Oscillation of clock and clock controlled genes induced by serum shock in human breast epithelial and breast cancer cells: regulation by melatonin.

This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells.

Melatonin inhibits fatty acid transport in inguinal fat pads of hepatoma 7288CTC-bearing and normal Buffalo rats via receptor-mediated signal transduction.

Melatonin inhibits fatty acid uptake and linoleic acid-dependent growth in hepatoma 7288CTC in vivo in Buffalo rats. In this study we measured the effects of melatonin on arteriovenous differences for fatty acids across inguinal fat pads in fed and fasted rats to determine if fatty acid transport in white adipose tissue was also affected by melatonin. Intravenous infusion of melatonin in fasted tumor-bearing rats in vivo simultaneously and rapidly inhibited both fatty acid release from fat pads and fatty acid uptake by the tumors. Perfusion of fat pads in situ in normal rats with melatonin (0.1 nM) inhibited fatty acid release (fasted rats) and uptake (fed rats). Fatty acid transport was restored by addition of any of the following: a melatonin receptor antagonist (S 20928, 1.0 nM), pertussis toxin (0.5 microg/ml), forskolin (1 microM) or 8-Br-cAMP (10 microM). We conclude that fatty acid transport in inguinal fat pads requires cAMP and that melatonin inhibits this transport via a melatonin receptor-mediated, Gi protein-coupled signal transduction pathway. Melatonin has both anticachectic and lipid homeostatic actions in the white adipose tissue of inguinal fat pads.

Polyunsaturated fatty acids, melatonin, and cancer prevention.

Many nutritional, hormonal, and environmental factors affect carcinogenesis and growth of established tumors in rodents. In some cases, these factors may either enhance or attenuate the neoplastic process. Recent experiments performed in our laboratory using tissue-isolated rat hepatoma 7288CTC in vivo or during perfusion in situ have demonstrated new interactions among four of these factors. Two agents, dietary linoleic acid (C18:2n6) and "light at night," enhanced tumor growth, and two others, melatonin and n3 fatty acids, attenuated growth. Linoleic acid stimulated tumor growth because it is converted by hepatoma 7288CTC to the mitogen, 13-hydroxyoctadecadienoic acid (13-HODE). Melatonin, the neurohormone synthesized and secreted at night by the pineal gland, and dietary n3 fatty acids are potent antitumor agents. Both inhibited tumor linoleic acid uptake and 13-HODE formation. Artificial light, specifically "light at night," increased tumor growth because it suppressed melatonin synthesis and enhanced 13-HODE formation. Melatonin and n3 fatty acids acted via similar or identical G(i) protein-coupled signal transduction pathways, except that melatonin receptors and putative n3 fatty acid receptors were used. The results link the four factors in a common mechanism and provide new insights into the roles of dietary n6 and n3 polyunsaturated fatty acid intake, "light at night," and melatonin in cancer prevention in humans.

Mechanism for the antitumor and anticachectic effects of n-3 fatty acids.

Dietary intake of the n-6 fatty acid (FA) linoleic acid (LA) has a strong growth-promoting effect on many rodent tumors and human tumor xenografts grown in immunodeficient rodents. n-3 FAs such as alpha-linolenic and eicosapentaenoic acids (EPAs), which differ from LA and arachidonic acid, respectively, by only a single double bond in the n-3 position, are recognized cancer chemopreventive and anticachectic agents. Understanding how this seemingly small structural difference leads to such remarkable functional differences has been a challenge. In a previous study, we showed that LA uptake, [3H]thymidine incorporation into DNA, and total DNA content were decreased in tissue-isolated hepatoma 7288CTC perfused in situ with arterial blood containing alpha-linolenic acid, EPA, or docosahexaenoic acids. The Ki for the inhibition of LA uptake and [3H]thymidine incorporation by alpha-linolenic acid was 0.18 and 0.25 mM, respectively. Here we show that the addition of alpha-linolenic acid or EPA to arterial blood inhibits tumor FA uptake, including LA, and the subsequent conversion of LA to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) in vivo and during perfusion in situ. [3H]Thymidine incorporation during perfusion in situ was also inhibited. Addition of 13-HODE to the arterial blood reversed the inhibition of [3H]thymidine incorporation but had no effect on FA uptake. These two n-3 FAs also inhibited FA transport in inguinal fat pads in vivo and during perfusion in situ in fed (FA uptake) and fasted (FA release) rats. The effects of EPA and talinolenic acid on transport of saturated, monounsaturated, and n-6 polyunsaturated FAs in hepatoma 7288CTC and inguinal fat pads during perfusion in situ were reversed by the addition of forskolin (1 microM), pertussis toxin (0.5 microg/ml), or 8-bromo-cyclic AMP (10 microM) to the arterial blood. We conclude that the antitumor and anticachectic effects of n-3 FAs on hepatoma 7288CTC and inguinal fat pads in vivo result from an inhibition of FA transport. These inhibitions are mediated by a putative n-3 FA receptor via a Gi protein-coupled signal transduction pathway that decreases intracellular cyclic AMP. A specific decrease in LA uptake and its conversion to the mitogen 13-HODE causes the tumor growth inhibition.

Preparation of the inguinal fat pad for perfusion in situ in the rat: a surgical technique that preserves continuous blood flow.

We developed a surgical technique for preparing the inguinal fat pad of rats for perfusion that preserves continuous blood flow to the tissue. Fatty acid uptake from the fat pads of fed rats (46.1 +/- 2.1% of arterial supply, n = 82) and release from those of fasted (48 h) animals (51.3 +/- 3.1% of supply, n = 69) occurred principally via the free fatty acid component of the blood; levels of triglycerides, cholestryl esters, and phospholipids did not change significantly. Venous blood flow in perfusions using blood from fed rats was 83.3 +/- 1.7 mL/min and 83.1 +/- 1.5 microL/min in experiments involving fasted donors. Values for arterial (A) and venous (V) pH (A, 7.41 +/- 0.03; V, 7.32 +/- 0.04), pO2 (A, 151.6 +/- 15.7 mm Hg; V, 34.7 +/- 9.2 mm Hg), pCO2 (A, 30.8 +/- 6.6 mm Hg; V, 58.2 +/- 5.2 mm Hg), and hematocrit (A, 44.6 +/- 1.2%; V, 45.7 +/- 1.2%) were unchanged throughout the course of the perfusions. Fat pad and blood perfusates were maintained at 37 degrees C. Tissue homogenates revealed that the total fatty acid content of fat pads from fed rats (333.5 +/- 0.3 mg/g tissue) differed significantly from that in fasted animals (260.7 +/- 0.7 mg/g tissue; P < 0.001). Our technique likely will have many uses in the study of lipid transport and metabolism, hyperlipidemia, and cancer-associated cachexia.

Dim light during darkness stimulates tumor progression by enhancing tumor fatty acid uptake and metabolism.

Tumor linoleic acid uptake and metabolism, and growth are suppressed by melatonin, the synthesis of which is inhibited by light. Linoleic acid, via its mitogenic metabolite 13-hydroxyoctadecadienoic acid (13-HODE) is an important growth stimulant of rat hepatoma 7288CTC. Here we compared the effects of an alternating light:dark cycle (12L:12D), dim light (0.25 lux) present during the dark phase of a diurnal light cycle, and constant light on growth and fatty acid metabolism in hepatoma 7288CTC. Our results show that dim light suppressed melatonin release by the pineal gland, increased tumor linoleic acid uptake and 13-HODE production, and promoted tumor growth as effectively as did constant light.

Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events.

The growth of rat hepatoma 7288CTC in vivo is stimulated by the uptake of linoleic acid (LA) and its metabolism to 13-hydroxyoctadecadienoic acid (13-HODE), an important mitogenic signaling molecule within this tumor. Conversely, the growth of a variety of experimental cancers in vivo is inhibited by either physiological or pharmacological levels of the pineal gland hormone melatonin, although the mechanism(s) are unknown. We tested the hypothesis that the mechanism of melatonin's anticancer action in vivo involves the inhibition of tumor LA uptake and metabolism to 13-HODE in hepatoma 7288CTC. Tumor uptake of LA and release of 13-HODE, measured in tissue-isolated rat hepatoma 7288CTC at 4-h intervals over a 24-h period, were highest during the light phase and lowest during the mid-dark phase, when plasma melatonin levels were lowest and highest, respectively. Pinealectomy eliminated this rhythm of tumor LA uptake and 13-HODE production, indicating that it was driven by the circadian melatonin rhythm. Perfusion of tissue-isolated tumors in situ with melatonin (1 nM) rapidly and reversibly inhibited the uptake of plasma fatty acids (FAs), including LA, and its metabolism to 13-HODE. These inhibitory effects of melatonin on tumor FA uptake and 13-HODE release were completely reversed by perfusion of tumors in situ with melatonin receptor antagonist S-20928, pertussis toxin, forskolin, or 8-bromo-cAMP. Perfusion of tumors in situ with melatonin also decreased tumor [3H]thymidine incorporation and DNA content; these effects on DNA synthesis were also prevented by the coperfusion of tumors with melatonin and S-20928, pertussis toxin, forskolin, 8-Br-cAMP, or 13-HODE. Pinealectomy stimulated tumor growth, LA uptake and metabolism to 13-HODE, and FA storage in hepatoma 7288CTC, whereas melatonin administration (200 microg/day) was inhibitory in vivo. Northern blot analysis revealed that, compared with normal liver tissue, hepatoma 7288CTC overexpressed mRNA transcripts for a plasma membrane-associated FA transport protein (FATP). FATP mRNA expression was unaffected by the treatment of tumor-bearing rats with daily afternoon melatonin injections or exposure to constant light. These results support a novel mechanism of tumor growth inhibition by melatonin involving a melatonin receptor-mediated suppression of cAMP levels, resulting in diminished tumor FA transport, possibly via decreased FATP function. The inhibition of these signal transduction events by melatonin culminates in the suppression of LA uptake, LA metabolism to the mitogenic signaling molecule 13-HODE, and cancer growth.

13-Hydroxyoctadecadienoic acid is the mitogenic signal for linoleic acid-dependent growth in rat hepatoma 7288CTC in vivo.

Growth of hepatoma 7288CTC in male Buffalo rats is directly dependent on uptake of linoleic acid (LA) from the arterial blood. One to 5% of the LA taken up is converted to 13-hydroxyoctadecadienoic acid (HODE), an agent that enhances epidermal growth factor-dependent mitogenesis. The role of 13-HODE in LA-dependent growth of solid tumors is not known. In this study, we examined LA uptake and 13-HODE formation on growth of tissue-isolated hepatoma 7288CTC in vivo and on [3H]thymidine incorporation and DNA content during perfusion in situ. Fatty acid uptake and metabolite release were determined from arteriovenous difference measurements. Tumor-bearing and blood donor rats were fed either LA-sufficient or -deficient diets. Hepatoma 7288CTC removed LA from the arterial blood and released 13-HODE [and a small amount of 13-ketooctadecadienoic acid (KODE)] into the venous blood both in vivo and during perfusion. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) did not affect tumor LA uptake, but inhibited release of 13-HODE and 13-KODE in vivo and during perfusion, suppressed growth in vivo, and inhibited [3H]thymidine incorporation during perfusion. The addition of 13-HODE to the nordihydroguaiaretic acid-containing whole blood perfusate increased the rate of [3H]thymidine incorporation 10 times and nearly doubled tumor DNA content; the addition of 13-KODE or 9-HODE had no effect. 13-HODE and 13-KODE were not released from tumors growing in rats fed a LA-deficient diet, and the rates of tumor growth in vivo and [3H]thymidine incorporation during perfusion were decreased. The addition of 13-HODE to the LA-deficient blood perfusate promoted tumor 13-HODE uptake and a dose-dependent increase in [3H]thymidine incorporation and tumor DNA content. These results provide strong evidence that 13-HODE is the mitogenic signal responsible for LA-dependent growth in hepatoma 7288CTC in vivo.

New actions of melatonin on tumor metabolism and growth.

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.

Light contamination during the dark phase in "photoperiodically controlled" animal rooms: effect on tumor growth and metabolism in rats.

Enhanced neoplastic growth and metabolism have been reported in animals maintained in a constant light (24L:0D) environment. Results from this laboratory indicate that tumor growth is directly dependent upon increased ambient blood concentrations of arachidonic and linoleic acids, particularly linoleic acid. Tumor linoleic acid utilization and production if its putative mitogenic metabolite, 13-hydroxyoctadecadienoic acid (13-HODE), are suppressed by the circadian neurohormone melatonin, the production of which is itself regulated by light in all mammals. This study was performed to determine whether minimal light contamination (0.2 lux) in an animal room during an otherwise normal dark phase may disrupt normal circadian production of melatonin and affect tumor growth and metabolism. Animals of groups I (12L:12D), II (12L:12-h light-contaminated dark phase), and III (24L:0D) had plasma total fatty acid (TFA), linoleic acid (LA), and melatonin concentrations measured prior to tumor implantation; groups I and II had daily cycles in plasma TFA and LA values, whereas group III had constant values throughout the day. The integrated mean TFA and LA values for the entire day were similar in all groups. Although group-I animals had a normal nocturnal surge of melatonin (127.0 pg/ml) at 2400 h, the nocturnal amplitude was suppressed in group-II animals (16.0 pg/ml); circadian variation in melatonin concentration was not seen in group-III animals (7.4 pg/ml). At 12 weeks of age, rats had the Morris hepatoma 7288CTC implanted as "tissue-isolated" tumors grown subcutaneously. Latency to onset of palpable tumor mass for groups I, II, and III was 11, 9, and 5 days respectively. Tumor growth rates were 0.72 +/- 0.09, 1.30 +/- 0.15, and 1.48 +/- 0.17 g/d (mean +/- SD, n = 6/group) in groups I, II, and III respectively. Arteriovenous difference measurements for TFA and LA across the tumors were 4.22 +/- 0.89 and 0.83 +/- 0.18 (group I), 8.26 +/- 0.66 and 1.64 +/- 0.13 (group II), and 7.10 +/- 0.78 and 1.50 +/- 0.16 (group III)/min/g, and groups II and III were significantly different from group I (P < 0.05). Tumor TFA and LA contents were 14.3 +/- 1.7 and 1.8 +/- 0.3 (group I), 52.9 +/- 5.5 and 7.9 +/- 0.8 (group II), and 106.0 +/- 12.0 and 18.5 +/- 2.4 (group III) micrograms/g and were significantly different from each other (P < 0.001). Production of 13-HODE by the hepatomas in groups I, II, and III was 35.5 +/- 6.3, 109.6 +/- 10.6, and 196.2 +/- 34.9 ng/min/g respectively, values which also were significantly different among groups (P < 0.001). The results indicate that minimal light contamination of only 0.2 lux during an otherwise normal dark phase inhibits host melatonin secretion and increases the rate of tumor growth and lipid uptake and metabolism. These data suggest that great care must be taken to prevent "light-leaks" in animal rooms during the dark phase of a diurnal cycle because such contamination may adversely affect the outcome of tumor growth investigations.

Dietary linoleic acid intake controls the arterial blood plasma concentration and the rates of growth and linoleic acid uptake and metabolism in hepatoma 7288CTC in Buffalo rats.

In this study, we tested the hypothesis that dietary linoleic acid intake controls the arterial blood plasma linoleic acid concentration and the rates of tumor growth and linoleic acid metabolism in vivo. Seven groups of young male Buffalo rats (11-21 rats/group) were given free access to semipurified diets containing different amounts of corn and/or olive oils. Four other groups (7-11 rats/group) were 30% energy-restricted. Each experiment included periods for rat growth and plasma lipid stabilization (6 wk), measurement of mean daily arterial blood plasma fatty acid concentrations (3 wk), surgical implantation of a subcutaneous tissue-isolated hepatoma 7288CTC, tumor growth and harvest (2-4 wk). Linoleic + arachidonic acid (P = 0.007) and oleic acid (P = 0.002) concentrations in arterial blood plasma were increased as dietary intake of linoleic and oleic acids was increased, respectively. In rats given free access to food, tumor growth was directly dependent on the plasma concentrations of linoleic (P < 0.001) and arachidonic acids (P = 0.04). Tumor growth in energy-restricted rats was dependent only on the linoleic acid concentration (P = 0.008). Energy restriction itself caused a growth inhibition independent of plasma linoleic acid. The linoleic acid and total fatty acid concentrations of tumor triacylglycerols were directly dependent on the plasma linoleic acid concentration in rats given free access to food (P = 0.009). Hepatoma 7288CTC (both in vivo and during perfusion in situ) supported a dose-dependent conversion (P < 0.001) of plasma linoleic acid to the mitogen, 13-hydroxy-9, 11-octadecadienoic acid. We conclude that increased arterial blood plasma linoleic acid concentrations, caused by increased dietary intakes, specifically stimulate growth, lipid storage and linoleic acid metabolism in hepatoma 7288CTC in vivo.

Lactate release and uptake in hepatoma 7288CTC perfused in situ with L-[(U)-14C]lactate or D-[(U)-14C]glucose.

Arteriovenous differences (AVD) for glucose and lactic acid measured across tissue-isolated rat tumors in vivo have shown that individual tumors with similar rates of glucose consumption may either release or utilize lactic acid. The experiments described here investigated the relationships among arterial blood lactate concentrations and tumor lactate and glucose balances. AVDs for lactate, pyruvate, glucose, 14CO2, PO2, PCO2, pH, and lactate specific activities were measured across 17 tissue-isolated 7288CTC hepatomas perfused in situ with arterial blood containing 2.5 to 14.4 mmol/L lactate and either L-[(U)-14C]lactic acid or D-[(U)-14C]glucose. Measurements were made over a range of blood flow rates from 60% to 200% of the mean in vivo rate, 0.11 mL/min. Data collected during steady states were compared by regression analysis. Tumor lactate balance and the arterial blood lactate concentration were directly related (r = .895, n = 22, P < .01). Net negative and positive balances occurred below and above approximately 6.5 mmol/L arterial blood lactate, respectively. The mean intratumor lactate concentration for all tumors was 6.9 +/- 1.0 mmol/L (mean +/- SD, n = 13). Rates of 14C-lactate oxidation to 14CO2 (r = .716, n = 18, P < .01) and tumor venous/arterial blood 14C-lactate specific activity ratios (r = .845, n = 19, P < .01) were low during lactate release and were increased during lactate uptake. Total arterial blood lactate removal estimated from chemical and isotopic analyses was 23.1% +/- 11% and 43.0% +/- 16% (P < .05), respectively, for six lactate-utilizing tumors. Perfusions performed with 14C-glucose showed that approximately 50% of the glucose consumed during net negative lactate balance was released as 14C-lactate to the tumor venous blood, whereas only 5% was released as 14C-lactate during net positive lactate balance. The data support the following conclusions: Arterial blood lactate controls net lactate balance in solid tumors; high concentrations increase uptake. Lactate uptake inhibits lactate formation from glucose without changing the glucose balance. Lactate is release during net lactate uptake. Since lactate uptake may exceed glucose uptake, arterial blood lactate can be a substrate for tumor energy metabolism and growth.

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in adrenal cortex mitochondria and microsomes.

3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of delta 5-3 beta-hydroxysteroids to delta 4-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD+ and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD+, was inhibited by reduction of intramitochondrial NAD(P)+, and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). However, mersalyl blocked the effects of exogenous NADH and NAD+ and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD+ activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD+ and contained none or 2.9-7.3 nmol NAD+/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD+ for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD+ was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD+. The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD+ or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.

Uptake of plasma lipids by tissue-isolated hepatomas 7288CTC and 7777 in vivo.

The uptake of myristic (C14:0), palmitic (C16:0), palmitoleic (C:16,N-7), stearic (C18:0), oleic (C18:1,N-9), linoleic (C18:2,N-6) and arachidonic (C20:4,N-6) acids from plasma free fatty acids (FFA), triglycerides (TGA), phospholipids (PL) and cholesterol esters (CE) was measured in tissue-isolated hepatomas 7288CTC and 7777 in vivo. Adult tumour-bearing Buffalo rats were fed a normal chow diet ad libitum and were subjected to darkness from 1800 to 0600 h. Arterial plasma levels of FFA, TGA, PL and CE were increased during the dark period without change in fatty acid compositions. Arteriovenous difference measurements of tumour lipid uptake were performed between 0600 and 0900 h and included both high (dark) and low (light) arterial blood lipid concentrations. The rate of lipid uptake from each lipid class was directly dependent on the rate of supply of the lipid to the tumour. The efficiency of uptake, however, depended on the type of plasma lipid and the tumour. During one pass of arterial blood, hepatoma 7288CTC (n = 5 to 13) removed 46, 33, 36 and 31%, and hepatoma 7777 (n = 7 to 9) removed 48, 50, 52 and 49% of the fatty acids supplied in FFA, TGA, PL and CE, respectively. Perfusion of tissue-isolated tumours in situ with donor blood containing plasma free (1-14C)palmitic acid showed that 14C-palmitic acid was removed from the arterial blood and was incorporated into tumour lipids and that 14CO2 was released into the tumour venous blood. Uptake of the seven fatty acids over a 24 h period was greatest from PL greater than TGA greater than FFA greater than CE and was estimated to total 18.1 +/- 3.5 mg fatty acids g-1 for hepatoma 7288CTC and 25.9 +/- 3.5 mg fatty acids g-1 for hepatoma 7777. Both hepatoma 7288CTC and 7777 grew at a rate of about 1 g day-1 and contained 13.4 +/- 2.5 and 10.6 +/- 3.9 mg of these 7 fatty acids g-1 tumour wet weight, respectively. We conclude that these two tumours obtain all of the fatty acids needed for daily growth from host arterial blood.

The effect of omega-6 and omega-3 fatty acids on 3H-thymidine incorporation in hepatoma 7288CTC perfused in situ.

Ingestion of diets containing corn oil or marine fish oils is known to increase or decrease, respectively, the growth of transplantable rodent tumours. The active agents in these oils have been identified as linoleic acid (in corn oil) and omega-3 fatty acids (in marine oils), but it is still not known how they influence the tumour growth processes. In these experiments we examined the effects of plasma free omega-6 and omega-3 fatty acids on the rate of 3H-thymidine incorporation in tissue-isolated hepatoma 7288CTC perfused in situ. Host Buffalo rats were fed an essential fatty acid-deficient diet. Plasma and tumours in these animals contained low endogenous levels of both omega-6 and omega-3 fatty acids. Perfusion of these tumours for 2 h with donor whole blood containing added omega-6 free fatty acids, including 0.5 mM linoleic (C18:2,N-6), gamma-linolenic (C18:3,N-6), dihomo-gamma-linolenic (C20:3,N-6) or arachidonic acids (C20:4,N-6), increased the rate of 3H-thymidine incorporation. Linoleic acid was about three times more effective than the other omega-6 fatty acids. Typical hyperbolic substrate-saturation curves were observed as the plasma free linoleate or arachidonate concentration was increased. When perfused alone plasma free omega-3 fatty acids had no effect on tumour 3H-thymidine incorporation, but in the presence of linoleic acid the omega-3 fatty acids, alpha-linolenic (C18:3,N-3) and eicosapentaenoic (C20:5,N-3), competitively inhibited both tumour linoleate uptake and the stimulative effect on 3H-thymidine incorporation. The results suggest that the ambient plasma free linoleic and arachidonic acid concentrations in host arterial blood directly influence the rate of tumour DNA synthesis. Plasma free omega-3 fatty acids appear to modulate the effect of linoleic acid by competitively inhibiting its uptake. These relationships could explain the actions of dietary linoleic and omega-3 fatty acids on tumour growth in vivo.

Identification of linoleic and arachidonic acids as the factors in hyperlipemic blood that increase [3H]thymidine incorporation in hepatoma 7288CTC perfused in situ.

Tumor growth and the incorporation of [3H]thymidine into tumor DNA in vivo are increased about 3 times in adult rats (greater than 250 g) after 1 to 2 days of starvation or the induction of diabetes with streptozotocin. These tumor growth responses require hyperlipemia and are reversed by refeeding or insulin treatment, respectively. They do not occur in young tumor-bearing rats (less than about 150 g) that lack appreciable fat stores. A direct relationship between the increased rates of both [3H]thymidine incorporation and tumor growth and host hyperlipemia suggests that tumor cell renewal in vivo in fed rats is limited by substances that are present in hyperlipemic blood. In this study we used a procedure for perfusion of solid tumors in situ to measure the sensitivity of tumor [3H]thymidine incorporation to hyperlipemic blood and to identify the rate-limiting substances. Tissue-isolated Morris hepatomas (7288CTC) growing in young or adult Buffalo rats were perfused with blood from donor rats. Hyperlipemic blood for perfusion was obtained from 2-day starved tumor-bearing (Buffalo) or non-tumor-bearing (Buffalo or Lewis) rats. At the end of the perfusions the tumors were labeled with a pulse of [3H]thymidine (2 microCi/g estimated tumor wet weight). [3H]Thymidine incorporation in tumors growing in fed adult rats was increased from 80 +/- 5 (SD) dpm/micrograms DNA at zero time (before perfusion) to 209 +/- 9 dpm/micrograms DNA (n = 3) after perfusion for 3 h. Tumors growing in fed or starved young rats showed similar responses, and hyperlipemic blood from non-tumor-bearing rats was as effective as hyperlipemic blood from tumor-bearing rats. Perfusion of tumors growing in starved rats with normolipemic blood from fed adult rats decreased [3H]thymidine incorporation from 211 +/- 13 dpm/micrograms DNA before perfusion to 68 +/- 9 dpm/micrograms DNA (n = 3) after perfusion for 3 h. Cells, plasma, and plasma subfractions from hyperlipemic blood were reconstituted to whole blood using plasma, cells, and whole blood, respectively, from fed rats and the mixtures were perfused into tumors growing in fed adult rats. Mixtures containing hyperlipemic plasma, lipid extracts (ethanol:acetone, 1:1) of hyperlipemic plasma, or albumin from hyperlipemic plasma increased tumor [3H]thymidine incorporation. Free fatty acid concentrations were increased about five times in hyperlipemic plasma and perfusion of tumors with normolipemic blood containing added linoleic and arachidonic acids increased [3H]thymidine incorporation. Blood mixtures containing palmitic, stearic, and oleic acids were inactive.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo nutrient uptake by head and neck cancers.

The arteriovenous uptake differences for glucose, lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate were measured in vessels across squamous cell carcinomas in ten selected patients with tumors of the head and neck. All tumors measured took up glucose, pyruvate, beta-hydroxybutyrate, and acetoacetate. They could either produce, take up, or maintain a constant level of lactate. These tumors demonstrate a heterogeneous affinity for various metabolites, including ketone bodies which are elevated in the nutritionally depleted, tumor-burdened patient.

Stimulation of tumor growth in adult rats in vivo during acute streptozotocin-induced diabetes.

The effects of acute diabetes mellitus on the growth of Morris hepatoma 7288CTC and Jensen sarcoma were studied in fed, young (less than 200 g), and adult (greater than 250 g) rats. Animals were matched for tumor size and growth; the rates of tumor growth were the same in fed, young and adult nondiabetic rats. Diabetes was induced by the i.v. injection of streptozotocin (65 mg/kg total body weight) into tumor-bearing rats and changes in arterial blood nutrient concentrations were compared to changes in the rates of tumor growth and DNA synthesis. In young rats acute diabetes did not increase the blood concentrations of the fat store-derived nutrients and did not increase the rate of tumor growth. In adult rats, however, acute diabetes raised the arterial blood free fatty acid, glycerol, triglyceride, and ketone body concentrations to high levels and increased the rate of tumor growth about three times over that observed in untreated rats. Progress curves for the mobilization of host fat stores and for incorporation of [methyl-3H]thymidine into tumor DNA during the onset of diabetes showed that these activities were closely correlated in adult rats. Both processes began to increase 2 to 4 h after streptozotocin treatment, reached an initial peak at 12 to 16 h, decreased to a low point at 18 to 20 h, and then increased again to the new steady state after 23 to 24 h. The results indicate that the rate of tumor growth in rats in vivo is limited by the availability of a substance(s) present in the hyperlipemic blood of adult diabetic rats. The tight relationship between host lipolysis and tumor growth suggests that the substance(s) is derived from host fat stores.