RA,Cordon-Cardo C,Thaler HT,Dannenberg AJ. Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism. Clin Cancer Res 2005; 11: 19992007.
  • 33
    Waskewich C,Blumenthal RD,Li H,Stein R,Goldenberg DM,Burton J. Celecoxib exhibits the greatest potency amongst cyclooxygenase (COX) inhibitors for growth inhibition of COX-2-negative hematopoietic and epithelial cell lines. Cancer Res 2002; 62: 202933.
  • 34
    Tsujii M,Kawano S,DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997; 94: 333640.
  • 35
    Masferrer JL,Leahy KM,Koki AT,Zweifel BS,Settle SL,Woerner BM,Edwards DA,Flickinger AG,Moore RJ,Seibert K. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 2000; 60: 130611.
  • 36
    Lin HI,Chu SJ,Wang D,Feng NH. Pharmacological modulation of TNF production in macrophages. J Microbiol Immunol Infect 2004; 37: 815.
  • 37
    Lehmann JM,Lenhard JM,Oliver BB,Ringold GM,Kliewer SA. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem 1997; 272: 340610.
  • 38
    Mahadevan V,Hart IR. Tumour angiogenesis and metastasis. Eur J Cancer 1991; 27: 67980.
  • 39
    Bikfalvi A. Significance of angiogenesis in tumour progression and metastasis. Eur J Cancer 1995; 31: 11014.
  • 40
    Oitate M,Hirota T,Koyama K,Inoue S,Kawai K,Ikeda T. Covalent binding of radioactivity from [14C]rofecoxib, but not [14C]celecoxib or [14C]CS-706, to the arterial elastin of rats. Drug Metab Dispos 2006; 34: 141722.
  • 41
    Tanaka Y,Eda H,Tanaka T,Udagawa T,Ishikawa T,Horii I,Ishitsuka H,Kataoka T,Taguchi T. Experimental cancer cachexia induced by transplantable colon 26 adenocarcinoma in mice. Cancer Res 1990; 50: 22905.
  • 42
    Lorite MJ,Cariuk P,Tisdale MJ. Induction of muscle protein degradation by a tumour factor. Br J Cancer 1997; 76: 103540.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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