Cytosolic phospholipase A2, cyclo-oxygenases and arachidonate in human stomach tumours.

Human stomach tumours usually form more prostaglandins (PGs) than their associated normal mucosa/submucosa, but the mechanisms are not fully understood. The key enzymes are cytosolic phospholipase A2 (cPLA2, Mr 85,000) and the cyclo-oxygenases (COXs) which exist in constitutive (COX-1) and inducible forms (COX-2). In human stomach tumours and associated macroscopically normal tissues, we determined the fatty acid composition by gas chromatography, amounts of cPLA2, COX-1 and COX-2 by immunoblotting with specific antibodies and cPLA2 enzyme activity using a tritiated substrate. Although compared to normal mucosa there was less arachidonate in tumours (P < 0.05), the arachidonate/total fatty acid ratio was higher. Mean amounts of cPLA2 and COX-1 and cPLA2 activity were similar in tumours and normal mucosa. However, substantial amounts of COX-2 were found in the tumours but not in the mucosa, which may explain why many gastric tumours form increased amounts of PGs.

Elevated aggrecan mRNA in early murine osteoarthritis.

Male STR/ort mice develop osteoarthritis in the tibial articular cartilage. Low grade histological lesions first appear between 10-20 weeks of age. A previous study showed that the level of aggrecan, the major cartilage proteoglycan, is approximately twofold greater in the tibial cartilage of 16-19-week-old STR/ort mice compared with that in normal cartilage in control CBA mice. In the present investigation aggrecan gene transcription was investigated in 20-week-old STR/ort and CBA tibial cartilage using a quantitative reverse transcription-polymerase chain reaction (RT-PCR). The amount of aggrecan cDNA obtained from the STR/ort medial and lateral plateau was 2.8- and 4.6-fold greater per milligram of wet cartilage than that from the CBA tibial plateau. The difference was not due to differences in cellularity of tibial cartilage in the two strains and indicates that aggrecan gene transcription is elevated in early osteoarthritis.

Articular cartilage proteoglycans in osteoarthritic STR/Ort mice.

Compared to controls, the lateral and medial tibial articular cartilage chondroitin sulfate (CS) content in male STR/Ort mice was elevated between 8 and 19 weeks of age, fell at 24-26 weeks and increased again thereafter. The CS cartilage content of CBA mice remained relatively unchanged. Cartilage CS content was measured in female CBA and STR/Ort mice and in male and female Balb C mice, all at 18 weeks of age. In every case the content was much lower than that found in male STR/Ort mice. CS unsaturated disaccharides were analysed by capillary electrophoresis after digestion of the glycosaminoglycans with chondroitinase ABC. Chondroitin-4-sulfate (C4S) was the predominant isomer at all ages in both strains. The C4S:C6S isomer ratio in CBA mice was much higher in the lateral than the medial cartilage. This difference was much less marked in STR/Ort knee joints: these mice have relatively more C6S than CBA mice. Proteoglycans, extracted from STR/Ort and CBA male mouse cartilage, were characterized by large pore gel electrophoresis and Western blotting. All cartilages contained two slow mobility bands identified as aggrecan by the reactivity with the mab 1C6. Both bands contained C4S and C6S chains. A third band of faster mobility contained only C4S and was 1C6 negative. It was present in both strains. Thus the STR/Ort cartilage contained a normal spectrum of murine articular cartilage proteoglycans.

A ruthenium-103 red dot blot assay specific for nanogram quantities of sulfated glycosaminoglycans.

Ruthenium-103 red has been used previously to detect nanogram quantities of glycosaminoglycans after they have been separated by electrophoresis on cellulose diacetate. We have applied the critical electrolyte principle to the binding of this dye to polyanions. This eliminates interaction with nucleic acids and hyaluronan. After samples are digested with chondroitinase ABC the method allows the measurement of chondroitin sulfates and heparan sulfates at the 2-ng level in dot blots of tissue extracts.

The effect of nifedipine alone or combined with cytotoxic chemotherapy on the mouse NC carcinoma in-vitro and in-vivo.

Effects of the calcium antagonist nifedipine on the response of the murine NC carcinoma has been examined alone and together with cytotoxic chemotherapy in-vitro and in-vivo. The cytotoxic drug combination of methotrexate and melphalan, or nifedipine alone (0.2-25 micrograms mL-1), caused a concentration-related reduction of NC cell growth in culture. At the lower concentrations, combination to the cytotoxic drugs with nifedipine resulted in an addition of the separate drug effects, but with drug concentrations that on their own approached maximal effectiveness the combined response was less than additive. NC tumours were excised from mice 14 days after inoculation s.c. with NC cells, weighed, and extracted for prostanoids. Mouse survival was determined up to day 121, and cancer spread was recorded postmortem. Nifedipine 1, 5 or 10 mg kg-1 had little or no effect on the tumour weight, tumour prostanoid content, metastasis to the lymph nodes or lungs, or on the increase of mouse longevity by the cytotoxic drugs.

The effects of dipyridamole and indomethacin on methotrexate cytotoxicity in LoVo human colon cancer cells.

Dipyridamole and indomethacin were studied for their effects in the in-vitro response of LoVo colon cancer cells to methotrexate (MTX) using a dye elution method. Dipyridamole 0.5-5 micrograms mL-1 or indomethacin 1 microgram mL-1 alone had little or no effect on cell growth. The tumour cells were refractory to even high concentrations of MTX (2.5-10 micrograms mL-1) alone or with indomethacin 1 microgram mL-1. In contrast, dipyridamole 0.5-5 micrograms mL-1 sensitized the cells to MTX 5 micrograms mL-1 (their growth was reduced by 25 to 69%), possibly by inhibiting thymidine salvage.

Effects of gastrointestinal hormones on the growth of human intestinal epithelial cells in vitro.

The growth of cultured epithelium like cells from human normal embryonic intestine was studied in response to various hormones using a method that quantifies the number of cells by the amount of dye that they bind after fixation. Gastrin and neurotensin in the pg/ml range and higher caused small increases in cell growth. Glucagon and VIP were stimulatory in the low ng/ml range, whereas somatostatin and bombesin had no effect at the lower concentrations but were stimulatory at the highest concentration tested (10 and 100 ng/ml respectively). Secretin and pancreozymin (cholecystokinin) seemed to be ineffective.

Studies on the mechanism by which indomethacin increases the anticancer effect of methotrexate.

The effect of indomethacin on the response of the NC carcinoma to methotrexate has been examined in vivo and in vitro. Survival was prolonged in mice treated with indomethacin 1.25 mg kg-1 twice daily plus methotrexate 4 mg kg-1 daily, compared to mice given either drug alone or controls. Indomethacin 1 microgram ml-1 increased the killing of cultured NC cells by methotrexate. This was not due to displacement of methotrexate from binding sites on the serum proteins. Nor was it due (entirely) to inhibition of prostaglandin synthesis, since flurbiprofen did not mimic the effect. Inhibition of cyclic AMP phosphodiesterase seems unlikely to explain the effect of indomethacin since theophylline had little or no effect on NC cell killing by methotrexate. Indomethacin 1 microgram ml-1 increased the accumulation of tritium in NC cells incubated with [3H]-methotrexate. In contrast, with normal epithelial cells from human embryonic intestine, indomethacin 1 microgram ml-1 did not alter the cytotoxicity of methotrexate or the accumulation of tritium during incubation with [3H]-methotrexate. The beneficial interaction between indomethacin and methotrexate may have therapeutic potential in man.

A new method for studying cell growth in suspension, and its use to show that indomethacin enhances cell killing by methotrexate.

Microturbidimetry has been used to measure the growth of cells in suspension. Disaggregated mouse NC carcinoma cells in culture medium were added to the wells of a microtitre test plate and incubated. Absorbance of 600 nm light was measured daily for 4 days using a microplate reader. As the cells grew, light absorbance increased. Methotrexate 2-40 ng ml-1 reduced cell growth; this effect was increased by indomethacin 1 microgram ml-1, possibly by displacing methotrexate from its binding by serum protein or by enhancing cell uptake of methotrexate. Similar results were obtained by conventional clonogenic assays. The new technique offers simplicity, better reproducibility, and substantial savings in time and cost.

The biological activities of phthalate esters on rat gastric muscle.

Monoethylhexyl phthalate, at concentrations that can occur in blood stored in plastic bags (0.1-0.5 mg/ml), reduced contractions of rat isolated gastric fundus to PGE2 and acetylcholine; the diethyl compound was less effective. In contrast, dibutyl phthalate (1 and 10 micrograms/ml) and, to a lesser extent di-isobutyl phthalate, increased the muscle tone. These results are discussed in relation to blood transfusion, and to structural similarities between phthalates and prostaglandins.

The effect of garlic extracts on contractions of rat gastric fundus and human platelet aggregation.

Garlic has been extracted and separated chromatographically into various fractions which show different degrees of activity as inhibitors of platelet aggregation and smooth muscle. The most potent smooth muscle inhibitor fraction had little activity on platelet aggregation, but microgram ml-1 concentrations greatly reduced the contractions of rat gastric fundus to prostaglandin E2 and acetylcholine. Material in this fraction may contribute to some of the claimed therapeutic effects of garlic involving smooth muscle. Its identity is not known, but is different from allyl sulphide, dimethyl sulphide and diallyl disulphide. These compounds eluted earlier on liquid chromatography than the most active fraction, and they showed only modest inhibitory activity against prostaglandin E2 and acetylcholine on rat fundus.