Normal Liver Cell Line Research Articles

Design, Synthesis, and Biological Evaluation of a Class of TEMPOmodified Naphthalimide Derivatives with Good Selectivity for Tumor Cells

Background: Naphthalimides have been receiving continuous attention for their outstanding performance in treating cancer. However, as a class of cytotoxic anti-tumor agents, their toxic side effects greatly limit their clinical application. Therefore, developing naphthalimide reagents with good selectivity for tumor cells is of great importance. Methods: A class of TEMPO-modified naphthalimide derivatives substituted with oxygen at the 6- position were designed, synthesized, and evaluated for the anti-tumor activity in vitro against three cancer cell lines (A549, MCF-7, and HeLa) as well as one normal human liver cell line (QSG-7701) by MTT assay. HRMS (or MS) and EPR spectra were used to identify the structures of target compounds. The UV–vis titration absorption spectra were performed to study the interaction between compounds and DNA. Results: The target compounds NT3, NT9, NT10, NT11, and NT12 exhibited moderate inhibition to all the three tested tumor cell lines, while NT1, NT2, and NT4 showed selectivity against A549, All the target compounds did not show measurable activities in QSG-7701, which imply the target compounds are likely to overcome the nonspecific toxicity against normal cells. UV-vis spectra showed this class of compounds could effectively intercalate into DNA and selectively bind to G-C base pairs. Conclusion: A novel class of TEMPO-modified naphthalimide derivatives substituted with oxygen at 6- position and the preliminary discussion on their SARs provide promising opportunities to guide further research on naphthalimide derivatives as anti-tumor agents.

Harnessing the potential of de-sulfated heparin for targeted drug delivery: A three-component approach exemplified by conjugation with galactose and paclitaxel

Heparin, an anticoagulant with a century-long history of use, has been investigated over the past decade as a potential drug delivery vehicle. Despite its safety and efficacy, its interactions with many proteins through specific sulfate patterns can complicate drug delivery by mediating diverse biological functions. Here, we present the synthesis of a three-component drug delivery system comprising de-sulfated heparin as the carrier, galactose as the targeting moiety, and paclitaxel as the therapeutic drug. Removal of sulfates eliminated most of its anticoagulant effects in all intermediates. Through coupling with galactose and paclitaxel, the system improved the solubility of the drug and achieved selective targeting and efficient drug delivery to HepG2 cells, a liver carcinoma cell line with high galactose receptor expression. While the three-component system exhibited a slightly higher IC50 value than native paclitaxel, demonstrating its efficacy as a drug carrier, the IC50 value for the normal human liver cell line QSG7701 was significantly higher, indicating its selectivity and safety. Our study introduces a novel approach utilizing desulfated heparin as a carrier, warranting further investigation to unlock its potential in targeted drug delivery strategies.

In vitro α-amylase/α-glucosidase, cytotoxicity and radical scavenging potential of Hypoxishemerocallidea synthesized magnesium oxide nanoparticles

The prevalence of diabetes has continued to increase over the past decade. Medicinal extract-synthesized nanoformulations incorporating trace elements found in the body have emerged as alternative therapeutic technology for the treatment of diabetes. In this study, magnesium oxide (MgO) nanoparticles were prepared using Hypoxishemerocallidea (HH) and investigated for their cytotoxicity, antioxidant, and antidiabetic potentials. MgO NPs were characterized by transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and zeta-potential techniques. TEM analysis confirmed the 2D nanosheet-like morphology of the nanoparticles with particle size (diameter; 300 nm and length of about 1 µm) while the FTIR spectra showed functional groups correlating to the bioactive compounds of HH in the MgO NPs. Moderate antioxidant activity of MgO NPs was observed against DPPH (IC50 = 57.35 ± 0.28 µg/mL) and ABTS (IC50 = 52.08 ± 0.24 µg/mL). The nanoparticles were shown to be less toxic to normal embryonic (HEK293) and human liver (HEPG2) cell lines, with IC50 of 48.63 ± 0.73 and 32.39 ± 0.95 µg/mL, respectively compared to a known cytotoxic drug, doxorubicin which exhibited IC50 of 2.70 ± 0.32 and 8.62 ± 0.57 µg/mL respectively. Besides, the anti-hyperglycemic potential of the MgO NPs as demonstrated by α-amylase and α-glucosidase activities were significantly high with IC50 values of 33.03 ± 1.43 and 52.38 ± 3.06 µg/mL, respectively. These results were compared with an FDA-approved antidiabetic reference standard Acarbose, which exhibited IC50 values of 24.54 ± 1.55 and 6.54 ± 0.27 µg/mL. The results indicated that the HH bioinspired MgO NPs are capable of inhibiting meditators of diabetes and oxidative stress. This study further suggests that the MgO NPs synthesized using HH could be a good candidate for the management of diabetes and other inflammatory diseases due to their strong enzyme inhibition, efficient antioxidant properties, and biocompatibility.

Bioassay-guided isolation of anti-leukemic steroids from Aglaia abbreviata by inducing apoptosis

The strategy of bioactivity-guided isolation is widely used to obtain active compounds as quickly as possible. Thus, the inhibitory effects on human erythroleukemia cells (HEL) were applied to guide the isolation of the anti-leukemic compounds from Aglaia abbreviata. As a result, 19 compounds (16 steroids, two phenol derivatives, and a rare C12 chain nor-sesquiterpenoid), including 13 new compounds, were isolated and identified based on spectroscopic data analysis, single-crystal X-ray diffraction data, and electronic circular dichroism (ECD) calculations. Among them, 9 steroids exhibited good selective anti-leukemic activity against HEL and K562 (human chronic myeloid leukemia cells) cells with IC50 values between 2.29 ± 0.18 μM and 19.58 ± 0.13 μM. Notably, all the active compounds had relatively lower toxicity on the normal human liver cell line (HL-7702). Furthermore, five compounds (1, 4, 8, 10, and 19) displayed good anti-inflammatory effects, with IC50 values between 7.15 ± 0.16 and 27.1 ± 0.37 μM. An α,β-unsaturated ketone or a 5,6Δ double bond was crucial for improving anti-leukemic effect from the structure–activity relationship analysis. The compound with the most potential, 14 was selected for the preliminary mechanistic study. Compound 14 can induce apoptosis and cause cell cycle arrest. The expression of the marker proteins, such as PARP and caspase 3, were notably effected by this compound, thus inducing apoptosis. In conclusion, our investigation implied that compound 14 may serve as a potential anti-leukemia agent.

Structure characterization and cryoprotective activity of an exopolysaccharide from Pseudoalteromonas sp. LP6-12-2

Non-toxic natural antifreeze agents are urgently needed in many fields. Exopolysaccharides produced by psychrophilic bacteria from harsh habitats, such as Antarctica, to battle cold environments are a significant source of biological antifreeze agents. In this study, a homogeneous exopolysaccharide (EPS0-1) was extracted from Antarctica Pseudoalteromonas sp. LP6-12-2. Using High-performance gel permeation chromatography and High-efficiency anion chromatogram, respectively, the homogeneity, molecular weight, and monosaccharide composition of EPS0-1 were examined. EPS0-1 with a molecular weight (Mw) of 4351.31 kDa was an α-configuration polysaccharide containing uronic acid, amino and sulfuric acid groups, and composed of galactosamine hydrochloride, rhamnose, glucosamine hydrochloride, galactose, glucose, mannose, galacturonic acid, and glucuronic acid with rates of 1.40:9.40:4.90:32.0:37.2:6.50:4.10:4.40, respectively. Congo red test and I2-KI reaction found that the triple helix structure of EPS0-1 polysaccharide chain was weakly bound internally, with a longer side chain and/or more branches. The smooth and dense surface of EPS0-1 under Atomic force microscopy and Scanning electron microscopy revealed that it was large polysaccharide particles. Freeze-thaw cycle and freezing point determination experiment showed that EPS0-1 may significantly increase E. Coli's freezing resistance and decrease the solution's freezing point, respectively. EPS0-1 was capable of increasing the viscosity of the solution and displaying a high degree of thermal stability, which was proved by the rheological properties. And it was not hazardous to human normal liver cell line (LO2) in a specific concentration range. Therefore, the findings of this investigation established the basis for EPS0-1 as a potential antifreeze and food functional component.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Crosstalk of cuproptosis-related prognostic signature and competing endogenous RNAs regulation in hepatocellular carcinoma.

Cuproptosis is a new type of programmed cell death involved in the regulation of neuroendocrine tumors, immune microenvironment, and substance metabolism. However, the role of cuproptosis-related genes (CRGs) in Hepatocellular carcinoma (HCC) remains unclear. Through multiple bioinformatics analysis, we constructed a prognostic gene model and competing endogenous RNA (ceRNA) network. The correlation between CRGs and prognosis, immune infiltration, immune checkpoints, microsatellite instability (MSI) and tumor mutational burden (TMB) was analyzed by Kaplan-Meier curve, univariate Cox, multivariate regression, and Spearman's analysis in HCC patients. Besides, the qRT-PCR and immunohistochemistry assays were used to determine prognostic CRGs mRNA and protein expression in HCC. We established a novel 3-gene signature related to CRGs for evaluating the prognosis of HCC patients. HCC patients with high risk scores had a poor prognosis with an area under the curve of 0.737, 0.646, and 0.634 on 1-year, 3-year, and 5-year receiver operating characteristic curves. Significant correlation was observed between prognostic CRGs and immune infiltration, immune checkpoints, MSI and TMB. We also developed five ceRNA networks to regulate the occurrence and progression of HCC. CDKN2A, DLAT, and PDHA1 protein expression was up-regulated in HCC versus normal tissues. Besides, the mRNA expression levels of CDKN2A, DLAT, GLS, and PDHA1 were elevated in the HCC cell lines compared to the normal liver cell lines. This novel prognostic CRGs signature could be accurately predict the prognosis of patients with HCC. The ceRNA regulatory network might be potential prognostic biomarkers and therapeutic targets for HCC patients.

Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma

PurposeThis study was based on hepatocellular carcinoma (HCC) patients of early-stage to explore the diagnostic capability and possible production causes of anti-GNAS autoantibody. MethodsWe evaluated the frequency of anti-GNAS autoantibody in sera from patients with early-stage HCC by enzyme-linked immunosorbent assay (ELISA) and the expression of GNAS protein in early-stage HCC tissues by immunohistochemistry. Western blotting (WB) and real-time polymerase chain reaction (RT-PCR) were utilized to examine the expressions of GNAS protein and mRNA in cell lines. GEO and International Cancer Genome Consortium (ICGC) databases were inquired to explore mRNA expression and mutation of GNAS in HCC tissues. ResultsThe positive rates of anti-GNAS autoantibody in HCC patients at clinical stage I (78.1 %) and clinical stage II (57.1 %) were all significantly higher than that in healthy control (20 %). There was also a significant difference in GNAS protein expression between HCC and its adjacent normal liver tissues. The results from WB and RT-PCR showed a significant difference at the mRNA level but no statistical difference at the protein level between HCC and normal liver cell lines. The difference in mRNA level between HCC and adjacent normal liver tissues was verified to be significant. Furthermore, the ICGC database demonstrated a 10.6 % mutation frequency for GNAS in HCC patients. ConclusionThe coordination of elevated anti-GNAS autoantibody, high expression of GNAS in the mRNA and protein levels in HCC, and high frequency of GNAS mutation indicates that anti-GNAS autoantibody may be used as an early indicator of HCC.

(E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol alleviates inflammatory responses in LPS-induced mice liver sepsis through inhibition of STAT3 phosphorylation

Sepsis is a life-threatening disease with limited treatment options, and the inflammatory process represents an important factor affecting its progression. Many studies have demonstrated the critical roles of signal transducer and activator of transcription 3 (STAT3) in sepsis pathophysiology and pro-inflammatory responses. Inhibition of STAT3 activity may therefore represent a promising treatment option for sepsis. We here used a mouse model to demonstrate that (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) treatment prevented the liver sepsis-related mortality induced by 30 mg/kg lipopolysaccharide (LPS) treatment and reduced LPS-induced increase in alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels, all of which are markers of liver sepsis progression. These recovery effects were associated with decreased LPS-induced STAT3, p65, and JAK1 phosphorylation and proinflammatory cytokine (interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha) level; expression of cyclooxygenase-2 and induced nitric oxide synthase were also reduced by MMPP. In an in vitro study using the normal liver cell line THLE-2, MMPP treatment prevented the LPS-induced increase of STAT3, p65, and JAK1 phosphorylation and inflammatory protein expression in a dose-dependent manner, and this effect was enhanced by combination treatment with MMPP and STAT3 inhibitor. The results clearly indicate that MMPP treatment prevents LPS-induced mortality by inhibiting the inflammatory response via STAT3 activity inhibition. Thus, MMPP represents a novel agent for alleviating LPS-induced liver sepsis.

Sonosensitizer Nanoplatforms Augmented Sonodynamic Therapy-Sensitizing Shikonin-Induced Necroptosis Against Hepatocellular Carcinoma.

Apoptosis resistance of hepatocellular carcinoma (HCC) often leads to treatment failure. Nonetheless, overcoming the resistance of HCC to apoptosis by inducing necroptosis of tumor cells to bypass the apoptotic pathway may be a promising treatment strategy. Sonodynamic therapy (SDT) has broad prospects in disease treatment because of its noninvasive characteristic and spatiotemporal control. The combination of SDT and shikonin in the treatment of HCC is expected to be a new tumor treatment method that can overcome apoptosis resistance. In this study, the antitumor effect was evaluated using normal liver cell line WRL68, HCC cell line HepG2 and HepG2 xenograft mouse models. Indocyanine green (ICG) was loaded on nanobubbles (NBs) to construct ICG-loaded nanobubbles (ICG-NBs). Combined sonosensitizer nanoplatforms with ultrasound (US) to achieve efficient SDT, the combination of SDT and shikonin in treating HCC can activate shikonin-induced necroptosis. As a result, tumor cells that produced apoptosis resistance were destroyed by necroptosis. The results indicated a successful preparation of ICG-NBs with a uniform particle size of 273.0 ± 118.9 nm spherical structures. ICG-NB-mediated SDT, in combination with shikonin treatment, inhibited the viability, invasion, and migration of tumor cells. SDT + shikonin treatment group caused a substantial increase in necroptotic cells. The increased degree of tumor necrosis and the upregulated expression of receptor-interacting protein 3 kinase were observed in vivo studies, which indicated that the antitumor effect was accompanied by enhanced necroptosis in the SDT + shikonin treatment group. ICG-NB-mediated SDT combined with shikonin inhibits the growth of HCC by increasing the necroptosis of tumor cells. Therefore, this combination therapy is a promising treatment strategy against the specific cancer.

Synthesis, characterization, antiproliferative activity and DNA binding calculation of substituted-phenyl-terpyridine copper(II) nitrate complexes

Ten 4′- (R-phenyl) -2,2′: 6′, 2′ – terpyridine ligands (R = hydrogen (L1), hydroxyl (L2), methoxyl (L3), methylsulfonyl (L4), methyl (L5), phenyl (L6), fluoro (L7), chloro (L8), bromo (L9), and iodo (L10)) were synthesized. The reaction of these ligands with copper(II) nitrate led to complexes 1–10. The characterization of 1–10 was carried out by means of mass spectrometry, elemental analysis, infrared spectroscopy and X-ray single crystal diffraction. Four cell lines including esophageal cancer cell line (Eca-109), human liver cancer cell line (Bel-7402), human breast cancer cell line (SIHa) and human normal liver cell line (HL-7702) were selected to carry out antiproliferation and cytotoxicity experiments in vitro. The results showed that the complexes have strong inhibitory ability on the growth of tumor cells. In order to study the anticancer mechanism of the complexes, the binding mode and binding ability of the complexes with DNA were further determined and discussed with UV–Vis spectroscopy and circular dichroism. The effects of the lowest binding energy and hydrogen bond on the binding were studied using molecular docking calculation.

Phenolics and Terpenoids Profiling in Diverse Loquat Fruit Varieties and Systematic Assessment of Their Mitigation of Alcohol-Induced Oxidative Stress.

To compare and investigate the phenolic compounds in the peel and flesh of loquat (Eriobotrya japonica) and evaluate their ability to protect against alcohol-induced liver oxidative stress, we employed a combination of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry (HRMS) to qualitatively and quantitatively analyze 22 phenolics and 2 terpenoid compounds in loquat peel and flesh extracts (extraction with 95% ethanol). Among these, six compounds were identified for the first time in loquat, revealing distinct distribution patterns based on variety and tissue. Various chemical models, such as DPPH, FRAP, ORAC, and ABTS, were used to assess free radical scavenging and metal ion reduction capabilities. The results indicate that peel extracts exhibited higher antioxidant capacity compared with flesh extracts. Using a normal mouse liver cell line, AML-12, we explored the protective effects of loquat extracts and individual compounds against ethanol-induced oxidative stress. The findings demonstrate the enhanced cell viability and the induction of antioxidant enzyme activity through the modulation of Nrf2 and Keap1 gene expression. In a C57/BL6 mouse model of alcohol-induced liver damage, loquat extract was found to alleviate liver injury induced by alcohol. The restoration of perturbed serum liver health indicators underscored the efficacy of loquat extract in reclaiming equilibrium. The culmination of these findings significantly bolsters the foundational knowledge necessary to explore the utilization of loquat fruit extract in the creation of health-focused products.

Upregulation of FAM134B inhibits endoplasmic reticulum stress-related degradation protein expression and promotes hepatocellular carcinogenesis.

Endoplasmic reticulum (ER) stress can stimulate the proliferation and metastasis of hepatocellular carcinoma (HCC) cells while hindering apoptosis and immune system function, but the molecular mechanism of ER stress in HCC has yet to be fully studied. We aim to investigate the molecular mechanism by which FAM134B inhibits autophagy of HCC cells by reducing the expression of ER stress-related degradation proteins. Clinical samples were collected for this study. Normal liver cell lines HL7702 and Hep3B and Huh7 HCC cell lines were cultured. Construction of FAM134B knockdown cell line. Cell proliferation was measured using the CCK-8 assay, while cell migration and invasion capabilities were detected using the plate colony formation assay. Flow cytometry was used to detect the apoptosis rate. Transmission electron microscopy was used to observe the formation of autophagosomes. qRT-PCR and WB detective expression changes related to autophagy proteins. Finally, the expression of the relevant proteins was observed by immunohistochemistry. The expression of FAM134B was significantly increased in human liver cancer tissue and HCC cell lines Hep3B and Huh7. After the lentiviral vector was transfected into Hep3B cells with sh-FAM134B, results showed that sh-FAM134B could effectively inhibit Hep3B cell proliferation and promote HCC cell apoptosis. Meanwhile, sh-FAM134B could effectively induce the autophagy of Hep3B liver cancer cells. Immunohistochemistry results showed that sh-FAM134B could effectively induce ER stress. FAM134B inhibits HCC cell autophagy and promotes the progression of liver cancer by inhibiting the expression of ER stress-related degradation factors such as DERL2, EDEM1, SEL1L and HRD1.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

Prognostic value and immunological role of CSNK1D in human cancers.

CSNK1D, also known as CK1δ, is a crucial gene involved in various biological processes such as cell cycle, transcriptional regulation, apoptosis, cell polarity, and cell motility. It is associated with different cancers and neurodegenerative diseases. This study aimed to investigate the role of CSNK1D in multiple human cancers, particularly hepatocellular carcinoma (HCC), through in vitro experiments. The research utilized various online resources and databases like UCSC, NCBI, GEPIA2, HPA, cBioPortal, SangerBox, UALCAN, and TCGA for analyzing CSNK1D expression, prognosis significance, immune features, and gene alterations in cancers. RT-PCR was employed to evaluate CSNK1D expression in normal liver and liver cancer cell lines. In vitro experiments, including CCK-8, Edu, wound healing, and Transwell assays, were conducted to assess CSNK1D's biological function in HCC cells. Results demonstrated consistent upregulation of CSNK1D in various tumors. Heightened CSNK1D expression correlated with reduced overall survival and disease-free survival rates in different cancer patient cohorts. Significant associations were found between CSNK1D expression levels and immune cell infiltrations, immune checkpoint inhibitors, tumor mutation burden, and microsatellite instability across multiple malignancies. Notably, statistical analyses using TCGA and ICGC data identified CSNK1D as a robust and independent prognostic biomarker in HCC. Inhibiting CSNK1D expression effectively hindered cell proliferation, migration, and invasion in cellular experiments. In conclusion, this study suggests that CSNK1D may serve as a biomarker for tumor prognosis and immunotherapy. It influences the proliferation and metastasis of HCC cells.

Chemo-/Regio-Selective Synthesis of Novel Functionalized Spiro[pyrrolidine-2,3'-oxindoles] under Microwave Irradiation and Their Anticancer Activity.

A novel series of nitrostyrene-based spirooxindoles were synthesized via the reaction of substituted isatins 1a-b, a number of α-amino acids 2a-e and (E)-2-aryl-1-nitroethenes 3a-e in a chemo/regio-selective manner using [3+2] cycloaddition (Huisgen) reaction under microwave irradiation conditions. The structure elucidation of all the synthesized spirooxindoles were done using 1H and 13C NMR and HRMS spectral analysis. The single crystal X-ray crystallographic study of compound 4l was used to assign the stereochemical arrangements of the groups around the pyrrolidine ring in spiro[pyrrolidine-2,3'-oxindoles] skeleton. The in vitro anticancer activity of spiro[pyrrolidine-2,3'-oxindoles] analogs 4a-w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines shows promising results. Out of the 23 synthesized spiro[pyrrolidine-2,3'-oxindoles], while five compounds (4c, 4f, 4m, 4q, 4t) (IC50 = 34.99-47.92 µM; SI = 0.96-2.43) displayed significant in vitro anticancer activity against human lung (A549) cancer cell lines, six compounds (4c, 4f, 4k, 4m, 4q, 4t) (IC50 = 41.56-86.53 µM; SI = 0.49-0.99) displayed promising in vitro anticancer activity against human liver (HepG2) cancer cell lines. In the case of lung (A549) cancer cell lines, these compounds were recognized to be more efficient and selective than standard reference artemisinin (IC50 = 100 µM) and chloroquine (IC50 = 100 µM; SI: 0.03). However, none of them were found to be active as compared to artesunic acid [IC50 = 9.85 µM; SI = 0.76 against lung (A549) cancer cell line and IC50 = 4.09 µM; SI = 2.01 against liver (HepG2) cancer cell line].

Silencing of keratin 15 impairs viability and mobility while facilitating the doxorubicin chemosensitivity by inactivating the β‑catenin pathway in liver cancer.

Keratin 15 (KRT15) regulates the invasion as well as the stemness and is associated with tumor size and metastasis of several gastrointestinal cancers apart from liver cancer. The present study aimed to explore the effect of KRT15 knockdown on liver cancer malignant behaviors and its interaction with the β-catenin pathway. Small interfering (si)-KRT15 and si-negative control (NC) were transfected into liver cancer cell lines, followed by the addition or not of CHIR-99021 (a β-catenin agonist). Cell viability, invasion, apoptosis, and the half maximal inhibitory concentration (IC50) value of doxorubicin (Dox) were then assessed. The present study illustrated that KRT15 gene and protein expression levels were upregulated in most liver cancer cell lines (Huh7, PLC, Hep3B and HepG2) compared to the normal liver cell line THLE-2. si-KRT15 reduced cell viability and invasive cell count while promoting the apoptosis rate in Huh7 and HepG2 cells. In addition, si-KRT15 also reduced the IC50 value of Dox. Furthermore, si-KRT15 inactivated the β-catenin pathway as reflected by β-catenin, cyclin D1 and c-Myc expression levels in Huh7 and HepG2 cells. Subsequently, CHIR-99021 treatment increased the cell viability and invasive cell count while reducing the apoptosis rate in Huh7 and HepG2 cells. Concurrently, the IC50 value of Dox was also increased. Notably, CHIR-99021 treatment attenuated the effect of si-KRT15 on mediating the aforementioned Huh7 and HepG2 cell malignant behaviors and Dox chemosensitivity. In conclusion, KRT15 knockdown suppressed viability and mobility but facilitated Dox chemosensitivity via inactivating the β-catenin pathway in liver cancer, suggesting its potential as a target for liver cancer treatment.

The Preventive Mechanism of Anserine on Tert-Butyl Hydroperoxide-Induced Liver Injury in L-02 Cells via Regulating the Keap1-Nrf2 and JNK-Caspase-3 Signaling Pathways.

Anserine is a naturally occurring histidine dipeptide with significant antioxidant activities. This study aimed to investigate the preventive mechanism of anserine on tert-butyl hydroperoxide (TBHP)-induced liver damage in a normal human liver cell line (L-02 cells). The L-02 cells were pretreated with anserine (10, 20, and 40 mmol/L) and then induced with 400 μmol/L of TBHP for 4 h. The results showed that the survival rates of L-02 cells and the contents of GSH were significantly increased with the pretreatment of anserine; the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the extracellular fluid were sharply decreased; and the formation of reactive oxygen species (ROS), nuclear fragmentation, and apoptosis were significantly inhibited. In addition, anserine could bind to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) with a binding force of -7.2 kcal/mol; the protein expressions of nuclear factor-erythroid 2-related factor-2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and Bcl-2 were upregulated by anserine in TBHP-induced L-02 cells, with the downregulation of p-JNK and caspase-3. In conclusion, anserine might alleviated liver injury in L-02 cells via regulating related proteins in the Keap1-Nrf2 and JNK-Caspase-3 signaling pathways.

Restoration and preservation effects of mung bean antioxidant peptides on H2O2-induced WRL-68 cells via Keap1-Nrf2 pathway.

Mung bean antioxidant peptides (MBAPs) were prepared from mung bean protein hydrolysate, and four peptide sequences including Ser-Asp-Arg-Thr-Gln-Ala-Pro-His (~953 Da), Ser-His-Pro-Gly-Asp-Phe-Thr-Pro-Val (~956 Da), Ser-Asp-Arg-Trp-Phe (~710 Da), and Leu-Asp-Arg-Gln-Leu (~644 Da) were identified. The effects of MBAPs on the oxidation-induced normal human liver cell line WRL-68 were analyzed to determine the mechanism protecting the oxidation-induced injury. The results showed that the cells were subjected to certain oxidative damage by H2O2 induction, as evidenced by decreased cell number and viability, overproduction of intracellular ROS, and decreased mitochondrial membrane potential. Compared with the H2O2-induced group, the MBAP-treated oxidation-induced group exhibited significantly higher cell number and viability, and the intracellular ROS was similar to that of the control group, suggesting that MBAP scavenges excessive intracellular free radicals after acting on the oxidation-induced cells. Combined with Western blotting results, it was concluded that the MBAP-treated oxidation-induced group also significantly promoted the expression of proteins related to the kelch-like ech-related protein 1 (Keap1)/ nuclear factor e2-related factor 2 (Nrf2) signaling pathway, which resulted in an approximately 2-fold increase in antioxidant enzymes, and a decrease in malondialdehyde content of approximately 55% compared to oxidatively-induced cells, leading to the recovery of both cell morphology and viability. These results suggest that MBAPs scavenge intracellular free radicals and improve oxidative stress in hepatocytes through the expression of Keap1/Nrf2 pathway-related protein, thereby reducing oxidative attack on the liver. Therefore, MBAP is applied as a nutritional ingredient in the functional food field, and this study provides a theoretical basis for the high utilization of mung bean proteins.

Biological activities of purified prolidase from pathogenic E.coli

Prolidase is a ubiquitous enzyme that plays a chief role in the metabolism of proline-rich proteins. The goal of this study extraction and purify prolidase from E. coli and evaluate some applications such as anti-biofilm and anticancer. Hundred stool Samples were collected from infants with breastfeeding, non-vomiting, and non-diarrhea to isolate E.coli bacteria. A 16S rRNA gene (585 bp) was found in all isolates of E. coli via PCR identification. Depending on the qualitative method on prolidase agar, only 40 (80%) isolates could produce prolidase from 50 isolates that were considered non-pathogens, then only 32 isolates revealed different levels in prolidase production with specific activity equal to (2.1U/mg) of E.coli. MS12. Sucrose, casein, and 40Co were the chosen isolate's best conditions for producing prolidase. Cold acetone precipitation and dialysis were used to extract the enzyme, and DEAE-cellulose and the Sephadex G-150 column were used in purification with specific activity (2 U/ml) and (6.6 U/mg) protein. Prolidase showed the highest effect on biofilm at 500 μg/ml concentration against P. aeruginosa, then E. coli, 65% and 60.3% respectively. Brain Tumor Cell Line (A127), Colorectal Adenocarcinoma cells (CaCo-2), and Normal embryonic liver cell line (WRL-68) were used to test the prolidase effect on these cell lines. An assay of MTT was used to detect the inhibiter concentration (IC50) values and cytotoxic effect of purified prolidase. Keywords: E.coli, Prolidase, purification, antibiofilm, anticancer activity