Asymmetric Nanopore Sensor for Logic Detection of Dam and M.SssI Methyltransferases in Combination of DNA Walker and Autocatalytic Hybridization Reaction.

The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure HD underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

Combination of transcriptomic and proteomic approaches helps unravel the mechanisms of luteolin in inducing liver cancer cell death via targeting AKT1 and SRC.

Introduction: Luteolin, a natural compound commonly used in traditional Chinese medicine, shows clinical potential as an anti-liver cancer agent. The mechanisms underlying the anti-liver cancer effect of luteolin are limited versus those reported for other cancers. Accordingly, this study was conducted to bridge the existing knowledge gap. Methods: Transcriptomic and proteomic analyses of the response of the hepatocellular carcinoma cell line HuH-7 to luteolin were conducted, and a possible pathway was elucidated using confocal laser scanning microscopy (CLSM), flow cytometry, western blotting, qRT-PCR and bio-layer interferometry assay to systematically explore the possible mechanisms underlying the inhibition of the proliferation of liver cancer cells by luteolin. Results and Discussion: Results showed that luteolin significantly inhibited HuH-7 cell proliferation. Transcriptomic and proteomic analyses collectively revealed that luteolin could promote cell cycle arrest and apoptosis in HuH-7 cells through transcription factors p53, nuclear factor kappa B (NF-κB), FOXO, ATF2, and TCF/LEF via AKT1, as well as the KEAP-NRF and SRC-STAT3 pathways. Furthermore, AKT1 and SRC were identified as the 2 targets of luteolin. Nuclear translocation of transcription factors p53 and NF-κB were affected by luteolin administration. Additionally, AKT1 activity affected normal metabolism in HuH-7 cells and resulted in the accumulation of reactive oxygen species, which activated MOMP and further promoted apoptosis. Our results systematically elucidate the mechanism of luteolin in inhibiting the proliferation of liver cancer cells, mainly through cell cycle arrest and apoptosis via targeting AKT1 and SRC.

Nrf2-dependent hepatoprotective effect of ellagic acid in titanium dioxide nanoparticles-induced liver injury.

BACKGROUND AND AIMS: Previous studies suggest that titanium dioxide nanoparticles (TiO2 NPs) induce liver injury, possibly due to oxidative stress and inflammation. Ellagic acid (EA) is a dietary polyphenol extracted from natural sources and possesses antioxidant and anti-inflammatory properties. Nonetheless, the efficacy of EA in mitigating liver injury induced by TiO2 NPs remains to be elucidated. METHODS: Primary hepatocytes and L02 cells were cultured with 45 µM EA and 10 µg/ml TiO2 NPs. Mice were orally administered TiO2 NPs (150 mg kg-1) and EA (25/50/100 mg kg-1) for eight weeks. sulforaphane (SFN) as a positive control to evaluate the inhibitory effect of EA on TiO2 NP-induced liver injury (SFN 10 mg kg-1). RNA sequencing (RNA-seq) was employed to elucidate the mechanisms underlying oxidative stress, inflammation, and liver fibrosis. RESULTS: We assessed the impact of EA on cytotoxicity, oxidative stress, inflammation, and fibrosis in both cells and mice exposed to TiO2 NPs for an extended period. Our findings indicated that EA had a protective effect on TiO2 NP-exposed hepatocytes, reducing cytotoxicity, oxidative stress, and inflammation. Furthermore, EA treatment markedly reduced serum aminotransferase levels in mice exposed to TiO2 NPs. Furthermore, EA treatment notably reduced hepatic stress response, inflammation, and fibrosis in mice. The treatment of EA demonstrates non-inferiority compared to SFN. The protective effects of EA were attributed to the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), EA promoted the translocation and phosphorylation of Nrf2, as indicated by the finding that Nfe2l2 shRNA and inhibition of Nrf2 by ML385 reversed the EA-induced hepatoprotective effects in TiO2 NP-exposed hepatocytes and mice. CONCLUSION: EA significantly mitigated liver injury induced by TiO2 NPs. Importantly, we identified that the nuclear translocation and phosphorylation of Nrf2 are the primary mechanisms through which EA alleviates liver injury resulting from exposure to TiO2 NPs. As a natural activator of Nrf2, EA emerges as a promising therapeutic candidate for treating TiO2 NPs-induced liver injury, further enhancing our understanding of its potential as a hepatoprotective agent and its underlying molecular mechanisms.

Microwave-activated Cu-doped zirconium metal-organic framework for a highly effective combination of microwave dynamic and thermal therapy.

Percutaneous microwave ablation (PMA) is a thermoablative method used as a minimally invasive treatment for liver cancer. However, the application of PMA is limited by its insufficient ROS generation efficiency and thermal effects. Herein, a new microwave-activated Cu-doped zirconium metal-organic framework (MOF) (CuZr MOF) used for enhanced PMA has a significantly improved microwave sensitizing effect. Owing to the strong inelastic collisions between ions confined in numerous micropores, CuZr MOF has strong microwave sensitivity and high thermal conversion efficiency, which can significantly improve microwave thermal therapy (MTT). Moreover, because of the existence of Cu2+ ions, a further benefit of CuZr MOF is their Fenton-like activity, in particular, microwaves used as an excitation source for microwave dynamic therapy (MDT) can improve the Fenton-like reaction to maximize the synergistic effectiveness of cancer therapy. Importantly, CuZr MOF can inhibit the production of heat shock proteins (HSPs) by producing abundant ROS to enhance tumor destruction. Mechanistically, we found that CuZr MOF + MW treatment modulates ferroptosis-mediated tumor cell death by targeting the HMOX1/GPX4 axis. In summary, this study develops a novel CuZr MOF microwave sensitizer with great potential for synergistic treatment of liver cancer by MTT and MDT.

The multikinase inhibitor axitinib in the treatment of advanced hepatocellular carcinoma: the current clinical applications and the molecular mechanisms.

Advanced hepatocellular carcinoma (HCC) is a formidable public health problem with limited curable treatment options. Axitinib, an oral tyrosine kinase inhibitor, is a potent and selective second-generation inhibitor of vascular endothelial growth factor receptor (VEGFR) 1, 2, and 3. This anti-angiogenic drug was found to have promising activity in various solid tumors, including advanced HCC. At present, however, there is no relevant review article that summarizes the exact roles of axitinib in advanced HCC. In this review, 24 eligible studies (seven studies in the ClinicalTrials, eight experimental studies, and nine clinical trials) were included for further evaluation. The included randomized or single-arm phase II trials indicated that axitinib could not prolong the overall survival compared to the placebo for the treatment of advanced HCC, but improvements in progression free survival and time to tumor progression were observed. Experimental studies showed that the biochemical effects of axitinib in HCC might be regulated by its associated genes and affected signaling cascades (e.g. VEGFR2/PAK1, CYP1A2, CaMKII/ERK, Akt/mTor, and miR-509-3p/PDGFRA). FDA approved sorafenib combined with nivolumab (an inhibitor of PD-1/PD-L1) as the first line regimen for the treatment of advanced HCC. Since both axitinib and sorafenib are tyrosine kinase inhibitors as well as the VEGFR inhibitors, axitinib combined with anti-PDL-1/PD-1 antibodies may also exhibit tremendous potential in anti-tumoral effects for advanced HCC. The present review highlights the current clinical applications and the molecular mechanisms of axitinib in advanced HCC. To move toward clinical applications by combining axitinib and other treatments in advanced HCC, more studies are still warranted in the near future.

PIK3R3 is upregulated in liver cancer and activates Akt signaling to control cancer growth by regulation of CDKN1C and SMC1A.

BACKGROUND: Liver cancer is a highly malignant disease and the third leading cause of cancer death worldwide. Abnormal activation of PI3K/Akt signaling is common in cancer, but whether phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) plays a role in liver cancer is largely unexplored. METHODS: We determined the expression of PIK3R3 in liver cancer by using TCGA data and our clinical samples and knocked it down by siRNA or overexpressing it by the lentivirus vector system. We also investigated the function of PIK3R3 by colony formation, 5-Ethynyl-2-Deoxyuridine, flow cytometry assay, and subcutaneous xenograft model. The downstream of PIK3R3 was explored by RNA sequence and rescue assays. RESULTS: We found that PIK3R3 was significantly upregulated in liver cancer and correlated with prognosis. PIK3R3 promoted liver cancer growth in vitro and in vivo by controlling cell proliferation and cell cycle. RNA sequence revealed that hundreds of genes were dysregulated upon PIK3R3 knockdown in liver cancer cells. CDKN1C, a cyclin-dependent kinase inhibitor, was significantly upregulated by PIK3R3 knockdown, and CDKN1C siRNA rescued the impaired tumor cell growth. SMC1A was partially responsible for PIK3R3 regulated function, and SMC1A overexpression rescued the impaired tumor cell growth in liver cancer cells. Immunoprecipitation demonstrated there is indirect interaction between PIK3R3 and CNKN1C or SMC1A. Importantly, we verified that PIK3R3-activated Akt signaling determined the expression of CDKN1C and SMC1A, two downstream of PIK3R3 in liver cancer cells. CONCLUSION: PIK3R3 is upregulated in liver cancer and activates Akt signaling to control cancer growth by regulation of CDNK1C and SMC1A. Targeting PIK3R3 could be a promising treatment strategy for liver cancer that deserves further investigation.

Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification.

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

The mechanism of flavonoids from Cyclocarya paliurus on inhibiting liver cancer based on in vitro experiments and network pharmacology.

Introduction: Cyclocarya paliurus (Batal.) Iljinsk., a subtropical tree belonging to the family Juglandaceae, is rich in polysaccharides, flavonoids, and terpenoids. It has important pharmacological effects such as lowering blood lipids, blood sugar, and blood pressure. However, little has been discerned regarding anti tumor effects and their potential mechanisms. Method: In vitro cell culture experiments were used to test the effect of C. paliurus total flavonoids (CTFs) extract on apoptosis mechanisms in HepG2 cells. Network pharmacology was applied to further explore the effects of CTFs on liver cancer as well as the mechanisms through which these effects might be achieved. Both 3 hydroxyflavone and luteolin were randomly selected to verify the effect on inducing apoptosis and inhibiting the proliferation of HepG2 cells. Results and Discussion: Network pharmacological analysis was applied to these 62 compounds and their targets, and 13 flavonoids were further screened for their potential anti liver cancer activity. These 13 flavonoids included: tangeretin, baicalein, 7,3'-dihydroxyflavone, velutin, 3-hydroxyflavone, chrysin, kumatakenin, tricin, luteolin, chrysoeriol, apigenin, pinocembrin, and butin. Together, these flavonoids were predicted to interact with AKT1, MAPK3, PIK3CA, EGFR, MAP2K1, SRC, IGF1R, IKBKB, MET, and MAPK14. It was predicted that the inhibitory effect on hepatocellular carcinoma would be accomplished by regulation of core proteins relating to such KEGG pathways as cancer, PI3K-Akt, proteoglycans in cancer, microRNAs in cancer, and endocrine resistance via core target proteins. Both 3-hydroxyflavone and luteolin were demonstrated to induce apoptosis and inhibit the proliferation of HepG2 cells. Our study provides scientific evidence supporting the use of CTFs for the treatment of liver cancer.

Roles of hypoxia-inducible factor in hepatocellular carcinoma under local ablation therapies.

Hepatocellular carcinoma (HCC) is one of the most common digestive malignancies. HCC It ranges as the fifth most common cause of cancer mortality worldwide. While The prognosis of metastatic or advanced HCC is still quite poor. Recently, locoregional treatment, especially local ablation therapies, plays an important role in the treatment of HCC. Radiofrequency ablation (RFA) and high-intensity focused ultrasound (HIFU) ablation are the most common-used methods effective and feasible for treating HCC. However, the molecular mechanisms underlying the actions of ablation in the treatments for HCC and the HCC recurrence after ablation still are poorly understood. Hypoxia-inducible factor (HIF), the key gene switch for adaptive responses to hypoxia, has been found to play an essential role in the rapid aggressive recurrence of HCC after ablation treatment. In this review, we summarized the current evidence of the roles of HIF in the treatment of HCC with ablation. Fifteen relevant studies were included and further analyzed. Among them, three clinical studies suggested that HIF-1α might serve as a crucial role in the RAF treatment of HCC or the local recurrence of HCC after RFA. The remainder included experimental studies demonstrated that HIF-1, 2α might target the different molecules (e.g., BNIP3, CA-IX, and arginase-1) and signaling cascades (e.g., VEGFA/EphA2 pathway), constituting a complex network that promoted HCC invasion and metastasis after ablation. Currently, the inhibitors of HIF have been developed, providing important proof of targeting HIF for the prevention of HCC recurrence after IRFA and HIFU ablation. Further confirmation by prospective clinical and in-depth experimental studies is still warranted to illustrate the effects of HIF in HCC recurrence followed ablation treatment in the future.

The roles of RNA N6-methyladenosine in esophageal cancer.

Esophageal cancer is a malignant tumour with a high degree of malignancy and high mortality. Its pathogenesis and treatment strategy remain unclear. N6-methyladenosine (m6A) is important for various biological functions in RNA modification and is currently being investigated extensively. It plays an essential role in RNA modification. m6A modification is a dynamic process that reversibly regulates the target RNA through its regulatory factors and plays an important role in several diseases, especially cancer. However, the role of m6A in esophageal cancer remains elusive. RNA modification and splicing are regulated by RNA methylation regulators called 'writers' (methyltransferases), 'erasers' (demethylases) and 'readers' (modified RNA-binding proteins). These regulatory factors recognise and bind to RNA methylation sites, regulate biological functions such as RNA splicing and translation and influence the occurrence, development, invasion and metastasis of tumours. Considering the importance of m6A modification, we reviewed the regulatory mechanisms, biological functions and therapeutic prospects of m6A RNA methylation regulators in esophageal cancer.