: Melatonin is a highly evolutionary conserved endogenous molecule that is mainly produced by the pineal gland, but also by other nonendocrine organs, of most mammals including man. In the recent ...years, a variety of anti‐inflammatory and antioxidant effects have been observed when melatonin is applied exogenously under both in vivo and in vitro conditions. A number of studies suggest that this indole may exert its anti‐inflammatory effects through the regulation of different molecular pathways. It has been documented that melatonin inhibits the expression of the isoforms of inducible nitric oxide synthase and cyclooxygenase and limits the production of excessive amounts of nitric oxide, prostanoids, and leukotrienes, as well as other mediators of the inflammatory process such as cytokines, chemokines, and adhesion molecules. Melatonin’s anti‐inflammatory effects are related to the modulation of a number of transcription factors such as nuclear factor kappa B, hypoxia‐inducible factor, nuclear factor erythroid 2‐related factor 2, and others. Melatonin’s effects on the DNA‐binding capacity of transcription factors may be regulated through the inhibition of protein kinases involved in signal transduction, such as mitogen‐activated protein kinases. This review summarizes recent research data focusing on the modulation of the expression of different inflammatory mediators by melatonin and the effects on cell signaling pathways responsible for the indole’s anti‐inflammatory activity. Although there are a numerous published reports that have analyzed melatonin’s anti‐inflammatory properties, further studies are necessary to elucidate its complex regulatory mechanisms in different cellular types and tissues.
Endoplasmic reticulum (ER) is a dynamic organelle that participates in a number of cellular functions by controlling lipid metabolism, calcium stores, and proteostasis. Under stressful situations, ...the ER environment is compromised, and protein maturation is impaired; this causes misfolded proteins to accumulate and a characteristic stress response named unfolded protein response (UPR). UPR protects cells from stress and contributes to cellular homeostasis re‐establishment; however, during prolonged ER stress, UPR activation promotes cell death. ER stressors can modulate autophagy which in turn, depending of the situation, induces cell survival or death. Interactions of different autophagy‐ and apoptosis‐related proteins and also common signaling pathways have been found, suggesting an interplay between these cellular processes, although their dynamic features are still unknown. A number of pathologies including metabolic, neurodegenerative and cardiovascular diseases, cancer, inflammation, and viral infections are associated with ER stress, leading to a growing interest in targeting components of the UPR as a therapeutic strategy. Melatonin has a variety of antioxidant, anti‐inflammatory, and antitumor effects. As such, it modulates apoptosis and autophagy in cancer cells, neurodegeneration and the development of liver diseases as well as other pathologies. Here, we review the effects of melatonin on the main ER stress mechanisms, focusing on its ability to regulate the autophagic and apoptotic processes. As the number of studies that have analyzed ER stress modulation by this indole remains limited, further research is necessary for a better understanding of the crosstalk between ER stress, autophagy, and apoptosis and to clearly delineate the mechanisms by which melatonin modulates these responses.
Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor‐acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive ...response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nmol/L to 50 μmol/L) in the presence of melatonin (1 and 2 mmol/L) was assessed in HCC cell lines HepG2, HuH7, and Hep3B. Cell viability was reduced by sorafenib from 1 μmol/L in HepG2 or HuH7 cells, and 2.5 μmol/L in Hep3B cells. Co‐administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co‐administration of 2.5 μmol/L sorafenib and 1 mmol/L melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin‐2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage, and BAX expression. These results demonstrate that the pro‐oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib.
Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence and its metastatic potential. Extracellular matrix degradation by matrix ...metalloproteinases (MMPs) has been connected with cancer cell invasion, and it has been suggested that inhibition of MMPs by synthetic and natural inhibitors may be of great importance in the HCC therapies. Melatonin, the main product of the pineal gland, exerts antiproliferative, proapoptotic, and antiangiogenic properties in HepG2 human hepatocellular cells, and exhibits anti‐invasive and antimetastatic activities by suppressing the enzymatic activity of MMP‐9 in different tumor types. However, the underlying mechanism of anti‐invasive activity in HCC models has not been fully elucidated. Here, we demonstrate that 1 mm melatonin dosage reduced in IL‐1β‐induced HepG2 cells MMP‐9 gelatinase activity and inhibited cell invasion and motility through downregulation of MMP‐9 gene expression and upregulation of the MMP‐9‐specific inhibitor tissue inhibitor of metalloproteinases (TIMP)‐1. No significant changes were observed in the expression and activity of MMP‐2, the other proteinase implicated in matrix collagen degradation, and its tissue inhibitor, TIMP‐2. Also, melatonin significantly suppressed IL‐1β‐induced nuclear factor‐kappaB (NF‐κB) translocation and transcriptional activity. In summary, we demonstrate that melatonin modulates motility and invasiveness of HepG2 cell in vitro through a molecular mechanism that involves TIMP‐1 upregulation and attenuation of MMP‐9 expression and activity via NF‐κB signal pathway inhibition.
Sorafenib, a multikinase inhibitor with antiproliferative, antiangiogenic, and proapoptotic properties, constitutes the only effective first-line drug approved for the treatment of advanced ...hepatocellular carcinoma (HCC). Despite its capacity to increase survival in HCC patients, its success is quite low in the long term owing to the development of resistant cells through several mechanisms. Among these mechanisms, the antiangiogenic effects of sustained sorafenib treatment induce a reduction of microvessel density, promoting intratumoral hypoxia and hypoxia-inducible factors (HIFs)-mediated cellular responses that favor the selection of resistant cells adapted to the hypoxic microenvironment. Clinical data have demonstrated that overexpressed HIF-1α and HIF-2α in HCC patients are reliable markers of a poor prognosis. Thus, the combination of current sorafenib treatment with gene therapy or inhibitors against HIFs have been documented as promising approaches to overcome sorafenib resistance both in vitro and in vivo. Because the depletion of one HIF-α subunit elevates the expression of the other HIF-α isoform through a compensatory loop, targeting both HIF-1α and HIF-2α would be a more interesting strategy than therapies that discriminate among HIF-α isoforms. In conclusion, there is a marked correlation between the hypoxic microenvironment and sorafenib resistance, suggesting that targeting HIFs is a promising way to increase the efficiency of treatment.
Autophagy is a process that maintains homeostasis during stress, although it also contributes to cell death under specific contexts. Ceramides have emerged as important effectors in the regulation of ...autophagy, mediating the crosstalk with apoptosis. Melatonin induces apoptosis of cancer cells; however, its role in autophagy and ceramide metabolism has yet to be clearly elucidated. This study was aimed to evaluate the effect of melatonin administration on autophagy and ceramide metabolism and its possible link with melatonin‐induced apoptotic cell death in hepatocarcinoma (HCC) cells. Melatonin (2 mm) transiently induced autophagy in HepG2 cells through JNK phosphorylation, characterized by increased Beclin‐1 expression, p62 degradation, and LC3II and LAMP‐2 colocalization, which translated in decreased cell viability. Moreover, ATG5 silencing sensitized HepG2 cells to melatonin‐induced apoptosis, suggesting a dual role of autophagy in cell death. Melatonin enhanced ceramide levels through both de novo synthesis and acid sphingomyelinase (ASMase) stimulation. Serine palmitoyltransferase (SPT) inhibition with myriocin prevented melatonin‐induced autophagy and ASMase inhibition with imipramine‐impaired autophagy flux. However, ASMase inhibition partially protected HepG2 cells against melatonin, while SPT inhibition significantly enhanced cell death. Findings suggest a crosstalk between SPT‐mediated ceramide generation and autophagy in protecting against melatonin, while specific ASMase‐induced ceramide production participates in melatonin‐mediated cell death. Thus, dual blocking of SPT and autophagy emerges as a potential strategy to potentiate the apoptotic effects of melatonin in liver cancer cells.
Quercetin is a flavonoid present in fruits, vegetables and plants with antioxidant, anti-inflammatory and anticancer properties. Its beneficial activities have been demonstrated in different human ...pathologies, including hepatoprotective effects against liver disorders. High mortality and late diagnosis of the primary liver tumor hepatocarcinoma (HCC) makes this cancer an interesting target for the study of quercetin effects. Our aim was to systematically review antitumor activities of quercetin in HCC preclinical studies employing single, encapsulated, combined or derived quercetin forms. Literature search was conducted in PubMed, Scopus and Web of Science (WOS), and 39 studies were finally included. We found that 17 articles evaluated quercetin effects alone, six used encapsulated strategy, 10 combined this flavonoid, two decided to co-encapsulate it and only four studied effects of quercetin derivatives, highlighting that only nine included in vivo models. Results evidence the quercetin antiproliferative and proapoptotic properties against HCC either alone and with the mentioned strategies; nevertheless, few investigations assessed specific activities on different processes related with cancer progression. Overall, further studies including animal models are needed to deeper investigate the precise mechanisms of action of quercetin as antitumor agent, as well as the potential of novel strategies aimed to improve quercetin effects in HCC.
The multikinase inhibitor sorafenib is, at present, the only drug approved for the treatment of hepatocellular carcinoma (HCC), one of the most lethal types of cancer worldwide. However, the increase ...in the number of sorafenib tumor resistant cells reduces efficiency. A better knowledge of the intracellular mechanism of the drug leading to reduced cell survival could help to improve the benefits of sorafenib therapy. Autophagy is a bulk cellular degradation process activated in a broad range of stress situations, which allows cells to degrade misfolded proteins or dysfunctional organelles. This cellular route can induce survival or death, depending on cell status and media signals. Sorafenib, alone or in combination with other drugs is able to induce autophagy, but cell response to the drug depends on the complex integrative crosstalk of different intracellular signals. In cancerous cells, autophagy can be regulated by different cellular pathways (Akt-related mammalian target of rapamycin (mTOR) inhibition, 5' AMP-activated protein kinase (AMPK) induction, dissociation of B-cell lymphoma 2 (Bcl-2) family proteins from Beclin-1), or effects of some miRNAs. Inhibition of mTOR signaling by sorafenib and diminished interaction between Beclin-1 and myeloid cell leukemia 1 (Mcl-1) have been related to induction of autophagy in HCC. Furthermore, changes in some miRNAs, such as miR-30α, are able to modulate autophagy and modify sensitivity in sorafenib-resistant cells. However, although AMPK phosphorylation by sorafenib seems to play a role in the antiproliferative action of the drug, it does not relate with modulation of autophagy. In this review, we present an updated overview of the effects of sorafenib on autophagy and its related activation pathways, analyzing in detail the involvement of autophagy on sorafenib sensitivity and resistance.
Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of ...endoplasmic reticulum (ER) stress, c‐Jun‐N‐terminal kinase (JNK), Akt, and 5′AMP‐activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3–12 hr) ER stress characterized by an increase of Ser51P‐eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185P‐JNK1/2/JNK1/2, Thr172P‐AMPKα, Ser413P‐Foxo3a, Thr308P‐AKt/AKt and Thr32P‐Foxo3a/Foxo3a ratios, and reduction of Ser2481P‐mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim
EL, Beclin‐1, Bcl‐xL, Bcl‐2, autophagy markers, and reduction of myeloid cell leukemia‐1 (Mcl‐1) expression. The progressive increase of CHOP expression, and reduction of Thr308P‐AKt/AKt and Ser473P‐AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim
EL expression and caspase‐3 activity (24 hr). Small interfering‐RNA (si‐RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase‐3 in sorafenib‐treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor‐derived xenograft model. In conclusion, the early sorafenib‐induced ER stress and regulation of JNK and AMPK‐dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK‐CHOP‐dependent rise of Bim
EL, which was associated with the shift from autophagy to apoptosis. The kinetic of Bim
EL expression profile might also be related to the tight balance between AKt‐ and AMPK‐related signaling leading to Foxo3a‐dependent BIM
EL upregulation.
The early sorafenib‐induced endoplasmic reticulum (ER) stress and regulation of JNK and AMPK‐dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK‐CHOP‐dependent rise of BimEL, which was associated with the shift from autophagy to apoptosis.
Early acquisition of sorafenib resistance is responsible for the dismal prognosis of advanced hepatocarcinoma (HCC). Autophagy, a catabolic process involved in liver homeostasis, has been associated ...with chemosensitivity modulation. Forkhead box O3 (FOXO3) is a transcription factor linked to HCC pathogenesis whose role on autophagy-related sorafenib resistance remains controversial. Here, we unraveled the linkage between autophagy and sorafenib resistance in HCC, focusing on the implication of FOXO3 and its potential modulation by regorafenib. We worked with two HepG2-derived sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3) and checked HCC patient data from the UALCAN database. Resistant cells displayed an enhanced basal autophagic flux compared to HepG2, showing higher autophagolysosome content and autophagy markers levels. Pharmacological inhibition of autophagy boosted HepG2S1 and HepG2S3 apoptosis and subG1 cells, but reduced viability, indicating the cytoprotective role of autophagy. HCC samples displayed higher FOXO3 levels, being associated with shorter survival and autophagic genes expression. Consistently, chemoresistant in vitro models showed significant FOXO3 upregulation. FOXO3 knockdown suppressed autophagy and caused resistant cell death, demonstrating that overactivation of such pro-survival autophagy during sorafenib resistance is FOXO3-dependent; a cytoprotective mechanism that the second-line drug regorafenib successfully abolished. Therefore, targeting FOXO3-mediated autophagy could significantly improve the clinical efficacy of sorafenib.
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