Journal: Nutrients

Article Title: Nicotinamide Adenine Dinucleotide Precursor Suppresses Hepatocellular Cancer Progression in Mice

doi: 10.3390/nu15061447

Figure Lengend Snippet: NR supplementation inhibited hematogenous multi-organ metastasis of HCC cells in mice. ( A ) Schematic of the hematogenous metastatic neoplasm model. ( B ) Trends in body weight of mice during the intervention. ( C ) Differences in weight changes between NR and control group after 37 days of intervention. Dotted line represented the line (y = 0). ( D ) The activity of HepG2-GL cells in vivo throughout the intervention, quantified by bioluminescent signals (Total Flux, p/s) of the whole mice body. Dotted line showed the bioluminescence signals on day 0. ( E ) The metastasis of HepG2-GL cells in vivo after 37 days of intervention shown by noninvasive bioluminescence imaging. ( F ) The metastasis of HepG2-GL cells in lungs after 37 days of intervention shown by bioluminescence imaging. ( G ) Quantification of the image in ( F ) with bioluminescent signals (Total Flux, p/s) of the lungs. ( H ) Lung-to-body weight ratios at the endpoint of the intervention. ( I ) Representative images of lungs dissected from mice per group. In the upper row, the arrow points to the macroscopic tumor nodule. In the lower row are scanning pictures of H&E staining sections, scale bars: 1 mm. ( J ) Representative H&E staining section of tumor metastasis to the bone and liver of mouse in the control group; scale bars: 200 μm (top row), 100 μm (bottom row). n = 7/group; Red dots: control mice; blue dots: NR-treated mice. * p < 0.05 compared to the control group. p -values were calculated using two-way RM ANOVA ( B ), unpaired t test and Welch’s correction ( C , H ), two-way RM ANOVA and Sidak’s multiple comparisons test ( D ), and Mann – Whitney test ( G ). NR, nicotinamide riboside.

Article Snippet: To establish the GFP and luciferase-labeled HepG2 cell line (HepG2-GL), HepG2 cells were transduced by ultra-purified lentivirus particles containing a plasmid encoding luciferase and eGFP (EX-hLUC-Lv201, Genecopoeia, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, In Vivo, Imaging, Staining, MANN-WHITNEY

Journal: Nutrients

Article Title: Nicotinamide Adenine Dinucleotide Precursor Suppresses Hepatocellular Cancer Progression in Mice

doi: 10.3390/nu15061447

Figure Lengend Snippet: NR inhibited migration and invasion of HepG2 cells. ( A ) The cell viability of LO2 and HepG2 cells after 48-h NR treatment. ( B ) Wound-healing assay showing cell migration of HepG2 cells after 48 h. ( C ) Transwell assays showing cell migration and invasion of HepG2 cells after 24 h. n = 3; * p < 0.05, ** p < 0.01 compared to the TGF-β group. p -values were calculated using Kruskal – Wallis test and Dunn’s multiple comparisons test ( A ), one-way ANOVA and Tukey’s multiple comparisons test (( B , C )-migration), and one-way ANOVA and Tukey’s multiple comparisons test (( C )-invasion). NR, nicotinamide riboside; TGF-β, transforming growth factor-β.

Article Snippet: To establish the GFP and luciferase-labeled HepG2 cell line (HepG2-GL), HepG2 cells were transduced by ultra-purified lentivirus particles containing a plasmid encoding luciferase and eGFP (EX-hLUC-Lv201, Genecopoeia, Rockville, MD, USA) following the manufacturer’s protocol.

Techniques: Migration, Wound Healing Assay

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: Validation of the production and targeting of DBCO‐scFv in the HepG2/C3A cell model. (A) Sandwich ELISAs were performed to detect serial dilutions of scFv that bound to ASGR1. scFv was then detected using an HRP‐conjugated anti‐6X His tag antibody, and the absorbance at 450 nm was quantified using an absorbance microplate reader. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of scFv to ASGR1. (B) Confocal microscopy images (630×) showed that after 30 min, scFv, which was stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody, specifically targeted ASGR1 expressed in the membrane of HepG2/C3A cells. ASGR1 was gradually internalized by the cells as the incubation time increased (0.5, 1, 3, and 6 h). Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (C) Quantification of the mean fluorescence intensity of the scFv targeting ASGR1 in HepG2/C3A cells incubated for different durations (0.5, 1, 3, and 6 h). The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (D) Different molar ratios of galactose to scFv were mixed and added to ELISA wells coated with the ASGR1 antigen. The scFv signal was gradually inhibited concomitant with increasing proportions of galactose. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (E) Confocal microscopy images (×400) showing that scFv (1 µM) stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells and that the targeting was inhibited by preincubation with galactose (500 mM) for 1 h. Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (F) The quantification results revealed that the mean fluorescence intensity of the scFv (1 µM) targeting ASGR1 in HepG2/C3A cells was inhibited after preincubation with galactose (500 mM) for 1 h. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (G) Schematic representation of DBCO‐scFv production through the conjugation of scFv (S19C) and DBCO‐PEG4‐maleimide by a site‐specific cysteine‐cyclooctyne reaction. (H) Sandwich ELISAs were performed to detect serial dilutions of DBCO‐scFv bound to ASGR1. DBCO‐scFv was detected with biotin‐PEG3‐azide (10 µM) via a click reaction and subsequently interacted with streptavidin HRP and TMB. The absorbance was measured at 450 nm by an absorbance microplate reader. Three replicates per sample were performed. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of DBCO‐scFv for ASGR1. (I) SDS‐PAGE of DBCO‒scFv conjugates was performed under nonreducing conditions. Coomassie blue staining (left) and fluorescence image (right) of the SDS‐PAGE gel of the DBCO‒scFv conjugates. M: marker ladder. Lane 1: scFv protein control group. Lane 2: Calfluor 488 azide control group. Lane 3: scFv + Calfluor 488 azide control group. Lane 4: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:1:1. Lane 5: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:2:2. Lane 6: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:4:4. Lane 7: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 2:4:4. Lane 8: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 4:4:4. (J) Confocal microscopy images (×400) showing that after 30 min, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells. DBCO‐scFv was gradually internalized by the cells after a prolonged incubation time (6 h). Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. (K) The mean fluorescence intensity was significantly higher ( p < 0.01) in the DBCO‐scFv‐treated HepG2/C3A cells than in the control cells without scFv treatment. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (L) Confocal microscopy images (×630) showing that after 3 h of incubation, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 and was internalized into the cytoplasm by HepG2/C3A cells. Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. All data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Biomarker Discovery, Microplate Reader Absorbance Measurement, Binding Assay, Confocal Microscopy, Staining, Membrane, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay, Conjugation Assay, SDS Page, Marker, Control

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: CAR‐sEVs significantly enhanced targeting efficacy in the HepG2/C3A cell model. (A) Schematic representation of the conjugation of N₃‐sEVs with DBCO‐Cy5 (red) and DBCO‐AF488‐scFv (green) using click chemistry to generate CAR‐sEVs. (B) Flow cytometry histograms showing the fluorescence intensity of HepG2/C3A cells treated with increasing doses (10⁷, 10⁸, or 10⁹ particles) of CAR‐sEVs or N 3 ‐sEVs for 18 h. The fluorescence was detected in the Alexa Fluor 488 and PE‐Cy5 channels. Sham treatment (no sEVs) was included as a control. (C) Quantification of targeting efficiency (%) of N 3 ‐sEVs and CAR‐sEVs at different doses (10⁷, 10⁸, or 10⁹ particles). (D) Confocal microscopy images (630×) showing the cellular uptake of N₃‐sEVs and CAR‐sEVs in HepG2/C3A cells at various time points (0.5, 1, 2, 4, and 6 h). The images illustrate the nuclei (DAPI, blue), sEV particles with azido groups (DBCO‐Cy5, red), and DBCO‐scFv (labelled with Alexa Fluor‐488, green). The merged DIC (differential interference contrast) images to fluorescent images. Scale bar = 10 µm. (E) Flow cytometry was used to measure the mean fluorescence intensity in HepG2/C3A cells treated with N₃‐sEVs (grey) or CAR‐sEVs (red) over 6 h. All data are presented as the mean ± SEM. *** p < 0.001.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Conjugation Assay, Flow Cytometry, Fluorescence, Control, Confocal Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

doi: 10.1002/jev2.70044

Figure Lengend Snippet: Compared to unmodified sEVs, CAR‐sEVs enhanced the therapeutic effects against the APAP challenge in C3A cells. (A) (i) APAP dose‐dependently reduced the viability of HepG2/C3A cells, with an IC50 of 10 mM APAP. (ii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iv) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (v) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (vi) Compared with corresponding doses of sEVs, CAR‐sEVs significantly increased the viability of HepG2/C3A cells in a dose‐dependent manner after 48 h of incubation in the presence of APAP (10 mM). Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) The heatmap displays the predicted interactions between pcMSC‐sEVs miRNAs and genes of interest categorized into four major functional groups: anti‐inflammation (TGF‐β1, TGF‐β2, TGF‐β3, IL‐6, IL‐1β, TNF‐α, and NKB1), liver regeneration promotion (PTEN, MKK4, MFAP4), anti‐apoptosis (caspase‐3, caspase‐7, caspase‐8, caspase‐9, Bax and Bim), and anti‐fibrosis (COL1A1 and COL3A1). TargetScanHuman 8.0 was used to calculate the targetability score, with only the top miRNAs having a TargetScore context++ score of ≤ −0.1 selected. The miRNAs were selected using TargetScanHuman 8.0, with a TargetScore context++ score ≤ −0.1. The miRNA expression is shown as log2 RPM (reads per million), with higher values indicating greater miRNA expression. (C) (i) The line chart showing the highly expressed miRNAs significantly involved in specific biological processes of negative regulation of hepatocyte proliferation (GO:2000346) and positive regulation of the apoptotic process (GO:0043065). (ii) The Venn diagram illustrates the overlap of miRNAs involved in two biological processes. The overlapping area represents 15 miRNAs predicted to target genes involved in both processes. (iii) The top miRNAs from this overlap are listed in the table below.

Article Snippet: The human hepatoblastoma cell line C3A (HepG2/C3A) was obtained from the Bioresource Collection and Research Center (BCRC), Taiwan.

Techniques: Incubation, Cell Viability Assay, Functional Assay, Expressing

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose-derived mesenchymal stem cells and their conditioned media on hepatocellular carcinoma cell proliferation and apoptosis. HCC cells (2 × 10 5 ) were seeded in six-well coculture plates in the presence or absence of ADMSCs and ADMSC CM, undiluted or diluted 1:5 or 1:25 for 48 h. The proliferation of HepG2 and PLC-PRF-5 HCC cell lines were measured by (A) cell count assay and (B) WST-8 proliferation tests; The apoptosis of HepG2 (C) and PLC-PRF (D) cells co-cultured as above was measured by flow cytometry using Annexin V/PI test kit; C and D: Two representative experiments of apoptosis in HepG2 and PLC-PRF cells, respectively; E: The average rate of apoptosis in HepG2 and PLC-PRF-5 cells induced by ADMSCs, ADMSC CM. Data are representative of three independent experiments, each repeated in triplicate. All data are represented as mean ± SD ( a P < 0.05, b P < 0.01, c P < 0.001). CTR: Control; ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The human HCC cell lines (HepG2/C3A/HB-8065, PLC-PRF-5/CRL-8024) were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States) in 2015.

Techniques: Derivative Assay, Cell Counting, Cell Culture, Flow Cytometry, Control

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose derived mesenchymal stem cells and their conditioned media on of hepatocellular carcinoma cell migration and invasion. HCC cells (2 × 10 5 ) were seeded in six- well co-culture plates in the presence or absence of ADMSCs (at ADSMCs: HCC ratio of 1:1 or 1:2) or ADMSC CM, undiluted or diluted 1:5 or 1:25. The migration of (A) HepG2 and (B) PLC-PRF-5 cells was assessed by wound healing assay. The migration rate at 24 h is represented in (C); D: A Transwell migration assay was performed to confirm the results of the wound healing assay; E: HCC cell invasiveness was measured by Transwell invasion assay. In the Transwell migration and invasion assay, 3 × 10 5 HCC cells alone, co-cultured with ADMSCs, or treated with ADMSC CM were seeded into the apical chamber of Transwell plates and allowed to migrate or invade through the uncoated polycarbonate membrane or collagen-coated polycarbonate membrane, respectively (8 μm pore size) to the lower chamber for 24 h or 48 h, respectively. The migratory or invasive cells were stained with crystal violet cell stain solution and extracted using an extraction solution provided in the kit. The level of migration and invasion was measured using a plate reader at the absorbance of 560 nm. Values shown are representative of five independent experiments, each performed in triplicate. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). CTR: Control; ADMSC: Adipose derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The human HCC cell lines (HepG2/C3A/HB-8065, PLC-PRF-5/CRL-8024) were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States) in 2015.

Techniques: Derivative Assay, Migration, Co-Culture Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Invasion Assay, Cell Culture, Membrane, Pore Size, Staining, Extraction, Control

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose-derived mesenchymal stem cells and adipose-derived mesenchymal stem cell conditioned media on tissue inhibitor metalloproteinase mRNA levels in hepatocellular carcinoma cells. HCC cells (2 × 10 5 ) were seeded in six-well co-culture plates in the presence or absence of ADMSCs or of undiluted ADMSC conditioned media (CM). The mRNA levels of TIMP-1, TIMP-2, and TIMP-3 in HepG2 (A) and PLC-PRF-5 (B) cells after the removal of ADMSCs in the case of coculture was measured by quantitative PCR. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The human HCC cell lines (HepG2/C3A/HB-8065, PLC-PRF-5/CRL-8024) were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States) in 2015.

Techniques: Derivative Assay, Co-Culture Assay, Real-time Polymerase Chain Reaction

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Expression of tumor suppressor genes and oncogenes in hepatocellular carcinoma cells. HCC cells (2 × 10 5 ) were seeded in six-well co-culture plates in the presence or absence of ADMSCs or undiluted ADMSC CM for 48 h. The mRNA expression of the tumor suppressor genes P53/RB, oncogene c-Myc and the enzymatic component of telomerase hTERT were assessed by RT-PCR in (A) HepG2 and (B) PLC-PRF-5 cells after the removal of ADMSCs in the case of co-culture. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The human HCC cell lines (HepG2/C3A/HB-8065, PLC-PRF-5/CRL-8024) were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States) in 2015.

Techniques: Expressing, Co-Culture Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay