Journal: Journal of Cancer

Article Title: Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation

doi: 10.7150/jca.32167

Figure Lengend Snippet: Fenretinide inhibits cell growth and induces apoptosis of HL-60 and Kasumi-1 cells. a) Cell viability. Cells are treated with fenretinide as indicated for 48 h. Cell viability is assessed according to the CCK-8 assay. b-c) Fenretinide induces the apoptosis of both HL-60 and Kasumi-1 cells. Cells are treated with fenretinide as indicated for 48 h. Apoptotic cells are tested using flow cytometry. d-e) HL-60 cells are treated with fenretinide at the concentrations of 0, 2.5, 5 and 10 μM for 12 h. The cell cycle is detected by flow cytometry. f) Cell lysates are collected for Western blotting, so as to analyze the expression of related cyclins. g) Grey values of cyclins expression in f), using Quantity One 4.6.2. All data represents the means from three independent experiments. *p≤0.05; **p≤ 0.01; ***p≤0.001 vs control.

Article Snippet: Fenretinide was purchased from MedChem Express, dissolved in DMSO and stored at -80°C.

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Expressing, Control

Journal: Journal of Cancer

Article Title: Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation

doi: 10.7150/jca.32167

Figure Lengend Snippet: Fenretinide-induced apoptosis is mediated by NR4A1 and the mitochondria-dependent pathway. a) NR4A1 mRNA expression is measured using qRT-PCR. HL-60 and Kasumi-1 cells are incubated with fenretinide for different time periods and at various concentrations. Cells are harvested to extract RNA, followed by subsequent reverse transcription into cDNA. b) HL-60 and Kasumi-1 cells are treated with 10 μM fenretinide for different time periods. Then, cell lysates re subjected to Western blotting to analyze NR4A1 and the mitochondria-mediated pathway-related proteins. All data represent the means of 3 independent experiments. c) Grey value of protein expression in b). d) HL-60 and Kasumi-1 cells are treated with fenretinide at various concentrations, and the processing method is the same as b). All data are representative of 3 independent experiments. e) Grey value of protein expression in d). f) The NR4A1 level is measured using Western blotting after HL-60 cells are transfected with siCTRL or siNR4A1 for 24 h. The blots represent the means of three independent experiments. g) HL-60 cells are transfected with siCTRL or siNR4A1 for 12 h and then treated with 10 μM fenretinide for 12 h. siCTRL is used as the negative control. Apoptotic cells are tested using flow cytometry. Data are presented as means ± SD. *p≤0.05; **p≤0.01. h) HL-60 cells are transfected with siCTRL or siNR4A1 for 12h and then treated with 10 μM fenretinide for 12 h. Later, cell lysates are collected for Western blotting to determine the expression of apoptosis-related proteins. i) Grey value of protein expression in h). All data are expressed as mean ± SEM of tests carried out in triplicate.

Article Snippet: Fenretinide was purchased from MedChem Express, dissolved in DMSO and stored at -80°C.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Reverse Transcription, Western Blot, Transfection, Negative Control, Flow Cytometry

Journal: Journal of Cancer

Article Title: Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation

doi: 10.7150/jca.32167

Figure Lengend Snippet: Fenretinide induces the translocation of NR4A1 into mitochondria and the transformation of Bcl-2. a) HL-60 cells are treated with 10 μM fenretinide in the presence or absence of LMB for 48 h. Cell lysates are then collected for Western blotting, so as to examine the expression of the cleaved PARP and caspase-3. β-actin is used as a loading control. b) Grey value of protein expression in a). c) HL-60 cells are treated with or without 10 μM fenretinide for 12 h, and then the mitochondria and nuclei are extracted to test the NR4A1 expression levels. PARP and porin are used as the loading controls. d) Grey value of NR4A1 expression in c). e) HL-60 cells are treated with 10 μM fenretinide for 12 h. Cell lysates are immunoprecipitated with an anti-Bcl-2 antibody (IP Bcl-2), and NR4A1 is detected using Western blotting. Inputs of cell lysates without IP process are used as the positive controls. f) HL-60 cells are treated with 10 μM fenretinide for 12 h. Immunofluorescence staining of Bcl-2 BH3 is performed as described in Materials and methods. The images shown are under 1000× magnification. Scale bars, 20 μm. The images are representative of three independent experiments.

Article Snippet: Fenretinide was purchased from MedChem Express, dissolved in DMSO and stored at -80°C.

Techniques: Translocation Assay, Transformation Assay, Western Blot, Expressing, Control, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of Cancer

Article Title: Fenretinide-induced Apoptosis of Acute Myeloid Leukemia Cells via NR4A1 Translocation into Mitochondria and Bcl-2 Transformation

doi: 10.7150/jca.32167

Figure Lengend Snippet: Fenretinide + ketoconazole is found to be active against mouse xenografts. NOD/SCID mice that are subcutaneously injected with HL-60 cells are given gavage of fenretinide + KETO, KETO alone or NS for 1 week. a) Comparison of tumor images. b) Tumor volume is compared among the fenretinide + KETO, KETO alone and NS groups. c) NR4A1 expression in tumors is analyzed through Western blotting. The blots are representative of three independent experiments. d) TUNEL assay is carried out according to the TUNEL apoptosis assay. The images shown are under 400× magnification. Scale bars, 20 μm.The number of apoptosis-positive cells is counted in five high-power fields (400× magnification), and the mean percentage of apoptotic cells is recorded. **p<0.001.

Article Snippet: Fenretinide was purchased from MedChem Express, dissolved in DMSO and stored at -80°C.

Techniques: Injection, Comparison, Expressing, Western Blot, TUNEL Assay, Apoptosis Assay

Journal: iScience

Article Title: A transcriptome-based risk model in sepsis enables prognostic prediction and drug repositioning

doi: 10.1016/j.isci.2024.111277

Figure Lengend Snippet: Identified fenretinide (Fen) and meloxicam (Melo) as potential sepsis therapeutics (A) Based on DEGs between HR and LR subgroups, the top 20 potential agents were screened via L1000FWD for sepsis therapy. (B) Schematic of the animal experimental design. Following drug administration for three consecutive days, LPS or PBS (Control) was given by intraperitoneal injection. For survival test, mice were monitored every 12 h till 72 h. For cytokines detection, blood biochemistry, and HE staining, samples were collected after 6 h and 24 h of LPS injection. (C) Survival rates of LPS, Fen-treated, and Melo-treated septic mice were recorded over 72 h. (D) At the 6-h time point, the levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in plasma were detected by Elisa tests. (E–G) At the 12-h time point, blood biochemistry tests were performed for (E) kidney (BUN and CREA), (F) heart (LDH), and (G) liver (ALT and AST) function. (H) The HE staining of lung, liver, kidney, and spleen tissues, with a red arrow indicating pathological areas. Magnification: 100×. Size bar: 100 μm. All results were represented as means with error bars representing SEM ( n = 3). Statistical significance was calculated by ANOVA using Dunnett’s test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The candidate drugs fenretinide (30 mg/kg, TargetMol T1872) and meloxicam (30 mg/kg, TargetMol T0826 ) were at 48h, 24h, and 0h before LPS injection.

Techniques: Control, Injection, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: A transcriptome-based risk model in sepsis enables prognostic prediction and drug repositioning

doi: 10.1016/j.isci.2024.111277

Figure Lengend Snippet:

Article Snippet: The candidate drugs fenretinide (30 mg/kg, TargetMol T1872) and meloxicam (30 mg/kg, TargetMol T0826 ) were at 48h, 24h, and 0h before LPS injection.

Techniques: Recombinant, SYBR Green Assay, Software

Journal: The Journal of Clinical Investigation

Article Title: Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity

doi: 10.1172/JCI66413

Figure Lengend Snippet: (A) AM580 (100 μM) and 4-HPR (100 μM) specifically evoked [Ca2+]i responses in HEK293T cells transfected with recombinant TRPV1. Neither retinoid had an effect on HEK293T cells transfected with TRPA1, TRPV3, or TRPM8. The y axis refers to a net increase of relative fluorescence units (RFU) induced by AM580 and 4-HPR after subtraction of the baseline response to vehicle alone. (B) Left: representative traces showing that AM580-activated TRPV1-mediated current in a concentration-dependent manner. Right: representative current–voltage (I–V) curves taken at specified time points from the traces on the left illustrate that an outwardly rectifying whole-cell current was evoked by AM580 at 1 μM (b), whereas 10 μM (c) AM580 activated a current with a linear I-V relationship in a TRPV1-expressing HEK293T cell (a refers to the baseline response). (C) An endogenous retinoid, 9-cis-RA, also activated TRPV1 current in a concentration-dependent manner similar to that activated by AM580 (a refers to the baseline response; b and c indicate responses activated by 10 and 30 μM 9-cis-RA, respectively). (D) Both AM580 (10 μM, n = 5) and 9-cis-RA (10 μM, n = 4) increased single channel open probability (nPo) in inside-out patches excised from TRPV1-expressing HEK293T cells (*P < 0.05). (E) Synthetic retinoids activate TRPV1 in a concentration-dependent manner. (F) Summary of effects of selected endogenous retinoids on TRPV1-expressing cells. Currents were recorded at a membrane potential of –60 mV. n = 5–10 per concentration for all retinoids examined.

Article Snippet: β-carotene, and all trans-retinal, retinol, acitretin, capsaicin, and AMG9810 were purchased from Sigma-Aldrich; 9-cis-retinoic acid and 13-cis-retinoic acid were from MP Biomedicals LLC; AM580 was from Tocris; all trans-retinoic acid and 4-HPR were from Enzo Bioscience; bexarotene was from CT-BEX ChemieTek; tazarotene was from Selleckchem; AITC was from ACROS; AGN 193109 was from Santa Cruz Biotechnology Inc.; papain and collagenase (type 2) were from Worthington.

Techniques: Transfection, Recombinant, Fluorescence, Concentration Assay, Expressing

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Acquired resistance upon long-term 4-HPR exposure . (A, left) R cell lines were continuously exposed to different 4-HPR concentrations (0.5, 3, 5, and 10 μM; cell lines named R0.5, R3, R5, and R10, respectively) and resistance verified by comparison to 48 h drug-withdrawn R cells. Data are the average ± SD of three independent experiments performed in quadruplicates, n = 12. (A, right) Parental CCRF-CEM cells and developed R cell lines were exposed to 10 μM 4-HPR and viability estimated after 48 h of drug exposure. (n = 12). ** P < 0.01; ANOVA plus Tamhane post-hoc analysis. (B) Comparative cell proliferation was determined by BrdU labeling as specified in Materials and Methods. Data are average ± SD of four independent experiments. * P < 0.05; Kruskal Wallis and Mann-Whitney test for pair-wise comparison.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Comparison, Labeling, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Effect of selected insults on the viability of 4-HPR-sensitive and resistant leukemia cell lines . 4-HPR-sensitive (CCRF-CEM) and derived resistant cell lines (R0.5 and R10) were exposed to several drugs or UV radiation, and cytotoxicity tested after 48 h by the XTT assay. Data are average ± SD of three independent experiments performed in quadruplicates (n = 12); * P < 0.05, ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Derivative Assay, XTT Assay

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative endogenous SL pattern in 4-HPR-sensitive and resistant leukemia cell lines . Cell were grown with (R cell lines) or without (CCRF-CEM) 4-HPR. Endogenous TOTAL (sum of the different fatty acid chain-length species) dhCer (A, left panel) and Cer (A, right panel) levels, as well as dhSph (B), TOTAL GluCer, and LactCer (C) levels were determined by LC/MS. Data are average ± SD of four (A, B) or two (C) independent experiments. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane or Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative analysis of dihydroceramide desaturase (DES) activity . Cells were incubated with the unnatural pyridinium dhCer (C12-PyrdhCer) alone or with 4-HPR (5 μM) for the selected time points and conversion into C12-PyrCer measured by LC/MS. Data are average ± SD of two independent values. ** P < 0.01; ANOVA plus Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative SL profile analysis upon drug withdrawal . R10 cells were incubated in 4-HPR-free medium for 48 h (WD = without drug) or long-term (~3 weeks) and endogenous SL profiles analyzed by LC-MS. Significance was based on R10 values. Data are average ± SD of 2-4 independent values. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane or Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Effect of drug withdrawal on cell resistance . (A) Parental sensitive CCRF-CEM cells, resistant R10, and long-term drug withdrawn resistant (R10 longWD) cells were treated and cell death determined by annexin V-FITC/propidium iodide staining at the selected time points. Values represent the percentage of cells in each quadrant. Representative data from two independent experiments are shown. (B) A portion of each sample for SL profile analysis (Figure 5) was saved for XTT assay estimation of resistance. Briefly, R10 cells after 48 h and long-term drug withdrawal were subjected to 48 h treatment with 3 or 10 μM 4-HPR. All data were related to the corresponding untreated cells. Statistical analysis was related to R10 data. Data are average ± SD of 12 values from to three independent experiments. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Staining, XTT Assay

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Analysis of 4-HPR-mediated alterations in dhSph and Sph phosphorylation . Externally added unnatural dhSph (17C-dhSph) and Sph (17C-Sph) analogues were used to estimate the secondary effects of 4-HPR-induced DES inhibition. CCRF-CEM cells were exposed to 4-HPR (10 μM) for 1 h and unnatural 17C species added for an additional 30 min incubation, as described in Methods. The effect on endogenous SLs (A) and unnatural dhSph species (B,C) analyzed by LC/MS. Comparative exogenous and endogenous SL profiles among parental (CCRF-CEM) and resistant cell lines (R0.5, R10) (D, E) were analyzed by the same methodology. Data are average ± SD of an experiment performed in duplicate.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Phospho-proteomics, Analogues, Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Toxicity mediated by the combination of 4-HPR with SL modulators . Parental-sensitive (CCRF-CEM) and derived-resistant (R0.5, R5, R10) cells were co-treated with 4-HPR (1 μM), DHS (5 μM; unnatural dhSph analogue), PPMP (10 μM; glucosylceramide synthase inhibitor), SAF (4 μM; another unnatural dhSph analogue), and/or SKI-II (5-20 μM) and cytotoxicity estimated after 48 h exposure using the XTT assay. Data are average ± SD of at least two independent experiments performed in quadruplicate (n ≥ 8); ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Derivative Assay, XTT Assay

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: Chemical structure of 4HPR and its effect on EGF-induced neoplastic transformation and signal transduction in JB6 Cl41 cells. (A) Chemical structure of 4HPR. (B) 4HPR inhibits EGF-induced anchorage-independent growth of JB6 Cl41 cells. Data are shown as means ± standard deviation of values from three independent experiments and the asterisk indicates a significant (* P < 0.01) decrease in colony formation in cells treated with 4HPR compared with the DMSO-treated group. (C) Cytotoxic effects of 4HPR on JB6 Cl41 cells. An MTS assay was used after treatment of cells with 4HPR for 24 or 48h, respectively. (D) 4HPR inhibits signal transduction in JB6 Cl41 cells. After starvation in serum-free medium for 24h, cells were treated with 4HPR at the indicated concentration for 2h and then stimulated with EGF (20ng/ml) for 15min. Cells were then harvested and protein levels were determined by western blot analysis.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: Transformation Assay, Transduction, Standard Deviation, MTS Assay, Concentration Assay, Western Blot

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: 4HPR inhibits mTOR kinase activity by directly binding with mTOR. A, 4HPR inhibits mTOR in vitro kinase activity in a concentration-dependent manner. An inactive p70S6K (A) or inactive Akt1 protein (B) was used as the substrate, respectively, for in vitro kinase assays with active mTOR and 100 µM ATP. Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and detected by western blotting. (C) proposed binding model of 4HPR-mTOR. 4HPR is shown in stick model and protein residues are shown in line model. 4HPR formed hydrogen bonds with Ser2165 and Lys2187. Several residues, including Tyr2225, Val2240, Met2345, Leu2354 and Ile2356 displayed strong hydrophobic interactions with the major retinal part of 4HPR. The figures were generated with VMD and Maestro. (D and E) 4HPR binds directly to mTOR in vitro (D) and ex vivo (E). Sepharose 4B was used for binding and pull-down assays as described in section ‘Materials and methods’. Lane 1 is input control (mTOR protein standard); lane 2 is the negative control, indicating no binding between mTOR and Sepharose 4B beads; and lane 3 shows that mTOR binds with 4HPR-Sepharose 4B beads.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: Activity Assay, Binding Assay, In Vitro, Concentration Assay, Polyacrylamide Gel Electrophoresis, Western Blot, Generated, Ex Vivo, Negative Control

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: Effects of 4HPR on anchorage-independent growth and mTOR signaling in lung cancer cells. (A–D) 4HPR inhibits anchorage-independent growth in a panel of NSCLC cell lines, including A549 cells (A), H520 cells (B), H1650 cells (C) and HCC827 cells (D). Data are shown as means ± standard deviation and the asterisk indicates a significant (* P < 0.01) decrease in colony formation in cells treated with 4HPR compared with the dimethyl sulfoxide –treated group. (E and F) 4HPR inhibits mTOR signaling in A549 cells (E) and H520 cells (F). Cells were starved in serum-free medium for 24h, and then treated with 4HPR at the indicated concentration for 2h. After stimulation with EGF (20ng/ml) for 15min, cells were harvested and protein levels were determined by western blot analysis.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: Standard Deviation, Concentration Assay, Western Blot

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: 4HPR targets both mTORC1 and mTORC2. (A) Lysates of A549 cells were subjected to immunoprecipitation using a raptor antibody followed by western blotting using anti-mTOR and antiraptor. (B) Lysates of A549 cells were subjected to immunoprecipitation using a rictor antibody followed by western blotting using anti-mTOR and antiraptor.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Western Blot

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: Knockdown of mTOR in A549 cells decreases sensitivity to 4HPR. (A) Efficiency of mTOR shRNA in A549 cells. (B) Anchorage-independent growth of A549 cells transfected with mock shRNA or mTOR shRNA . (C) Sensitivity of A549 cells transfected with mock shRNA or mTOR shRNA to treatment with 4HPR.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: shRNA, Transfection

Journal: Carcinogenesis

Article Title: Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo

doi: 10.1093/carcin/bgs234

Figure Lengend Snippet: Effect of 4HPR on lung cancer growth and mTOR targets in an A549 xenograft mouse model. (A) 4HPR significantly suppresses lung cancer cell growth. The average tumor volume of vehicle-treated control mice and 4HPR-treated mice plotted over 29 days after tumor cell injection. Data are shown as means ±standard error of measurements. The P values indicate a significant inhibition by 4HPR on tumor growth (*P < 0.05; **P < 0.01). (B) 4HPR has no effect on mouse body weight. Body weights from treated or untreated groups of mice were measured once a week. (C) H&E staining and immunohistochemical analysis of tumor tissues. Treated or untreated groups of mice were euthanized and tumors extracted. Lung cancer tissue slides were prepared with paraffin sections after fixation with formalin and then stained with H&E or antiphospho-p70S6K (T389). (D) quantification of expression of phospho-p70S6K (T389) in tumor tissues using the Image-Pro Plus software program and data are shown as integrated optical density units. (E) 4HPR inhibits mTOR-targeted protein expression in A549 lung cancer tissues. The tumor tissues from groups treated with vehicle, 10 mg 4HPR/kg body weight (B.W.), or 40 mg 4HPR/kg B.W. were immunoblotted with antibodies to detect phosphorylated S6 (S235, S236). Expression of β-actin was used to verify equal protein loading.

Article Snippet: After culturing for various times, the cytotoxicity of 4HPR was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.

Techniques: Injection, Inhibition, Staining, Immunohistochemical staining, Expressing, Software