Regulation of apoptosis and cell proliferation by Sorafenib in Hepatocellular carcinoma

Motivation: Hepatocellular carcinoma (HCC), which represents more than 90% of primary liver cancers, is the sixth most common type of cancer with 749.000 new cases each year and the third cause of death associated with cancer. At early stages of HCC, the tumor might be cured by treatments as resection, ablation or liver transplantation. However, there are no effective therapies for patients with advanced HCC, who represents two thirds of diagnosed patients. In this context, Sorafenib, an oral multikinase inhibitor of some targets like Raf, VEGFR and PDGFR, among others, is the only therapeutic option in advanced HCC. Sorafenib has important actions inhibiting the cell proliferation and apoptosis in tumor hepatocytes, developing limited side effects but, more fundamentally, disease stabilization and increasing the survival rate to 9.2 months. The importance of this work is based on the lack of curative therapies in advanced HCC. Bearing in mind that illness is a major global health problem, is really important to carry out researches for finding new mechanisms, other targets and improvements of this drug in order to enhance the therapeutic options of patients, especially who suffer advanced HCC. Results: On the one hand, we find that Sorafenib decreases the Mcl-1 expression and increases the pMcl-1 (pSer159 + pThr193) leading to an increment of intrinsic apoptosis. In this context, not only does Sorafenib promote the activity of caspase-3 and caspasa-9, but also alters mitochondrial membrane potential in different HCC cell lines. On the other hand, Sorafenib decreases the cell proliferation. Taking into account all these results, Sorafenib has a relevant anti-tumor activity in in vitro studies.Conclusions: Sorafenib has an anti-tumor activity by activating cell death and blocking cell proliferation. The promotion of intrinsic apoptosis is due to the activation of caspase-3 and caspase-9 as well as inhibition the anti-apoptotic action of Mcl-1 and changes in the mitochondrial membrane potential

Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells

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.Ministerio de Economía y Competitividad BFU2016‐75352‐PInstituto de Salud Carlos III PI15/00034, PI13/ 00021, PI16/00090, PI14/01349Ministerio de Educación FPU16/05127, FPU12/01433, FPU13/01237Junta de Andalucía CTS-6264, PI-00025-2013, PI-0127-2013, PI-0198-201

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.We thank the Institute of Health Carlos III (ISCiii) cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF) (PI13/00021, P16/00090 and PI19/01266), as well as the Andalusian Ministry of the Economy, Innovation, Science and Employment (CTS-6264) and Andalusian Ministry of Health (PI13/00025, PI16/0198, PIP-0215-2020 and PI-0216-2020) for their financial support to J.M. We also thank the Spanish Ministry of Economy and Competitiveness (MINECO) cofinanced by the ERDF (BFU2016-75352-P AEI/FEDER, EU) for their financial support to J.d.l.C. We thank Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and cofinanced by the ERDF for their financial support. E.N-V. acknowledges IFI18/00014 fellowship from the ISCiii. P.d.l.C-O. acknowledges FPU17/00026 fellowship from the Spanish Ministry of Education (MEC). L.C. acknowledges FPU16/05127 fellowship from the MEC

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, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-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 cellsEspaña Ministry of Economy and Competitiveness (MINECO) cofinanced by the ERDF (BFU2016-75352-P AEI/FEDER, EU

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.This study was funded by Institute of Health Carlos III (ISCiii) (PI16/00090, PI19/00838 and PI19/01266), Spanish Ministry of Economy and Competitiveness (BFU2016-80006-P), Andalusian Ministry of Economy, Innovation, Science and Employment (BIO-216 and CTS-6264), Andalusian Ministry of Equality, Health and Social Policies (PI-0198-2016) and Valencian Ministry of Education, Culture and Sports (PROMETEO/2019/027). P de la C-O was supported by FPU predoctoral fellowship (FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E N-V was supported by the the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii. We thank the Biomedical Research Network Center for Cardiovascular Diseases (CIBERcv), and the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and co-financed by European Regional Development Fund (ERDF) "A way to achieve Europe" for their financial support

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).Financial support was provided by the Spanish Ministry of Economy and Competitiveness (CTQ2016-78580-C2-1-R to N.K.) and the Institute of Health Carlos III (ISCIII) (PI13/00021, PI16/00090, and PI19/01266 to J.M.) both cofinanced by the European Regional Development Fund (ERDF) from FEDER and the European Social Fund (ESF), as well by the Andalusian Ministry of Economy, Science and Innovation (P07-FQM-2774 to N.K., CV20-04221 to N.K. P20_00882 to N.K. and CTS-6264 to J.M.), the PAIDI Program from the Andalusian Government (FQM-313 to N.K., CV20-04221 to N.K., P20_00882 to N.K., P20_00882 to N.K., and CTS-0664 to J.M.), the Andalusian Ministry of Health (PI-00025-2013, and PI-0198-2016 to J.M.), and the CSIC (CSIC–COV19-047). We thank the Biomedical Research Network Center for Liver and Digestive Diseases founded by the ISCIII and cofinanced by FEDER “A way to achieve Europe” and ERDF for their financial support. The COST action CA-18132 “Functional Glyconanomaterials for the Development of Diagnostic and Targeted Therapeutic Probe” is also acknowledged. E.R.B., C.C.A., and P. de la C.-O. were supported by FPU predoctoral fellowship (FPU15/04267, FPU17/00190, and FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E.N.-V. was supported by the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii.Peer reviewe

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-1β 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

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.Instituto de Salud Carlos III PI13/00021Ministerio de Economía y Competitividad BFU2012-32056Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía BIO-0216Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía CTS-6264Consejería de Salud, Junta de Andalucía PI13/ 0002

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).This research was funded by the Institute of Health Carlos III (ISCiii) (PI19/01266) and Andalusian Ministry of Health (PI-0216-2020). P de la C-O was supported by the FPU predoctoral fellowship (FPU17/00026) from the Ministry of Education, Culture and Sports. E N-V was supported by the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from the ISCiii. S D-B was supported by the “Rio Hortega” postdoctoral contract (CM19/00151) from the ISCiii. We thank the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd), founded by the ISCIII and co-financed by European Regional Development Fund “A way to achieve Europe” ERDF for their financial support.Ye