Multi-Omics profiling identifies aldehyde dehydrogenase 2 as a critical mediator in the crosstalk between Treg-mediated immunosuppression microenvironment and hepatocellular carcinoma.

Dysregulation of the aldehyde dehydrogenase (ALDH) family has been implicated in various pathological conditions, including cancer. However, a systematic evaluation of ALDH alterations and their therapeutic relevance in hepatocellular carcinoma (HCC) remains lacking. Herein, we found that 15 of 19 ALDHs were transcriptionally dysregulated in HCC tissues compared to normal liver tissues. A four gene signature, including ALDH2, ALDH5A1, ALDH6A1, and ALDH8A1, robustly predicted prognosis and defined a high-risk subgroup exhibiting immunosuppressive features like regulatory T cell (Tregs) infiltration. Single-cell profiling revealed selective overexpression of tumor necrosis factor receptor superfamily member 18 (TNFRSF18) on Tregs, upregulated in high-risk HCC patients. We identified ALDH2 as a tumor suppressor in HCC, with three novel phosphorylation sites mediated by protein kinase C zeta that enhanced enzymatic activity. Mechanistically, ALDH2 suppressed Tregs differentiation by inhibiting ß-catenin/TGF-ß1 signaling in HCC. Collectively, our integrated multi-omics analysis defines an ALDH-Tregs-TNFRSF18 axis that contributes to HCC pathogenesis and represents potential therapeutic targets for this aggressive malignancy.

Six immune-related promising biomarkers may promote hepatocellular carcinoma prognosis: a bioinformatics analysis and experimental validation.

BACKGROUND: Abnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear. METHODS: Differentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis. RESULTS: We identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression. CONCLUSIONS: Briefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression.

Antinuclear antibody (ANA) status predicts immune-related adverse events in liver cancer patients undergoing anti-PD-1 therapy.

Immune-related adverse events (irAEs) clinically resemble autoimmune diseases, indicating autoantibodies could be potential biomarkers for the prediction of irAEs. This study aimed to assess the predictive value of peripheral blood antinuclear antibody (ANA) status for irAEs, considering the time and severity of irAEs, as well as treatment outcome in liver cancer patients administered anti-PD-1 therapy. Ninety-three patients with advanced primary liver cancer administered anti-PD-1 treatment were analyzed retrospectively. They were divided into the ANA positive (ANA+, titer ≥ 1:100) and negative (ANA-, titer < 1:100) groups. Development of irAEs, progression-free survival (PFS), and overall survival (OS) were assessed. Compared with ANA- patients, ANA+ cases were more prone to develop irAEs (43.3% vs. 19.2%, P = 0.031). With the increase of ANA titers, the frequency of irAEs increased. The time interval between anti-PD-1 therapy and the onset of irAEs was significantly shorter in ANA+ patients compared with the ANA- group (median, 1.7 months vs. 5.0 months, P = 0.022). Moreover, the time between anti-PD-1 therapy and irAE occurrence decreased with increasing ANA titer. In addition, PFS and OS were decreased in ANA+ patients compared with the ANA- group (median PFS, 2.8 months vs. 4.2 months, P = 0.043; median OS, 21.1 months vs. not reached, P = 0.041). IrAEs occur at higher frequency in ANA+ liver cancer patients undergoing anti-PD-1 therapy. ANA titer could help predict irAE development and treatment outcome in these patients.

circRanGAP1/miR-27b-3p/NRAS Axis may promote the progression of hepatocellular Carcinoma.

BACKGROUND: Though circular RNAs (circRNAs) are the key regulators in tumor carcinogenesis, they remain largely unexplored in hepatocellular carcinoma (HCC). METHODS: The expression of RanGAP1-derived circRNAs (circ_0063531, circ_0063534, circ_0063513, circ_0063518, circ_0063507, circ_0063723) were evaluated in eight paired HCC and normal tissues, and the correlation between circRanGAP1 (circ_0063531) expression and clinicopathological characteristics in 40 HCC patients was determined. The association between miR-27b-3p and circRanGAP1 or NRAS was predicted using bioinformatics analysis. The expression of circRanGAP1, miR-27b-3p, and NRAS were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The potential oncogenic role of circ-RanGAP1 was assessed using CCK-8, colony formation, transwell assays in vitro, subcutaneous tumor mouse model, vein tail metastatic model, and orthotopically implanted intrahepatic HCC model in vivo. Luciferase reporter and RNA immunoprecipitation (RIP) assays were used to explore the binding site between miR-27b-3p and circ-RanGAP1 or NRAS. Protein expression was detected using western blotting. The localization of miR-27b-3p and circ-RanGAP1 was investigated using fluorescence in situ hybridization (FISH). The level of immune infiltration was assessed by bioinformatics analysis, flow cytometry, and orthotopically implanted intrahepatic HCC models. RESULTS: Here, we found elevated circRanGAP1 in the cells and clinical tissues of patients with HCC. Increased circRanGAP1 levels are associated with enlarged tumors and the advanced stage of TNM. CircRanGAP1 promotes the growth, migration, and HCC cell invasion, concurrently with the growth and metastasis of tumors in-vivo. Moreover, circRanGAP1 is mainly located inside the cytoplasm. Mechanistically, circRanGAP1 as an oncogene promotes HCC progression by miR-27b-3p/NRAS/ERK axis, furthermore, affects the infiltration level of tumor-associated macrophages probably by sponging miR-27b-3p. Immune infiltration analysis shows that NRAS is positively correlated with the levels of CD68+ tumor-associated macrophages in HCC samples and that NRAS and CD68 are related to the poor outcome of HCC. CONCLUSION: These results reveal that circRanGAP1 is a HCC oncogene that function by the miR-27b-3p/NRAS/ERK axis and regulates the infiltration levels of tumor-associated macrophages by sponging miR-27b-3p. Therefore, circRANGAP1/ NRAS axis may be an important potential treatment target against HCC.

Nalidixic acid potentiates the antitumor activity in sorafenib-resistant hepatocellular carcinoma via the tumor immune microenvironment analysis.

Sorafenib resistance is often developed and impedes the benefits of clinical therapy in hepatocellular carcinoma (HCC) patients. However, the relationship between sorafenib resistance and tumor immune environment and adjuvant drugs for sorafenib-resistant HCC are not systemically identified. This study first analyzed the expression profiles of sorafenib-resistant HCC cells to explore immune cell infiltration levels and differentially expressed immune-related genes (DEIRGs). The prognostic value of DEIRGs was analyzed using Cox regression and Kaplan-Meier analysis based on The Cancer Genome Atlas. The primary immune cells infiltrated in sorafenib-resistant HCC mice were explored using flow cytometry (FCM). Finally, small-molecule drugs for sorafenib-resistant HCC treatment were screened and validated by experiments. The CIBERSORT algorithm and mice model showed that macrophages and neutrophils are highly infiltrated, while CD8+ T cells are downregulated in sorafenib-resistant HCC. Totally, 34 DEIRGs were obtained from sorafenib-resistant and control groups, which were highly enriched in immune-associated biological processes and pathways. NR6A1, CXCL5, C3, and TGFB1 were further identified as prognostic markers for HCC patients. Finally, nalidixic acid was identified as a promising antagonist for sorafenib-resistant HCC treatment. Collectively, our study reveals the tumor immune microenvironment changes and explores a promising adjuvant drug to overcome sorafenib resistance in HCC.

lncRNA-AC079061.1/VIPR1 axis may suppress the development of hepatocellular carcinoma: a bioinformatics analysis and experimental validation.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant tumors to threaten human life, and the survival rate remains low due to delayed diagnosis. Meanwhile, lncRNAs have great potential for application in tumor prognosis, therefore relevant research in hepatocellular carcinoma is indispensable. METHODS: Based on the EZH2 expression, the differentially expressed lncRNAs DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) were identified in hepatocellular carcinoma by using the TCGA database. Bioinformatics technology was utilized to determine the effect of key genes in HCC progression. The methylation and immune infiltration analyses were performed to explore the underlying function of hub genes. Finally, cellular function experiments were performed to investigate the association between identified genes and biological phenotypes in HCC. RESULTS: lncRNA-AC079061.1, hsa-miR-765, and VIPR1 were identified as independent factors that affect the prognosis of hepatocellular carcinoma. The immune infiltration analyses revealed that lncRNA-AC079061.1 can alter the immune microenvironment and thus inhibit the development of HCC by regulating the expression of an immune-related gene (VIPR1). Methylation analyses demonstrated that VIPR1 expression is negatively related to the methylation level in HCC. Experimental results suggested that lncRNA-AC079061.1 and VIPR1 were frequently downregulated in HCC cells, while hsa-miR-765 was significantly upregulated. Moreover, the lncRNA-AC079061.1/VIPR1 axis suppressed the proliferation and invasion of HCC cells. CONCLUSION: The present study identified the lncRNA-AC079061.1/VIPR1 axis as a novel biomarker that inhibited the proliferation and invasion of hepatocellular carcinoma, affecting the ultimate disease outcome.

An SCD1-dependent mechanoresponsive pathway promotes HCC invasion and metastasis through lipid metabolic reprogramming.

Matrix stiffness promotes hepatocellular carcinoma (HCC) metastasis. This study examined the contribution of lipid metabolic reprogramming to matrix stiffness-induced HCC metastasis. HCC cells were cultured on mechanically tunable polyacrylamide gels and subjected to lipidomic analysis. The key enzyme that responded to matrix stiffness and regulated lipid metabolism was identified. The comparative lipidomic screening revealed that stearoyl-CoA desaturase 1 (SCD1) is a mechanoresponsive enzyme that reprogrammed HCC cell lipid metabolism. The genetic and pharmacological inhibition of SCD1 expression/activity altered the cellular lipid composition, which in turn impaired plasma membrane fluidity and inhibited in vitro invasive motility of HCC cells in response to high matrix stiffness. Knockdown of SCD1 suppressed HCC invasion and metastasis in vivo. Conversely, the overexpression of SCD1 or exogenous administration of its product oleic acid augmented plasma membrane fluidity and rescued in vitro invasive migration in HCC cells cultured on soft substrates, mimicking the effects imposed by high matrix stiffness. In human HCC tissues, collagen content, a marker of increasing matrix stiffness, and increased expression of SCD1 together predicted poor survival of HCC patients. An SCD1-dependent mechanoresponsive pathway that responds to increasing matrix stiffness in the tumor microenvironment promotes HCC invasion and metastasis through lipid metabolic reprogramming.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

ITGB1-DT/ARNTL2 axis may be a novel biomarker in lung adenocarcinoma: a bioinformatics analysis and experimental validation.

BACKGROUND: Lung cancer is one of the most lethal malignant tumors that endangers human health. Lung adenocarcinoma (LUAD) has increased dramatically in recent decades, accounting for nearly 40% of all lung cancer cases. Increasing evidence points to the importance of the competitive endogenous RNA (ceRNA) intrinsic mechanism in various human cancers. However, behavioral characteristics of the ceRNA network in lung adenocarcinoma need further study. METHODS: Groups based on SLC2A1 expression were used in this study to identify associated ceRNA networks and potential prognostic markers in lung adenocarcinoma. The Cancer Genome Atlas (TCGA) database was used to obtain the patients' lncRNA, miRNA, and mRNA expression profiles, as well as clinical data. Informatics techniques were used to investigate the effect of hub genes on prognosis. The Cox regression analyses were performed to evaluate the prognostic effect of hub genes. The methylation, GSEA, and immune infiltration analyses were utilized to explore the potential mechanisms of the hub gene. The CCK-8, transwell, and colony formation assays were performed to detect the proliferation and invasion of lung cancer cells. RESULTS: We eventually identified the ITGB1-DT/ARNTL2 axis as an independent fact may promote lung adenocarcinoma progression. Furthermore, methylation analysis revealed that hypo-methylation may cause the dysregulated ITGB1-DT/ARNTL2 axis, and immune infiltration analysis revealed that the ITGB1-DT/ARNTL2 axis may affect the immune microenvironment and the progression of lung adenocarcinoma. The CCK-8, transwell, and colonu formation assays suggested that ITGB1-DT/ARNTL2 promotes the progression of lung adenocarcinoma. And hsa-miR-30b-3p reversed the ITGB1/ARNTL2-mediated oncogenic processes. CONCLUSION: Our study identified the ITGB1-DT/ARNTL2 axis as a novel prognostic biomarker affects the prognosis of lung adenocarcinoma.

The effect of NH4+ on phosphoenolpyruvate carboxykinase gene expression, metabolic flux and citrate content of citrus juice sacs.

Citrate is one of the most important metabolites determining the flavour of citrus fruit. It has been reported that nitrogen supply may have an impact on acid level of fruit. Here, the relationship between nitrogen metabolism and citrate catabolism was studied in pumelo juice sacs. Differences in metabolites, gene expression and flux distributions were analyzed in juice sacs incubated in medium with and without NH4+. Compared with those incubated with NH4+, juice sacs under nitrogen deficiency exhibited enhanced flux through phosphoenolpyruvate carboxykinase (PEPCK) and accelerated consumption of citrate, while the other two TCA cycle efflux points, through malic enzyme (ME) and glutamate dehydrogenase (GDH), were both repressed. Consistent with the estimated fluxes, the expression of PEPCK1 was upregulated under nitrogen deficiency, while that of GDH1, GDH2, NAD-ME1 and NADP-ME2 were all repressed. Thus, we propose that PEPCK1 contributes to citrate degradation under nitrogen limitation.

Suppressing DRP1-mediated mitochondrial fission and mitophagy increases mitochondrial apoptosis of hepatocellular carcinoma cells in the setting of hypoxia.

Transarterial embolization/transarterial chemoembolization (TAE/TACE) is the acceptable palliative treatment for hepatocellular carcinoma (HCC), mainly through ischemic necrosis induced by arterial embolization. However, how HCC cells survive under such ischemic hypoxic condition remains unclear, which can be exploited to potentiate TAE/TACE treatment. We hypothesized that targeting mitophagy can increase HCC cell apoptosis during hypoxia. HCC cells were subjected to hypoxia and then mitophagy was quantified. The role of dynamin-related protein 1 (DRP1) in hypoxia-induced HCC mitophagy was determined. Moreover, the synergistic effect of hypoxia and DRP1 inhibitor on HCC apoptosis was assessed in vitro and in vivo. Clinical association between DRP1 expression and outcome for HCC patients was validated. HCC cells that survived hypoxia showed significantly increased DRP1-mediated mitochondrial fission and mitophagy compared with cells in normoxia. Hypoxia induced mitophagy in surviving HCC cells by enhancing DRP1 expression and its translocation into the mitochondria and excessive mitochondrial fission into fragments. Blocking the DRP1 heightened the possibility of hypoxic cytotoxicity to HCC cells due to impaired mitophagy and increased the mitochondrial apoptosis, which involved decreased in mitochondrial membrane potential and mitochondrial release of apoptosis-inducing factor and cytochrome c. Additionally, DRP1 inhibitor Mdivi-1 suppressed the in vivo growth of hypoxia-exposed HCC cells. High expression of DRP1 was significantly associated with shorter survival in HCC patients. In conclusion, our results demonstrate that blocking DRP1-mediated mitochondrial fission and mitophagy increases the incidence of mitochondrial apoptosis of HCC cells during hypoxia, suggesting the new approach of targeting mitophagy to potentiate TAE/TACE.

Norepinephrine-stimulated HSCs secrete sFRP1 to promote HCC progression following chronic stress via augmentation of a Wnt16B/ß-catenin positive feedback loop.

BACKGROUND: Sustained adrenergic signaling secondary to chronic stress promotes cancer progression; however, the underlying mechanisms for this phenomenon remain unclear. Hepatocellular carcinoma (HCC) frequently develops within fibrotic livers rich in activated hepatic stellate cells (HSCs). Here, we examined whether the stress hormone norepinephrine (NE) could accelerate HCC progression by modulating HSCs activities. METHODS: HCC cells were exposed to conditioned medium (CM) from NE-stimulated HSCs. The changes in cell migration and invasion, epithelial-mesenchymal transition, parameters of cell proliferation, and levels of cancer stem cell markers were analyzed. Moreover, the in vivo tumor progression of HCC cells inoculated with HSCs was studied in nude mice subjected to chronic restraint stress. RESULTS: CM from NE-treated HSCs significantly promoted cell migration and invasion, epithelial-mesenchymal transition (EMT), and expression of cell proliferation-related genes and cancer stem cell markers in HCC cells. These pro-tumoral effects were markedly reduced by depleting secreted frizzled related protein 1 (sFRP1) in CM. The pro-tumoral functions of sFRP1 were dependent on ß-catenin activation, and sFRP1 augmented the binding of Wnt16B to its receptor FZD7, resulting in enhanced ß-catenin activity. Additionally, sFRP1 enhanced Wnt16B expression, reinforcing an autocrine feedback loop of Wnt16B/ß-catenin signaling. The expression of sFRP1 in HSCs promoted HCC progression in an in vivo model under chronic restraint stress, which was largely attenuated by sFRP1 knockdown. CONCLUSIONS: We identify a new mechanism by which chronic stress promotes HCC progression. In this model, NE activates HSCs to secrete sFRP1, which cooperates with a Wnt16B/ß-catenin positive feedback loop. Our findings have therapeutic implications for the treatment of chronic stress-promoted HCC progression.

Suppression of DRP1­mediated mitophagy increases the apoptosis of hepatocellular carcinoma cells in the setting of chemotherapy.

The efficacy of chemotherapy for hepatocellular carcinoma (HCC) remains unsatisfactory, primarily due to inherent self­defense mechanisms (e.g., mitophagy and autophagy). In the present study, we aimed to explore the pro­apoptotic effects of targeting mitophagy to potentiate the efficacy of chemotherapy for HCC. HCC cells were subjected to cisplatin, after which cisplatin­induced mitophagy was quantified by immunofluorescence. Mdivi­1, a specific dynamin­related protein 1 (DRP1) inhibitor, was used to study the role of DRP1 in cisplatin­induced HCC mitophagy. The synergistic effect of cisplatin and the DRP1 inhibitor on HCC was assessed in vitro and in vivo. Accordingly, cisplatin induced mitophagy in surviving HCC cells by activating DRP1. The DRP1 inhibitor (Mdivi­1) increased the apoptosis of cisplatin­treated HCC cells by targeting mitophagy. Mechanistically, Mdivi­1 upregulated Bax and downregulated Bcl­xL, leading to an increase in mitochondrial membrane permeability and subsequent release of cytochrome c from mitochondria into the cytosol, thereby aggravating cisplatin­induced apoptosis in HCC cells. Moreover, Mdivi­1 acted synergistically with cisplatin to suppress HCC xenograft growth in vivo. Our results indicate that targeting cisplatin­mediated mitophagy increases HCC apoptosis via DRP1 inhibition, providing preclinical proof of concept for combination therapy targeting mitophagy to potentiate the efficacy of chemotherapy.

Long non-coding RNA PSMA3-AS1 promotes malignant phenotypes of esophageal cancer by modulating the miR-101/EZH2 axis as a ceRNA.

BACKGROUNDS: Emerging evidences has demonstrated that dysregulation of long non-coding RNAs (lncRNAs) is critically involved in esophageal squamous cell carcinoma (ESCC) progression. However, the function of lncRNA PSMA3-AS1 in ESCC is unclear. Therefore, we aimed to explore the functions and potential mechanisms of PSMA3-AS1 in ESCC cells progression. RESULTS: Here, we found that PSMA3-AS1 expression was significantly up-regulated in ESCC tissues. Forced PSMA3-AS1 expression was correlated with tumor size, distant metastasis, and poor prognosis in ESCC patients. Functionally, PSMA3-AS1-overexpression promoted ESCC cells proliferation, invasion, and migration in vitro. Mechanistically, PSMA3-AS1 up-regulated EZH2 expression by competitively binding to miR-101. CONCLUSION: PSMA3-AS1 is significantly up-regulated in ESCC tissues, and the PSMA3-AS1/miR-101/EZH2 axis plays a critical role in ESCC progression. Taken together, our results may provide promising targets for ESCC therapy. METHODS: PSMA3-AS1 and miR-101 expression were explored using qRT-PCR in ESCC tissues and cell lines. Immunohistochemistry assays were carried out to analyze EZH2 (enhancer of zeste homolog) protein expression. RIP, dual-luciferase reporter, fluorescence in situ hybridization, and biotin pull-down assays were used to detect the interactions of PSMA3-AS1, miR-101 and EZH2. The biological functions of PSMA3-AS1 in PSMA3-AS1-altered cells were explored using CCK-8, colony formation, wound healing, and transwell assays in vitro.

Intermittent hypoxia alleviates increased VEGF and pro-angiogenic potential in liver cancer cells.

Vascular endothelial growth factor (VEGF) is an important angiogenic factor. The VEGF rebound induced by hypoxia following transarterial embolization/chemoembolization for primary liver cancer is associated with treatment failure and poor survival rates in patients. The present study investigated the ability of intermittent hypoxia to alleviate the acute hypoxia-induced increase of VEGF and decrease the pro-angiogenic potential of liver cancer cells. The liver cancer cells were exposed to normoxia, or acute or intermittent hypoxia, and the expression of VEGF was determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The pro-angiogenic effects of acute or intermittent hypoxia-exposed liver cancer cells on endothelial cells were assessed in vitro and in vivo. The expression of VEGF in the liver cancer cells exposed to intermittent hypoxia was significantly lower than that in cells exposed to acute hypoxia. Compared with conditioned medium (CM) from acute hypoxia-exposed liver cancer cells, the CM from intermittent hypoxia-exposed liver cancer cells showed markedly less promotion of proliferation and tube formation in endothelial cells. Activation of the reactive oxygen species (ROS)/NF-κB/hypoxia-inducible factor-1α/VEGF signaling pathway was increased in the liver cancer cells exposed to acute hypoxia. Exposure to ROS scavenger N-acetyl-cysteine or NF-κB inhibitor PDTC inhibited the activation of the above pathway and the expression of VEGF induced by acute hypoxia. The in vivo pro-angiogenic effects of intermittent hypoxia-exposed liver cancer cells on endothelial cells were significantly reduced compared with those of acute hypoxia-exposed liver cancer cells. Intermittent hypoxia may alleviate the acute hypoxia-induced increase of VEGF and decrease the pro-angiogenic potential of liver cancer cells, suggesting a novel treatment strategy.

Activated hepatic stellate cells promote progression of post-heat residual hepatocellular carcinoma from autophagic survival to proliferation.

BACKGROUND: Microscopic residual tumor often occurs after thermal ablation for medium-large hepatocellular carcinoma (HCC), leading to early aggressive recurrence or late relapse during follow-up. The mechanism how microscopic residual HCC cells survive sublethal heat stress and develop rapid outgrowth remains poorly understood. METHODS: HCC cells were exposed to sublethal heat treatment and co-cultured with conditioned media from activated HSCs (HSC-CM). Changes of cell proliferation, parameters of cell autophagy and activation of signaling pathways in heat-treated residual HCC cells were analyzed. An HCC orthotopic model was subjected to partial thermal ablation and antitumor effects of a combined treatment regimen were studied. RESULTS: HCC cells survived sublethal heat stress via activation of autophagy. HSC-CM enhanced autophagic survival within 24 h and then promoted proliferation of heat-treated residual HCC cells through HGF/c-Met signaling. Inhibition of autophagy or c-Met increased apoptosis of heat-treated residual HCC cells and reversed the protective effect of HSC-CM. HGF modulated biological status in autophagic survival or proliferation of heat-treated residual HCC through HGF/c-Met/ERK signaling and downstream components of ATG5/Beclin1 or cyclinD1. In an animal model, inhibiting autophagy in combination with c-Met inhibitor significantly thwarted tumor progression of residual HCC after incomplete thermal ablation via the suppressed autophagy, the decreased proliferation and the increased apoptosis. CONCLUSIONS: Activated HSCs promote progression of residual HCC cells after sublethal heat treatment from autophagic survival to proliferation via HGF/c-Met signaling. A combined treatment regimen of inhibiting autophagy and c-Met signaling could be used to suppress tumor progression of residual HCC after incomplete thermal ablation.

Periostin involved in the activated hepatic stellate cells-induced progression of residual hepatocellular carcinoma after sublethal heat treatment: its role and potential for therapeutic inhibition.

BACKGROUND: Incomplete thermal ablation may induce invasiveness of hepatocellular carcinoma (HCC). Here, we investigated whether activated hepatic stellate cells (HSCs) would accelerate the progression of residual HCC after sublethal heat treatment, and thus sought to identify the potential targets. METHODS: Hepatocellular carcinoma cells were exposed to sublethal heat treatment and then cultured with the conditioned medium from activated HSCs (HSC-CM). The cell proliferation, migration, invasion and parameters of epithelial-mesenchymal transition (EMT) were analyzed. In vivo tumor progression of heat-treated residual HCC cells inoculated with activated HSCs was studied in nude mice. RESULTS: HSC-CM significantly enhanced the proliferation, motility, invasion, prominent EMT activation and decreased apoptosis of heat-exposed residual HCC cells. These increased malignant phenotypes were markedly attenuated by neutralizing periostin (POSTN) in HSC-CM. Furthermore, exogenous POSTN administration exerted the similar effects of HSC-CM on heat-treated residual HCC cells. POSTN induced the prominent activation of p52Shc and ERK1/2 via integrin ß1 in heat-exposed residual HCC cells. Vitamin D analog calcipotriol blocked POSTN secretion from activated HSCs. Calcipotriol plus cisplatin significantly suppressed the activated HSCs-enhanced tumor progression of heat-treated residual HCC cells via the inhibited POSTN expression and the increased apoptosis. CONCLUSIONS: Activated HSCs promote the tumor progression of heat-treated residual HCC through the release of POSTN, which could be inhibited by calcipotriol. Calcipotriol plus cisplatin could be used to thwart the accelerated progression of residual HCC after suboptimal heat treatment.

Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment.

BACKGROUND: Accelerated malignant behaviors induced by insufficient thermal ablation have been increasingly reported, however, the exact mechanisms are still unclear. Here, we investigated the importance of the extracellular matrix (ECM) in modulating the progression of residual hepatocellular carcinoma (HCC) after heat treatment. METHODS: Heat-exposed residual HCC cells were cultured in different ECM gels. We used basement membrane gel (Matrigel) to simulate the normal microenvironment and collagen I to model the pathological stromal ECM. The alterations of morphology and parameters of proliferation, epithelial-mesenchymal transition (EMT) and stemness were analyzed in vitro and in vivo. RESULTS: Increased collagen I deposition was observed at the periablational zone after incomplete RFA of HCC in a xenograft model. The markers of cell proliferation, EMT, motility and progenitor-like traits of heat-exposed residual HCC cells were significantly induced by collagen I as compared to Matrigel (p values all < 0.05). Importantly, collagen I induced the activation of ERK phosphorylation in heat-exposed residual HCC cells. ERK1/2 inhibitor reversed the collagen I-promoted ERK phosphorylation, cell proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells in vitro. Moreover, collagen I promoted the in vivo tumor progression of heat-exposed residual HCC cells, and sorafenib markedly reversed the collagen I-mediated protumor effects. CONCLUSIONS: Our findings demonstrate that collagen I could enhance the aggressive progression of residual HCC cells after suboptimal heat treatment and sorafenib may be a treatment approach to thwart this process.

Light-Induced Activation of c-Met Signalling by Photocontrolled DNA Assembly.

Optical manipulation appears to be a powerful tool for spatiotemporally controlling a variety of cellular functions. Herein, a photocontrolled DNA assembly approach is described which enables light-induced activation of cellular signal transduction by triggering protein dimerization (c-Met signalling in this case). Three kinds of DNA probes are designed, including a pair of receptor recognition probes with adaptors and a blocker probe with a photocleavable linker (PC-linker). By implementing PC-linkers in blocker probes, the designed DNA probes response to light irradiation, which then induces the assembly of receptor recognition probes through adaptor complementing. Consequently, light-mediated DNA assembly promotes the dimerization of c-Met receptors, resulting in activation of c-Met signalling. It is demonstrated that the proposed photocontrolled DNA assembly approach is effective for regulating c-Met signalling and modulating cellular behaviours, such as cell proliferation and migration. Therefore, this simple approach may offer a promising strategy to manipulate cell signalling pathways precisely in living cells.

Kiwifruit-like Persistent Luminescent Nanoparticles with High-Performance and in Situ Activable Near-Infrared Persistent Luminescence for Long-Term in Vivo Bioimaging.

Persistent luminescence nanoparticles (PLNPs) have great potential for bioimaging because they can eliminate the tissue autofluorescence and improve the signal-to-noise ratio significantly. High-temperature calcination is a necessary process for the PLNPs to achieve high luminescence intensity and long afterglow time. However, high-temperature calcination usually results in uncontrollable morphology and poor homogeneity of PLNPs, which greatly limit their applications. Therefore, there is still a high demand to find a suitable method for synthesizing PLNPs with high luminescence intensity and long afterglow time while maintaining their monodispersed morphology. Herein, we report a facile silica template method to synthesize PLNPs with a kiwifruit-like structure that can tolerate high-temperature calcination. The as-prepared kiwifruit-like SiO2@ZnGa2O4:Cr3+@SiO2 PLNPs have enhanced near-infrared persistent luminescence, uniform morphology and size, and good biocompatibility. Moreover, the SiO2@ZnGa2O4:Cr3+@SiO2 PLNPs can be repeatedly activated by soft X-rays in situ and emit near-infrared persistent luminescence with long decay time, holding great potential for deep-tissue and long-term in vivo bioimaging. We believe that this study will open new perspectives for synthesizing high-performance PLNPs for optical imaging and diversified applications.