Anticancer

Celecoxib and 2,5-Dimethylcelecoxib (DM-celecoxib) inhibit proliferation and induced apoptosis in human colon cancer cell line HCT‐116.[1]
First, we examined the effect of celecoxib and DM‐celecoxib on the proliferation and apoptosis of the human colon cancer cell line HCT‐116, which expresses the wild‐type APC and a mutant form of β‐catenin. Both compounds inhibited HTC‐116 proliferation in a dose‐dependent manner (Fig. 1a). As we previously reported, celecoxib induces apoptosis, assessed by caspase‐3 activity measurement. As shown in Figure 1(b), DM‐celecoxib was also able to significantly elevate caspase‐3 activity.

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Celecoxib and DM‐celecoxib accelerate TCF7L2 degradation in HCT‐116 cells[1]
We previously reported that celecoxib induced degradation of TCF7L2, a key transcription factor in the Wnt/β‐catenin signaling pathway, in HCT‐116 cells. As shown in Figure 2(a,b), not only celecoxib but also DM‐celecoxib suppressed TCF7L2 expression in dose‐ and time‐dependent manners. As the effect of 50 μM DM‐celecoxib was almost comparable with 100 μM celecoxib, we mainly used these concentrations in the following experiments. Next, we examined the effect of the proteasome inhibitor MG132 on celecoxib‐ or DM‐celecoxib‐induced TCF7L2 reduction. Cells were treated with or without 10 μM MG132 for 1 h and then incubated in the presence or absence of 100 μM celecoxib or 50 μM DM‐celecoxib for 6 h. As shown in Figure 2(c), pretreatment with MG132 significantly attenuated the effects of celecoxib and DM‐celecoxib, indicating that both compounds accelerated the proteasome‐dependent degradation of TCF7L2.

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Celecoxib and DM‐celecoxib inhibit TCF‐dependent transcription in HCT‐116 cells[1]
As celecoxib and DM‐celecoxib induced TCF7L2 degradation in HCT‐116 cells, we examined the effects of celecoxib and DM‐celecoxib on TCF‐dependent transcription activity using the TOPflash assay. Celecoxib strongly inhibited TOPflash activity without affecting FOPflash (negative control) activity, confirming our previous results. 2,5‐Dimethylcelecoxib also showed a similar response (Fig. 3a). These results suggest that not only celecoxib but also DM‐celecoxib inhibit the transcription activity of the Wnt/β‐catenin signaling pathway target genes through TCF7L2 degradation.

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Effects of celecoxib and DM‐celecoxib on DLD‐1 cells[1]
We next examined whether celecoxib and DM‐celecoxib were able to inhibit cell proliferation in DLD‐1 cells, expressing wild‐type β‐catenin and a mutant form of APC. Both compounds clearly suppressed cell proliferation in a dose‐dependent manner (Fig. 4a), similar to that observed in HCT‐116 cells. Moreover, similar to their effects on HCT‐116 cells, both compounds markedly reduced the expression levels of TCF7L2, cyclin D1, and survivin, in DLD‐1 cells (Fig. 4b).

Effect of celecoxib and 2,5-Dimethylcelecoxib (DM-celecoxib) on oxidative stress‐induced cancers in Mutyh–/– mice [1]
We then carried out in vivo experiments. First, we examined whether, after oral administration, the plasma concentrations of celecoxib and DM‐celecoxib reached levels to show antiproliferative effects on tumor cells. We measured the drug concentrations in wild‐type C57BL/6J mice using an HPLC system. The concentration of celecoxib reached the maximal level (45.3 ± 6.3 μg/mL; n = 3) 2 h after treatment; the concentration of DM‐celecoxib reached the maximal level (110.7 ± 14.3 μg/mL; n = 3) more rapidly, within 1 h of treatment (Fig. 5). Because the EC50 values of celecoxib and DM‐celecoxib calculated from the in vitro antiproliferative assay (Figs 1a,4a) were approximately 15–19 μg/mL and 12–16 μg/mL, respectively, we hypothesized that the plasma concentrations of these compounds may be high enough to induce antiproliferative effects in vivo.[1]
To investigate the in vivo effects of celecoxib and 2,5-Dimethylcelecoxib (DM-celecoxib) , we used mice deficient for MUTYH (Mutyh −/−), an enzyme that prevents the formation of oxidative stress‐induced DNA damage. MUTYH deficiency has been associated with the development of colorectal adenomas and carcinomas in humans. We previously reported that the occurrence of oxidative stress‐induced carcinomas in the small intestine was dramatically increased in Mutyh −/− mice compared to normal mice.29, 35, 36 Twelve weeks of treatment with 0.2% KBrO3, a strong oxidant, induced the development of numerous intestinal carcinomas in Mutyh −/− mice. To evaluate the effect of the two compounds on these carcinomas, celecoxib, DM‐celecoxib, or the vehicle were given orally to Mutyh −/− mice for 4 weeks. Treatment with celecoxib or DM‐celecoxib markedly reduced the number of intestinal carcinomas (Fig. 6), and this was more pronounced in the case of large carcinomas with a diameter >1.0 mm (Fig. 6b). [1]
We also examined whether long‐term treatment with celecoxib and 2,5-Dimethylcelecoxib (DM-celecoxib) showed adverse effects on general animal condition. For this purpose, we measured mice body weight and peripheral blood cell counts, and we observed their general appearance and activity. Treatment with celecoxib or DM‐celecoxib did not affect their appearance, activity, white blood cell count, platelet numbers, or body weight of mice. Red blood cell count and hemoglobin concentrations tended to be elevated after celecoxib or DM‐celecoxib treatment, but the changes were not statistically significant (Table 1).

Measurement of celecoxib and 2,5-Dimethylcelecoxib (DM-celecoxib) plasma concentrations [1]
Murine blood samples were collected by cardiac puncture at the indicated times and plasma was isolated by centrifugation at 500g for 15 min. The plasma concentrations of celecoxib and DM‐celecoxib were determined by a reverse phase HPLC system, as previously described34 with a slight modification. Briefly, the plasma samples (200 μL) containing 500 ng caffeine as an internal standard were mixed with 200 μL chloroform. After mixing, the solution was centrifuged at 13 000g for 5 min, and the organic phase was then separated and evaporated. The obtained residue was dissolved in 80 μL mobile phase (methanol:water = 72:28, v/v) and an aliquot (50 μL) was then injected into a column for separation. The running time was 10 min, and the flow rate 1.0 mL/min. Samples were measured with a UV detector operating at 254 nm. A calibration curve was prepared by plotting the ratios of celecoxib or DM‐celecoxib areas normalized to that of the internal standard.

Cell proliferation assay [1]
HCT‐116 or DLD‐1 cells were seeded into 24‐well plates (5 × 104 cells/well) and treated with various concentrations of celecoxib or 2,5-Dimethylcelecoxib (DM-celecoxib) for the indicated periods. Cells were harvested by addition of trypsin/EDTA and counted using an automated cell counter (TC10; Bio‐Rad, Tokyo, Japan).

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Caspase‐3 activity assay [1]
Caspase‐3 activity was assayed using a cysteine protease protein 32/caspase‐3 colorimetric protease assay kit, following manufacturer's instructions.

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Western blot analysis [1]
Samples were separated with 12% SDS‐PAGE, and then transferred to a PVDF membrane using a semidry transfer system (1 h at 12 V). Proteins of interest were detected after incubation with primary and secondary antibodies, and were visualized using a detection reagent. Densitometric analysis was carried out using ImageJ software.

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Luciferase reporter assay [1]
TOPFlash (a TCF reporter plasmid) and FOPFlash (a TOPflash negative control) were used. Cells were cotransfected with luciferase reporter plasmids and pRL‐SV40, a Renilla luciferase expression plasmid (transfection efficiency control), using Lipofectamine Plus reagent. After 24 h, cells were stimulated with celecoxib or DM‐celecoxib for the indicated periods. Luciferase activity was determined with a luminometer and normalized against Renilla luciferase activity.

Intestinal tumor model Intestinal tumors (adenomas and carcinomas) were induced in Mutyh −/− mice by a method previously reported.29, 35, 36 Briefly, KBrO3 dissolved in water at a concentration of 2 g/L was given to 4‐week‐old mice for 12 weeks. At 16 weeks of age, mice were randomly divided into five groups (male:female = 1:1). The indicated amounts of celecoxib or DM‐celecoxib suspended in a 0.25% methylcellulose solution were given orally to mice in the test groups for 5 days/week over 4 weeks. Control mice received the vehicle only (methylcellulose). The body weight of the mice was monitored weekly. At 20 weeks of age, all mice were killed and blood and intestinal samples were collected. Blood cell counts were determined by a Celltac‐α MEK‐6358 (Nihon Kohden, Tokyo, Japan). Intestines were fixed in 4% formaldehyde, and the tumors were scrutinized under a microscope. Images of the tumors were analyzed using ImageJ software. For immunohistochemical analysis, tumors of 1.0–2.0 mm diameter that had developed 3.0–5.0 cm distal from the pylorus, were resected from formaldehyde‐fixed intestines. Samples were embedded in paraffin and subjected to immunohistochemical staining. Briefly, the sections were incubated with the anti‐TCF7L2 antibody (1:1000 dilution), the anti‐cyclin D1 antibody (1:100 dilution), or anti‐survivin antibody (1:100 dilution) overnight at 4°C, followed by incubation with the secondary antibody (Histofine; Nichirei, Tokyo, Japan) for 1 h. The sections were then analyzed with a Biozero microscope (Keyence, Osaka, Japan). For Western blot analysis, mucosal strips (2 cm to the bottom, from the pylorus) were homogenized in Laemmli's sample buffer immediately after resection. The same amount of proteins underwent electrophoresis and Western blotting.

[1]. Celecoxib and 2,5-dimethylcelecoxib inhibit intestinal cancer growth by suppressing the Wnt/β-catenin signaling pathway. Cancer Sci. 2017 Jan;108(1):108-115.

We previously reported that celecoxib, a selective COX-2 inhibitor, strongly inhibited human colon cancer cell proliferation by suppressing the Wnt/β-catenin signaling pathway. 2,5-Dimethylcelecoxib (DM-celecoxib), a celecoxib analog that does not inhibit COX-2, has also been reported to have an antitumor effect. In the present study, we elucidated whether DM-celecoxib inhibits intestinal cancer growth, and its underlying mechanism of action. First, we compared the effect of DM-celecoxib with that of celecoxib on the human colon cancer cell lines HCT-116 and DLD-1. 2,5-Dimethylcelecoxib suppressed cell proliferation and inhibited T-cell factor 7-like 2 expression with almost the same strength as celecoxib. 2,5-Dimethylcelecoxib also inhibited the T-cell factor-dependent transcription activity and suppressed the expression of Wnt/β-catenin target gene products cyclin D1 and survivin. Subsequently, we compared the in vivo effects of celecoxib and DM-celecoxib using the Mutyh-/- mouse model, in which oxidative stress induces multiple intestinal carcinomas. Serum concentrations of orally administered celecoxib and DM-celecoxib elevated to the levels enough to suppress cancer cell proliferation. Repeated treatment with celecoxib and DM-celecoxib markedly reduced the number and size of the carcinomas without showing toxicity. These results suggest that the central mechanism for the anticancer effect of celecoxib derivatives is the suppression of the Wnt/β-catenin signaling pathway but not the inhibition of COX-2, and that DM-celecoxib might be a better lead compound candidate than celecoxib for the development of novel anticancer drugs.[1]

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Our results indicate that both celecoxib and DM‐celecoxib inhibited proliferation and induced apoptosis in HCT‐116 cells, independent of their actions on COX‐2. Thus, the results obtained from the in vitro and in vivo experiments clearly show that the inhibition of COX‐2 is not essential for the antitumor effect of celecoxib, and that not only celecoxib but also DM‐celecoxib can inhibit tumor growth, even under the Wnt/β‐catenin signaling pathway activated condition, because they directly inhibit TCF7L2‐mediated transcription. Although it appeared that DM‐celecoxib showed slightly stronger effects compared with celecoxib in vitro and celecoxib showed slightly stronger effects in vivo, the effects resulting from the same concentration of these drugs were not significantly different, except for the effect on cyclin D1 expression in vitro (Fig. 3b). Therefore, the structural difference between celecoxib and DM‐celecoxib may not have significant influence on the ability of these drugs to inhibit the Wnt/β‐catenin signaling pathway by the degradation of TCF7L2. Further studies are required to elucidate the mechanism by which these drugs induce the degradation of TCF7L2.[1]

C18H16F3N3O2S

395.40

395.091

C, 54.68; H, 4.08; F, 14.41; N, 10.63; O, 8.09; S, 8.11

457639-26-8

169590-42-5

11545682

White to off-white solid powder

1.4±0.1 g/cm3

516.7±60.0 °C at 760 mmHg

266.3±32.9 °C

0.0±1.3 mmHg at 25°C

1.600

4.67

1

7

3

27

614

0

CC1=CC(=C(C)C=C1)C2=CC(=NN2C3=CC=C(C=C3)S(=O)(=O)N)C(F)(F)F

NTFOSUUWGCDXEF-UHFFFAOYSA-N

InChI=1S/C18H16F3N3O2S/c1-11-3-4-12(2)15(9-11)16-10-17(18(19,20)21)23-24(16)13-5-7-14(8-6-13)27(22,25)26/h3-10H,1-2H3,(H2,22,25,26)

4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide

457639-26-8; 2,5-Dimethyl Celecoxib; 2,5-dimethylcelecoxib; 2,5-dimethyl-celecoxib; 4-(5-(2,5-Dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide; Dimethyl Celecoxib; 2,5-DimethylCelecoxib-d4; 4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)