VDAC1-interacting molecules promote cell death in cancer organoids through mitochondrial-dependent metabolic interference.

The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico-to-in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions.

The protease activated receptor 2 - CCAAT/enhancer-binding protein beta - SerpinB3 axis inhibition as a novel strategy for the treatment of non-alcoholic steatohepatitis.

OBJECTIVE: The serine protease inhibitor SerpinB3 has been described as critical mediator of liver fibrosis and it has been recently proposed as an additional hepatokine involved in NASH development and insulin resistance. Protease Activated Receptor 2 has been identified as a novel regulator of hepatic metabolism. A targeted therapeutic strategy for NASH has been investigated, using 1-Piperidine Propionic Acid (1-PPA), since this compound has been recently proposed as both Protease Activated Receptor 2 and SerpinB3 inhibitor. METHODS: The effect of SerpinB3 on inflammation and fibrosis genes was assessed in human macrophage and stellate cell lines. Transgenic mice, either overexpressing SerpinB3 or carrying Serpinb3 deletion and their relative wild type strains, were used in experimental NASH models. Subgroups of SerpinB3 transgenic mice and their controls were also injected with 1-PPA to assess the efficacy of this compound in NASH inhibition. RESULTS: 1-PPA did not present significant cell and organ toxicity and was able to inhibit SerpinB3 and PAR2 in a dose-dependent manner. This effect was associated to a parallel reduction of the synthesis of the molecules induced by endogenous SerpinB3 or by its paracrine effects both in vitro and in vivo, leading to inhibition of lipid accumulation, inflammation and fibrosis in experimental NASH. At mechanistic level, the antiprotease activity of SerpinB3 was found essential for PAR2 activation, determining upregulation of the CCAAT Enhancer Binding Protein beta (C/EBP-ß), another pivotal regulator of metabolism, inflammation and fibrosis, which in turn determined SerpinB3 synthesis. CONCLUSIONS: 1-PPA treatment was able to inhibit the PAR2 - C/EBP-ß - SerpinB3 axis and to protect from NASH development and progression, supporting the potential use of a similar approach for a targeted therapy of NASH.

The polymorphic variant of SerpinB3 (SerpinB3-PD) is associated with faster cirrhosis decompensation.

BACKGROUND: SerpinB3 is a cysteine protease inhibitor involved in liver disease progression due to its proinflammatory and profibrogenic properties. The polymorphic variant SerpinB3-PD (SB3-PD), presents a substitution in its reactive centre loop, determining the gain of function. AIMS: To disclose the clinical characteristics of a cohort of patients with cirrhosis in relation to the presence of SB3-PD and to assess the effect of this genetic variant on fibrogenic and inflammatory cytokines in vitro. METHODS: We assessed SB3 polymorphism in 90 patients with cirrhosis, prospectively followed up in our referral centre. We used HepG2 and HuH-7 cells transfected to overexpress either wild-type SB3 (SB3-WT) or SB3-PD to assess their endogenous effect, while LX2 and THP-1 cells were treated with exogenous SB3-WT or SB3-PD proteins. RESULTS: Patients carrying SB3-PD had more severe portal hypertension and higher MELD scores, than patients carrying SB3-WT. In multivariate analysis, SB3-PD was an independent predictor of cirrhosis complications. Patients with SB3-PD polymorphism presented with more severe liver fibrosis and inflammatory features. Hepatoma cells overexpressing SB3-PD showed higher TGF-ß1 expression than controls. The addition of recombinant SB3-PD induced an up-regulation of TGF-ß1 in LX2 cells and a more prominent inflammatory profile in THP-1 cells, compared to the effect of SB3-WT protein. CONCLUSIONS: The polymorphic variant SB3-PD is highly effective in determining activation of TGF-ß1 and inflammation in vitro. Patients with cirrhosis who carry SB3-PD polymorphism may be more prone to develop severe liver disease progression. However, further validation studies are warranted to support the in vivo relevance of this polymorphism.

Hepatocellular Carcinoma: Latest Research in Pathogenesis, Detection and Treatment.

The most common form of primary liver malignancy is hepatocellular carcinoma (HCC) [...].

Chronic Liver Disease: Latest Research in Pathogenesis, Detection and Treatment.

Chronic liver disease (CLD) is a major global health threat and has emerged as a leading cause of human death [...].

SerpinB3 Upregulates Low-Density Lipoprotein Receptor-Related Protein (LRP) Family Members, Leading to Wnt Signaling Activation and Increased Cell Survival and Invasiveness.

Abnormal activation of the Wnt-ß-catenin signaling cascade is involved in tumor growth and dissemination. SerpinB3 has been shown to induce ß-catenin, and both molecules are overexpressed in tumors, particularly in those with poor prognoses. The aim of this study was to evaluate the ability of SerpinB3 to modulate the Wnt pathway in liver cancer and in monocytic cells, the main type of inflammatory cells in the tumor microenvironment. The Wnt cascade, Wnt co-receptors, and low-density lipoprotein receptor-related protein (LRP) members were analyzed in different cell lines and human monocytes in the presence or absence of SerpinB3. The Wnt-ß-catenin axis was also evaluated in liver tumors induced in mice with different extents of SeprinB3 expression. In monocytic cells, SerpinB3 induced a significant upregulation of Wnt-1/7, nuclear ß-catenin, and c-Myc, which are associated with increased cell lifespan and proliferation. In liver tumors in mice, the expression of ß-catenin was significantly correlated with the presence of SerpinB3. In hepatoma cells, Wnt co-receptors LRP-5/6 and LRP-1, implicated in cell survival and invasiveness, were upregulated by SerpinB3. The LRP pan-inhibitor RAP not only induced a decrease in LRP expression, but also a dose-dependent reduction in SerpinB3-induced invasiveness. In conclusion, SerpinB3 determines the activation of the Wnt canonical pathway and cell invasiveness through the upregulation of LRP family members.

Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition.

SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this study was to generate antibodies directed against different SerpinB3 epitopes to better investigate their biological role. Five exposed epitopes were identified using the software DNASTAR Lasergene and the corresponding synthetic peptides were used for NZW rabbit immunization. Anti-P#2 and anti-P#4 antibodies were able to recognize both SerpinB3 and SerpinB4 by ELISA. Anti-P#5 antibody, produced against the reactive site loop of SerpinB3, showed the greatest specific reactivity for human SerpinB3. This antibody was able to recognize SerpinB3 at nuclear level, while anti-P#3 antibody recognized SerpinB3 only at cytoplasmic level, both by immunofluorescence and by immunohistochemistry. The biological activity of each antibody preparation was assessed in HepG2 cells overexpressing SerpinB3 and anti-P#5 antibody reduced proliferation by 12% cell and cell invasion by 75%, while trivial results were obtained with the other antibody preparations. These findings indicate that the reactive site loop of SerpinB3 is essential for the invasiveness features induced by this serpin and it could become a novel druggable target.

Low-Dose Acetylsalicylic Acid and Mitochondria-Targeted Antioxidant Mitoquinone Attenuate Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. NAFLD can evolve from simple fatty liver to non-alcoholic steatohepatitis (NASH), and ultimately, to cirrhosis. Inflammation and oxidative stress, promoted by mitochondrial dysfunction, play a crucial role in the onset and development of NASH. To date, no therapy has been approved for NAFLD and NASH. The aim of this study is to evaluate if the anti-inflammatory activity of acetylsalicylic acid (ASA) and the mitochondria-targeted antioxidant effect of mitoquinone could hinder the progression of non-alcoholic steatohepatitis. In mice, fatty liver was induced through the administration of a deficient in methionine and choline and rich in fat diet. Two experimental groups were treated orally with ASA or mitoquinone. Histopathologic evaluation of steatosis and inflammation was performed; the hepatic expression of genes associated with inflammation, oxidative stress, and fibrosis was evaluated; the protein expression of IL-10, cyclooxygenase 2, superoxide dismutase 1, and glutathione peroxidase 1 in the liver was analyzed; a quantitative analysis of 15-epi-lipoxin A4 in liver homogenates was performed. Mitoquinone and ASA significantly reduced liver steatosis and inflammation by decreasing the expression of TNFα, IL-6, Serpinb3, and cyclooxygenase 1 and 2 and restoring the anti-inflammatory IL-10. Treatment with mitoquinone and ASA increased the gene and protein expression of antioxidants, i.e., catalase, superoxide dismutase 1, and glutathione peroxidase 1, and decreased the expression of profibrogenic genes. ASA normalized the levels of 15-epi-Lipoxin A4. In mice fed with a deficient in methionine and choline and rich in fat diet, mitoquinone and ASA reduce steatosis and necroinflammation and may represent two effective novel strategies for the treatment of non-alcoholic steatohepatitis.

Liver Organoids as an In Vitro Model to Study Primary Liver Cancer.

Primary liver cancers (PLC), including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are among the leading causes of cancer-related mortality worldwide. Bi-dimensional in vitro models are unable to recapitulate the key features of PLC; consequently, recent advancements in three-dimensional in vitro systems, such as organoids, opened up new avenues for the development of innovative models for studying tumour's pathological mechanisms. Liver organoids show self-assembly and self-renewal capabilities, retaining essential aspects of their respective in vivo tissue and allowing modelling diseases and personalized treatment development. In this review, we will discuss the current advances in the field of liver organoids focusing on existing development protocols and possible applications in regenerative medicine and drug discovery.

SerpinB3 administration protects liver against ischemia-reperfusion injury.

We have investigated the change in SerpinB3 during hepatic ischemia and the potential role of its antiprotease activity in cell protection by the administration of wild-type SerpinB3 (SerpinB3-WT) or active loop-deleted recombinant SerpinB3 protein (SerpinB3-D) in a rat model of ischemia (60 min)/reperfusion (60 min) (I/R). A time-dependent increase of SerpinB3, both at transcription and protein level, was found in ischemic livers after 60, 120 and 180 min. SerpinB3-WT decreased polymorphonuclear cell infiltration and serum enzymes and increased ATP when compared with I/R group. These events were not obtained using SerpinB3-D. No significant changes in both liver SerpinB3 mRNA and protein were found in all I/R groups considered. The present data show that the administration of SerpinB3-WT reduced the I/R injury and this effect appears to be dependent on its anti-protease activity.

Semisolid Wet Sol-Gel Silica/Hydroxypropyl Methyl Cellulose Formulation for Slow Release of Serpin B3 Promotes Wound Healing In Vivo.

Foot ulcerations are a disabling complication of diabetes and no treatment is currently available based on disease mechanisms. The protein serpin B3 (SB3) was identified as a positive biomarker of successful diabetic wound healing; therefore, its exogenous administration may promote healing. The topical administration of SB3 is challenging due to its protein nature. Physical entrapment in wet sol-gel silica can stabilize the protein's conformation and permit its sustained delivery. However, irreversible syneresis and poor viscoelastic properties hamper wet sol-gel silica application as a semisolid vehicle. To overcome these limits, a sol-gel silica/hydroxypropylmethylcellulose (HPMC) hydrogel blend was developed. SB3 entrapped in 8% SiO2 wet sol-gel silica preserved its structure, was stabilized against denaturation, and was slowly released for at least three days. Blending a silica gel with an HPMC-glycerol (metolose-G) hydrogel permitted spreadability without affecting the protein's release kinetics. When administered in vivo, SB3 in silica/metolose-G-but not in solution or in metolose-G alone-accelerated wound healing in SB3 knockout and diabetic mouse models. The results confirmed that SB3 is a new pharmacological option for the treatment of chronic ulcers, especially when formulated in a slow-releasing vehicle. Silica-metolose-G represents a novel type of semisolid dosage form which could also be applied for the formulation of other bioactive proteins.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1ß and reactive oxygen species along with that of TGFß and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression.

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis.

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Engineered EVs for Oxidative Stress Protection.

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

Prone Positioning Is Safe and May Reduce the Rate of Intubation in Selected COVID-19 Patients Receiving High-Flow Nasal Oxygen Therapy.

BACKGROUND: Patients with COVID-19 may experience hypoxemic Acute Respiratory Failure (hARF) requiring O2-therapy by High-Flow Nasal Cannula (HFNO). Although Prone Positioning (PP) may improve oxygenation in COVID-19 non-intubated patients, the results on its clinical efficacy are controversial. The present study aims to prospectively investigate whether PP may reduce the need for endotracheal intubation (ETI) in patients with COVID-19 receiving HFNO. METHODS: All consecutive unselected adult patients with bilateral lung opacities on chest X-ray receiving HFNO after admission to a SARS-CoV-2 Respiratory Intermediate Care Unit (RICU) were considered eligible. Patients who successfully passed an initial PP trial (success group) underwent PP for periods ≥ 2 h twice a day, while receiving HFNO. The study's primary endpoint was the intubation rate during the stay in the RICU. RESULTS: Ninety-three patients were included in the study. PP was feasible and safe in 50 (54%) patients. Sixteen (17.2%) patients received ETI and 27 (29%) escalated respiratory support, resulting in a mortality rate of 9/93 (9.7%). The length of hospital stay was 18 (6-75) days. In 41/50 (80%) of subjects who passed the trial and underwent PP, its use was associated with clinical benefit and survival without escalation of therapy. CONCLUSIONS: PP is feasible and safe in over 50% of COVID-19 patients receiving HFNO for hARF. Randomized trials are required to confirm that PP has the potential to reduce intubation rate.

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc-/- mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc-/- showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.