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

Oxidative stress influence on renal dysfunction in patients with obstructive jaundice: A case and control prospective study

Obstructive Jaundice (OJ) is associated with a significant risk of developing acute renal failure (ARF). The involvement of oxidative stress in the development of cholestasis has been demonstrated in different experimental models. However, its role in the morbidity of human cholestasis is far to be elucidated. The aim of the study was the evaluation of oxidative stress markers in blood from patients with OJ and its relation to complications and benign/malignant evolution of cholestasis. Methods: A prospective cross-sectional study of 105 patients with OJ and 34 control subjects were included. Several markers of liver function and oxidative stress, such as lipoperoxides (LPO), as well as reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were assessed. Results: The patients with OJ showed a marked increase in plasma levels of LPO, SOD and GSH, while GSH-Px levels were decreased. The increase in lipid peroxidation products and the depletion of SOD activity in blood were also related to renal dysfunction. The highest level of LPO was associated with malignant etiology of the disease. The logistic regression analysis showed that the age of the patient and the levels of LPO in blood were predictors of renal dysfunction in OJ patients. Conclusions: This study demonstrates a correlation between oxidative stress and renal dysfunction patients with OJ.Instituto de Salud Carlos III PI02/015