Mitochondrial Dysfunction and Mitophagy in Type 2 Diabetes: Pathophysiology and Therapeutic Targets.

Significance: Type 2 diabetes mellitus, which is related to oxidative stress and mitochondrial dysfunction, is one of the most prevalent diseases in the world. In the past decade, alterations in autophagy have been shown to play a fundamental role in the development and control of type 2 diabetes. Further, mitophagy has been recognized as a key player in eliminating dysfunctional mitochondria in this disease. Recent Advances: Recently, much progress has been made in understanding the molecular events associated with oxidative stress, mitochondrial dysfunction, and alterations in autophagy and mitophagy in type 2 diabetes. Critical Issues: Despite increasing evidence of a relationship between mitochondrial dysfunction, oxidative stress, and alterations of autophagy and mitophagy and their role in the pathophysiolology of type 2 diabetes, effective therapeutic strategies to combat the disease through targeting mitochondria, autophagy, and mitophagy are yet to be implemented. Future Directions: This review provides a wide perspective of the existing literature concerning the complicated interplay between autophagy, mitophagy, and mitochondrial dysfunction in type 2 diabetes. Further, potential therapeutic targets based on these molecular mechanisms are explored. Antioxid. Redox Signal. 39, 278-320.

Hepatocellular carcinoma risk-stratification based on ASGR1 in circulating epithelial cells for cancer interception.

Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular carcinoma impedes stratifying patients based on their risk of developing cancer. The aim of this study was to evaluate phenotypic and genetic heterogeneity of circulating epithelial cells (CECs) based on asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as potential diagnostic and prognostic tools in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Methods: Peripheral blood samples were extracted from LC and HCC patients at different disease stages. CECs were isolated using positive immunomagnetic selection. Genetic and phenotypic characterization was validated by double immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization with miR-122-5p and CECs were visualized by confocal microscopy. Results: The presence of CECs increased HCC risk by 2.58-fold, however, this was only significant for patients with previous LC (p = 0.028) and not for those without prior LC (p = 0.23). Furthermore, the number of CECs lacking ASGR1 expression correlated significantly with HCC incidence and absence of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p = 0.021) whereas progression-free survival was influenced by the absence of ASGR1 expression. Conclusion: Identification and characterization of CECs by ASGR1 and/or miR-122-5p expression may be used as a risk-stratification tool in LC patients, as it was shown to be an independent prognostic and risk-stratification marker in LC and early disease stage HCC patients.

miR-200c-3p, miR-222-5p, and miR-512-3p Constitute a Biomarker Signature of Sorafenib Effectiveness in Advanced Hepatocellular Carcinoma.

BACKGROUND: Sorafenib constitutes a suitable treatment alternative for patients with advanced hepatocellular carcinoma (HCC) in whom atezolizumab + bevacizumab therapy is contraindicated. The aim of the study was the identification of a miRNA signature in liquid biopsy related to sorafenib response. METHODS: miRNAs were profiled in hepatoblastoma HepG2 cells and tested in animal models, extracellular vesicles (EVs), and plasma from HCC patients. RESULTS: Sorafenib altered the expression of 11 miRNAs in HepG2 cells. miR-200c-3p and miR-27a-3p exerted an anti-tumoral activity by decreasing cell migration and invasion, whereas miR-122-5p, miR-148b-3p, miR-194-5p, miR-222-5p, and miR-512-3p exerted pro-tumoral properties by increasing cell proliferation, migration, or invasion, or decreasing apoptosis. Sorafenib induced a change in EVs population with an increased number of larger EVs, and promoted an accumulation of miR-27a-3p, miR-122-5p, miR-148b-3p, miR-193b-3p, miR-194-5p, miR-200c-3p, and miR-375 into exosomes. In HCC patients, circulating miR-200c-3p baseline levels were associated with increased survival, whereas high levels of miR-222-5p and miR-512-3p after 1 month of sorafenib treatment were related to poor prognosis. The RNA sequencing revealed that miR-200c-3p was related to the regulation of cell growth and death, whereas miR-222-5p and miR-512-3p were related to metabolic control. CONCLUSIONS: The study showed that Sorafenib regulates a specific miRNA signature in which miR-200c-3p, miR-222-5p, and miR-512-3p bear prognostic value and contribute to treatment response.

Impact of nitric oxide in liver cancer microenvironment.

The pro- or antitumoral properties of nitric oxide (NO) are dependent on local concentration, redox state, cellular status, duration of exposure and compartmentalization of NO generation. The intricate network of the tumor microenvironment (TME) is constituted by tumor cells, stromal and immune cells surrounded by active components of extracellular matrix that influence the biological behavior and, consequently, the treatment and prognosis of cancer. The review describes critical events in the crosstalk of cellular and stromal components in the TME, with special emphasis in the impact of NO generation in the regulation of hepatocellular carcinoma (HCC). The increased expression of nitric oxide synthase (NOS) in tumors and NO-end products in plasma have been associated with poor prognosis of cancer. We have assessed the level of the different isoforms of NOS in tumors and its relation to cell proliferation and death markers, and cell death receptor expression in tumors, and apoptotic markers and ligands of TNF-α receptor family in blood from a cohort of patients with HCC from different etiologies submitted to orthotopic liver transplantation (OLT). The high levels of NOS2 in tumors were associated with low plasma concentration of apoptotic markers (M30 and M65), FasL and TNF-α in HCV patients. By contrast, the low levels of NOS2 in tumors from alcohol-derived patients was associated with increased Trail-R1 expression in tumors, and circulating Trail levels compared to observed in plasma from HCV- and alcohol + HCV-derived patients. This study reinforces the association between increased NOS2 expression and potential risk of low patients' survival in HCC. However, a differential functional relevance of NOS expression in HCC seems to be influenced by etiologies.

Broad Transcriptomic Impact of Sorafenib and Its Relation to the Antitumoral Properties in Liver Cancer Cells.

Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma- (HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes.

Long non-coding RNA H19 as a biomarker for hepatocellular carcinoma.

BACKGROUND AND AIMS: Liver cancer stem cells (CSCs) could be involved in the carcinogenesis, recurrence, metastasis and chemoresistance of hepatocellular carcinoma (HCC). The aim of this study was to explore the role of lncRNA-H19 as a biomarker for liver cancer. METHODS: LncRNA-H19 expression levels and the functional assays were conducted in EpCAM+ CD133+ CSCs and C57BL/6J mice fed with a high-fat high-cholesterol carbohydrate (HFHCC) or standard diet for 52 weeks. Liver tissue and plasma samples from patients with cirrhosis, with or without HCC, were used for the analyses of gene expression and circulating lncRNA-H19 levels in an estimation and validation cohort. RESULTS: EpCAM+ CD133+ cells showed a stem cell-like phenotype, self-renewal capacity, upregulation of pluripotent gene expression and overexpressed lncRNA-H19 (p < .001). Suppression of lncRNA-H19 by antisense oligonucleotide treatment significantly reduced the self-renewal capacity (p < .001). EpCAM, CD133 and lncRNA-h19 expression increased accordingly with disease progression in HFHCC-fed mice (p < .05) and also in liver tissue from HCC patients (p = .0082). Circulating lncRNA-H19 levels were significantly increased in HCC patients in both cohorts (p = .013; p < .0001). In addition, lncRNA-H19 levels increased accordingly with BCLC staging (p < .0001) and decreased after a partial and complete therapeutic response (p < .05). In addition, patients with cirrhosis who developed HCC during follow-up showed higher lncRNA-H19 levels (p = .0025). CONCLUSION: LncRNA-H19 expression was increased in CSCs, in liver tissue and plasma of patients with HCC and decreased after partial/complete therapeutic response. Those patients who developed HCC during the follow-up showed higher levels of lncRNA-H19. LncRNA-H19 could constitute a new biomarker of HCC.

The Role of Non-Coding RNAs in Autophagy During Carcinogenesis.

Macroautophagy (autophagy herein) is a cellular stress response and a survival pathway involved in self-renewal and quality control processes to maintain cellular homeostasis. The alteration of autophagy has been implicated in numerous diseases such as cancer where it plays a dual role. Autophagy serves as a tumor suppressor in the early phases of cancer formation with the restoration of homeostasis and eliminating cellular altered constituents, yet in later phases, autophagy may support and/or facilitate tumor growth, metastasis and may contribute to treatment resistance. Key components of autophagy interact with either pro- and anti-apoptotic factors regulating the proximity of tumor cells to apoptotic cliff promoting cell survival. Autophagy is regulated by key cell signaling pathways such as Akt (protein kinase B, PKB), mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) involved in cell survival and metabolism. The expression of critical members of upstream cell signaling, as well as those directly involved in the autophagic and apoptotic machineries are regulated by microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Consequently, non-coding RNAs play a relevant role in carcinogenesis and treatment response in cancer. The review is an update of the current knowledge in the regulation by miRNA and lncRNA of the autophagic components and their functional impact to provide an integrated and comprehensive regulatory network of autophagy in cancer.

Role of Nitric Oxide in Gene Expression Regulation during Cancer: Epigenetic Modifications and Non-Coding RNAs.

Nitric oxide (NO) has been identified and described as a dual mediator in cancer according to dose-, time- and compartment-dependent NO generation. The present review addresses the different epigenetic mechanisms, such as histone modifications and non-coding RNAs (ncRNAs), miRNA and lncRNA, which regulate directly or indirectly nitric oxide synthase (NOS) expression and NO production, impacting all hallmarks of the oncogenic process. Among lncRNA, HEIH and UCA1 develop their oncogenic functions by inhibiting their target miRNAs and consequently reversing the inhibition of NOS and promoting tumor proliferation. The connection between miRNAs and NO is also involved in two important features in cancer, such as the tumor microenvironment that includes key cellular components such as tumor-associated macrophages (TAMs), cancer associated fibroblasts (CAFs) and cancer stem cells (CSCs).

Inhibition of the NLRP3 inflammasome prevents ovarian aging.

Inflammation is a hallmark of aging and is negatively affecting female fertility. In this study, we evaluate the role of the NLRP3 inflammasome in ovarian aging and female fertility. Age-dependent increased expression of NLRP3 in the ovary was observed in WT mice during reproductive aging. High expression of NLRP3, caspase-1, and IL-1ß was also observed in granulosa cells from patients with ovarian insufficiency. Ablation of NLRP3 improved the survival and pregnancy rates and increased anti-Müllerian hormone levels and autophagy rates in ovaries. Deficiency of NLRP3 also reduced serum FSH and estradiol levels. Consistent with these results, pharmacological inhibition of NLRP3 using a direct NLRP3 inhibitor, MCC950, improved fertility in female mice to levels comparable to those of Nlrp3-/- mice. These results suggest that the NLRP3 inflammasome is implicated in the age-dependent loss of female fertility and position this inflammasome as a potential new therapeutic target for the treatment of infertility.

PDA-Based Glyconanomicelles for Hepatocellular Carcinoma Cells Active Targeting Via Mannose and Asialoglycoprotein Receptors.

Hepatocellular carcinoma (HCC) is the sixth most common neoplasia and the fourth most common cause of cancer-related mortality worldwide. Sorafenib is the first-line molecular therapy for patients in an advanced stage of HCC. However, the recommended clinical dose of Sorafenib is associated with several complications, which derive from its lack of cell specificity and its very low water solubility. To circumvent these drawbacks, in the present study we developed two sugar-coated polydiacetylene-based nanomicelles-Sorafenib carriers targeting mannose and asialoglycoprotein receptors (MR and ASGPR, respectively). The strategies allowed the inducement of apoptosis and reduction of cell proliferation at a nanomolar, instead of micromolar, range in liver cancer cells. The study showed that, contrary to literature data, Sorafenib included into the pMicMan (Man = mannose) vector (targeting MR) is more efficient than pMicGal (Gal = galactose) (targeting ASGPR). Indeed, pMicMan increased the endosomal incorporation with an increased intracellular Sorafenib concentration that induced apoptosis and reduced cell proliferation at a low concentration range (10-20 nM).

Mechanisms of action of metformin in type 2 diabetes: Effects on mitochondria and leukocyte-endothelium interactions.

Type 2 diabetes (T2D) is a very prevalent, multisystemic, chronic metabolic disorder closely related to atherosclerosis and cardiovascular diseases. It is characterised by mitochondrial dysfunction and the presence of oxidative stress. Metformin is one of the safest and most effective anti-hyperglycaemic agents currently employed as first-line oral therapy for T2D. It has demonstrated additional beneficial effects, unrelated to its hypoglycaemic action, on weight loss and several diseases, such as cancer, cardiovascular disorders and metabolic diseases, including thyroid diseases. Despite the vast clinical experience gained over several decades of use, the mechanism of action of metformin is still not fully understood. This review provides an overview of the existing literature concerning the beneficial mitochondrial and vascular effects of metformin, which it exerts by diminishing oxidative stress and reducing leukocyte-endothelium interactions. Specifically, we describe the molecular mechanisms involved in metformin's effect on gluconeogenesis, its capacity to interfere with major metabolic pathways (AMPK and mTORC1), its action on mitochondria and its antioxidant effects. We also discuss potential targets for therapeutic intervention based on these molecular actions.

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.

Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells.

BACKGROUND/AIMS: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. METHODS: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. RESULTS: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172P-Cdk4/Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR-720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. CONCLUSION: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cells.

Differential effectiveness of tyrosine kinase inhibitors in 2D/3D culture according to cell differentiation, p53 status and mitochondrial respiration in liver cancer cells.

Sorafenib and Regorafenib are the recommended first- and second-line therapies in patients with advanced hepatocellular carcinoma (HCC). Lenvatinib and Cabozantinib have shown non-inferior antitumoral activities compared with the corresponding recommended therapies. The clinical trials have established recommended doses for each treatment that lead different blood concentrations in patients for Sorafenib (10 µM), Regorafenib (1 µM), Lenvatinib (0.1 µM), and Cabozantinib (1 µM). However, very low response rates are observed in patients attributed to intrinsic resistances or upregulation of survival signaling. The aim of the study was the comparative dose-response analysis of the drugs (0-100 µM) in well-differentiated (HepG2, Hep3B, and Huh7), moderately (SNU423), and poorly (SNU449) differentiated liver cancer cells in 2D/3D cultures. Cells harbors wild-type p53 (HepG2), non-sense p53 mutation (Hep3B), inframe p53 gene deletion (SNU423), and p53 point mutation (Huh7 and SNU449). The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose-response analysis in 2D cultures showed Sorafenib and Regorafenib were increasingly effective in reducing cell proliferation, and inducing apoptosis in well-differentiated and expressing wild-type p53 in HCC cells. Lenvatinib and Cabozantinib were particularly effective in moderately to poorly differentiated cells with mutated or lacking p53 that have lower basal oxygen consumption rate (OCR), ATP, and maximal respiration capacity than observed in differentiated HCC cells. Sorafenib and Regorafenib downregulated, and Lenvatinib and Cabozantinib upregulated epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor receptor (c-Met) in HepG2 cells. Conclusions: Sorafenib and Regorafenib were especially active in well-differentiated cells, with wild-type p53 and increased mitochondrial respiration. By contrast, Lenvatinib and Cabozantinib appeared more effective in moderately to poorly differentiated cells with mutated p53 and low mitochondrial respiration. The development of strategies that allow us to deliver increased doses in tumors might potentially enhance the effectiveness of the treatments.

Downregulation of thioredoxin-1-dependent CD95 S-nitrosation by Sorafenib reduces liver cancer.

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.

Mitochondria and T2D: Role of Autophagy, ER Stress, and Inflammasome.

Type 2 diabetes (T2D) is one of the main current threats to human health. Both T2D and its numerous clinical complications are related to mitochondrial dysfunction and oxidative stress. Over the past decade, great progress has been made in extending our knowledge about the signaling events regulated by mitochondria. However, the links among mitochondrial impairment, oxidative stress, autophagy, endoplasmic reticulum (ER) stress, and activation of the inflammasome still need to be clarified. In light of this deficit, we aim to provide a review of the existing literature concerning the complicated crosstalk between mitochondrial impairment, autophagy, ER stress, and the inflammasome in the molecular pathogenesis of T2D.

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.

Dose-dependent regulation of mitochondrial function and cell death pathway by sorafenib in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.