Antitumoral properties of Sorafenib, Regorafenib, Cabozantinib and Lenvatinib in 3D tumor liver cell culture

Motivation: Most researchs to find effective therapies to treat cancer has been done in cell lines grown in monolayer that don´t take into account the three-dimensional structure of the tumor or the interactions between the tumor cells. In cell cultures in two dimensions, all cells are exposed in the same way to the therapeutic agent, so the results are not very precise.For this reason, it is important to develop cell culture techniques in which it is possible simulate in vivo conditions to predict more exactly the behavior and cellular interactions within the tumor. Like tumors, spheroids are three-dimensional aggregates of cancer cells that naturally form regions of hypoxia.Methods: To carry out this study we have used three different cell lines: HepG2, Hep3B and Huh7. First, the cells were cultured in 96-well plates coated with agarose. The spheroids were collected on day 5, 8 (when treated with the different drugs), 10, 12 and 15. The collected spheroids were fixed with paraformaldehyde, passed through a paraffination cycle, were introduced in paraffin blocks and cut with the microtome. Other spheroids were disintegrated by trypsinization to determine the number of cells and lysates to be able to determine caspase-3 activity. The parameters that we have measured have been apoptosis, cell proliferation, cell viability, cell death, hypoxia and cell growth. In addition, was carried out a study of the expression of  different growth factor receptors such as EGFR, VGFR, FGFR ans PDGFR by immunodetection.Results: Sorafenib and Regorafenib induced cell death and reduced cell proliferation both in 2D and 3D cultured HCC lines. These effects are higher than those observed with Cabozantinib and Lenvatinib in the same conditions. The expression of some receptors such as EGFR is reduced in the cells treated for 24 hours with sorafenib and regorafenib but no changes are observed in the cells treated with lenvatinib and cabozantinib with respect to the control.Conclusions: The observed resistance of Lenvatinib and Cabozantinib treatment to the induction of cell death and cell cycle arrest in comparison with that observed with Sorafenib and Regorafenib may be related to the induction of EGFR-dependent pathway

Extracellular vesicles secretion by Lenvatinib and Sorafenib in HepG2 cells and their effect on cell death and proliferation

Motivation: Sorafenib, which acts on the RAF / MEK / ERK pathways through the inhibition of Raf kinase and different tyrosine kinases (VEGFR2, PDGFR, c-Kit receptors), is the drug currently used as a first-line treatment in hepatocellular carcinomas of advanced stage. It has recently been shown that Lenvatinib, another multi-kinase inhibitor, also improves mean progression-free survival and mean time to cancer progression. This finding motivated us to study the possible antiproliferative effects of Lenvatinib compared to Sorafenib, in addition to the secretion profile of extracellular vesicles in HepG2 cultures due to its recognized role in tumor progression and metastasis.Methods: To determine the percentage of proliferating cells in culture, the incorporation of bromodeoxyuridine (BrdU) was used as a marker, while the analysis of the apoptotic activity was done through a colorimetric test that allows detecting the amount of caspase 3/7 existing in culture. It is well known that there is a connection between apoptosis and autophagy, so we decided to study the changes that occurred in the latter process after treatment. For this, the level of expression of LC3-II was determined through an SDS-PAGE coupled to a Western-Blot analysis. The changes produced in the expression of VEGFR-2 and EGFR were also monitored and, finally, the secretion profile of extracellular vesicles was studied through the analysis of the expression of different markers (Lamp1, E-Selectin, CD63, TSG101, Grp78, GM130, Annexin V and Prohibitin) in fractions enriched in exosomes, extracellular vesicles and apoptotic bodies.Results and Conclusions: The results for the group treated with Sorafenib reproduced what has been described so far in the literature referring to hepatocellular carcinoma: decrease in cell proliferation caused by the downregulation of the expression of different growth factors (EGFR and VEGFR-2) and increase of cell death by apoptosis. However, Lenvatinib did not reproduce the pattern we expected for an antineoplastic drug, since it increased cell proliferation. With respect to the secretion profile of extracellular vesicles, no convincing results were obtained. We think that this could be due to the capacity of separation of the different fractions of the protocol used or to the difficulty of obtaining, from them, high amounts of proteins to proceed to its analysis by WB

Obesity dependent metabolic signatures associated with nonalcoholic fatty liver disease progression

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultra-performance liquid chromatography coupled to mass spectrometry (UPLCMS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual’s level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values = 0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention

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.Ministerio de Ciencia e Innovación 10.13039/501100011033Junta de Andalucía PIP-0215-2020, PI-0216-2020Universidad de Sevilla US-138087

Effect of Methyltrienolone on the Metabolic Disorders in Rat Model of Alloxan-Induced Diabetes

Aim: The aim of the study was to investigate the restoration of metabolic imbalance related with deficiency of insulin by the exogenous androgen supplementation in the experimental model of alloxan-induced diabetes in Wistar male rats. Methods: The experimental diabetes was induced by a single intraperitoneal administration of alloxan. The concentrations of glucose, immunereactive insulin, corticosterone, testosterone and estradiol were examined in blood, the intensity of DNA and RNA synthesis and androgen receptor expression were studied in the liver tissue – at 15th, 30th and 45th days of alloxan-induced diabetes. The synthetic androgen methyltrienolone was administered to rats with 30-days diabetes during 15 days. All data were compared to control group received solvent. Results: The induction of diabetes increased the concentrations of glucose, corticosterone and estradiol while decreases insulin and testosterone concentration in blood as well as DNA/RNA synthesis and androgen receptors expression in hepatocytes. The administration of exogenous androgen significantly restored the metabolic imbalance and the expression of androgen receptors and increased DNA/RNA synthesis in liver cells maintained close to control level. Conclusion: The administration of methyltrienolone reduced the effect of “diabetic stress” and restored the hormonal dysfunction induced by alloxan

Thioredoxin Downregulation Enhances Sorafenib Effects in Hepatocarcinoma Cells

Sorafenib is the first-line recommended therapy for patients with advanced hepatocarcinoma (HCC) in de-differentiation stage (presenting epithelial–mesenchymal transition, EMT). We studied the role of the thioredoxin system (Trx1/TrxR1) in the sensitivity or resistance of HCC cells to the treatment with Sorafenib. As a model, we used a set of three established HCC cell lines with different degrees of de-differentiation as occurs in metastasis. By quantitative proteomics, we found that the expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers in these cell lines. Treatment with Sorafenib induced thiol redox reductive changes in critical elements of oncogenic pathways in all three cell lines but induced drastic proteome reprograming only in HCC cell lines of intermediate stage. Trx1 downregulation counteracted the thiol reductive effect of Sorafenib on Signal Transducer and Activator of Transcription 3 (STAT3) but not on Mitogen-Activated Protein Kinase (MAPK) or Protein Kinase B (Akt) and transformed advanced HCC cells into Sorafenib-sensitive cells. Ten targets of the combined Sorafenib–siRNATrx1 treatment were identified that showed a gradually changing expression trend in parallel to changes in the expression of canonical EMT markers, likely as a result of the activation of Hippo signaling. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.Ministerio de Economía y Competitividad BFU2016-80006-PInstituto de Salud Carlos III (ISCIII) PI13/00021 y PI16/00090Junta de Andalucía Consejería de Economía, Innovación, Ciencia y Empleo BIO-0216 y CTS-6264Junta de Andalucía Consejería de Igualdad, Salud y Políticas Sociales PI-00025-2013 y PI-0198-201

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

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

Este artículo pertenece a un número especial: Autophagy in CancerAutophagy 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 samplesInstitute de Salud Carlos III (ISCIII) y Fondos FEDER de la UE PI14/01085 y PI17/00093Ministerio de Ciencia, Innovación y Universidades RTI2018-096748-B-100 to N.A.Ministerio de Ciencia, Innovación y Universidades FPU17/00026Consejería de Igualdad, Salud y Políticas Sociales PI-0198-2016Fondos FEDER de la UE NORTE-01-0145-FEDER-000013 y los proyectos POCI-01-0145-FEDER-028159 y POCI-01-0145-FEDER-03078