Reticulon-1C Involvement in Muscle Regeneration.

Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca2+ homeostasis are implicated in many adverse conditions affecting skeletal muscle. Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury.

Reticulon Homology Domain-Containing Proteins and ER-Phagy.

The endoplasmic reticulum (ER) is a dynamic membrane system comprising different and interconnected subdomains. The ER structure changes in response to different stress conditions through the activation of a selective autophagic pathway called ER-phagy. This represents a quality control mechanism for ER turnover and component recycling. Several ER-resident proteins have been indicated as receptors for ER-phagy; among these, there are proteins characterized by the presence of a reticulon homology domain (RHD). RHD-containing proteins promote ER fragmentation by a mechanism that involves LC3 binding and lysosome delivery. Moreover, the presence of a correct RHD structure is closely related to their capability to regulate ER shape and morphology by curvature induction and membrane remodeling. Deregulation of the ER-selective autophagic pathway due to defects in proteins with RHD has been implicated in several human diseases, infectious and neurodegenerative diseases in particular, as well as in cancer development. While the molecular mechanisms and the physiological role of ER-phagy are not yet fully understood, it is quite clear that this process is involved in different cellular signaling pathways and has an impact in several human pathologies.

Modulation of autophagy by RTN-1C: role in autophagosome biogenesis.

The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and to determine the cell's fate under stress conditions. Among the known proteins that regulate ER structure and function there is Reticulon-1C (RTN-1C), a member of the reticulon family localized primarily on the ER membrane. We previously demonstrated that RTN-1C expression affects ER function and stress condition. ER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling. Based on these evidences, we have investigated the impact of RTN-1C modulation on autophagy induction. Interestingly we found that reticulon overexpression is able to activate autophagic machinery and its silencing results in a significative inhibition of both basal and induced autophagic response. Using different experimental approaches we demonstrated that RTN-1C colocalizes with ATG16L and LC3II on the autophagosomes. Considering the key role of reticulon proteins in the control of ER membrane shaping and homeostasis, our data suggest the participation of RTN-1C in the autophagic vesicle biogenesis at the level of the ER compartment. Our data indicate a new mechanism by which this structural ER protein modulates cellular stress, that is at the basis of different autophagy-related pathologies.

The spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A.

The exogenous administration of spermidine promotes longevity in many model organisms. It has been proposed that this anti-age activity of spermidine is related to this polyamine's ability to promote autophagy. Since spermidine is the substrate for the eIF5A post-translational modification by hypusination, we asked ourselves whether mature eIF5A may represent the link between spermidine and autophagy induction. To test this hypothesis, we inhibited the conversion of native eIF5A by a pharmacological approach, using the N1-guanyl-1,7-diamineoheptane (GC7), a spermidine analogue which competitively and reversibly inhibits deoxyhypusine synthase (DHS). In addition, we also employed genetic approaches by ablating both the eIF5A protein itself and DHS, the rate limiting enzyme catalyzing the conversion of lysine to hypusine. Collectively the data presented in this study demonstrate that the mature eIF5A (hypusinated form) is not involved in the autophagic pathway and that the inhibitor of DHS, GC7, produces off-target effect(s) resulting in marked induction of basal autophagy. These data are relevant in light of the fact that GC7 is considered a potent and selective inhibitor of DHS and is a potential candidate drug for cancer, diabetes and HIV therapy.

Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin.

In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma.

Aloe-emodin as antiproliferative and differentiating agent on human U937 monoblastic leukemia cells.

AIMS: Aloe-emodin (AE), a plant derived anthraquinone, has been shown to have anticancer activity in various human cancer cell lines. We have recently reported that AE possesses a differentiative potential on melanoma cells. The purpose of this study was to investigate the possible modulation of defined markers of monocytic differentiation of AE on human U937 cell line. MAIN METHODS: U937 cells differentiation has been confirmed unequivocally by Griess and nitroblue tetrazolium reduction assays, protoporphyrin IX accumulation, expression of CD14 and CD11b surface antigens, phagocytic activity, migration and attachment ability. The effect on polyamine metabolism, apoptosis and cytokine production was also investigated. KEY FINDINGS: We showed that AE-treated U937 cells exhibit a noticeably rise in transglutaminase activity. This enhanced enzyme activity correlates with AE-induced growth arrest and differentiation to functionally mature monocytes. SIGNIFICANCE: Taken together, the results reported here show that AE can promote the macrophage differentiation of U937 cells, suggesting that this anthraquinone could be a potential candidate as a differentiation-inducing selective agent for therapeutic treatment of leukemia.

The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy.

Autophagy is an evolutionary conserved catabolic process involved in several physiological and pathological processes such as cancer and neurodegeneration. Autophagy initiation signaling requires both the ULK1 kinase and the BECLIN 1-VPS34 core complex to generate autophagosomes, double-membraned vesicles that transfer cellular contents to lysosomes. In this study, we show that the BECLIN 1-VPS34 complex is tethered to the cytoskeleton through an interaction between the BECLIN 1-interacting protein AMBRA1 and dynein light chains 1/2. When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from dynein. Its subsequent relocalization to the endoplasmic reticulum enables autophagosome nucleation. Therefore, AMBRA1 constitutes a direct regulatory link between ULK1 and BECLIN 1-VPS34, which is required for core complex positioning and activity within the cell. Moreover, our results demonstrate that in addition to a function for microtubules in mediating autophagosome transport, there is a strict and regulatory relationship between cytoskeleton dynamics and autophagosome formation.

Antitumor properties of aloe-emodin and induction of transglutaminase 2 activity in B16-F10 melanoma cells.

AIMS: Aloe-emodin (AE), a natural hydroxyanthraquinone compound, has been reported as a potential anticancer agent. We studied the antineoplastic properties of AE on highly metastatic B16-F10 melanoma murine cells. MAIN METHODS: Cell proliferation was assessed by cell counting and viability was investigated using MTT and Trypan Bleu exclusion tests. As a growth marker, we determined intracellular polyamine levels by high performance liquid chromatography. Then, we evaluated transglutaminase 2 (TG2) activity, protoporphyrin IX accumulation and melanin content as differentiative markers. Tyrosinase activity was checked by DOPA-staining assay. The antimetastatic effect of AE was evaluated by means of a series of in vitro metastatic assays, including aggregation, wound healing migration, adhesion, 3D-invasion, circular invasion and the Boyden chamber invasion assays. Gelatin zymography was performed to evaluate metalloproteinase activities. KEY FINDINGS: Our results demonstrated inhibitory effects of AE on melanoma cell proliferation and invasion power, accompanied by the stimulation of cell differentiation parameters. Cell differentiation correlated with a remarkable increase of the activity of the transamidating form of TG2, with a significative enhancement of cell adhesion and aggregation. Impaired invasion was paralleled by the decrease of the secretion of matrix metalloproteinase-9. SIGNIFICANCE: The overall data confirm a remarkable antiproliferative, antimetastatic and differentiative capability of this anthraquinone. Results suggest that AE appears particularly promising for its potential application in the newborn differentiation therapy of cancer.

Transglutaminase 2 is involved in autophagosome maturation.

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Fenretinide: a p53-independent way to kill cancer cells.

The synthetic retinoid fenretinide [N-(4 hydroxyphenyl)retinamide] induces apoptosis of cancer cells and acts synergistically with chemotherapeutic drugs, thus providing opportunities for novel approaches to cancer therapy. The upstream signaling events induced by fenretinide include an increase in intracellular levels of ceramide, which is subsequently metabolized to GD3. This ganglioside triggers the activation of 12-Lox (12-lipoxygenase) leading to oxidative stress and apoptosis via the induction of the transcription factor Gadd153 and the Bcl-2-family member protein Bak. Increased evidence suggests that the apoptotic pathway activated by fenretinide is p53-independent and this may represent a novel way to treat tumors resistant to DNA-damaging chemotherapeutic agents. Therefore, fenretinide offers increased clinical benefit as a novel agent for cancer therapy, able to complement the action of existing chemotherapeutic treatment regimes. Furthermore, synergy between fenretinide and chemotherapeutic drugs may facilitate the use of chemotherapeutic drugs at lower concentrations, with possible reduction in treatment-associated morbidity.

Growth and DNA damage-inducible transcription factor 153 mediates apoptosis in response to fenretinide but not synergy between fenretinide and chemotherapeutic drugs in neuroblastoma.

Fenretinide induces apoptosis of neuroblastoma cells in vitro and interacts synergistically with the chemotherapeutic drugs cisplatin and etoposide. The stress-inducible transcription factor known as growth and DNA damage (GADD)-inducible transcription factor 153 is induced in response to fenretinide and in other cell types modulates apoptosis via pro- and antiapoptotic members of the BCL2 family. Because BCL2-family proteins are important in apoptosis induced by chemotherapeutic drugs, GADD153 may be a key mediator of synergy between fenretinide and chemotherapeutic drugs. To investigate this, GADD153 cDNA in sense and antisense orientations was stably transfected into SH-SY5Y neuroblastoma cells using a tetracycline-inducible vector. Increased expression of GADD153 raised the background level of apoptosis and increased apoptosis induced by fenretinide or the chemotherapeutic drugs cisplatin and etoposide. However, there was no increase in synergy between fenretinide and chemotherapeutic drugs. Conversely, expression of antisense-GADD153 virtually abolished the induction of apoptosis in response to fenretinide but overall had no significant effect on apoptosis induced by chemotherapeutic drugs. The effect of antisense-GADD153 on synergy between chemotherapeutic drugs and fenretinide varied with the drug used: there was no effect on synergy between fenretinide and cisplatin, but the combination of fenretinide with etoposide became antagonistic. These results suggest that mechanisms mediating synergy between fenretinide and chemotherapeutic drugs lie upstream of GADD153.

Bak: a downstream mediator of fenretinide-induced apoptosis of SH-SY5Y neuroblastoma cells.

Unlike 13-cis-retinoic acid, the synthetic retinoid fenretinide [N-(4-hydroxyphenyl)retinamide] induces apoptosis of neuroblastoma cells by mechanisms involving retinoic acid receptors and oxidative stress. After screening a cDNA array for apoptosis-related genes, the Bcl2-related protein Bak was identified as a fenretinide-inducible gene in SH-SY5Y neuroblastoma cells, and this was confirmed by Western blotting and flow cytometry. Although fenretinide acts synergistically in vitro with chemotherapeutic drugs, these drugs did not induce Bak expression. Retinoic acid receptor antagonists did not block the induction of Bak by fenretinide. Conversely, Bak induction was blocked by the antioxidant vitamin C. Overexpression of Bak increased apoptosis in both the presence and absence of fenretinide, whereas expression of antisense Bak inhibited fenretinide-induced apoptosis. Bak expression was also induced in cells overexpressing the stress-induced transcription factor GADD153, but Bak expression was inhibited in cells expressing an antisense GADD153 construct. These results suggest that Bak is a downstream mediator of an oxidative stress pathway leading to apoptosis of SH-SY5Y neuroblastoma cells in response to fenretinide.

Induction of GADD153 and Bak: novel molecular targets of fenretinide-induced apoptosis of neuroblastoma.

Unlike 13-cis retinoic acid, the synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. The stress-induced transcription factor GADD153 and the Bcl2-related protein Bak are upregulated in response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists do not block the induction of GADD153 or Bak by fenretinide. Conversely, the induction of GADD153 and Bak is blocked by antioxidants. Neither GADD153 or Bak were induced by chemotherapeutic agents but over expression of GADD153 results in increased sensitivity to fenretinide-induced apoptosis. Therefore, fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterised by the reactive oxygen species-dependent induction of GADD153 and Bak. The targeting of GADD153 and Bak in neuroblastoma cells may be novel pathways for the development of drugs inducing apoptosis of neuroblastoma with improved tumour specificity.

Neuroleukin inhibition sensitises neuronal cells to caspase-dependent apoptosis.

Neuroleukin (NLK) is a multifunctional protein, involved in neuronal growth, glucose metabolism, cell motility, and differentiation. Expressed in the brain, it supports the growth of embryonic spinal, skeletal motor, and sensory neurons. We have previously demonstrated that NLK is up-regulated in the brain during Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats. In order to study the biological role of NLK, we have generated an inducible rat pheochromocytoma PC12 cell line in which the expression of NLK is selectively down-regulated by antisense strategy. We show here that the block of NLK commits PC12 cells to caspase-dependent apoptosis. This priming effect elicited by NLK inhibition is independent from the differentiation state of the neuronal cells. These results suggest a general protective role of NLK in the control of cell death in neuronal cells.

GADD153 and 12-lipoxygenase mediate fenretinide-induced apoptosis of neuroblastoma.

The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apoptotic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A(2) and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.