Light: An Extrinsic Factor Influencing Animal-based Research.

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.

Dietary Linoleic Acid: An Omega-6 Fatty Acid Essential for Liver Regeneration in Buffalo Rats.

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.

Vivarium Lighting as an Important Extrinsic Factor Influencing Animal-based Research.

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.

A Method for Perfusion of Tissue-Isolated Human Tumor Xenografts in Nude Rats to Investigate the Oncostatic Role of the Physiological Nocturnal Melatonin Signal.

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.

A Method for Growing Tissue-Isolated Human Tumor Xenografts in Nude Rats for Melatonin/Cancer Studies.

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.

Artificial Light at Night Reduces Anxiety-like Behavior in Female Mice with Exacerbated Mammary Tumor Growth.

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 × 105 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.

Dietary Melatonin and Omega-3 Fatty Acids Induce Human Cancer Xenograft Regression In Vivo in Rats by Suppressing Linoleic Acid Uptake and Metabolism.

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.

Effects of Daytime Blue-Enriched LED Light on Physiologic Parameters of Three Common Mouse Strains Maintained on an IVC System.

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.

Relevance of Electrical Light on Circadian, Neuroendocrine, and Neurobehavioral Regulation in Laboratory Animal Facilities.

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.

Influence of Daytime LED Light Exposure on Circadian Regulatory Dynamics of Metabolism and Physiology in Mice.

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 3H-thymidine incorporation, fat content) in major organ systems were significantly lower and activation of major metabolic signaling pathways (mTOR, GSK3ß, 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.

Epigenetic inhibition of the tumor suppressor ARHI by light at night-induced circadian melatonin disruption mediates STAT3-driven paclitaxel resistance in breast cancer.

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 MT1 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.

Effect of Daytime Blue-enriched LED Light on the Nighttime Circadian Melatonin Inhibition of Hepatoma 7288CTC Warburg Effect and Progression.

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.

Development and Characterization of a Novel Congenic Rat Strain for Obesity and Cancer Research.

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.

Health consequences of electric lighting practices in the modern world: A report on the National Toxicology Program's workshop on shift work at night, artificial light at night, and circadian disruption.

The invention of electric light has facilitated a society in which people work, sleep, eat, and play at all hours of the 24-hour day. Although electric light clearly has benefited humankind, exposures to electric light, especially light at night (LAN), may disrupt sleep and biological processes controlled by endogenous circadian clocks, potentially resulting in adverse health outcomes. Many of the studies evaluating adverse health effects have been conducted among night- and rotating-shift workers, because this scenario gives rise to significant exposure to LAN. Because of the complexity of this topic, the National Toxicology Program convened an expert panel at a public workshop entitled "Shift Work at Night, Artificial Light at Night, and Circadian Disruption" to obtain input on conducting literature-based health hazard assessments and to identify data gaps and research needs. The Panel suggested describing light both as a direct effector of endogenous circadian clocks and rhythms and as an enabler of additional activities or behaviors that may lead to circadian disruption, such as night-shift work and atypical and inconsistent sleep-wake patterns that can lead to social jet lag. Future studies should more comprehensively characterize and measure the relevant light-related exposures and link these exposures to both time-independent biomarkers of circadian disruption and biomarkers of adverse health outcomes. This information should lead to improvements in human epidemiological and animal or in vitro models, more rigorous health hazard assessments, and intervention strategies to minimize the occurrence of adverse health outcomes due to these exposures.

Effect of Isoflurane Anesthesia on Circadian Metabolism and Physiology in Rats.

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.

Effects of Daytime Exposure to Light from Blue-Enriched Light-Emitting Diodes on the Nighttime Melatonin Amplitude and Circadian Regulation of Rodent Metabolism and Physiology.

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, pO2, pCO2, 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.

Effects of Colored Enrichment Devices on Circadian Metabolism and Physiology in Male Sprague-Dawley Rats.

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.

Melatonin Represses Metastasis in Her2-Postive Human Breast Cancer Cells by Suppressing RSK2 Expression.

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.

Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma.

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, GSK3ß 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.