For cell culture, aspirin was purchased from Rhodia and indomethacin was from Spectrum Chemical (Gardena, CA, USA). For the animal study, aspirin (uncoated) was purchased from Walgreens (Deerfield, IL, USA). Aspirin-PC and Indomethacin-PC were prepared as described below for the cell culture and animal studies.

We used established procedures to prepare our PC-associated test drug formulations for cell culture and intragastric dosing (20,21). For cell culture, the Aspirin-PC stock solution was prepared as described previously (9). Briefly, the aspirin was firstly dissolved in the serum-free culture medium at 10 mmol/l and then combined with an equimolar amount of purified soy phosphatidylcholine/PC (S-100; Lipoid LLC, Newark, NJ, USA), which was previously dissolved in chloroform and then blown dry under nitrogen. The tubes were then sonicated at room temperature in a bath-type sonicator for 20 min until a homogenous suspension was obtained (Fig. 1 for the chemical structures of aspirin, indomethacin and soy PC). In the animal experiments, we used different procedures preparing Aspirin-PC as described previously (9,22). To make Indomethacin-PC stock for both cell culture and animal study, 8 gram of indomethacin (acid form) and 16 gram of Lipoid S-100 were subsequently dissolved into 60 ml of Acetone (Thermo Fisher Scientific, Inc., Fair Lawn, NJ, USA) in a 500-ml flat bottom round flask in 40°C water bath. Then the flask was connected to a rota-vaporator and vacuum-processed for 14–16 h to remove the acetone. Finally, the Indomethacin-PC was collected in a brown glass jar and kept at 4°C. To prepare the Indomethacin-PC solution for oral administration, the drug was weighed, and deionized distilled water added to a glass vial to the desired concentration and sonicated for 20 min at room temperature.

Murine colon cancer cells (MC-26) were obtained from the NIH National Cancer Institute. The cell line was cultured in suggested growth medium with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Tests for mycoplasma were negative and were conducted with the MycoAlert Mycoplasma Detection Kit from Lonza (Rockland, ME, USA). This cell line is known to express COX-2 (14).

MC-26 cells were preincubated with the drugs at a concentration range from 0 to 1.0 mmol/l (aspirin/Aspirin-PC) or 0 to 50 µmol/l (indomethacin/Indomethacin-PC) for 15 min to promote optimal exposure to our test-drugs, prior to pipetting the cells onto 48-well plates at a density of 2×10³ cells/well, and cultured at 37°C in a mixture of 5% CO₂ and 95% air. The cells were then cultured in the above growth medium in the presence and absence of the test drug formulations for 8 days with one medium change on the 4th day, at which time the culture medium was collected into 1.5 ml Eppendorf tubes and centrifuged at high speed for 10 min. Then the supernatant was collected for prostaglandin (PGE₂) assay as a measure of COX-2 activity. Cells on day 8 were used for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay as a measure of cell number as outlined below.

MTT (purchased from Sigma-Aldrich; Merck KGaA) was added to the culture media of cells at a final concentration of 0.5 mg/ml for 4 h. The purple formazan product was then extracted into a solvent (90% isopropanol, 0.2% sodium dodecyl sulfate, and 0.01 mol/l HCl) which was then collected from the wells and read at an absorbance of 570 nm, as previously described (22).

Young adult (20–24 g) male BALB/c mice were supplied by Harlan Laboratories, Inc. (Envigo, Indianapolis, IN, USA) and housed in the Center for Laboratory Animal Medicine and Care (CLAMC) facility at The University of Texas Health Science Center at Houston (UTHealth). Mice were maintained in accordance and compliance with policies approved by the Animal Welfare Committee (AWC), the Institutional Animal Care and Use Committee (IACUC) for The University of Texas Health Science Center at Houston (UTHealth). This facility is approved by the PHS and AAALAC.

On day one, mice were anesthetized and subjected to a laparotomy under isoflurane anesthesia as previously described (14,15,23), in order to inoculate their splenic capsules with 2×10⁵ cells/ml, 0.1 ml per mouse. Following the method of Yao et al cited above, immediately after cancer cell implantation, the mice were randomly divided into five treatment groups and treated once daily orally with vehicle (saline), aspirin (20 mg/kg), Aspirin-PC (20 mg ASA+20 mg PC/kg), indomethacin (2 mg/kg), or Indomethacin-PC (2 mg indomethacin + 4 mg PC/kg) and this treatment was continued daily for 28 days. A non-cancer group was also included as control. Thereafter, the mice were sacrificed and tissues were collected for analysis of tumor growth (spleen weight), possible GI injury due to NSAID (hematocrit), and metastatic cancer cell spread (liver weight and nodule number), plus fecal hemoglobin was assessed for evidence of GI bleeding, serum levels of Thromboxane B₂ (TXB₂) were assayed as a measure of NSAID pharmacologic action (inhibition of platelet COX-1 activity), and spleen tissue was assayed for PGE₂ as a measure of COX-2 activity.

Fecal hemoglobin (Hb) was monitored by collecting the fecal droppings at regular intervals from the bedding and storing them at −20°C until the day of analysis. The feces were weighed, and then distilled water was added at a 1:10 feces (g): Water (ml) ratio. After standing for 1 h, the feces were disrupted into a homogenous suspension by vortexing for 2 min and then the Hb analyzed by a previously described method (24).

The animal serum was analyzed by using the thromboxane B₂ EIA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's specifications. Blood of individual mice was collected at the end of the experiment under terminal anesthesia following a protocol for cardiac puncture, and serum was separated within 1 h following blood collection by centrifugation at 500 × g for 10 min, and then aliquoted and stored at −80°C for subsequent testing at a 1/200 dilution.

The MC-26 cell medium collected on day 4 of culture, and the excised animal spleen tissue were analyzed by using the Prostaglandin E₂ EIA kit (Cayman Chemical) according to the manufacturer's specifications. Splenic tumor tissues were homogenized in methanol, followed by SPE (C18) purification as suggested by the manufacturer's instruction. The final extract was resuspended in buffer and tested.

Statistical analyses were performed using the statistics application StatView 5.01 (SAS Institute Inc., Cary, NC, USA). Values are expressed as the mean ± standard error of the mean, and were evaluated by ANOVA followed by Fisher's PLSD test. A two-tailed value of P<0.05 was considered to indicate statistically significant differences.

The effects of our test drugs on the growth of MC-26 colon cancer cell line were examined over an 8-day culture period (Fig. 2A and B). Aspirin alone had an inhibitory effect on the growth of the cells only at the highest concentration of 1 mmol/l, while the PC complexed aspirin, Aspirin-PC, showed significant inhibition at the much lower concentration of 25 µmol/l (Fig. 2A). All of the Aspirin-PC concentrations gave significantly lower cell growth than the comparable doses of aspirin alone. In comparison, the other tested NSAID, indomethacin, was much more potent than aspirin with a significant inhibition of the cancer cell growth at a concentration of 20 µmol/l, and Indomethacin-PC was inhibitory at an even lower concentration of 8 µmol/l (Fig. 2B). The Indomethacin-PC concentrations of 8–50 µmol/l were significantly more effective at cell growth inhibition than comparable doses of indomethacin alone.

The expression of PGE₂ in culture medium (Fig. 2C and D) did not parallel the effects on cell growth for either NSAID. Aspirin alone (Fig. 2C) had no apparent effect on PGE₂ levels, while Aspirin-PC was significantly inhibitory at concentrations of 0.4–1 mmol/l, which was higher than the level that inhibited cell growth. In contrast, indomethacin alone (Fig. 2D) was a potent inhibitor of PGE₂, even at the lowest concentration tested. Once again, Indomethacin-PC was even more potent than the unmodified NSAID, with significantly lower levels of PGE₂ than indomethacin at every concentration. These concentrations of both indomethacin and Indomethacin-PC that inhibited PGE₂ levels were considerably lower than the concentrations that affected cell growth.

As described above, the MC-26 colon cancer study in mice was terminated after four weeks of cancer cell implantation and animal dosing. This time allowed for greater cancer cell growth as evidenced by spleen weights in vehicle-treated mice (20 mg/g body weight), compared to that of the non-cancer control mice (3.3 mg/g body weight). Treatment with indomethacin, Indomethacin-PC or Aspirin-PC gave clear and significant reductions (P<0.05) in splenic tumor nodules and spleen weights (Fig. 3A). However, aspirin alone was not protective in this model at the dose tested. Since a previous unpublished animal study showed PC alone had no effect on cancer cell growth in this model, we did not include a PC alone treatment group in this experiment.

An analysis of liver tissue revealed the presence of a number of metastatic tumor nodules (Fig. 3B), which tended to be reduced by treatment with indomethacin, Indomethacin-PC or Aspirin-PC, but not aspirin alone, similar to the spleen weight results. However, there were too few liver nodules to see a significant difference and the liver organs weights did not show differences either (not shown).

Assessments of GI bleeding showed no differences between treatment groups, with hematocrits in a normal range of 0.43 to 0.47 and fecal hemoglobin also showing minimal alterations (0.62 to 0.88 mg Hb/g feces) (Table I).

To verify that the NSAIDs used in this study were pharmacologically active, serum was analyzed for COX-1 activity by measurement of TXB₂ formed from platelets during blood clotting. Fig. 3C shows 80–90% inhibition of TXB₂ by all treatments, including aspirin alone, supporting the NSAIDs' ability to inhibit prostaglandin formation, a primary action of this class of drugs. It was noted that there was no difference between TXB₂ levels in non-cancer controls and vehicle (cancer) controls, suggesting there are no cancer-driven differences in platelet counts and/or activity. This lack of a difference was confirmed by platelet counts in a sampling of animals where measured values were between 366 to 634×10³/µl.

Spleen tissue levels of PGE₂ in Saline-treated cancer controls (Fig. 3D) were elevated over non-cancer Controls (~35%), but not significantly so, by the infiltration of cancer cells, as the Saline group was not different from Control (P=0.0708). There were apparent reductions of PGE₂ by all test NSAIDs, with indomethacin and Indomethacin-PC inducing the greatest inhibition (~84%). Aspirin and Aspirin-PC gave similar reductions of PGE₂ (~36% vs. saline-treated controls), although Aspirin-PC just missed the level of significance (P=0.0545).