Liver Tumor Cells Research Articles

Influence of the physico-chemical bioink composition on the printability and cell biological properties in 3D-bioprinting of a liver tumor cell line.

The selection of a suitable matrix material is crucial for the development of functional, biomimetic tissue and organ models. When these tissue models are fabricated with 3D-bioprinting technology, the requirements do not only include the biological functionality and physico-chemical properties, but also the printability. In our work, we therefore present a detailed study of seven different bioinks with the focus on a functional liver carcinoma model. Agarose, gelatin, collagen and their blends were selected as materials based on their benefits for 3D cell culture and Drop-on-Demand (DoD) bioprinting. The formulations were characterized for their mechanical (G' of 10-350Pa) and rheological (viscosity 2-200Pa*s) properties as well as albumin diffusivity (8-50μm2/s). The cellular behavior was exemplarily shown for HepG2 cells by monitoring viability, proliferation and morphology over 14days, while the printability on a microvalve DoD printer was evaluated by drop volume monitoring in flight (100-250nl), camera imaging of the wetting behavior and microscopy of the effective drop diameter (700µm and more). We did not observe negative effects on cell viability or proliferation, which is due to the very low shear stresses inside the nozzle (200-500Pa). With our method, we could identify the strengths and weaknesses of each material, resulting in a material portfolio. By specifically selecting certain materials or blends, cell migration and possible interaction with other cells can be directed as indicated by the results of our cellular experiments.

Batroxin I: A Novel Bradykinin-Potentiating Peptide with Cytotoxic Activity Isolated from Bothrops atrox Snake Venom

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.

Role of LGMN in tumor development and its progression and connection with the tumor microenvironment.

Legumain (LGMN) has been demonstrated to be overexpressed not just in breast, prostatic, and liver tumor cells, but also in the macrophages that compose the tumor microenvironment. This supports the idea that LGMN is a pivotal protein in regulating tumor development, invasion, and dissemination. Targeting LGMN with siRNA or chemotherapeutic medicines and peptides can suppress cancer cell proliferation in culture and reduce tumor growth in vivo. Furthermore, legumain can be used as a marker for cancer detection and targeting due to its expression being significantly lower in normal cells compared to tumors or tumor-associated macrophages (TAMs). Tumor formation is influenced by aberrant expression of proteins and alterations in cellular architecture, but the tumor microenvironment is a crucial deciding factor. Legumain (LGMN) is an in vivo-active cysteine protease that catalyzes the degradation of numerous proteins. Its precise biological mechanism encompasses a number of routes, including effects on tumor-associated macrophage and neovascular endothelium in the tumor microenvironment. The purpose of this work is to establish a rationale for thoroughly investigating the function of LGMN in the tumor microenvironment and discovering novel tumor early diagnosis markers and therapeutic targets by reviewing the function of LGMN in tumor genesis and progression and its relationship with tumor milieu.

Antagonism between wild-type and mutant β-catenin controls hepatoblastoma differentiation via fascin-1

β-catenin is a well-known effector of the Wnt pathway, and a key player in cadherin-mediated cell adhesion. Oncogenic mutations of β-catenin are very frequent in paediatric liver primary tumours. Those mutations are mostly heterozygous, which allows the co-expression of wild-type (WT) and mutated β-catenins in tumour cells. We investigated the interplay between WT and mutated β-catenins in liver tumour cells, and searched for new actors of the β-catenin pathway. Using an RNAi strategy in β-catenin-mutated hepatoblastoma (HB) cells, we dissociated the structural and transcriptional activities of β-catenin, which are carried mainly by WT and mutated proteins, respectively. Their impact was characterised using transcriptomic and functional analyses. We studied mice that develop liver tumours upon activation of β-catenin in hepatocytes (APCKO and β-cateninΔexon3 mice). We used transcriptomic data from mouse and human HB specimens, and used immunohistochemistry to analyse samples. We highlighted an antagonistic role of WT and mutated β-catenins with regard to hepatocyte differentiation, as attested by alterations in the expression of hepatocyte markers and the formation of bile canaliculi. We characterised fascin-1 as a transcriptional target of mutated β-catenin involved in tumour cell differentiation. Using mouse models, we found that fascin-1 is highly expressed in undifferentiated tumours. Finally, we found that fascin-1 is a specific marker of primitive cells including embryonal and blastemal cells in human HBs. Fascin-1 expression is linked to a loss of differentiation and polarity of hepatocytes. We present fascin-1 as a previously unrecognised factor in the modulation of hepatocyte differentiation associated with β-catenin pathway alteration in the liver, and as a new potential target in HB. The FSCN1 gene, encoding fascin-1, was reported to be a metastasis-related gene in various cancers. Herein, we uncover its expression in poor-prognosis hepatoblastomas, a paediatric liver cancer. We show that fascin-1 expression is driven by the mutated beta-catenin in liver tumour cells. We provide new insights on the impact of fascin-1 expression on tumour cell differentiation. We highlight fascin-1 as a marker of immature cells in mouse and human hepatoblastomas.

Integration of chemotherapy and phototherapy based on a pH/ROS/NIR multi-responsive polymer-modified MSN drug delivery system for improved antitumor cells efficacy

Due to the difference of the intracellular microenvironment between tumor and normal cells, many stimuli-responsive drug carriers have been developed for precise drug delivery and improved treatment effect. In this paper, a multi responsive drug delivery system based on mesoporous silica nanoparticle (MSN) was prepared. A pH/ROS-sensitive polymer was designed and used to attach to the MSN surface, which acted as a smart nano-valve to prevent premature leakage of drugs. The carrier has been proved to release drugs selectively in simulated tumor microenvironment with lower pH and higher ROS content. And, by co-loading a photosensitizer with antitumor drug, the carrier could produce exogenous ROS under the near-infrared light irradiation, resulting in much quicker removing of the polymer and more drug release. Furtherly, the antitumor effect was evaluated by CCK-8 assay using normal and tumor liver cells. In short, the results of drug release and cell experiments both showed that the carriers had excellent anti-tumor cells effects.

Design, synthesis, and antitumor evaluation of trimethoxyflavonoid with arylurea structure against hepatocellular carcinoma.

Simultaneous targeting of tumor vasculature and inhibitors of tumor cell glycolysis may be a promising antitumor strategy. Here, we reported the total synthesis and biological evaluation of A-ring arylurea flavonoid derivatives with B-ring trimethoxy group, which exhibited potent antitumor activity against a variety of tumor cells in vitro. Most of the derivatives showed in vitro antitumor activity on HepG-2, HGC-27, MDA-MB-231, and A549 cells. Among them, compounds 8e, 8f, 8g, 8h, 8j, and 8l also exhibited significant anti-proliferation effects on liver tumor cell subtypes BEL-7402 and SMMC-7721. Compound 8l had the lowest IC50 value (5.61 ± 0.39 μM) on HepG-2 cells, and showed the effects of inhibiting colony formation, arresting the cell cycle in G0 /G1 phase, and inducing apoptosis in a concentration-dependent manner. In addition, the toxicity of compound 8l on human normal cells LO2 and GES-1 was lower than that of sorafenib. The inhibitory effects of compound 8l on the expression of glycolytic rate-limiting enzymes HKII, PFK-1, PKM2 and vascular endothelial growth factorwere further evaluated. Corresponding reduction in intracellular lactate was also detected after compound 8 treatment. Our results support an antitumor strategy targeting tumor vasculature and glycolysis to discover and develop a new generation of antitumor drugs.

YAP1 suppression inhibits autophagy and improves the efficacy of anti-PD-1 immunotherapy in hepatocellular carcinoma

Anti-PD-1 immunotherapy is a promising treatment for hepatocellular carcinoma (HCC), but some patients with HCC do not experience clinical benefits. Autophagy promotes tumor progression and participates in drug resistance. Previous studies have revealed that suppressing the expression level of Yes-associated protein 1 (YAP1) improves anti-PD-1 therapy efficacy. Therefore, the relationship between YAP1 expression and autophagy activity during anti-PD-1 treatment was investigated in this study. A positive correlation was found between the expression level of YAP1 and LC3B by analyzing The Cancer Genome Atlas (TCGA), UALCAN databases, and HCC tissue microarray. Meanwhile, YAP1 expression and autophagy constituted positive feedback, in which YAP1 inhibition decreased the autophagy activity in liver tumor cells by hepatocyte-specific Yap1 knockout mice. Further, anti-PD-1 treatment increased autophagy and YAP1 expression levels in the cancer tissues from DEN/TCPOBOP-induced liver cancer mice. Finally, Yap1 knockout suppressed autophagy and improved anti-PD-1 therapy efficacy in hepatocyte-specific Yap1 knockout mice with liver tumors. These results suggested that YAP1 suppression was sensitized to anti-PD-1 treatment and inhibited autophagy activity in liver tumor cells. YAP1 is a promising target for improving the efficacy of anti-PD-1 immunotherapy in HCC.

Abstract A008: An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Tumor progression is monitored with MRI, chest CT, and serum levels of Alpha fetoprotein. Unfortunately, these assays involve exposing children to radiation and anesthesia and are not always accurate. The goal of this work is to develop and validate a liquid biopsy test for circulating tumor cells (CTCs) with a panel of HB-specific markers to provide a less invasive and more accurate way of assessing disease. Methods: We first explored accumulation of the far-red fluorescent dye indocyanine green (ICG) in widely available cell lines (HepG2, Huh-6, Hep3B, HepRG, 293T, SH-SY5Y, A549), our own HB patient-derived cell line (HB17), and primary fibroblasts. We assessed ICG accumulation with fluorescence microscopy and flow cytometry, including standard flow cytometry on a BD Symphony instrument and imaging flow cytometry on the Amnis ImageStream X MK II instrument. Based on this data, we then developed a new method for quantifying CTC burden from patient whole blood samples using HB-specific markers ICG, Glypican-3 (GPC3), and DAPI with fluorescence microscopy and flow cytometry readouts. We tested this panel with cell lines and non-cancer control blood samples, as well as blood samples from mice harboring intrahepatic patient-derived xenograft (PDX) tumors representing HB. Finally, we used this panel to analyze whole blood samples for CTC burden with a cohort of HB patients and correlated with patient characteristics and outcomes. Results: First, we showed that ICG accumulation is specific to liver cancer cells (HepG2, Huh-6, Hep3B, HB17), compared to non-malignant liver cells (HepRG), non-liver solid tumor cells (SH-SY5Y, A549), and non-malignant cells (293T, primary fibroblasts). In addition, we found that ICG+ liver tumor cells remain positive for at least 96 hours after uptake. We also showed that ICG accumulation can be used to identify HepG2 HB cells in a mixed population of cells down to a dilution of 1:100 (2.7% ICG+ in the HepG2/SH-SY5Y mix versus 1.9% ICG+ in the SH-SY5Y control). Flow cytometry with HepG2 and A549 cell lines with the ICG/GPC3/DAPI panel showed 94.4% of HepG2 cells were triple ICG+/GPC3+/DAPI+ while 0% of A549 cells were triple ICG+/GPC3+/DAPI+. With non-cancer control blood samples, we identified less than 1 ICG+ cell per ml of blood. We showed the presence of triple ICG+/GPC3+/DAPI+ cells in the blood of animals harboring PDX tumors. Most importantly, with a low-risk HB patient sample, we found 2.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood; with a high-risk sample we counted 17.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood. Conclusions: This work shows the translation from the use of ICG to identify HB lesions in patients during surgery to its role in a novel liquid biopsy test for CTCs, which has the real potential to improve the standard of care by offering a more accurate and less invasive means of monitoring disease. Citation Format: Andres F. Espinoza, Roma H. Patel, Bryan W. Armbruster, Pavan Kureti, Saiabhiroop R. Govindu, Sanjeev A. Vasudevan, Sarah E. Woodfield. An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A008.

Bifunctional cancer cell-based vaccine concomitantly drives direct tumor killing and antitumor immunity.

The administration of inactivated tumor cells is known to induce a potent antitumor immune response; however, the efficacy of such an approach is limited by its inability to kill tumor cells before inducing the immune responses. Unlike inactivated tumor cells, living tumor cells have the ability to track and target tumors. Here, we developed a bifunctional whole cancer cell-based therapeutic with direct tumor killing and immunostimulatory roles. We repurposed the tumor cells from interferon-β (IFN-β) sensitive to resistant using CRISPR-Cas9 by knocking out the IFN-β-specific receptor and subsequently engineered them to release immunomodulatory agents IFN-β and granulocyte-macrophage colony-stimulating factor. These engineered therapeutic tumor cells (ThTCs) eliminated established glioblastoma tumors in mice by inducing caspase-mediated cancer cell apoptosis, down-regulating cancer-associated fibroblast-expressed platelet-derived growth factor receptor β, and activating antitumor immune cell trafficking and antigen-specific T cell activation signaling. This mechanism-based efficacy of ThTCs translated into a survival benefit and long-term immunity in primary, recurrent, and metastatic cancer models in immunocompetent and humanized mice. The incorporation of a double kill-switch comprising herpes simplex virus-1 thymidine kinase and rapamycin-activated caspase 9 in ThTCs ensured the safety of our approach. Arming naturally neoantigen-rich tumor cells with bifunctional therapeutics represents a promising cell-based immunotherapy for solid tumors and establishes a road map toward clinical translation.

XBP1‐elicited environment by chemotherapy potentiates repopulation of tongue cancer cells by enhancing miR‐22/lncRNA/KAT6B‐dependent NF‐κB signalling

Tumour repopulation initiated by residual tumour cells in response to cytotoxic therapy has been described clinically and biologically, but the mechanisms are unclear. Here, we aimed to investigate the mechanisms for the tumour-promoting effect in dying cells and for tumour repopulation in surviving tongue cancer cells. Tumour repopulation in vitro and in vivo was represented by luciferase activities. The differentially expressed cytokines in the conditioned medium (CM) were identified using a cytokine array. Gain or loss of function was investigated using inhibitors, neutralising antibodies, shRNAs and ectopic overexpression strategies. We found that dying tumour cells undergoing cytotoxic therapy increase the growth of living tongue cancer cells in vitro and in vivo. Dying tumour cells create amphiregulin (AREG)- and basicfibroblast growth factor (bFGF)-based extracellular environments via cytotoxic treatment-induced endoplasmic reticulum stress. This environment stimulates growth by activating lysine acetyltransferase 6B (KAT6B)-dependent nuclear factor-kappa B (NF-κB) signalling in living tumour cells. As direct targets of NF-κB, miR-22 targets KAT6B to repress its expression, but long noncoding RNAs (lncRNAs) (XLOC_003973 and XLOC_010383) counter the effect of miR-22 to enhance KAT6B expression. Moreover, we detected increased AREG and bFGF protein levels in the blood of tongue cancer patients with X-box binding protein-1 (XBP1) activation in tumours under cytotoxic therapy and found that XBP1 activation is associated with poor prognosis of patients. We also detected activation of miR-22/lncRNA/KAT6B/NF-κB signalling in recurrent cancers compared to paired primary tongue cancers. We identified the molecular mechanisms of cell death-induced tumour repopulation in tongue cancer. Such insights provide new avenues to identify predictive biomarkers and effective strategies to address cancer progression.

Robust nontarget DNA-triggered catalytic hairpin assembly amplification strategy for the improved sensing of microRNA in complex biological matrices.

A simple but robust fluorescence strategy based on a nontarget DNA-triggered catalytic hairpin assembly (CHA) was constructed to probe microRNA-21 (miR-21). A short ssDNA rather than degradable target miRNA was employed as an initiator. Two molecular beacons needed to assist the CHA process were simplified to avoid unfavorable nonspecific interactions. In the presence of the target, the initiator was released from a partially duplex and triggered the cyclic CHA reaction, resulting in a significantly amplified optical readout. A wide linear range from 0.1 pM to 1000 pM for the sensing of miR-21 in buffer was achieved with a low detection limit of 0.76 pM. Fortunately, this strategy demonstrated an obviously improved performance for miR-21 detection in diluted serum. The fluorescence signals were enhanced remarkably and the sensitivity was further improved to 0.12 pM in 10% serum. The stability for miR-21 quantification and the capability for the analysis of single nucleotide polymorphisms (SNPs) were also improved greatly. More importantly, the biosensor could be applied to image miR-21 in different living tumor cells with high resolution, illustrating its promising potential for the assay of miRNAs in various complex situations for early-stage disease diagnosis and biological studies in cells.

Orthometric multicolor encoded hybridization chain reaction amplifiers for multiplexed microRNA profiling in living cells.

Multiplexed microRNA (miRNA) profiling of more than four types in living cells is challenging due to fluorescent spectral overlap, representing a significant limitation in studying the complex interactions related to the occurrence and development of diseases. Herein, we report a multiplexed fluorescent imaging strategy based on an orthometric multicolor encoded hybridization chain reaction amplifier named multi-HCR. The targeting miRNA can trigger this multi-HCR strategy due to the specific sequence recognition, and then its self-assembly to amplify the programmability signals. We take the four-colored chain amplifiers, showing that the multi-HCR can form 15 combinations simultaneously. In a living process of hypoxia-induced apoptosis and autophagy under complicated mitochondria and endoplasmic reticulum stress, the multi-HCR demonstrates excellent performance in detecting eight different miRNA changes. The multi-HCR provides a robust strategy for simultaneously profiling multiplexed miRNA biomarkers in studying complicated cellular processes.

Twelve-week treadmill endurance training in mice is associated with upregulation of interleukin-15 and natural killer cell activation and increases apoptosis rate in Hepa1-6 cell-derived mouse hepatomas.

Regular exercise reduces the risk of malignancy and decreases the recurrence of cancer. However, the mechanisms behind this protection remain to be elucidated. Natural killer (NK) cells are lymphocytes of the innate immune system, which play essential roles in immune defense and effectively prevent cancer metastasis. Physical exercise can increase the activity of NK cells. Interleukin-15 (IL-15) is the best-studied cytokine activator of NK cells, and it was shown to have many positive functional effects on NK cells to improve antitumor responses. The aim of this study was to clarify the possible important mechanisms behind endurance exercise-induced changes in NK cell function, which may be highly correlated with IL-15. An animal model was used to study IL-15 expression level, tumor volume, cancer cell apoptosis, and NK cell infiltration after treadmill exercise. Although IL-15 was highly expressed in skeletal muscle, treadmill exercise further elevated IL-15 levels in plasma and muscle (P<0.05). In addition, tumor weight and volume of tumor-bearing mice were decreased (P<0.05), and liver tumor cell apoptosis was increased after 12 weeks of treadmill exercise (P<0.05). NK cell infiltration was upregulated in tumors from treadmill exercise mice, and the level of interferon-gamma (IFN-γ) and IL-15 were higher than in sedentary mice (P<0.05). The study indicated that regular endurance training can reduce cancer risk, which was related to increased IL-15 expression, activation of the immune killing effect of NK cells, and promotion of tumor cell apoptosis, which can ultimately control tumor growth.

Citotoxicidade de compostos isolados de Picrasma crenata (Vell.) Engl. em células tumorais animais (HTC)

Abstract The study aim was to evaluate the cytotoxic activity, using the MTT test [3-(4,5-Dimethilthiazol-2-yl)-2,5-diphenil tetrazolium bromide], from the crude extract of Picrasma crenata (Pau Tenente) and its isolated compounds, quassin and parain, in culture of rat liver tumor cells (HTC). The test was carried out exposing the cells for 24, 48 and 72 hours to concentrations of 5, 10, 50, 100, 200, 300, 400, 500 and 1000 μg of crude extract of Pau Tenente/mL of culture medium and 1, 5, 10, 15, 20, 40, 60, 80 and 100 μg of quassin or parain compounds/mL of culture medium. The absorbances averages results obtained showed that the crude extract did not present cytotoxicity for the HTC cells in all the concentrations and evaluated times. For quassin, the concentrations of 80 and 100 μg/mL were cytotoxic, after 72 hours of treatment. For parain, the concentrations of 1, 5, 20, 40, 60, 80 and 100 µg/mL, in 72 hours, were cytotoxic, revealing a new activity for this compound. Thus, the results demonstrate a first indication of the cytotoxic activity of compounds quassin and parain, adding an important social and economic value to them, and may have application in future research and in pharmaceutical industry.

Dihydroartemisinin broke the tumor immunosuppressive microenvironment by inhibiting YAP1 expression to enhance anti-PD-1 efficacy.

The efficacy of anti-PD-1 therapy is not as expected in hepatocellular carcinoma (HCC). YAP1 was overexpressed and activated in HCC. The mechanism of YAP1 in HCC immune escape is unclear. Anti-PD-1 treatment increased YAP1 expression in liver tumor cells, and exhausted CD4+ and CD8+ T cells in the blood and spleen of liver tumor mice. YAP1 knockdown suppressed PD-L1 expression, which was involved in JAK1/STAT1, 3 pathways. Moreover, Yap1 knockout elevated CD4+ and CD8+ T cells in liver tumor niche. Consistently, verteporfin, YAP1 inhibitor, decreased TGF-β and IFN-γ in liver tumor niche and exhausted CD8+ T cell in the spleen. DHA suppressed YAP1 expression and break immune evasion in liver tumor niche, characterized by decreased PD-L1 in liver tumor cells and increased CD8+ T cell infiltration. Furthermore, DHA combined with anti-PD-1 treatment promoted CD4+ T cell infiltration in the spleen and CD8+ T cell in tumor tissues of mice. In summary, YAP1 knockdown in liver tumor cells suppressed PD-L1 expression and recruited cytotoxic T lymphocytes (CTLs), leading to break immune evasion in tumor niche. Mechanistically, YAP1 knockdown suppressed PD-L1 expression, which was involved in JAK1/STAT1, 3 pathways. Finally, DHA inhibited YAP1 expression, which not only inhibited liver tumor proliferation but also break the immunosuppressive niche in liver tumor tissues and improve the effect of anti-PD-1 therapy.

Experimental evaluation of the antioxidant and antitumor activities of thyme and basil essential oils and their phenolic constituents: theoretical antioxidant evaluation

BackgroundIdentifying specific biological activities of natural products are of the main concerns worldwide for the use in safe functional food manufacture; essential oils and their components are good candidates in this respect. The present work aims to evaluate the biological activities of basil and thyme oils as well as their phenolic constituents. Using computational methods to predict biological activities are currently effective tools in minimizing and explaining experimental works.ResultsChemical composition of thyme and basil oils were determined using GC–MS. The identified phenolic components were thymol (28.21%) and carvacrol (0.47%) in thyme oil and eugenol (11.37%) in basil oil. The antioxidant activity of both oils and their phenolic constituents as expressed by EC50 value were 535.01, 134.37, 176.57, 407.89 and 2.29 µg/mL against DPPH and 131.95, 56.65, 57.15, 82.71and 32.80 µg/mL against hydrogen peroxide, respectively. The order of activity is basil oil > thyme oil while phenolic compound order is eugenol > thymol > carvacrol; reducing power showed the same order. Basil oil showed also higher and good antitumor activity where it reduces the surviving fraction to 38.4% of brain tumor cells (U251) and 61.3% of liver tumor cells (HEPG2) at concentration 10 µg/mL. The antioxidant activity were evaluated theoretically according to the main three mechanisms, Hydrogen-Atom-Transfer (HAT), Single Electron Transfer–Proton Transfer (SET-PT) and the Sequential Proton Loss Electron-Transfer (SPLET); the results proved the experimental order of antioxidant and biological activities, and explained the remarkably higher activities of basil oil and its main phenolic component, eugenol.ConclusionTheoretical calculation can be used successfully to explain and predict the experimental biological activity results. Basil oil and its main phenolic component, eugenol, were found effective as antioxidants. Basil oil was also efficient in reducing the surviving fraction of liver and brain cancer cells where it reduces brain cells even lower than cells treated by doxorubicin, a known anti-cancer agent; thus, basil oil and its main phenolic components, eugenol, can be used safely in food preservation and functional food production.Graphical

Induction of Apoptosis by Matrine Derivative ZS17 in Human Hepatocellular Carcinoma BEL-7402 and HepG2 Cells through ROS-JNK-P53 Signalling Pathway Activation.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and ranks third among cancer-related deaths worldwide. Using matrine as a lead compound, 12 matrine derivatives were designed and synthesised, and their antiproliferative activities were evaluated in four cancer cell lines. Eight of the twelve compounds showed strong antiproliferative activity, with an IC50 of &lt;10 μM. The compound ZS17 exhibited strong antiproliferative activity in hepatocellular carcinoma cell lines with IC50 values in the range of 3.014-3.388 μM, which was much lower than that of matrine. Furthermore, we explored the role of ZS17 in inducing apoptosis in HCC cells in vitro and in vivo, as well as possible mechanisms involved. ZS17 inhibited the proliferation of BEL-7402 and HepG2 cells in time- and dose-dependent manners. In addition, we found that ZS17 significantly induced apoptosis and ROS (reactive oxygen species) production, promoted JNK phosphorylation, activated p53, and activated the caspase signalling pathway. Furthermore, the antioxidant NAC, JNK inhibitor SP600125, and Si-JNK increased cell viability, re-established cell metastasis, and inhibited ZS17-induced apoptosis. An in vivo antitumour assay demonstrated that ZS17 significantly reduced the number of migrating HepG2 cells in zebrafish embryos and suppressed the growth of HepG2 xenografts in nude mice without any obvious side effects. Our study demonstrated that the ROS-JNK-P53 pathway plays an important role in the destruction of liver tumour cells by ZS17. Thus, ZS17 may represent a promising chemotherapeutic agent for the treatment of HCC patients.

Biosensor based on bimetallic/graphene composite for non-enzymatic detection of hydrogen peroxide in living tumor cells.

A highly sensitive electrochemical biosensor was manufactured with triple synergistic catalysis to detect hydrogen peroxide (H2 O2 ). In this study, a highly sensitive biosensor based on Prussian blue-chitosan/graphene-hemin nanomaterial/platinum and palladium nanoparticles (PB-CS/HGNs/Pt&Pd biosensor) was fabricated for the detection of H2 O2 . The materials described above were modified on the electrode surface and applied to catalyze the breakdown of hydrogen peroxide. The current response of the biosensor presented a linear relationship with H2 O2 concentration from 6 × 10-2 to 20μM (R2 = 0.9766) and with the logarithm of H2 O2 concentration from 20 to 9×103 μM (R2 = 0.9782), the low detection limit of 25nM was obtained at the signal/noise (S/N) ratio of 3. Besides, the biosensor showed an outstanding anti-interference ability and acceptable reproducibility. PB-CS/HGNs/Pt&Pd electrodes are effective in measuring H2 O2 from living tumor cells, which implies that the biosensor has the potential to assess reactive oxygen species in various living tumor cells.

STUDY OF BIOLOGICAL ACTIVITY OF DOXORUBICIN CONJUGATE WITH NACETYL-D-GALACTOSAMINE HYDRASONIC LINKER

Molecules containing the N-acetylgalactosamine residue are capable to bind to the asialoglycoprotein receptor, a protein presented on the surface of the liver tumor cells; therefore it is suggested that doxorubicin conjugates containing such fragment can be delivered to hepatocellular carcinoma cells as target, and then, upon hydrazone link splitting, in acidic environment of the tumor release the active substance.

Expression of programmed cell death-1 on neuroblastoma cells in TH-MYCN transgenic mice.

Immunotherapy may improve the poor prognosis of high-risk neuroblastoma. Programmed cell death-1 (PD-1) is expressed in several cancers. The tyrosine hydroxylase MYCN (TH-MYCN) transgenic mouse model is widely used in neuroblastoma research, but detailed information on its immunological background is lacking. Therefore, we studied the immunological tumor microenvironment and tumor cell surface antigen expression in homozygote and hemizygote mice and effects of antibody therapy against PD-1. CD4, CD8, CD11b, and CD11c expression in immune cells from retroperitoneal lymph nodes and spleen was analyzed by flow cytometry. Tumor cell surface antigen expression was confirmed, and data from homozygote and hemizygote mice were compared. Effects of anti-PD-1 antibody were evaluated. CD4-, CD8-, CD11b-, and CD11c-positive cells were not significantly different in homozygote and hemizygote mice, and CD11b- and CD11c-positive cells were identified in the tumor microenvironment in both. Tumor cells expressed PD-1, and anti-PD-1 antibody had anti-tumor effects and significantly reduced the percentage of living tumor cells in cultures after 2h. The immunological background is similar in homozygote and hemizygote TH-MYCN transgenic mice, and both have PD-1-positive tumor cells. Anti-PD-1 antibody suppresses tumor growth. This mouse model may be a useful for studying immunotherapy of neuroblastoma.