Comparative evaluation of ethyl acetate and n-Hexane extracts of Cannabis sativa L. leaves for muscle function restoration after peripheral nerve lesion.

Peripheral nerve injuries are one of those complex medical conditions for which a highly effective first-line treatment is currently missing. The use of natural compound as medicines to treat various disorders has a long history. Our previous research explored that crude Cannabis sativa L. accelerated the recovery of sensorimotor functions following nerve injury. The purpose of the current study was to investigate the effects of n-Hexane and ethyl acetate extracts of C. sativa L. leaves on the muscle function restoration in a mouse model after sciatic nerve injury. For this purpose, albino mice (n = 18) were equally divided into control and two treatment groups. The control group was fed on a plain diet while treatment groups were given a diet having n-Hexane (treatment 1) and ethyl acetate (treatment 2) extracts of C. sativa L. (10 mg/kg body weight), respectively. The hot plate test (M = 15.61, SD = 2.61, p = .001), grip strength (M = 68.32, SD = 3.22, p < .001), and sciatic functional index (SFI) (M = 11.59, SD = 6.54, p = .012) assessment indicated significant amelioration in treatment 1 as compared to treatment 2 group. Furthermore, muscle fiber cross-sectional area revealed a noticeable improvement (M = 182,319, SD = 35.80, p = .013) in treatment 1 while muscle mass ratio of Gastrocnemius (M = 0.64, SD = 0.08, p = .427) and Tibialis anterior (M = 0.57, SD = 0.04, p = .209) indicated nonsignificant change. A prominent increase in total antioxidant capacity (TAC) (M = 3.76, SD = 0.38, p < .001) and momentous decrease in total oxidant status (TOS) (M = 11.28, SD = 5.71, p < .001) along with blood glucose level indicated significant difference (M = 105.5, SD = 9.12, p < 0.001) in treatment 1 group. These results suggest that treatment 1 has the ability to speed up functional recovery after a peripheral nerve lesion. Further research is necessary, nevertheless, to better understand the extract's actual curative properties and the mechanisms that improve functional restoration.

Axonal Regeneration: Underlying Molecular Mechanisms and Potential Therapeutic Targets.

Axons in the peripheral nervous system have the ability to repair themselves after damage, whereas axons in the central nervous system are unable to do so. A common and important characteristic of damage to the spinal cord, brain, and peripheral nerves is the disruption of axonal regrowth. Interestingly, intrinsic growth factors play a significant role in the axonal regeneration of injured nerves. Various factors such as proteomic profile, microtubule stability, ribosomal location, and signalling pathways mark a line between the central and peripheral axons' capacity for self-renewal. Unfortunately, glial scar development, myelin-associated inhibitor molecules, lack of neurotrophic factors, and inflammatory reactions are among the factors that restrict axonal regeneration. Molecular pathways such as cAMP, MAPK, JAK/STAT, ATF3/CREB, BMP/SMAD, AKT/mTORC1/p70S6K, PI3K/AKT, GSK-3ß/CLASP, BDNF/Trk, Ras/ERK, integrin/FAK, RhoA/ROCK/LIMK, and POSTN/integrin are activated after nerve injury and are considered significant players in axonal regeneration. In addition to the aforementioned pathways, growth factors, microRNAs, and astrocytes are also commendable participants in regeneration. In this review, we discuss the detailed mechanism of each pathway along with key players that can be potentially valuable targets to help achieve quick axonal healing. We also identify the prospective targets that could help close knowledge gaps in the molecular pathways underlying regeneration and shed light on the creation of more powerful strategies to encourage axonal regeneration after nervous system injury.

Vitamin C supplementation ameliorates liver function profile and antiviral treatment response in Hepatitis C patients.

An imbalance between oxidative stress and antioxidative defence mediates a variety of diseases pathogenesis. The present study aims to assess the possible outcome of supplementation of oral vitamin-C (VC), an antioxidant, in Viral Hepatitis C (HCV) treatment as an adjuvant therapy. 200 HCV-patients were selected, 100 were given Vitamin-C (1000 mg/day) along with anti HCV treatment (sofosbuvir plus daclatasvir) while the other 100 took only anti-HCV treatment for 4weeks. The serum ascorbic acid (Vitamin-C) levels and functions of the liver were tested before and after the VC supplementation. HCV patients with relatively low serum ascorbic acid showed significant improvement after the intake of vitamin C. After 4 weeks of treatment, AST, ALP, albumin, and total, direct and indirect bilirubin were improved significantly in the VC group; whereas only ALT and indirect bilirubin were improved in both groups when associated with the control subjects. Comparing the two treatment groups at 4weeks; more effective and significant improvement was observed in ALT (p<0.01), AST (p<0.001), direct (p<0.01) and indirect bilirubin (p<0.001), total proteins (p<0.001) and albumin (p<0.05) in patients with VC supplementation on anti-viral treatment compared to only anti-viral treatment group. Thus, VC supplementation improves the antiviral therapy outcome by bestowing a beneficial effect in minimizing liver damage in HCV cases.

Synthesis, anticancer evaluation and molecular docking studies of methotrexate's novel Schiff base derivatives against malignant glioma cell lines.

Recent years have witnessed advancement in cancer research that has led to the development of improved cytotoxic therapies with reduced side effects. Methotrexate (MTX) is a commonly used anticancer drug having robust activity, but with serious side effects. Several derivatives of MTX have been reported by modification at different sites to reduce its side effects and enhance efficacy. The current work describes the development of active MTX Schiff base derivatives by treating MTX with several aldehydes viz 2-chlorobenzaldehyde, 3-nitrobenzaldehyde, 5-chloro-2-hydroxybenz-aldehyde, 2-hydroxy-5-nitrobenzaldehyde, 2-thiocarboxyaldehyde, trans-2-pentenal and glutaraldehyde. Newly synthesized derivatives were evaluated for their anticancer potential against human malignant glioma U87 (MG-U87) cell lines at different concentrations of 200 µM, 100 µM, 50 µM, 25 µM, 12.5 µm, 6.25 µm and 0 µM. MTX derivatives with 2-Chlorobenzaldehyde (IC50 ∼100 µM), 2-Thiocarboxyaldehyde (IC50 <200 µM) and 2- Pentenal (IC50 ∼250 µM) showed much better activity at 100 µM compared to 400 µM concentration of MTX. Molecular docking studies were performed that showed a good correlation with the results obtained from in vitro experiments. The excellent agreement between molecular modeling and growth inhibition assay shows that the binding mode hypothesis is justly close to the experimentally biological values, therefore, may prove helpful for further lead optimization and clinical trials.Communicated by Ramaswamy H. Sarma.

Dietary biomolecules as promising regenerative agents for peripheral nerve injury: An emerging nutraceutical-based therapeutic approach.

Peripheral nerve damage is a debilitating condition that can result in partial or complete functional loss as a result of axonal degeneration, as well as lifelong dependence. Many therapies have been imbued with a plethora of positive features while posing little risks. It is worth noting that these biomolecules work by activating several intrinsic pathways that are known to be important in peripheral nerve regeneration. Although the underlying mechanism is used for accurate and speedy functional recovery, none of them are without side effects. As a result, it is believed that effective therapy is currently lacking. The dietary biomolecules-based intervention, among other ways, is appealing, safe, and effective. Upregulation of transcription factors, neurotrophic factors, and growth factors such as NGF, GDNF, BDNF, and CTNF may occur as a result of these substances' dietary intake. Upregulation of the signaling pathways ERK, JNK, p38, and PKA has also been seen, which aids in axonal regeneration. Although several mechanistic approaches to understanding their involvement have been suggested, more work is needed to reveal the amazing properties of these biomolecules. We have discussed in this article that how different dietary biomolecules can help with functional recovery and regeneration after an injury. PRACTICAL APPLICATIONS: Based on the information known to date, we may conclude that treatment techniques for peripheral nerve injury have downsides, such as complications, donor shortages, adverse effects, unaffordability, and a lack of precision in efficacy. These difficulties cast doubt on their efficacy and raise severe concerns about the prescription. In this situation, the need for safe and effective therapeutic techniques is unavoidable, and dietary biomolecules appear to be a safe, cost-efficient, and effective way to promote nerve regeneration following an injury. The information on these biomolecules has been summarized here. Upregulation of transcription factors, neurotrophic factors, and growth factors, such as NGF, GDNF, BDNF, and CTNF, as well as the ERK, JNK, p38, and PKA, signaling pathways, may stimulate axonal regeneration.

Dysregulated Autophagy Leads to Oxidative Stress and Aberrant Expression of ABC Transporters in Women with Early Miscarriage.

Early miscarriage (EMC) is a devastating obstetrical complication. ATP-binding cassette (ABC) transporters mediate cholesterol transfer across the placenta and enhance cell survival by effluxing substrates from target cells in the presence of stressors. Recent evidence reports an intricate interplay between autophagy and ABC transporters. We hypothesized that dysregulated autophagy and oxidative stress (OS) in the placenta leads to abnormal expression of membrane transporters contributing to poor pregnancy survival in EMC. We determined mRNA and protein expression of autophagy genes (Beclin-1/Bcl-2/LC3I/LC3II/p62) and ABC transporters (ABCA1/ABCG1/ABCG2) in placentae from EMC patients (n = 20), term controls (n = 19), first trimester (n = 6), and term controls (n = 5) controls. Oxidative/antioxidant status and biomarkers of oxidative damage were evaluated in maternal serum and placentae from EMC and healthy controls. In EMC, placental expression of LC3II/LC3I as well as of the key autophagy regulatory proteins Beclin-1 and Bcl-2 were reduced, whereas p62 was increased. Both in the serum and placentae of EMC patients, total OS was elevated reflected by increased oxidative damage markers (8-OHdG/malondialdehyde/carbonyl formation) accompanied by diminished levels of total antioxidant status, catalase, and total glutathione. Furthermore, we found reduced ABCG1 and increased ABCG2 expression. These findings suggest that a decreased autophagy status triggers Bcl-2-dependent OS leading to macromolecule damage in EMC placentae. The decreased expression of ABCG1 contributes to reduced cholesterol export to the growing fetus. Increasing ABCG2 expression could represent a protective feedback mechanism under inhibited autophagy conditions. In conclusion, dysregulated autophagy combined with increased oxidative toxicity and aberrant expression of placental ABC transporters affects materno-fetal health in EMC.

Sex-Specific Regulation of Interferon-γ Cytotoxicity in Mouse Liver by Autophagy.

BACKGROUND AND AIMS: Interferon-γ (IFNγ) is a central activator of immune responses in the liver and other organs. IFNγ triggers tissue injury and inflammation in immune diseases, which occur predominantly in females for unknown reasons. Recent findings that autophagy regulates hepatotoxicity from proinflammatory cytokines led to an examination of whether defective hepatocyte autophagy underlies sex-specific liver injury and inflammation induced by IFNγ. APPROACH AND RESULTS: A lentiviral autophagy-related 5 (Atg5) knockdown was performed to decrease autophagy-sensitized alpha mouse liver (AML 12) hepatocytes to death from IFNγ in combination with IL-1ß or TNF. Death was necrosis attributable to impaired energy homeostasis and adenosine triphosphate depletion. Male mice with decreased autophagy from a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were resistant to IFNγ hepatotoxicity whereas female knockout mice developed liver injury and inflammation. Female mice had increased IFNγ-induced signal transducer and activator of transcription 1 (STAT1) levels compared to males. Blocking STAT1, but not interferon regulatory factor 1, signaling prevented IFNγ-induced hepatocyte death in autophagy-deficient AML12 cells and female mice. The mechanism of death is STAT1-induced overexpression of nitric oxide synthase 2 (NOS2) as in vitro hepatocyte death and in vivo liver injury were blocked by NOS2 inhibition. CONCLUSIONS: Decreased hepatocyte autophagy sensitizes mice to IFNγ-induced liver injury and inflammation through overactivation of STAT1 signaling that causes NOS2 overexpression. Hepatotoxicity is restricted to female mice, suggesting that sex-specific effects of defective autophagy may underlie the increased susceptibility of females to IFNγ-mediated immune diseases.

Strychnos nux-vomica L. seed preparation promotes functional recovery and attenuates oxidative stress in a mouse model of sciatic nerve crush injury.

BACKGROUND: Peripheral nerve injury is a debilitating condition that may lead to partial or complete motor, sensory and autonomic function loss and lacks effective therapy until date. Therefore, it is quite imperative to explore impending remedies for rapid and accurate functional retrieval following such conditions. Natural product-based intervention can prove effective to facilitate the process of functions regain. METHODS: Here, we investigated the effect of processed Strychnos nux-vomica seeds at a dose of 250 mg/kg body weight in a mouse model of induced Sciatic nerve lesion in promoting the recovery of the functions. A compression injury was induced in the Sciatic nerve of the right leg in the mice. Sensory function recovery was evaluated by hot-plate and formalin tests, whereas the motor function retrieval was assessed by measuring muscle grip strength, sciatic functional index, and muscle mass restoration. Oxidative stress and blood cell count were measured by biochemistry and haematological analyses. RESULTS: This study indicates that Strychnos nux-vomica seeds enhance the rate of recovery of both sensory and motor functions. It helps restore the muscle mass, attenuates total oxidant status and enhances the total anti-oxidant capacity of the biological system. Moreover, the treated animals manifested an enhanced glucose tolerance aptitude and augmented granulocyte and platelet counts. Improved oxidant control, enhanced glucose sensitivity and amended granulocyte and platelet counts are likely to contribute to the advantageous effects of Strychnos nux-vomica, and warrant further in-depth studies for deciphering possible mechanisms and identification of active constituent(s) responsible for these effects. CONCLUSION: Strychnos nux-vomica seed offers functional recovery promoting effects following a mechanical injury to the Sciatic nerve and the possible reasons behind this effect can be reduced oxidative stress and improved glycaemic control. Further and detailed investigations can unravel this mystery.

Decreased Hepatocyte Autophagy Leads to Synergistic IL-1ß and TNF Mouse Liver Injury and Inflammation.

BACKGROUND AND AIMS: The proinflammatory cytokine IL-1ß has been implicated in the pathophysiology of nonalcoholic and alcoholic steatohepatitis. How IL-1ß promotes liver injury in these diseases is unclear, as no IL-1ß receptor-linked death pathway has been identified. Autophagy functions in hepatocyte resistance to injury and death, and findings of decreased hepatic autophagy in many liver diseases suggest a role for impaired autophagy in disease pathogenesis. Recent findings that autophagy blocks mouse liver injury from lipopolysaccharide led to an examination of autophagy's function in hepatotoxicity from proinflammatory cytokines. APPROACH AND RESULTS: AML12 cells with decreased autophagy from a lentiviral autophagy-related 5 (Atg5) knockdown were resistant to toxicity from TNF, but sensitized to death from IL-1ß, which was markedly amplified by TNF co-treatment. IL-1ß/TNF death was necrosis by trypan blue and propidium iodide positivity, absence of mitochondrial death pathway and caspase activation, and failure of a caspase inhibitor or necrostatin-1s to prevent death. IL-1ß/TNF depleted autophagy-deficient cells of ATP, and ATP depletion and cell death were prevented by supplementation with the energy substrate pyruvate or oleate. Pharmacological inhibitors and genetic knockdown studies demonstrated that IL-1ß/TNF-induced necrosis resulted from lysosomal permeabilization and release of cathepsins B and L in autophagy-deficient cells. Mice with a tamoxifen-inducible, hepatocyte-specific Atg5 knockout were similarly sensitized to cathepsin-dependent hepatocellular injury and death from IL-1ß/TNF in combination, but neither IL-1ß nor TNF alone. Knockout mice had increased hepatic inflammation, and IL-1ß/TNF-treated, autophagy-deficient AML12 cells secreted exosomes with proinflammatory damage-associated molecular patterns. CONCLUSIONS: The findings delineate mechanisms by which decreased hepatocyte autophagy promotes IL-1ß/TNF-induced necrosis from impaired energy homeostasis and lysosomal permeabilization and inflammation through the secretion of exosomal damage-associated molecular patterns.

Neurada procumbens promotes functions regain in a mouse model of mechanically induced sciatic nerve injury.

Peripheral nerve injury is a complex condition which results in restricted physical activity. Despite the tremendous efforts to figure out effective remedies, the complete functional retrieval is still a goal to be achieved. So, the need of hour is the exploration of potential natural compounds to recover this functional loss. Here, we have investigated the role of a local plant "Neurada procumbens" in ameliorating the functional recovery after an induced nerve compression injury in a mouse model. A dose of N. procumbens (50mg/kg of body weight) was administered orally from the day of injury to onwards. The motor functional recovery was assessed by evaluating muscle grip strength and sciatic functional index; while the sensory functions were gauged by the hotplate test. The serological parameters were carried out to analyze the effect of N. procumbens on oxidative stress level. The recovery of sensory and motor functions was significantly improved and perceived earlier in the treatment group. Moreover, the elevated antioxidant level was statistically significant in the treatment group. These results indicate that the supplementation of N. procumbens accelerates functional recovery after sciatic nerve crush injury.

Supplementation of Cannabis sativa L. leaf powder accelerates functional recovery and ameliorates haemoglobin level following an induced injury to sciatic nerve in mouse model.

Peripheral nerve injury is a common condition with a multitude of signs and symptoms. The major consequence of injury is limited physical activity. Presently, we are lacking effective therapies for PNI and it is need of the hour is to explore potential remedies for the recovery of functional loss. Here, we have investigated the role of crude Cannabis sativa L. leaf powder in promoting functions recovery, in mouse model subjected to a traumatic sciatic nerve injury. A dose of 200mg/kg of the body weight per day was administered orally from the day of nerve crush till the end of the experiment. The motor functions were evaluated by measuring sciatic functional index, muscle grip strength and muscle mass; whereas the sensory functions were assessed by hotplate test. The haematology and serum analyses were carried out to estimate the effect of treatment on the systemic index and oxidative stress. The gain of motor functions was significantly improved and was early noticed in the treated mice. Restoration of muscle mass and elevated haemoglobin level were statistically significant in the treatment group. This study indicates that Cannabis sativa L. supplementation accelerates the motor functions recovery after nerve compression injury.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1ß. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Unsaturated fatty acids induce non-canonical autophagy.

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.

Coffee induces autophagy in vivo.

Epidemiological studies and clinical trials revealed that chronic consumption coffee is associated with the inhibition of several metabolic diseases as well as reduction in overall and cause-specific mortality. We show that both natural and decaffeinated brands of coffee similarly rapidly trigger autophagy in mice. One to 4 h after coffee consumption, we observed an increase in autophagic flux in all investigated organs (liver, muscle, heart) in vivo, as indicated by the increased lipidation of LC3B and the reduction of the abundance of the autophagic substrate sequestosome 1 (p62/SQSTM1). These changes were accompanied by the inhibition of the enzymatic activity of mammalian target of rapamycin complex 1 (mTORC1), leading to the reduced phosphorylation of p70(S6K), as well as by the global deacetylation of cellular proteins detectable by immunoblot. Immunohistochemical analyses of transgenic mice expressing a GFP-LC3B fusion protein confirmed the coffee-induced relocation of LC3B to autophagosomes, as well as general protein deacetylation. Altogether, these results indicate that coffee triggers 2 phenomena that are also induced by nutrient depletion, namely a reduction of protein acetylation coupled to an increase in autophagy. We speculate that polyphenols contained in coffee promote health by stimulating autophagy.

Regulation of autophagy by cytosolic acetyl-coenzyme A.

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy.

Direct interaction between STAT3 and EIF2AK2 controls fatty acid-induced autophagy.

A chemical screen designed to identify novel inducers of autophagy led to the discovery that signal transducer and activator of transcription 3 (STAT3) inhibitors can potently stimulate the autophagic flux. Although STAT3 is best known as a pro-inflammatory and oncogenic transcription factor, mechanistic analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear, pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase 2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA. This interaction, which could be recapitulated using recombinant proteins in pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus, STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e., Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter does so in cell-free systems in vitro. A further screen designed to identify EIF2AK2-dependent autophagy inducers revealed that several fatty acids including palmitate trigger autophagy via a pathway that involves the disruption of the STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These results reveal an unsuspected crosstalk between cellular metabolism (fatty acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and translational control (EIF2S1) that regulates autophagy.

Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity.

In a screen designed to identify novel inducers of autophagy, we discovered that STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic inhibition of STAT3 stimulated autophagy in vitro and in vivo, while overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable, and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2 EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic inhibition of STAT3 stimulated the activating phosphorylation of PKR and consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like palmitate. STAT3-targeting chemicals and palmitate caused the disruption of inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α phosphorylation, which facilitates autophagy induction. These results unravel an unsuspected mechanism of autophagy control that involves STAT3 and PKR as interacting partners.

Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins.

Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called "French paradox," i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines.

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.

Selective killing of p53-deficient cancer cells by SP600125.

The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53(+/+) and TP53(-/-) human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53(-/-) (but not TP53(+/+) ) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53(-/-) cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53(-/-) cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.