No abstract

Summary The potential involvement of lipoxygenase metabolites in the tumour growth stimulatory activity of arachidonic and linoleic acid has been studied using the 5-lipoxygenase inhibitors, BWA4C, BWB70C and Zileuton. In vitro the former two agents were relatively potent inhibitors of growth of murine adenocarcinomas (MACs) with IC50 values < 10 giM, whereas Zileuton was less effective. In vivo studies showed BWA4C to be an effective inhibitor of the growth of both the MAC26 and MAC16 tumours at dose levels between 5 and 25 mg kg-' (b.d.). The growth rate of the MAC26 tumour was also decreased by BWB70C at 25 mg kg-, whereas lower doses were either ineffective or stimulated tumour growth. This differential effect of the 5-lipoxygenases inhibitors on tumour growth may arise from effects on the 12-and 15-lipoxygenase pathways. To quantify the effect cells were labelled with [3H]arachidonic acid and the biosynthesis of 5-, 12-and 15-hydroxyeicosatetraenoic acid (HETE) was analysed by high-performance liquid chromatography. All three agents caused a decrease in 5-HETE production, although the effect was less pronounced with Zileuton. In MAC26 cells both BWA4C and BWB70C caused a decrease in 12-HETE formation whereas Zileuton had no effect on the other lipoxygenase pathways. The inhibitory effect of these agents on cell growth may result from an imbalance of metabolism of arachidonic acid between the 5-, 12-and 15-lipoxygenase pathways.

SummaryThe effect of a proteolysis-inducing factor (PIF), produced by cachexia-inducing tumours on glucose utilization by different tissues and the effect of pretreatment with the polyunsaturated fatty acid eicosapentaenoic acid (EPA), has been determined using the 2-deoxyglucose tracer technique. Mice receiving PIF showed a profound depression of body weight (2.3 g) over a 24-h period, which was completely abolished by pretreatment with a monoclonal antibody to PIF or by 3 days pretreatment with EPA at 500 mg kg -1 . Animals receiving PIF exhibited a marked hypoglycaemia, which was effectively reversed by both antibody and EPA pretreatment. There was an increase in glucose utilization by brain, heart and brown fat, but a decrease by kidney, white fat, diaphragm and gastrocnemius muscle after administration of PIF. Changes in organ glucose consumption were attenuated by either monoclonal antibody, EPA, or both. There was a decrease in 2-deoxyglucose uptake by C 2 C 12 myoblasts in vitro, which was attenuated by EPA. This suggests a direct effect of PIF on glucose uptake by skeletal muscle. These results suggest that in addition to a direct catabolic effect on skeletal muscle PIF has a profound effect on glucose utilization during cachexia.

S_mary }The effect of the polyunsaturated fatty acids (PUFAs) linoleic acid (LA) and arachidonic acid (AA) on the growth of two murine colon adenocarcinoma cell lines (MAC26 and MAC13) has been determined both in vitro and in vivo. When the serum concentration in the medium became growth limiting, low concentrations (18-33 pM) of both PUFAs were growth stimulatory to both cell lines, while higher concentrations were growth inhibitory. Growth stimulation by AA in both cell lines, and by LA in MAC13, was effectively inhibited by both the cyclo-oxygenase and lipoxygenase inhibitor indomethacin, and the lipoxygenase inhibitor BWA4C in a dose-dependent manner. The most effective inhibition was exerted by BWA4C, suggesting metabolism of both PUFAs through the lipoxygenase pathway for growth stimulation. In vivo studies using the MAC26 tumour showed a significant stimulation of tumour growth when LA was administered orally at concentrations higher than 0. Ci mmol-') were purchased from Amersham International (Amersham, UK). LA (99%) and AA (99%) were purchased as the free acids from Sigma (Poole, UK). RPMI-1640 tissue culture medium and fetal calf serum were purchased from Gibco (Paisley, UK). Fatty acids were complexed to sterile bovine serum albumin (fatty acid free) in water on an equal weight basis. The acid was neutralised with equimolar sodium bicarbonate and sonicated for 5 min to form micelles. Indomethacin was purchased from Sigma (Dorset, UK). BWA4C was kindly donated by L. Garland, Wellcome Research Laboratories, Kent, UK. The fatty acid composition of the rat and mouse breeding diet and arachis oil was determined by gas-liquid chromatographic (GIC) analysis of the fatty acids as the methyl esters as previously described (Hudson et al., 1993) and is given in Table I. Cell cultureThe MAC13 and MAC26 mouse colon adenocarcinoma cell lines were derived from the solid tumours and kindly donated by J. Double, University of Bradford, Bradford, UK. They were maintained in RPMI-1640 medium containing 10% fetal calf serum under an atmosphere of 5% carbon dioxide in air and were passaged twice a week. Cells for growth experiments were taken from logarithmically growing cultures and seeded at an initial cell density of 2 x 104 ml ' and cell counts were determined daily by means of a Coulter Electronic Particle Counter, model D.Correspondence: M.J. Tisdale.

The fluorinated pyrimidine nucleoside, 5′-deoxy-5-fluorouridine (5′-dFUrd) has been shown to effectively attenuate the progress of cachexia in the murine adenocarcinomas MAC16 and colon 26 as well as in the human uterine cervical carcinoma xenograft, Yumoto. Although concomitant inhibition of tumour growth was observed in all three models this was not sufficient to account for the preservation of body weight. An attempt has been made to correlate the anti-cachectic activity of 5′-dFUrd with the presence of a tumour produced proteolysis-inducing factor (PIF), thought to be responsible for the development of cachexia in the MAC16 model. Two variants of colon 26 adenocarcinoma were employed, clone 20 which produces profound cachexia, and clone 5 which produces no change in body weight in recipient animals. Mice bearing the colon 26, clone 20 variant showed evidence for the presence of PIF in tumour, serum and urine, while there was no evidence for the presence of PIF in tumour or body fluids of mice bearing the clone 5 tumours. Treatment of animals bearing the clone 20 variant with 5′-dF Urd led to the disappearance of PIF from the tumour, serum and urine concomitant with the attenuation of the development of cachexia. The human cervical carcinoma, Yumoto, which also induced cachexia in recipiant animals, showed expression of PIF in tumour, serum and urine in control and vehicle-treated mice, but was absent in mice treated with 5′-dFUrd. Thus in these experimental models cachexia appears to be correlated with the presence of PIF. © 2000 Cancer Research Campaign

Summary 2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase and thromboxane A2 synthase and a scavenger of active oxygen species, has been shown to exhibit profound antitumour activity against three established murine adenocarcinomas (MACs) that are generally refractory to standard cytotoxic agents. For the cachexia-inducing MAC16 tumour, optimal anti-tumour activity was seen at dose levels of 10 and 25 mg kg -day -, together with a reversal of cachexia and a doubling of the time to sacrifice of the animals through cachexia from 8 days to 17 days. The remaining tumour fragments showed extensive necrosis in regions distal from the blood supply. Growth of the MAC13 tumour was also effectively suppressed at dose levels between 5 and 50 mg kg-l day-1, resulting in a specific growth delay between 1.0 and 1.2. Growth of the MAC26 tumour was also inhibited in a concentration-related manner, with doses of 25-50 mg kg-' day-1 being optimal. Anti-tumour activity towards all three tumours at low dose levels of CV-6504 was effectively suppressed by concurrent administration of linoleic acid (1 g kg-' day-'), suggesting that inhibition of linoleate metabolism was responsible for the anti-tumour effect. Tumour sensitivity may be correlated with increased DT-diaphorase levels that are required to metabolise CV-6504 to the active hydroquinone, which inhibits 5-lipoxygenase activity.

Summary 2,3,5-Trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase, effectively suppressed growth of the MAC16 tumour in vivo and prevented the accompanying cachexia, when administered daily at a dose of 10 mg kg-'. There was a reduction in the tumour concentration of linoleic (LA), arachidonic (AA), oleic, stearic and palmitic acid. In order to elucidate the mechanism of the anti-tumour action, the effect of CV-6504 on the metabolism of AA through the 5-, 12-and 15-lipoxygenase pathways has been determined in cell lines sensitive (MAC16, MAC13, MAC26 and Caco-2) and resistant (A549 and DU-145) to CV-6504. Incubation of all cell lines with [3H]AA led to the appearance of [3H]5-, 12-and 1 5-HETE. Preincubation of MAC16, MAC13, MAC26 and Caco-2 with 10 gM CV-6504 inhibited the conversion of AA to 5-, 12-and 15-HETE, while in A549 and DU-145 cells there was no effect on metabolism through any lipoxygenase pathway. Two other cell lines, MDA-MB-231 and PC-3, sensitive to growth inhibition by CV-6504, are known to require LA for growth, while DU-145, which was insensitive to growth inhibition by CV-6504, showed no growth response to LA. These results suggest that some tumours are dependent on lipoxygenase metabolites of LA and AA for their continual growth, and interference with this pathway produces a specific growth inhibition.

Summary 2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone is an effective inhibitor of the growth of established murine adenocarcinomas (MACs) and is shortly to enter clinical investigation. When administered to mice bearing the MAC16 tumour, CV-6504 rapidly disappeared from the plasma and tissues and there was an accumulation of the sulphate and glucuronide metabolites. After 24 h, the concentration of free CV-6504 in the tumour (3.3 gM) was higher than that in the liver (0.24 /M) and equal to the IC50 value for the inhibition of the growth of MAC16 cells in vitro (3 pM). Products of metabolism of the polyunsaturated fatty acids (PUFAs), arachidonic (AA) or linoleic (LA) acid, through the lipoxygenase pathways have been shown to stimulate cell proliferation (Bandyopadhyay et al., 1988) and may also act as intermediaries in the mitogenic signalling by growth factors, such as epidermal growth factor (EGF) (Glasgow and Eling, 1990). Linoleic acid has been shown to induce DNA synthesis, c-fos, c-jun and c-myc mRNA expression and mitogen-activated protein kinase activation in vascular smooth muscle cells, and this effect was blocked by nordihydroguairetic acid, a potent inhibitor of the lipoxygenase system (Rao et al., 1995). Lipoxygenase inhibitors have also been shown to inhibit the growth of both rat (Lee and Ip, 1992) and mouse (Buckman et al., 1991) mammary tumour cells and HL60 human leukaemia cells (Simon et al., 1992).Our own studies have identified the 5-lipoxygenase inhibitor (Ohkawa et al., 1991a), 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), as an effective inhibitor of the growth of established murine adenocarcinomas (MAC) in vivo with a therapeutic index of at least 10 (Hussey et al., 1996). Such tumours are generally refractory to standard cytotoxic agents, suggesting a novel mechanism of tumour inhibition, and this agent is shortly to undergo clinical evaluation.CV-6504 undergoes rapid reduction by two-electron donating enzymes, such as DT-diaphorase, and the resulting hydroquinone inhibits both 5-lipoxygenase activity and lipid peroxidation on the basis of its antioxidant ability (Ohkawa et al., 199 lb) by reducing the ferric iron in the active site of the enzyme to the ferrous (resting state). Studies on the metabolism of CV-6504 by mice, rats, dogs and monkeys indicate reduction of the quinone ring and subsequent conjugation to yield the 1-and 4-glucuronides and the corresponding sulphates (Takeda Chemical Co., personal communication). These conjugates would not be capable of inhibiting 5-lipoxygenase by the suggested mechanism.In the present study the tumour and tissue levels of CV-