Chronic ethanol intake alters circadian period-responses to brief light pulses in rats

Although chronic alcohol intake is associated with widespread disruptions of sleep-wake cycles and other daily biological rhythms in both human alcoholics and experimental animals, the extent to which the chronobiological effects of alcohol are mediated by effects on the underlying circadian pacemaker remains unknown. Nevertheless, recent studies indicate that both adult and perinatal ethanol treatments may alter the free-running period and photic responsiveness of the circadian pacemaker. The present experiment was designed to further characterize the effects of chronic ethanol intake on the response of the rat circadian pacemaker to brief light pulses. Ethanol-treated and control animals were exposed to 15-min light pulses during either early or late subjective night on the first day of constant darkness following entrainment to a 12:12 light-dark cycle. Relative to pulses delivered during early subjective night and to “no-pulse” conditions, light pulses delivered during late subjective night resulted in period-shortening after-effects under constant darkness, but only in control animals, not in ethanol-treated animals. These results indicate that chronic ethanol intake reduces the responsiveness of the circadian pacemaker to acute photic stimulation, and suggest that the chronobiological disruptions seen in human alcoholics are due in part to alterations in circadian pacemaker function.     

GABA synchronizes clock cells within the suprachiasmatic circadian clock

The master clock in the suprachiasmatic nuclei (SCN) is composed of multiple, single-cell circadian clocks. We test the postulate that these individual "clock cells" can be synchronized to each other by the inhibitory transmitter gamma-aminobutyric acid (GABA). For these experiments, we monitored the firing rate rhythm of individual clock cells on fixed multielectrode plates in culture and tested the effects of GABA. The results show that the daily variation in responsiveness of the SCN to phase-shifting agents is manifested at the level of individual neurons. Moreover, GABA, acting through A-type receptors, can both phase shift and synchronize clock cells. We propose that GABA is an important synchronizer of SCN neurons in vivo.     

Aging alters circadian and light-induced expression of clock genes in golden hamsters

Aging alters numerous aspects of circadian biology, including the amplitude of rhythms generated by the suprachiasmatic nuclei (SCN) of the hypothalamus, the site of the central circadian pacemaker in mammals, and the response of the pacemaker to environmental stimuli such as light. Although previous studies have described molecular correlates of these behavioral changes, to date only 1 study in rats has attempted to determine if there are age-related changes in the expression of genes that comprise the circadian clock itself. We used in situ hybridization to examine the effects of age on the circadian pattern of expression of a subset of the genes that comprise the molecular machinery of the circadian clock in golden hamsters. Here we report that age alters the 24-h expression profile of Clock and its binding partner Bmal1 in the hamster SCN. There is no effect of age on the 24-h profile of either Per1 or Per2 when hamsters are housed in constant darkness. We also found that light pulses, which induce smaller phase shifts in old animals than in young, lead to decreased induction of Per1, but not of Per2, in the SCN of old hamsters.     

Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition.Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism.Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA.Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.     

Research on circadian clock genes in non-small-cell lung carcinoma

Circadian clock genes have become a hot topic in cancer research in recent years, and more and more studies are showing that clock genes are involved in regulating cell proliferation cycle and apoptosis of malignant tumors, neuroendocrine and immune function, and other processes. Lung cancer is a malignant tumor with increasing incidence worldwide. The pathogenesis of lung cancer is extremely complicated and includes genetic factors, living environment, and smoking, and the occurrence of lung cancer is related to the regulation of many oncogenes and tumor suppressor genes. But there are few studies on clock genes in lung cancer. Studies on clock genes may help to better understand the mechanism of lung cancer development for an improved treatment. The expressions of all 14 kinds of clock genes in adenocarcinoma (ADC) and squamous cell carcinoma (SCC), two main kinds of non-small-cell lung cancer (NSCLC), were studied based on integration and analysis of data from The Cancer Genome Atlas (TCGA) to show the association between clock gene expression and prognosis of cancer patients. Analysis of TCGA data indicated that overexpression of Cry2, BMAL1, and RORA with underexpression of Timeless and NPAS2 was associated with a favorable prognosis of ADC, and the expression of NPAS2 was associated with the time of patient survival. Additionally, the expression of Cry2 was related to TNM stage. In SCC, high expression of DEC1 was correlated with poor overall survival in patients and the expression of Timeless was associated with the time of patient survival. In NSCLC, circadian clock genes constitute cancer circadian rhythm by interacting with each other, showing that asynchrony with normal tissues, which collectively controlling the occurrence and development of NSCLC.     

The regulation of plant growth by the circadian clock

Circadian regulated changes in growth rates have been observed in numerous plants as well as in unicellular and multicellular algae. The circadian clock regulates a multitude of factors that affect growth in plants, such as water and carbon availability and light and hormone signalling pathways. The combination of high-resolution growth rate analyses with mutant and biochemical analysis is helping us elucidate the time-dependent interactions between these factors and discover the molecular mechanisms involved. At the molecular level, growth in plants is modulated through a complex regulatory network, in which the circadian clock acts at multiple levels.     

Circadian disruption alters mouse lung clock gene expression and lung mechanics

Most aspects of human physiology and behavior exhibit 24-h rhythms driven by a master circadian clock in the brain, which synchronizes peripheral clocks. Lung function and ventilation are subject to circadian regulation and exhibit circadian oscillations. Sleep disruption, which causes circadian disruption, is common in those with chronic lung disease, and in the general population; however, little is known about the effect on the lung of circadian disruption. We tested the hypothesis circadian disruption alters expression of clock genes in the lung and that this is associated with altered lung mechanics. Female and male mice were maintained on a 12:12-h light/dark cycle (control) or exposed for 4 wk to a shifting light regimen mimicking chronic jet lag (CJL). Airway resistance (Rn), tissue damping (G), and tissue elastance (H) did not differ between control and CJL females. Rn at positive end-expiratory pressure (PEEP) of 2 and 3 cmH(2)O was lower in CJL males compared with controls. G, H, and G/H did not differ between CJL and control males. Among CJL females, expression of clock genes, Bmal1 and Rev-erb alpha, was decreased; expression of their repressors, Per2 and Cry 2, was increased. Among CJL males, expression of Clock was decreased; Per 2 and Rev-erb alpha expression was increased. We conclude circadian disruption alters lung mechanics and clock gene expression and does so in a sexually dimorphic manner.     

Scheduled feeding alters the timing of the suprachiasmatic nucleus circadian clock in dexras1-deficient mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1(-/-)) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1(-/-) mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1(-/-) mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca ).     

Interactions of circadian clock genes with the hallmarks of cancer

The molecular machinery of the circadian clock regulates the expression of many genes and processes in the organism, allowing the adaptation of cellular activities to the daily light-dark cycles.Disruption of the circadian rhythm can lead to various pathologies, including cancer. Thus, disturbance of the normal circadian clock at both genetic and environmental levels has been described as an independent risk factor for cancer. In addition, researchers have proposed that circadian genes may have a tissue-dependent and/or context-dependent role in tumorigenesis and may function both as tumor suppressors and oncogenes.Finally, circadian clock core genes may trigger or at least be involved in different hallmarks of cancer. Hence, expanding the knowledge of the molecular basis of the circadian clock would be helpful to identify new prognostic markers of tumorigenesis and potential therapeutic targets.     

Chronic inflammation inhibits GH secretion and alters the serum insulin-like growth factor system in rats

Adjuvant-induced arthritis in rats is associated with growth failure, hypermetabolism and accelerated protein breakdown. The aim of this work was to study the effects of adjuvant-induced arthritis on GH and insulin-like growth factor-I (IGF-I). Arthritis was induced by an intradermal injection of complete Freund's adjuvant and rats were killed 18 and 22 days later. IGF-I and GH levels were measured by radioimmunoassay. Pituitary GH mRNA was analyzed by northern blot and IGF binding proteins (IGFBPs) by western blot. Arthritic rats showed a decrease in both serum and hepatic concentrations of IGF-I. On the contrary, arthritis increased the circulating IGFBPs. The serum concentration of IGF-I in the arthritic rats was negatively correlated with the body weight loss observed in these animals. Arthritis decreased the serum concentration of GH and this decrease seems to be due to an inhibition of GH synthesis, since pituitary GH mRNA content was decreased in arthritic rats (p<0.01). These data suggest that the decrease in body weight gain in arthritic rats may be, at least in part, secondary to the decrease in GH and IGF-I secretion. Furthermore, the increased serum IGFBPs may also be involved in the disease process.     

Downregulation of miR-16 promotes growth and motility by targeting HDGF in non-small cell lung cancer cells

MicroRNAs play important roles in the development and progression of non-small cell lung cancer (NSCLC). miR-16 functions as a tumor-suppressor and is inhibited in several malignancies. Herein, we validated that miR-16 is downregulated in NSCLC tissue samples and cell lines. Ectopic expression of miR-16 significantly inhibited cell proliferation and colony formation. Moreover, miR-16 suppressed cell migration and invasion in NSCLC cells. Hepatoma-derived growth factor (HDGF) was found to be a direct target of miR-16 in NSCLC cell lines. Rescue experiments showed that the suppressive effect of miR-16 on cell proliferation, colony formation, migration, and invasion is partially mediated by inhibiting HDGF expression. This study indicates that miR-16 might be associated with NSCLC progression, and suggests an essential role for miR-16 in NSCLC.     

Effects of altered photoperiod on circadian clock and lipid metabolism in rats

Disruption of circadian clock timekeeping due to changes in the photoperiod enhances the risk of lipid metabolism disorders and metabolic syndrome. However, the effects of altered photoperiods on the circadian clock and lipid metabolism are not well understood. To explore the effects of altered photoperiods, we developed a rat model where rats were exposed to either short-day or long-day conditions. Our findings demonstrated that altered photoperiods mediated circadian clocks by partly disrupting rhythmicity and shifting phase values of clock genes. We also showed that compared to long-day conditions, rats under short-day conditions exhibited more photoperiodic changes in a variety of physiological outputs related to lipid metabolism, such as significant increases in serum triglyceride (TG), high-density lipoprotein, and leptin levels, as well as increased body weight, fat:weight ratio, and hepatic TG levels. These increments were gained possibly through upregulated expression of forkhead box O1 (FoxO1), which partly mediates the expression of peroxisome proliferator-activated receptorα (PPARα) to increase the expression of phosphoenolpyruvate carboxykinase (PEPCK), peroxisome proliferator-activated receptor-g coactivator-1β (PGC1β), and fatty acid synthase (Fasn). In addition, the oscillation rhythms of FoxO1, PEPCK, PGC1β, and Fasn expression levels in the livers of rats exposed to a short-day photoperiod were more robust than those exposed to a long-day photoperiod. These findings suggest that a change in photoperiod can partly disrupt the circadian rhythmcity of clock genes, impair lipid metabolism, and promote obesity.     

NK cells and cancer

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.     

Exploring the role of circadian clock gene and association with cancer pathophysiology

ABSTRACT

Most of the processes that occur in the mind and body follow natural rhythms. Those with a cycle length of about one day are called circadian rhythms. These rhythms are driven by a system of self-sustained clocks and are entrained by environmental cues such as light-dark cycles as well as food intake. In mammals, the circadian clock system is hierarchically organized such that the master clock in the suprachiasmatic nuclei of the hypothalamus integrates environmental information and synchronizes the phase of oscillators in peripheral tissues.

The circadian system is responsible for regulating a variety of physiological and behavioral processes, including feeding behavior and energy metabolism. Studies revealed that the circadian clock system consists primarily of a set of clock genes. Several genes control the biological clock, including BMAL1, CLOCK (positive regulators), CRY1, CRY2, PER1, PER2, and PER3 (negative regulators) as indicators of the peripheral clock.

Circadian has increasingly become an important area of medical research, with hundreds of studies pointing to the body’s internal clocks as a factor in both health and disease. Thousands of biochemical processes from sleep and wakefulness to DNA repair are scheduled and dictated by these internal clocks. Cancer is an example of health problems where chronotherapy can be used to improve outcomes and deliver a higher quality of care to patients.

In this article, we will discuss knowledge about molecular mechanisms of the circadian clock and the role of clocks in physiology and pathophysiology of concerns.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

S100A4 is frequently overexpressed in lung cancer cells and promotes cell growth and cell motility

S100A4, a small calcium-binding protein belonging to the S100 protein family, is commonly overexpressed in a variety of tumor types and is widely accepted to associate with metastasis by regulating the motility and invasiveness of cancer cells. However, its biological role in lung carcinogenesis is largely unknown. In this study, we found that S100A4 was frequently overexpressed in lung cancer cells, irrespective of histological subtype. Then we performed knockdown and forced expression of S100A4 in lung cancer cell lines and found that specific knockdown of S100A4 effectively suppressed cell proliferation only in lung cancer cells with S100A4-overexpression; forced expression of S100A4 accelerated cell motility only in S100A4 low-expressing lung cancer cells. PRDM2 and VASH1, identified as novel upregulated genes by microarray after specific knockdown of S100A4 in pancreatic cancer, were also analyzed, and we found that PRDM2 was significantly upregulated after S100A4-knockdown in one of two analyzed S100A4-overexpressing lung cancer cells. Our present results suggest that S100A4 plays an important role in lung carcinogenesis by means of cell proliferation and motility by a pathway similar to that in pancreatic cancer.