The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage

Combined targeting of the p53 and pRb pathway in neuroblastoma does not lead to synergistic responses

BACKGROUND: Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutation but rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. METHODS AND RESULTS: We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. In vivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. CONCLUSION: We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas

Combined targeting of the p53 and pRb pathway in neuroblastoma does not lead to synergistic responses

BACKGROUND: Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutation but rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. METHODS AND RESULTS: We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. In vivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. CONCLUSION: We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas

Liver Regeneration After Portal Vein Embolization Using Absorbable and Permanent Embolization Materials in a Rabbit Model

Objective: To compare the safety and hypertrophy response after portal vein embolization (PVE) using 2 absorbable and 3 permanent embolization materials. Background: Portal vein embolization is used to increase future remnant liver volume preoperatively. Application of temporary, absorbable embolization materials could be advantageous in some situations, provided sufficient hypertrophy is achieved from the nonembolized lobe. Methods: Six groups of rabbits (n = 5) underwent PVE of 80% of the total liver volume using saline (sham), gelatin sponge, fibrin glue, polyvinyl alcohol particles with coils, n-butyl cyanoacrylate, or polidocanol. The rabbits were killed after 7 days. Portography, computed tomographic volumetry, Doppler ultrasonography, laboratory liver function and damage parameters (nonembolized) liver-to-body weight ratio, immunohistochemistry, and cytokine and growth factor tissue levels were assessed to examine the differences in the liver regeneration response. Results: Polidocanol was discontinued because of toxic reactions in 3 rabbits. Gelatin sponge was the only material that was absorbed after 7 days and resulted in less hypertrophy of the nonembolized lobe than the other 3 materials. There were no significant differences in hypertrophy response between the other 3 embolization groups. Volumetric data obtained from computed tomography were supported by liver-to-body weight ratio and the amount of proliferating hepatocytes. The volume gain of the nonembolized lobe was proportional to the volume loss of the embolized liver lobes. The number of Kupffer cells in the embolized liver lobe was significantly higher in the fibrin glue, polyvinyl alcohol particles with coils, and n-butyl cyanoacrylate groups than in the sham and gelatin sponge groups. However, the levels of interleukin-6, tumor necrosis factor-alpha, hepatocyte growth factor, and transforming growth factor-beta 1 were significantly lower. Conclusions: Temporary occlusion using gelatin sponge for PVE resulted in significantly less hypertrophy response than the use of permanent embolization materials. Except for polidocanol, none of the embolization materials exhibited evident hepatotoxicit

RaS–MAPK pathway-driven tumor progression is associated with loss of CIC and other genomic aberrations in neuroblastoma

Mutations affecting the RAS–MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS–MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS–MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS–MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS–MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo. These results show that the RAS–MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma. Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer

Inhibition of hypoxia-inducible factor 1 with acriflavine sensitizes hypoxic tumor cells to photodynamic therapy with zinc phthalocyanine-encapsulating cationic liposomes

Photodynamic therapy (PDT) is a tumor treatment modality in which a tumorlocalized photosensitizer is excited with light, which results in local production of reactive oxygen species, destruction of tumor vasculature, tumor hypoxia, tumor cell death, and induction of an anti-tumor immune response. However, pre-existing tumor hypoxia may desensitize tumors to PDT by activating the hypoxia-inducible factor 1 (HIF-1) survival pathway. Therefore, we hypothesized that inhibition of HIF-1 with acriflavine (ACF) would exacerbate cell death in human epidermoid carcinoma (A431) cells. PDT of A431 tumor cells was performed using newly developed and optimized PEGylated cationic liposomes containing the photosensitizer zinc phthalocyanine (ZnPC). Molecular docking revealed that ACF binds to the dimerization domain of HIF-1 alpha, and confocal microscopy confirmed translocation of ACF from the cytosol to the nucleus under hypoxia. HIF-1 was stabilized in hypoxic, but not normoxic, A431 cells following PDT. Inhibition of HIF-1 with ACF increased the extent of PDT-induced cell death under hypoxic conditions and reduced the expression of the HIF-1 target genes VEGF, PTGS2, and EDN1. Moreover, co-encapsulation of ACF in the aqueous core of ZnPC-containing liposomes yielded an adjuvant effect on PDT efficacy that was comparable to non-encapsulated ACF. In conclusion, HIF-1 contributes to A431 tumor cell survival following PDT with liposomal ZnPC. Inhibition of HIF-1 with free or liposomal ACF improves PDT efficac

High-throughput screening identifies idasanutlin as a resensitizing drug for venetoclax-resistant neuroblastoma cells

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Nonresistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies

Time-Dependent Impact of Irreversible Electroporation on Pathology and Ablation Size in the Porcine Liver: A 24-Hour Experimental Study

Irreversible electroporation causes cell death through low frequency, high voltage electrical pulses and is increasingly used to treat non-resectable cancers. A recent systematic review revealed that tissue damage through irreversible electroporation is time-dependent, but the impact of time on the ablation zone size remains unknown. Irreversible electroporation ablations were performed hourly during 24 consecutive hours in the peripheral liver of 2 anaesthetized domestic pigs using clinical treatment settings. Immediately after the 24th ablation, the livers were harvested and examined for tissue response in time based on macroscopic and microscopic pathology. The impact of time on these outcomes was assessed with Spearman rank correlation test. Ablation zones were sharply demarcated as early as 1 hour after treatment. During 24 hours, the ablation zones showed a significant increase in diameter (rs = 0.493, P = .014) and total surface (rs = 0.499, P = .013), whereas the impact of time on the homogeneous ablated area was not significant (rs = 0.172, P = .421). Therefore, the increase in size could mainly be attributed to an increase in the transition zone. Microscopically, the ablation zones showed progression in cell death and inflammation. This study assessed the dynamics of irreversible electroporation on the porcine liver during 24 consecutive hours and found that the pathological response (ie, cell death/inflammation), and ablation size continue to develop for at least 24 hours. Consequently, future studies on irreversible electroporation should prolong their observation period

High-throughput screening identifies idasanutlin as a resensitizing drug for venetoclax-resistant neuroblastoma cells

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Nonresistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies