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Dietary fish oil decreases growth of solid tumors in rodents. Mechanisms for this effect are not well defined. In rat hepatoma 7288CTC, short-term (1-2 h) treatment with eicosapentaenoic acid during perfusion in situ reduced fatty acid uptake and [3H]thymidine incorporation. To determine if dietary fish oil had this effect in vivo, 48 male Buffalo rats were implanted with tissue-isolated hepatoma 7288CTC and were divided into three groups: Diet I (8% olive oil/2% corn oil), Diet II (6% olive oil/2% corn oil/2% fish oil), or Diet III (3% olive oil/3% corn oil/4% fish oil). When tumors weighed 4 to 6 g rats were anesthetized and tumor fatty acid uptake and 13-hydroxyoctadecadienoic acid release were measured in vivo by arterial minus venous differences. Tumors were analyzed for cyclic adenosine monophosphate (CAMP), DNA content, and [3H]thymidine incorporation. Fish oil feeding significantly (P < 0.05) reduced tumor growth, cAMP content, fatty acid uptake, 13-hydroxyoctadecadienoic acid formation, DNA content, and [3H]thymidine incorporation. Addition of either pertussis toxin or 8-bromoadenosine-cAMP to the arterial blood reversed the inhibitions in tumors in rats fed diet II. These results provide in vivo evidence that dietary fish oil suppressed a specific linoleic acid-dependent, inhibitory G protein-coupled, growth promoting signaling pathway in rat hepatoma 7288CTC. [ABSTRACT FROM AUTHOR]

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use., Competing Interests: RJL has received research grant funding from Philips Lighting/Signify and honoraria from Samsung Electronics. GCB has received research grant funding from the Nova Institute, Toshiba Materials Science, Seoul Semiconductor, BIOS, Robern, and PhotoPharmics Inc. He has a current patent (USPTO 7678140 B2) that is licensed by Litebook Company Ltd. He is a paid member of the PhotoPharmics Scientific Advisory Board; has been a paid consultant by Lutron, Inc. and McCullough Hill LLC; and has received honoraria from the Institute for Functional Medicine. BNG has received funding from Tecniplast to travel and present at a symposium. RAH has received research grant funding from Philips Lighting/Signify and is a scientific advisor for the Good Light Group and Chrono@Work. JST is a co-founder and SAB member of Synchronicity Pharma. SNP has received consulting fees from NASA Ames and Sleep Standards., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Light is an environmental factor that is extrinsic to animals themselves and that exerts a profound influence on the regulation of circadian, neurohormonal, metabolic, and neurobehavioral systems of all animals, including research animals. These widespread biologic effects of light are mediated by distinct photoreceptors-rods and cones that comprise the conventional visual system and melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) of the nonvisual system that interact with the rods and cones. The rods and cones of the visual system, along with the ipRGCs of the nonvisual system, are species distinct in terms of opsins and opsin concentrations and interact with one another to provide vision and regulate circadian rhythms of neurohormonal and neurobehavioral responses to light. Here, we review a brief history of lighting technologies, the nature of light and circadian rhythms, our present understanding of mammalian photoreception, and current industry practices and standards. We also consider the implications of light for vivarium measurement, production, and technological application and provide simple recommendations on artificial lighting for use by regulatory authorities, lighting manufacturers, designers, engineers, researchers, and research animal care staff that ensure best practices for optimizing animal health and well-being and, ultimately, improving scientific outcomes.

Rodents are currently the most common animals used for hepatic surgical resection studies that investigate liver regeneration, chronic liver disease, acute liver failure, hepatic metastasis, hepatic function, and hepatic cancer. Our previous work has shown that dietary consumption of linoleic acid (LA) stimulates the growth of rodent and human tumors in vivo. Here we compared 3 diets - a 5% corn oil diet (control), a diet deficient in essential fatty acids (EFAD), and an EFAD supplemented with LA in amounts equal to those in the control diet (EFAD+LA). We hypothesized that consumption of the LA provided in the EFAD+LA diet would elevate plasma levels of LA and stimulate regeneration in rats after a 70% hepatectomy (HPX), and that regeneration would not occur in the EFAD rats. Each diet group was comprised of 30 male and 30 female Buffalo rats (BUFF/CrCrl). Rats were fed one of the 3 diets and water ad libitum. After 8 wk on the assigned diet, rats were underwent a 70% HPX. On days 4 and 21 after HPX, 30 male and 30 female rats from each diet group were anesthetized for in vivo study and then were euthanized for tissue collection. For the in vivo study, arterial and venous blood samples were collected from the liver. LA-, glucose-, and O₂ -uptake, and lactate- and CO₂ -output were significantly higher in LA-replete rats as compared with LA-deficient rats. After a 70% HPX, the remaining liver mass in control and EFAD+LA groups had doubled at day 4, reaching 60% of the original total weight, and had regenerated completely at day 21. However, no regeneration occurred in the EFAD group. At day 4 the portions of livers removed from the control and EFAD+LA groups had significantly higher content of LA, protein, cAMP, and DNA as compared with their livers on day 21. [³ H]thymidine incorporation into liver DNA was significantly higher in the 2 LA-replete groups, with male values greater than female values, as compared with LA-deficient group. These data indicate that liver regeneration after HPX is dependent on dietary LA. Understanding the mechanisms of LA-dependent liver regeneration in rats supports our current efforts to enhance successful surgical resection therapies in humans.

Light is an extrinsic factor that exerts widespread influence on the regulation of circadian, physiologic, hormonal, metabolic, and behavioral systems of all animals, including those used in research. These wide-ranging biologic effects of light are mediated by distinct photoreceptors, the melanopsin-containing intrinsically photosensitive retinal ganglion cells of the nonvisual system, which interact with the rods and cones of the conventional visual system. Here, we review the nature of light and circadian rhythms, current industry practices and standards, and our present understanding of the neurophysiology of the visual and nonvisual systems. We also consider the implications of this extrinsic factor for vivarium measurement, production, and technological application of light, and provide simple recommendations on artificial lighting for use by regulatory authorities, lighting manufacturers, designers, engineers, researchers, and research animal care staff that ensure best practices for optimizing animal health and wellbeing and, ultimately, improving scientific outcomes.

Light and lighting protocols of animal research facilities are critically important to the outcomes of biomedical research that uses animals. Previous studies from our laboratory showed that the wavelength (color) of light in animal housing areas affects the nocturnal melatonin signal that temporally coordinates circadian rhythms in rodents. Here, we tested the hypothesis that exposure to LED light enriched in the blue-appearing portion (460-480 nm) of the visible spectrum during the light phase (bLAD) influences circadian concentrations of select neuroendocrine hormones in adolescent Sprague-Dawley rats. Male and female rats (4 to 5 wk old) were housed on a novel IVC system under a 12L:12D in either cool-white fluorescent (control, n = 72) or bLAD (experimental, n = 72) lighting. Every third day, body weight and food and water consumption were measured. On Day 30, rats were anesthetized with ketamine/xylazine and terminal collection of arterial blood was performed to quantify serum concentrations of melatonin, corticosterone, insulin, and glucose at 6 circadian time points (0400, 0800, 1200, 1600, 2000, 2400). As compared with male and female rats housed under cool white fluorescent (CWF) lighting, rats in bLAD lighting showed a 6-fold higher peak in dark phase serum melatonin ( P < 0.05). Effects on serum corticosterone were sex dependent, as CWF and bLAD females had significantly higher corticosterone levels than did CWF and bLAD males, respectively. CWF and bLAD females had significantly higher serum glucose overall as compared with males. However, serum insulin was not affected by sex (M or F) or lighting conditions (CWF or bLAD). These data show that housing Sprague-Dawley rats under bLAD lighting conditions increases circadian peaks of melatonin without increasing serum levels of corticosterone, glucose or insulin, indicating less variation of circadian cycling of key neuroendocrine hormones in bLAD-exposed rats.

The tissue-isolated tumor model permits the investigation of melatonin's influence on human tumor growth and metabolism in laboratory rats in vivo. Here we describe a unique surgical technique for implanting and growing human tumor xenografts on a vascular stalk composed of the nude rat epigastric artery and vein that provides a continuous blood supply from a single source to the tissue-isolated tumor while insuring the absence of extraneous vascular connections. A variety of human tumor types may be implanted and grown utilizing this unique model that may provide a plethora of scientific data from a single tumor examined., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The tissue-isolated human tumor perfusion methodology enables the elucidation of physiological melatonin's oncostatic impact on cancer metabolism and physiology. Here we describe an apparatus and surgical technique for perfusing tissue-isolated human tumor xenografts in nude rats in situ that ensures continuous blood flow to and from the tissue. This system and methodology have proven quite successful in examining the receptor-mediated oncostatic effects of the physiological nocturnal melatonin signal on metabolism and physiology in a variety of epithelial and mesenchymal human tumors., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Artificial light at night (ALAN) is a pervasive phenomenon. Although initially assumed to be innocuous, recent research has demonstrated its deleterious effects on physiology and behavior. Exposure to ALAN is associated with disruptions to sleep/wake cycles, development of mood disorders, metabolic disorders, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. We hypothesize that exposure to ALAN accelerates mammary tumor growth and predict that ALAN exacerbates negative affective behaviors in tumor-bearing mice. Adult (>8 weeks) female C3H mice received a unilateral orthotropic injection of FM3A mouse mammary carcinoma cells (1.0 × 10 5 in 100 μL) into the fourth inguinal mammary gland. Nineteen days after tumor inoculation, mice were tested for sucrose preference (anhedonia-like behavior). The following day, mice were subjected to an open field test (anxiety-like behavior), followed by forced swim testing (depressive-like behavior). Regardless of tumor status, mice housed in ALAN increased body mass through the first ten days. Tumor-bearing ALAN-housed mice demonstrated reduced latency to tumor onset (day 5) and increased terminal tumor volume (day 21). Exposure to ALAN reduced sucrose preference independent of tumor status. Additionally, tumor-bearing mice housed in dark nights demonstrated significantly increased anxiety-like behavior that was normalized via housing in ALAN. Together, these data reaffirm the negative effects of ALAN on tumorigenesis and demonstrate the potential anxiolytic effect of ALAN in the presence of mammary tumors.

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [³H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.

Light has been a crucial part of everyday life since the beginning of time. Most recently, light-emitting diode (LED) light enriched in the blue-appearing portion of the visible spectrum (465 to 485 nm), which is more efficient in energy use, is becoming the normal lighting technology in facilities around the world. Previous reports revealed that blue-enriched LED light at day (bLAD) enhances animal health and wellbeing as compared with cool white fluorescent (CWF) lighting. We hypothesized that bLAD, compared with CWF light, has a positive influence on basic physiologic indices such as food consumption, water consumption, weight gain, nesting behavior, complete blood count, and blood chemistry profile. To test this, we allocated 360 mice into equal-sized groups by sex, strain (C3H/HeNCrl, C57BL/6NCrl, BALB/cAnNCrl), lighting conditions, and 6 blood collection time points ( n = 5 mice/sex/strain/lighting condition/time point). Food consumption, water consumption, body weight, nest location, and nest type were recorded every 3 d. At the end of the study, all mice were anesthetized over a period of 1 wk and blood was collected via cardiocentesis at 6 different time points. Overall, male C3H/HeNCrl consumed more food under bLAD conditions as compared with CWF conditions; male C3H/HeNCrl had lower cholesterol levels under bLAD conditions than under CWF conditions; female BALB/cAnNCrl mice had higher serum total protein under bLAD conditions than under CWF conditions; female C57BL/6NCrl mice had higher phosphorus levels under bLAD conditions than under CWF conditions, and female C3H/HeNCrl mice had a higher neutrophil count under bLAD conditions as compared with CWF conditions. Although sex and strain differences were found in various physiologic parameters under bLAD as compared with CWF lighting conditions, the differences were minimal. Thus, this study suggests that for these strains of mice, bLAD and CWF are largely equivalent with regard to indices of health and wellbeing, although some differences could affect research outcomes.

Light is a key extrinsic factor to be considered in operations and design of animal room facilities. Over the past four decades, many studies on typical laboratory animal populations have demonstrated impacts on neuroendocrine, neurobehavioral, and circadian physiology. These effects are regulated independently from the defined physiology for the visual system. The range of physiological responses that oscillate with the 24 hour rhythm of the day include sleep and wakefulness, body temperature, hormonal secretion, and a wide range of other physiological parameters. Melatonin has been the chief neuroendocrine hormone studied, but acute light-induced effects on corticosterone as well as other hormones have also been observed. Within the last two decades, a new photosensory system in the mammalian eye has been discovered. A small set of retinal ganglion cells, previously thought to function as a visual output neuron, have been shown to be directly photosensitive and act differently from the classic photoreceptors of the visual system. Understanding the effects of light on mammalian physiology and behavior must take into account how the classical visual photoreceptors and the newly discovered ipRGC photoreceptor systems interact. Scientists and facility managers need to appreciate lighting impacts on circadian, neuroendocrine, and neurobehavioral regulation in order to improve lighting of laboratory facilities to foster optimum health and well-being of animals., (© The Author(s) 2020. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Light is a potent biologic force that profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in animals. Previously we examined the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit more light in the blue-appearing portion of the visible spectrum (465 to 485 nm) than do broad-spectrum cool white fluorescent (CWF) light, on the nighttime melatonin amplitude and circadian regulation of metabolism and physiology. In the current studies, we tested the hypothesis that exposure to blue-enriched LED light at day (bLAD), compared with CWF, promotes the circadian regulation of neuroendocrine, metabolic, and physiologic parameters that are associated with optimizing homeostatic regulation of health and wellbeing in 3 mouse strains commonly used in biomedical research (C3H [melatonin-producing], C57BL/6, and BALB/c [melatonin-non-producing]). Compared with male and female mice housed for 12 wk under 12:12-h light:dark (LD) cycles in CWF light, C3H mice in bLAD evinced 6-fold higher peak plasma melatonin levels at the middark phase; in addition, high melatonin levels were prolonged 2 to 3 h into the light phase. C57BL/6 and BALB/c strains did not produce nighttime pineal melatonin. Body growth rates; dietary and water intakes; circadian rhythms of arterial blood corticosterone, insulin, leptin, glucose, and lactic acid; pO₂ and pCO₂; fatty acids; and metabolic indicators (cAMP, DNA, tissue DNA 3 H-thymidine incorporation, fat content) in major organ systems were significantly lower and activation of major metabolic signaling pathways (mTOR, GSK3β, and SIRT1) in skeletal muscle and liver were higher only in C3H mice in bLAD compared with CWF. These data show that exposure of C3H mice to bLAD compared with CWF has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing that may influence scientific outcomes. The absence of enhancement in amelatonic strains suggests hyperproduction of nighttime melatonin may be a key component of the physiology.

Disruption of circadian time structure and suppression of circadian nocturnal melatonin (MLT) production by exposure to dim light at night (dLAN), as occurs with night shift work and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of breast cancer and resistance to tamoxifen and doxorubicin. Melatonin inhibition of human breast cancer chemoresistance involves mechanisms including suppression of tumor metabolism and inhibition of kinases and transcription factors which are often activated in drug-resistant breast cancer. Signal transducer and activator of transcription 3 (STAT3), frequently overexpressed and activated in paclitaxel (PTX)-resistant breast cancer, promotes the expression of DNA methyltransferase one (DNMT1) to epigenetically suppress the transcription of tumor suppressor Aplasia Ras homolog one (ARHI) which can sequester STAT3 in the cytoplasm to block PTX resistance. We demonstrate that breast tumor xenografts in rats exposed to dLAN and circadian MLT disrupted express elevated levels of phosphorylated and acetylated STAT3, increased DNMT1, but reduced sirtuin 1 (SIRT1) and ARHI. Furthermore, MLT and/or SIRT1 administration blocked/reversed interleukin 6 (IL-6)-induced acetylation of STAT3 and its methylation of ARH1 to increase ARH1 mRNA expression in MCF-7 breast cancer cells. Finally, analyses of the I-SPY 1 trial demonstrate that elevated MT 1 receptor expression is significantly correlated with pathologic complete response following neo-adjuvant therapy in breast cancer patients. This is the first study to demonstrate circadian disruption of MLT by dLAN driving intrinsic resistance to PTX via epigenetic mechanisms increasing STAT3 expression and that MLT administration can reestablish sensitivity of breast tumors to PTX and drive tumor regression., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Liver cancer is the second leading cause of cancer death worldwide. Metabolic pathways within the liver and liver cancers are highly regulated by the central circadian clock in the suprachiasmatic nuclei (SCN). Daily light and dark cycles regulate the SCN-driven pineal production of the circadian anticancer hormone melatonin and temporally coordinate circadian rhythms of metabolism and physiology in mammals. In previous studies, we demonstrated that melatonin suppresses linoleic acid metabolism and the Warburg effect (aerobic glycolysis)in human breast cancer xenografts and that blue-enriched light (465-485 nm) from light-emitting diode lighting at daytime (bLAD) amplifies nighttime circadian melatonin levels in rats by 7-fold over cool white fluorescent (CWF) lighting. Here we tested the hypothesis that daytime exposure of tissue-isolated Morris hepatoma 7288CTC-bearing male rats to bLAD amplifies the nighttime melatonin signal to enhance the inhibition of tumor growth. Compared with rats housed under a 12:12-h light:dark cycle in CWF light, rats in bLAD light evinced a 7-fold higher peak plasma melatonin level at the mid-dark phase; in addition, high melatonin levels were prolonged until 4 h into the light phase. After implantation of tissue-isolated hepatoma 7288CTC xenografts, tumor growth rates were markedly delayed, and tumor cAMP levels, LA metabolism, the Warburg effect, and growth signaling activities were decreased in rats in bLAD compared with CWF daytime lighting. These data show that the increased nighttime circadian melatonin levels due to bLAD exposure decreases hepatoma metabolic, signaling, and proliferative activities beyond what occurs after normal melatonin signaling under CWF light.

The association between a Western Diet and colon cancer suggests that dietary factors and/or obesity may contribute to cancer progression. Our objective was to develop a new animal model of obesity and the associated pathophysiology to investigate human cancer independent of dietary components that induce obesity. A novel congenic rat strain was established by introducing the fa allele from the Zucker rat into the Rowett Nude rat to generate a "fatty nude rat". The obese phenotype was first characterized in the new model. To then examine the utility of this model, lean and obese rats were implanted with HT-29 human colon cancer xenografts and tumor growth monitored. Fatty nude rats were visibly obese and did not develop fasting hyperglycemia. Compared to lean rats, fatty nude rats developed fasting hyperinsulinemia, glucose intolerance, and insulin resistance. Colon cancer tumor growth rate and final weight were increased (P < 0.05) in fatty nude compared to lean rats. Final tumor weight was associated with p38 kinase phosphorylation (P < 0.01) in fatty nude rats. We have established a novel model of obesity and pre-type 2 diabetes that can be used to investigate human cancer and therapeutics in the context of obesity and its associated pathophysiology.

Isoflurane anesthesia alters the blood levels of several neuroendocrine hormones associated with normal metabolism and physiology and increases stress, but the effect of brief CO2 anesthesia on these parameters is unknown. In this study, we examined the effects of isoflurane (4%) compared with brief CO2 (70% CO2, 30% air) anesthesia on circadian rhythms of plasma measures of physiology and metabolism. Adult male Sprague-Dawley rats (Crl:SD; n = 6 per group) were maintained on a 12:12-h light:dark (300 lx; lights on, 0600) photoperiod. After 1 wk of acclimation, a series of 6 low-volume blood draws were collected by cardiocentesis under anesthesia using isoflurane (10 min or less) compared with CO2 (1 min or less) at a single circadian time point every 4 d (0400, 0800, 1200, 1600, 2000, or 2400) over 3 wk to assess arterial blood glucose, lactic acid, and potassium and plasma melatonin, leptin, insulin, total fatty acids, and corticosterone concentrations. Results revealed that plasma levels (mean ± SEM) of melatonin were low (11 ± 1 pg/mL) during the light phase in both groups but were significantly lower during the dark phase in the isoflurane group (48 ± 6 pg/mL) compared with the CO2 group (162 ± 18 pg/mL). In addition, prominent circadian rhythms of arterial plasma levels of corticosterone, glucose, total fatty acids, lactic acid, and potassium were altered in the isoflurane group compared with the CO2 group. These findings demonstrate that the normal circadian rhythms of endocrine physiology and metabolism observed during brief CO2 anesthesia in rats are markedly disrupted by isoflurane anesthesia.

The importance of the circadian/melatonin signal in suppressing the metastatic progression of breast and other cancers has been reported by numerous laboratories including our own. Currently, the mechanisms underlying the antimetastatic actions of melatonin have not been well established. In the present study, the antimetastatic actions of melatonin were evaluated and compared on the ERα-negative, Her2-positive SKBR-3 breast tumor cell line and ERα-positive MCF-7 cells overexpressing a constitutively active HER2.1 construct (MCF-7Her2.1 cells). Activation of Her2 is reported to induce the expression and/or phosphorylation-dependent activation of numerous kinases and transcription factors that drive drug resistance and metastasis in breast cancer. A key signaling node activated by the Her2/Mapk/Erk pathway is Rsk2, which has been shown to induce numerous signaling pathways associated with the development of epithelial-to-mesenchymal transition (EMT) and metastasis including: Creb, Stat3, cSrc, Fak, Pax, Fascin, and actin polymerization. The data demonstrate that melatonin (both endogenous and exogenous) significantly represses this invasive/metastatic phenotype through a mechanism that involves the suppression of EMT, either by promoting mesenchymal-to-epithelial transition, and/or by inhibiting key signaling pathways involved in later stages of metastasis. These data, combined with our earlier in vitro studies, support the concept that maintenance of elevated and extended duration of nocturnal melatonin levels plays a critical role in repressing the metastatic progression of breast cancer., Implications: Melatonin inhibition of Rsk2 represses the metastatic phenotype in breast cancer cells suppressing EMT or inhibiting other mechanisms that promote metastasis; disruption of the melatonin signal may promote metastatic progression in breast cancer. Mol Cancer Res; 14(11); 1159-69. ©2016 AACR., Competing Interests: The authors have no conflicts of interest to disclose., (©2016 American Association for Cancer Research.)

Leiomyosarcoma (LMS) represents a highly malignant, rare soft tissue sarcoma with high rates of morbidity and mortality. Previously, we demonstrated that tissue-isolated human LMS xenografts perfused in situ are highly sensitive to the direct anticancer effects of physiological nocturnal blood levels of melatonin which inhibited tumour cell proliferative activity, linoleic acid (LA) uptake and metabolism to 13-hydroxyoctadecadienoic acid (13-HODE). Here, we show the effects of low pharmacological blood concentrations of melatonin following oral ingestion of a melatonin supplement by healthy adult human female subjects on tumour proliferative activity, aerobic glycolysis (Warburg effect) and LA metabolic signalling in tissue-isolated LMS xenografts perfused in situ with this blood. Melatonin markedly suppressed aerobic glycolysis and induced a complete inhibition of tumour LA uptake, 13-HODE release, as well as significant reductions in tumour cAMP levels, DNA content and [(3) H]-thymidine incorporation into DNA. Furthermore, melatonin completely suppressed the phospho-activation of ERK 1/2, AKT, GSK3β and NF-kB (p65). The addition of S20928, a nonselective melatonin antagonist, reversed these melatonin inhibitory effects. Moreover, in in vitro cell culture studies, physiological concentrations of melatonin repressed cell proliferation and cell invasion. These results demonstrate that nocturnal melatonin directly inhibited tumour growth and invasion of human LMS via suppression of the Warburg effect, LA uptake and other related signalling mechanisms. An understanding of these novel signalling pathway(s) and their association with aerobic glycolysis and LA metabolism in human LMS may lead to new circadian-based therapies for the prevention and treatment of LMS and potentially other mesenchymally derived solid tumours., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Regular cycles of exposure to light and dark control pineal melatonin production and temporally coordinate circadian rhythms of metabolism and physiology in mammals. Previously we demonstrated that the peak circadian amplitude of nocturnal blood melatonin levels of rats were more than 6-fold higher after exposure to cool white fluorescent (CWF) light through blue-tinted (compared with clear) rodent cages. Here, we evaluated the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit light rich in the blue-appearing portion of the visible spectrum (465-485 nm), compared with standard broadspectrum CWF light, on melatonin levels during the subsequent dark phase and on plasma measures of metabolism and physiology. Compared with those in male rats under a 12:12-h light:dark cycle in CWF light, peak plasma melatonin levels at the middark phase (time, 2400) in rats under daytime LED light were over 7-fold higher, whereas midlight phase levels (1200) were low in both groups. Food and water intakes, body growth rate, and total fatty acid content of major metabolic tissues were markedly lower, whereas protein content was higher, in the LED group compared with CWF group. Circadian rhythms of arterial plasma levels of total fatty acids, glucose, lactic acid, pO 2 , pCO 2 , insulin, leptin, and corticosterone were generally lower in LED-exposed rats. Therefore, daytime exposure of rats to LED light with high blue emissions has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing and thus may influence scientific outcomes.

Environmental enrichment (EE) gives laboratory animals opportunities to engage in species-specific behaviors. However, the effects of EE devices on normal physiology and scientific outcomes must be evaluated. We hypothesized that the spectral transmittance (color) of light to which rats are exposed when inside colored enrichment devices (CED) affects the circadian rhythms of various plasma markers. Pair-housed male Crl:SD rats were maintained in ventilated racks under a 12:12-h light:dark environment (265.0 lx; lights on, 0600); room lighting intensity and schedule remained constant throughout the study. Treatment groups of 6 subjects were exposed for 25 d to a colored enrichment tunnel: amber, red, clear, or opaque. We measured the proportion of time rats spent inside their CED. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and analyzed for plasma melatonin, total fatty acids, and corticosterone. Rats spent more time in amber, red, and opaque CED than in clear tunnels. All tubes were used significantly less after blood draws had started, except for the clear tunnel, which showed no change in use from before blood sampling began. Normal peak nighttime melatonin concentrations showed significant disruption in the opaque CED group. Food and water intakes and body weight change in rats with red-tinted CED and total fatty acid concentrations in the opaque CED group differed from those in other groups. These results demonstrate that the color of CED altered normal circadian rhythms of plasma measures of metabolism and physiology in rats and therefore might influence the outcomes of scientific investigations.

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462-484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake-metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.

Chemotherapeutic resistance, particularly to doxorubicin (Dox), represents a major impediment to successfully treating breast cancer and is linked to elevated tumor metabolism and tumor over-expression and/or activation of various families of receptor- and non-receptor-associated tyrosine kinases. Disruption of circadian time structure and suppression of nocturnal melatonin production by dim light exposure at night (dLEN), as occurs with shift work, and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of an array of diseases, including breast cancer. Melatonin inhibits human breast cancer growth via mechanisms that include the suppression of tumor metabolism and inhibition of expression or phospho-activation of the receptor kinases AKT and ERK1/2 and various other kinases and transcription factors. We demonstrate in tissue-isolated estrogen receptor alpha-positive (ERα+) MCF-7 human breast cancer xenografts, grown in nude rats maintained on a light/dark cycle of LD 12:12 in which dLEN is present during the dark phase (suppressed endogenous nocturnal melatonin), a significant shortening of tumor latency-to-onset, increased tumor metabolism and growth, and complete intrinsic resistance to Dox therapy. Conversely, a LD 12:12 dLEN environment incorporating nocturnal melatonin replacement resulted in significantly lengthened tumor latency-to-onset, tumor regression, suppression of nighttime tumor metabolism, and kinase and transcription factor phosphorylation, while Dox sensitivity was completely restored. Melatonin acts as both a tumor metabolic inhibitor and circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to Dox and drive tumor regression, indicating that dLEN-induced circadian disruption of nocturnal melatonin production contributes to a complete loss of tumor sensitivity to Dox chemotherapy., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The present review discusses recent work on melatonin-mediated circadian regulation, the metabolic and molecular signaling mechanisms that are involved in human breast cancer growth, and the associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1 receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin suppresses ERα mRNA expression and ERα transcriptional activity via the MT1 receptor. Melatonin also regulates the transactivation of other members of the nuclear receptor superfamily, estrogen-metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (the Warburg effect) and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type-specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways, including inhibition of p38 MAPK and repression of epithelial-mesenchymal transition (EMT). Studies have demonstrated that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models has indicated that LEN-induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer and drives breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms that underpin the epidemiologic demonstration of elevated breast cancer risk in night-shift workers and other individuals who are increasingly exposed to LEN., (© 2015 Society for Endocrinology.)

Early studies on rodents showed that short-term exposure to high-intensity light (> 70 lx) above 600 nm (red-appearing) influences circadian neuroendocrine and metabolic physiology. Here we addressed the hypothesis that long-term, low-intensity red light exposure at night (rLEN) from a 'safelight' emitting no light below approximately 620 nm disrupts the nocturnal circadian melatonin signal as well as circadian rhythms in circulating metabolites, related regulatory hormones, and physi- ologic parameters. Male Sprague-Dawley rats (n = 12 per group) were maintained on control 12:12-h light:dark (300 lx; lights on, 0600) or experimental 12:12 rLEN (8.1 lx) lighting regimens. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-wk period to assess arterial plasma melatonin, total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin and corticosterone concentrations. Results revealed plasma melatonin levels (mean ± 1 SD) were high in the dark phase (197.5 ± 4.6 pg/mL) and low in the light phase (2.6 ± 1.2 pg/mL) of control condi- tions and significantly lower than controls under experimental conditions throughout the 24-h period (P < 0.001). Prominent circadian rhythms of plasma levels of total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were significantly (P < 0.05) disrupted under experimental conditions as compared with the corresponding entrained rhythms under control conditions. Therefore, chronic use of low-intensity rLEN from a common safelight disrupts the circadian organization of neuroendocrine, metabolic, and physiologic parameters indicative of animal health and wellbeing.

The central circadian clock within the suprachiasmatic nucleus (SCN) plays an important role in temporally organizing and coordinating many of the processes governing cancer cell proliferation and tumor growth in synchrony with the daily light/dark cycle which may contribute to endogenous cancer prevention. Bioenergetic substrates and molecular intermediates required for building tumor biomass each day are derived from both aerobic glycolysis (Warburg effect) and lipid metabolism. Using tissue-isolated human breast cancer xenografts grown in nude rats, we determined that circulating systemic factors in the host and the Warburg effect, linoleic acid uptake/metabolism and growth signaling activities in the tumor are dynamically regulated, coordinated and integrated within circadian time structure over a 24-hour light/dark cycle by SCN-driven nocturnal pineal production of the anticancer hormone melatonin. Dim light at night (LAN)-induced melatonin suppression disrupts this circadian-regulated host/cancer balance among several important cancer preventative signaling mechanisms, leading to hyperglycemia and hyperinsulinemia in the host and runaway aerobic glycolysis, lipid signaling and proliferative activity in the tumor.

Resistance to endocrine therapy is a major impediment to successful treatment of breast cancer. Preclinical and clinical evidence links resistance to antiestrogen drugs in breast cancer cells with the overexpression and/or activation of various pro-oncogenic tyrosine kinases. Disruption of circadian rhythms by night shift work or disturbed sleep-wake cycles may lead to an increased risk of breast cancer and other diseases. Moreover, light exposure at night (LEN) suppresses the nocturnal production of melatonin that inhibits breast cancer growth. In this study, we used a rat model of estrogen receptor (ERα(+)) MCF-7 tumor xenografts to demonstrate how altering light/dark cycles with dim LEN (dLEN) speed the development of breast tumors, increasing their metabolism and growth and conferring an intrinsic resistance to tamoxifen therapy. These characteristics were not observed in animals in which the circadian melatonin rhythm was not disrupted, or in animals subjected to dLEN if they received nocturnal melatonin replacement. Strikingly, our results also showed that melatonin acted both as a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to tamoxifen and tumor regression. Together, our findings show how dLEN-mediated disturbances in nocturnal melatonin production can render tumors insensitive to tamoxifen., (©2014 American Association for Cancer Research.)

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF-7 human breast cancer cells and several other estrogen receptor α (ERα)-positive human breast cancer cell lines. However, the MDA-MB-231 breast cancer cell line, an ERα-negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth-suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα-negative breast cancer cell lines including MDA-MB-231, BT-20, and SK-BR-3 cells. Although the MT1 G-protein-coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK-BR-3 cells without having any significant effect on the growth of MDA-MB-231 and BT-20 cells. We confirmed that the MT1-associated Gα proteins are expressed in MDA-MB-231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA-MB-231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The suprachiasmatic nucleus is synchronized by the light:dark cycle and is the master biologic clock that serves as a pacemaker to regulate circadian rhythms. We explored the hypothesis that spectral transmittance (tint) of light through caging alters circadian rhythms of endocrine and metabolic plasma constituents in nonpigmented Sprague-Dawley rats. Rats (Crl:SD; n = 12 per group) were housed in a 12:12-h light:dark environment (300 lx; 123.0 μ W/cm(2); lights on, 0600) in either clear-, amber-, blue-, or red-tinted rodent cages. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and measured for melatonin, total fatty acids, pH, glucose, lactic acid, corticosterone, insulin, and leptin. As expected, plasma melatonin levels were low during the light phase but higher during the dark phase in all groups; however, when compared with the clear-cage group, rats in amber-, blue-, and red-tinted cages had 29%, 74%, and 48%, respectively, greater total daily melatonin levels due to an increased duration and, in some cases, amplitude of the nocturnal melatonin signal. No differences were found in dietary and water intake, body growth rates, total fatty acids, pH, or glucose among groups. Disruptions in circadian rhythms, manifesting as alterations in phase timing, amplitude, or duration, occurred in the melatonin, lactic acid, corticosterone, insulin, and leptin levels of rats in tinted compared with clear cages. Therefore, the use of variously tinted animal cages significantly alters circadian rhythms in plasma measures of metabolism and physiology in laboratory rats, thus potentially altering the outcomes of scientific investigations.

Obesity is a chronic inflammation with increased serum levels of insulin, insulin-like growth factor 1 (IGF1), and interleukin-17 (IL-17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL-17-induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)-dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL-17-induced expression of C-X-C motif ligand 1 (Cxcl1) and C-C motif ligand 20 (Ccl20) in the Gsk3b(+/+) , but not in Gsk3b(-/-) mouse embryonic fibroblast (MEF) cells. IL-17 induced higher levels of Cxcl1 and Ccl20 in the Gsk3b(-/-) MEF cells, compared with the Gsk3b(+/+) MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P-GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL-17 alone or by a combination of insulin and IL-17. Melatonin's inhibitory effects were only observed in the Gsk3b(+/+) , but in not Gsk3b(-/-) MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il-6 that was induced by a combination of insulin and IL-17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il-6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL-17-mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Light entrains normal circadian rhythms of physiology and metabolism in all mammals. Previous studies from our laboratory demonstrated that spectral transmittance (color) of light passing through cages affects these responses in rats. Here, we addressed the hypothesis that red tint alters the circadian nocturnal melatonin signal and circadian oscillation of other metabolic and physiologic functions. Female nude rats (Hsd:RH-Foxn1(rnu); n = 12 per group) were maintained on a 12:12-h light (300 lx; 123.0 μW/cm(2); lights on 0600):dark regimen in standard polycarbonate translucent clear or red-tinted cages. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis over a 4-wk period. Plasma melatonin levels were low during the light phase (1.0 ± 0.2 pg/mL) in rats in both types of cages but were significantly lower in red-tinted (105.0 ± 2.4 pg/mL) compared with clear (154.8 ± 3.8 pg/mL) cages during the dark. Normal circadian rhythm of plasma total fatty acid was identical between groups. Although phase relationships of circadian rhythms in glucose, lactic acid, pO2, and pCO2 were identical between groups, the levels of these analytes were lower in rats in red-tinted compared with clear cages. Circadian rhythms of plasma corticosterone, insulin, and leptin were altered in terms of phasing, amplitude, and duration in rats in red-tinted compared with clear cages. These findings indicate that spectral transmittance through red-colored cages significantly affects circadian regulation of neuroendocrine, metabolic, and physiologic parameters, potentially influencing both laboratory animal health and wellbeing and scientific outcomes.

Light is potent in circadian, neuroendocrine, and neurobehavioral regulation, thereby having profound influence on the health and wellbeing of all mammals, including laboratory animals. We hypothesized that the spectral quality of light transmitted through colored compared with clear standard rodent cages alters circadian production of melatonin and temporal coordination of normal metabolic and physiologic activities. Female nude rats (Hsd:RH-Foxn1(rnu); n = 6 per group) were maintained on a 12:12-h light:dark regimen (300 lx; lights on, 0600) in standard translucent clear, amber, or blue rodent cages; intensity and duration of lighting were identical for all groups. Rats were assessed for arterial blood levels of pO(2) and pCO(2), melatonin, total fatty acid, glucose, lactic acid, insulin, leptin, and corticosterone concentrations at 6 circadian time points. Normal circadian rhythms of arterial blood pO(2) and pCO(2) were different in rats housed in cages that were blue compared with amber or clear. Plasma melatonin levels (mean ± 1 SD) were low (1.0 ± 0.2 pg/mL) during the light phase in all groups but higher at nighttime in rats in blue cages (928.2 ± 39.5 pg/mL) compared with amber (256.8 ± 6.6 pg/mL) and clear (154.8 ± 9.3 pg/mL) cages. Plasma daily rhythms of total fatty acid, glucose, lactic acid, leptin, insulin, and corticosterone were disrupted in rats housed in blue or amber compared with clear cages. Temporal coordination of circadian rhythms of physiology and metabolism can be altered markedly by changes in the spectral quality of light transmitted through colored standard rodent cages.

Disturbed sleep-wake cycle and circadian rhythmicity are associated with cancer, but the underlying mechanisms are unknown. Employing a tissue-isolated human breast xenograft tumor nude rat model, we observed that glycogen synthase kinase 3β (GSK3β), an enzyme critical in metabolism and cell proliferation/survival, exhibits a circadian rhythm of phosphorylation in human breast tumors. Exposure to light-at-night suppresses the nocturnal pineal melatonin synthesis, disrupting the circadian rhythm of GSK3β phosphorylation. Melatonin activates GSK3β by inhibiting the serine-threonine kinase Akt phosphorylation, inducing β-catenin degradation and inhibiting epithelial-to-mesenchymal transition, a fundamental process underlying cancer metastasis. Thus, chronic circadian disruption by light-at-night via occupational exposure or age-related sleep disturbances may contribute to cancer incidence and the metastatic spread of breast cancer by inhibiting GSK3β activity and driving epithelial-to-mesenchymal transition in breast cancer patients.

We present a new perfusion system and surgical technique for simultaneous perfusion of 2 tissue-isolated human cancer xenografts in nude rats by using donor blood that preserves a continuous flow. Adult, athymic nude rats (Hsd:RH-Foxn1(rnu)) were implanted with HeLa human cervical or HT29 colon adenocarcinomas and grown as tissue-isolated xenografts. When tumors reached an estimated weight of 5 to 6 g, rats were prepared for perfusion with donor blood and arteriovenous measurements. The surgical procedure required approximately 20 min to complete for each tumor, and tumors were perfused for a period of 150 min. Results showed that tumor venous blood flow, glucose uptake, lactic acid release, O(2) uptake and CO(2) production, uptake of total fatty acid and linoleic acid and conversion to the mitogen 13-HODE, cAMP levels, and activation of several marker kinases were all well within the normal physiologic, metabolic, and signaling parameters characteristic of individually perfused xenografts. This new perfusion system and technique reduced procedure time by more than 50%. These findings demonstrate that 2 human tumors can be perfused simultaneously in situ or ex vivo by using either rodent or human blood and suggest that the system may also be adapted for use in the dual perfusion of other organs. Advantages of this dual perfusion technique include decreased anesthesia time, decreased surgical manipulation, and increased efficiency, thereby potentially reducing the numbers of laboratory animals required for scientific investigations.

This review article discusses recent work on the melatonin-mediated circadian regulation and integration of molecular, dietary, and metabolic signaling mechanisms involved in human breast cancer growth and the consequences of circadian disruption by exposure to light at night (LAN). The antiproliferative effects of the circadian melatonin signal are mediated through a major mechanism involving the activation of MT(1) melatonin receptors expressed in human breast cancer cell lines and xenografts. In estrogen receptor (ERα+) human breast cancer cells, melatonin suppresses both ERα mRNA expression and estrogen-induced transcriptional activity of the ERα via MT(1) -induced activation of G(αi2) signaling and reduction of 3',5'-cyclic adenosine monophosphate (cAMP) levels. Melatonin also regulates the transactivation of additional members of the steroid hormone/nuclear receptor super-family, enzymes involved in estrogen metabolism, expression/activation of telomerase, and the expression of core clock and clock-related genes. The anti-invasive/anti-metastatic actions of melatonin involve the blockade of p38 phosphorylation and the expression of matrix metalloproteinases. Melatonin also inhibits the growth of human breast cancer xenografts via another critical pathway involving MT(1) -mediated suppression of cAMP leading to blockade of linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Down-regulation of 13-HODE reduces the activation of growth factor pathways supporting cell proliferation and survival. Experimental evidence in rats and humans indicating that LAN-induced circadian disruption of the nocturnal melatonin signal activates human breast cancer growth, metabolism, and signaling provides the strongest mechanistic support, thus far, for population and ecological studies demonstrating elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LAN., (© 2011 John Wiley & Sons A/S.)

This review article discusses recent work on the melatonin-mediated circadian regulation and integration of molecular and metabolic signaling mechanisms involved in human breast cancer growth and the associated consequences of circadian disruption by exposure to light-at-night (LAN). The anti-proliferative effects of the circadian melatonin signal are, in general, mediated through mechanisms involving the activation of MT(1) melatonin receptors expressed in human breast cancer cell lines and xenografts. In estrogen receptor-positive (ERα+) human breast cancer cells, melatonin suppresses both ERα mRNA expression and estrogen-induced transcriptional activity of the ERα via MT(1)-induced activation of G(αi2) signaling and reduction of cAMP levels. Melatonin also regulates the transcriptional activity of additional members of the nuclear receptor super-family, enzymes involved in estrogen metabolism, and the expression of core clock and clock-related genes. The anti-invasive/anti-metastatic actions of melatonin involve the blockade of p38 phosphorylation and matrix metalloproteinase expression. Melatonin also inhibits the growth of human breast cancer xenografts via MT(1)-mediated suppression of cAMP leading to a blockade of linoleic acid (LA) uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Down-regulation of 13-HODE reduces the activation of growth factor pathways supporting cell proliferation and survival. Finally, studies in both rats and humans indicate that light-at-night (LAN) induced circadian disruption of the nocturnal melatonin signal activates human breast cancer growth, metabolism, and signaling, providing the strongest mechanistic support, thus far, for epidemiological studies demonstrating the elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LAN.

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR(-)) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O(2), glucose, total fatty acid and CO(2) production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine-threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.

Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling.

Dark-phase light contamination can significantly disrupt chronobiologic rhythms, thereby potentially altering the endocrine physiology and metabolism of experimental animals and influencing the outcome of scientific investigations. We sought to determine whether exposure to low-level light contamination during the dark phase influenced the normally entrained circadian rhythms of various substances in plasma. Male Sprague-Dawley rats (n = 6 per group) were housed in photobiologic light-exposure chambers configured to create 1) a 12:12-h light:dark cycle without dark-phase light contamination (control condition; 123 μW/cm(2), lights on at 0600), 2) experimental exposure to a low level of light during the 12-h dark phase (with 0.02, 0.05, 0.06, or 0.08 μW/cm(2) light at night), or 3) constant bright light (123 μW/cm(2)). Dietary and water intakes were recorded daily. After 2 wk, rats underwent 6 low-volume blood draws at 4-h intervals (beginning at 0400) during both the light and dark phases. Circadian rhythms in dietary and water intake and levels of plasma total fatty acids and lipid fractions remained entrained during exposure to either control conditions or low-intensity light during the dark phase. However, these patterns were disrupted in rats exposed to constant bright light. Circadian patterns of plasma melatonin, glucose, lactic acid, and corticosterone were maintained in all rats except those exposed to constant bright light or the highest level of light during the dark phase. Therefore even minimal light contamination during the dark phase can disrupt normal circadian rhythms of endocrine metabolism and physiology and may alter the outcome of scientific investigations.

The circadian production of melatonin by the pineal gland during the night provides an inhibitory signal to tissue-isolated steroid receptor SR+ and - MCF-7 human breast cancer xenografts in female nude rats. A pivotal mechanism for melatonin's anticancer effects in vivo involves a melatonin receptor-mediated inhibition of linoleic acid (LA) uptake and its metabolism to mitogenically active 13-hydroxyoctadecadienoic acid (13-HODE). Exposure of (SR-) xenograft-bearing rats to increasing intensities of polychromatic white light at night suppresses melatonin while increasing tumor growth rates, DNA content, [3H]thymidine incorporation into DNA, LA uptake, 13-HODE formation, cAMP levels and ERK1/2 activation a dose-dependent manner. Similar effects occur in SR- human breast cancer xenografts perfused in situ with melatonin-depleted blood from healthy female subjects after their exposure to a single bright intensity (2800 lux) of polychromatic light at night. Additionally, SR- human breast cancer xenografts exhibit robust circadian rhythms of LA uptake, 13-HODE formation and proliferative activity. Exposure of xenograft-bearing rats to dim light at night results in the complete elimination of these rhythms which culminates in unfettered, high rates of tumor metabolism and growth. The organization of tumor metabolism and growth within circadian time structure by the oncostatic melatonin signal helps create a balance between the cancer and its host that is disrupted by host exposure to light at night. This biological mechanism may partially explain the higher risk of breast and other cancers in women working rotating night shifts and possibly others who also experience prolonged exposure to light at night.

Melatonin provides a circadian signal that regulates linoleic acid (LA)-dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth-stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). This study tested the hypothesis that both acute and long-term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue-isolated human leiomyosarcoma (LMS), a rare, mesenchymally-derived smooth muscle tissue sarcoma, via melatonin receptor-mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue-isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 +/- 0.02 g/day) versus control xenografts that continued to grow at 0.22 +/- 0.03 g/day over a 10-day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose-dependent suppression of tumor cAMP production, LA uptake, 13-HODE release, extracellular signal-regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [(3)H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-Br-cAMP. These results demonstrate that, as observed in epithelially-derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long-term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor-mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.

Development of a diet that provides adequate nutrition and effective cancer prevention is an important goal in nutrition and cancer research. A confounding aspect of dietary control of tumor growth is the fact that some nutrients may up-regulate tumor growth, whereas other nutrients and nonnutrients down-regulate growth. Both up- and down-regulators may be present in the same foodstuff. Identification of these substances, determination of their mechanisms of action and potencies, as well as the interactions among the different mechanisms are topics of ongoing research. In this review, we describe results obtained in vivo or during perfusion in situ using solid tissue-isolated rodent tumors and human cancer xenografts in nude rats. Linoleic acid (LA), an essential n-6 polyunsaturated fatty acid (PUFA), was identified as an agent in dietary fat that is responsible for an up-regulation of tumor growth in vivo. Tumor LA uptake, mediated by high intratumor cAMP, stimulated formation of the mitogen, 13-hydroxyoctadecadienoic acid (13-HODE) and also increased ERK1/2 phosphorylation, [(3)H]thymidine incorporation and growth. A mechanism for control of this growth-promoting pathway was revealed during studies of the effects of dietary nutrients and nonnutrients known to inhibit tumor growth. These included four groups of lipophilic agents: n-3 fatty acids, melatonin, conjugated LA isomers and trans fatty acids. Each of these agents activated an inhibitory G protein-coupled receptor-mediated pathway that specifically suppressed tumor uptake of saturated, monounsaturated and n-6 PUFAs, thereby inhibiting an early step in the LA-dependent growth-promoting pathway.

Melatonin and eicosapentaenoic and 10t,12c-conjugated linoleic acids suppress the growth-stimulating effects of linoleic acid (LA) and its metabolism to the mitogenic agent 13-(S)-hydroxyoctadecadienoic acid (13-(S)-HODE) in established rodent tumors and human cancer xenografts. Here we compared the effects of these 3 inhibitory agents on growth and LA uptake and metabolism in human FaDu squamous cell carcinoma xenografts perfused in situ in male nude rats. Results demonstrated that these agents caused rapid inhibition of LA uptake, tumor cAMP content, 13-(S)-HODE formation, extracellular signal-regulated kinase p44/ p42 (ERK 1/2) activity, mitogen-activated protein kinase kinase (MEK) activity, and [3H]thymidine incorporation into tumor DNA. Melatonin's inhibitory effects were reversible with either the melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-bromoadenosine-cAMP, suggesting that its growth-inhibitory effect occurs in vivo via a receptor-mediated, pertussis-toxin-sensitive pathway.

We developed an artificial lung and catheter system for perfusing tissue-isolated tumors in situ that dramatically minimizes perfusate delivery time. Our investigations demonstrated that the circadian neurohormone melatonin (MLT), eicosapentaenoic acid (EPA), and conjugated linoleic acid (CLA) inhibit growth and metabolism in several rodent and human tumors. These anticancer agents function in a receptor-mediated manner to suppress tumor uptake of linoleic acid (LA), the principal tumor growth-promoting fatty acid, and its conversion to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). Using this perfusion system and MCF-7 human breast xenografts, we examined the efficacy and timing of perfusate delivery to tumors. Tumors were perfused with rat donor blood to establish baseline LA uptake values; after 36 min of perfusion, we supplemented the perfusate with MLT, EPA, or CLA and collected arteriovenous whole-blood samples over 5-min intervals for a total perfusion period of 70 min. Arterial blood pH, pO2, and pCO2 (mean+/-33.7+/-1.9, and 59.8+/-1.9 mm Hg, respectively; none of these values varied during the perfusions. Tumor LA uptake and 13-HODE production were 1.06+/-0.28 microg/min/g and 1.38+/-0.02 ng/min/g, respectively, and were completely suppressed within 5 min after delivery of anticancer agents to the tissue. This new system provides rapid perfusate delivery for use with both normal and neoplastic tissues while maintaining normal physiologic tissue parameters.

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 microW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 microW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.

The type and content of dietary PUFAs have profound influences on the growth rate of transplantable human breast cancers in immunodeficient rodents. Diets enriched in linoleic acid (LA), an (n-6) fatty acid, stimulate tumor growth, whereas dietary fats containing (n-3) fatty acids slow such growth. Interactions between LA and (n-3) fatty acids capable of regulating cell proliferation in solid tumors in vivo are not yet well defined. Here we tested the hypothesis that plasma eicosapentaenoic acid (EPA), an (n-3) fatty acid, suppresses cell proliferation in MCF-7 human breast cancer xenografts via a pertussis toxin-sensitive reduction of intratumor cAMP, LA uptake, and formation of the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) from LA. Plasma fatty acid uptake and 13-HODE release were determined in control and EPA-treated xenografts from arteriovenous differences measured during perfusion in situ. Intratumor cAMP, extracellular signal-regulated kinase p44/p42 (ERK1/2) phosphorylation, and [3H]thymidine incorporation (TTI) were measured in tumors freeze-clamped at the end of the perfusions. Arterial blood containing EPA caused significant decreases (P < 0.05) in cAMP, uptake of SFA, monounsaturated fatty acids, and (n-6) PUFA, 13-HODE formation, ERK1/2 phosphorylation, and TTI in MCF-7 xenografts. These effects of EPA were reversed by the addition of either pertussis toxin or 8-bromoadenosine-cAMP to the EPA-containing arterial blood. Addition of 13-HODE to the EPA-containing arterial blood restored phosphorylated ERK1/2 and TTI but not FA uptake. The results suggest that EPA regulates cell proliferation in MCF-7 xenografts via a novel inhibitory G protein-coupled, (n-3) FFA receptor-mediated signal transduction pathway.