Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: F12 medium sensitizes lung cancer cells to iron-dependent lipid peroxidation . ( A, B ) Quantification by flow cytometry of oxidized BODIPY-C11 fluorescence in A549 (A) or H838 (B) cells that were cultured in RPMI or F12 medium and treated with 100 μM BSO or vehicle for 24 h. ( C ) Dose response curves for F12-cultured A549 and H838 cells treated with BSO in combination with 5 μM liproxstatin-1 (LIP-1), 5 μM ferrostatin-1 (FER-1) or 50 μM α-tocopherol (α-toco) for 72 h. ( D ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM deferoxamine (DFO) for A549 cells and 9 μM for H838 cells for 72 h. ( E ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM necrostatin-1 (NEC-1) or 10 μM ZVAD-FMK (ZVAD) for 72 h. ( F ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 4 μg/mL certolizumab (CER), 15 μM CU-CPT4a (C4a), 3 μM resatorvid (RST), or 50 μM necrostatin-1 (NEC-1) for 72 h (ND, not done). Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001.

Article Snippet: Drugs used were auranofin (A6733; Sigma-Aldrich), α-tocopherol (T3251; Sigma-Aldrich), bafilomycin A1 (SML1661; Sigma-Aldrich), certolizumab pegol (HYP9953; MedChemExpress), chloroquine (C6628; Sigma-Aldrich), CU-CPT4A (HY-108473; MedChemExpress), deferoxamine (D9533; Sigma-Aldrich), erastin (e7781; Sigma Aldrich), FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (HY-100410; MedChemExpress), ferrostatin-1 (SML0583; Sigma-Aldrich), l -buthionine-sulfoximine (b2515; Sigma-Aldrich), mito-TEMPO (HY–W001187; MedChemExpress), necrostatin-1 (N9037; Sigma-Aldrich), liproxstatin-1 (SML1414; Sigma-Aldrich), oligomycin (HY–N6782; MedChemExpress), puromycin dihydrochloride (A1113803; Gibco), rotenone (HY–B1756; MedChemExpress), RSL3 (SML2234; Sigma-Aldrich), resatorvid (HY-11109; MedChemExpress), and Z-VAD-FMK (V116; Sigma-Aldrich).

Techniques: Flow Cytometry, Fluorescence, Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Dual-Action Theranostic Nanoparticles Delivering Toll-Like Receptor 2 Inhibitors and Chemotherapy Target Breast Cancer Cells and the Tumor Microenvironment

doi: 10.2147/IJN.S556284

Figure Lengend Snippet: Schematic representation of the NP formulations. The PLGA NPs (left) consist of a PLGA matrix with embedded PEGylated phospholipids, some functionalized with a maleimide moiety for conjugation with targeting cyclic RGD peptides. The system also incorporates the therapeutic agent CU-CPT22, fluorescent probes (carboxyfluorescein or cyanine 5), and the MRI contrast agent Gd-DOTAMA. The liposome formulation (right) features a lipid bilayer formed by POPC and PEGylated phospholipids, also functionalized with maleimide moieties for cyclic RGD conjugation. Cholesterol is included to modulate membrane stability. Doxorubicin is encapsulated as the therapeutic agent, while fluorescent probes and the MRI contrast agent Prohance are integrated for imaging purposes.

Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000] (ammonium salt) (DPPE-PEG2000-methoxy), (1,2-dioleoyl-sn-glycero-3-poshosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (DOPE-CF) and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids Inc. CU-CPT22 was purchased from Tocris Bioscience.

Techniques: Conjugation Assay, Formulation, Membrane, Imaging

Journal: International Journal of Nanomedicine

Article Title: Dual-Action Theranostic Nanoparticles Delivering Toll-Like Receptor 2 Inhibitors and Chemotherapy Target Breast Cancer Cells and the Tumor Microenvironment

doi: 10.2147/IJN.S556284

Figure Lengend Snippet: PLGA-CU inhibits TLR2 signaling. ( A ) Representative FACS histogram of the expression of TLR2 and α v β 3 integrin on 4T1 and WT-874 cells. Light grey histograms show the background signal obtained by staining cells with an isotype control antibody; dark grey histograms show the signal obtained by staining with the specific monoclonal antibodies. ( B ) FACS analysis of PLGA-CU internalization into 4T1 and WT-874 cells incubated for 4 h at 37° C with 5 µM FITC-containing PLGA-CU or untargeted PLGA-CU, ie PLGA-CU not decorated with RGD. The graphs report the individual values and means ± SEM of the FITC MFI measured from two independent experiments. Representative FACS histograms are shown. ( C ) FACS analysis of PLGA-CU internalization into 4T1 and WT-874 cells incubated for 1 h at 37° C or 4° C with 5 µM FITC-containing PLGA-CU. An aliquot of cells was subjected to acidic stripping to remove membrane-bound PLGA-CU before analysis. The graphs report the individual values and means ± SEM of the FITC MFI measured from two independent experiments. Representative FACS histograms are shown. ( D ) FACS analysis of p65 NF-κB phosphorylation in WT-874 cells treated with PGN-SA (200 µg/mL) ± 5 µM CU-CPT22 or PLGA-CU for 24 h or left untreated. The individual values and means ± SEM of mean fluorescence intensity (MFI) from three independent experiments are reported, as well as representative FACS histograms where light grey histograms show the signal obtained with an isotype control antibody, dark grey histograms with the specific monoclonal antibody. **P < 0.01; ***P < 0.001, ****P < 0.0001, One-Way ANOVA.

Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000] (ammonium salt) (DPPE-PEG2000-methoxy), (1,2-dioleoyl-sn-glycero-3-poshosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (DOPE-CF) and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids Inc. CU-CPT22 was purchased from Tocris Bioscience.

Techniques: Expressing, Staining, Control, Bioprocessing, Incubation, Stripping Membranes, Membrane, Phospho-proteomics, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Dual-Action Theranostic Nanoparticles Delivering Toll-Like Receptor 2 Inhibitors and Chemotherapy Target Breast Cancer Cells and the Tumor Microenvironment

doi: 10.2147/IJN.S556284

Figure Lengend Snippet: PLGA-CU and LIPO-DOXO impair breast cancer cell viability. MTT assays on 4T1 and WT-874 cell lines treated for 48 h with ( A ) empty PLGA, PLGA-CU or free CU-CPT22 or ( C ) empty liposome, LIPO-DOXO or free doxorubicin at various concentrations. Cell viability of the same cell lines treated with ( B ) 5 μM PLGA-CU or CU-CPT22 for 48 h with or with ( D ) 1 μM LIPO-DOXO or doxorubicin, or with empty NPs (N = 3 independent experiments). ( E – G ) 4T1, WT-879, and MDA-MB-231 cells were treated with PLGA-CU, LIPO-DOXO or the free drugs for 48 h and analyzed using Annexin-V/propidium iodide FACS staining. Representative density plots are shown. The graphs show individual values and means ± SEM of the % of Annexin-V + from 6 independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001, ****P < 0.0001, One-Way ANOVA.

Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000] (ammonium salt) (DPPE-PEG2000-methoxy), (1,2-dioleoyl-sn-glycero-3-poshosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (DOPE-CF) and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids Inc. CU-CPT22 was purchased from Tocris Bioscience.

Techniques: Staining

Journal: International Journal of Nanomedicine

Article Title: Dual-Action Theranostic Nanoparticles Delivering Toll-Like Receptor 2 Inhibitors and Chemotherapy Target Breast Cancer Cells and the Tumor Microenvironment

doi: 10.2147/IJN.S556284

Figure Lengend Snippet: PLGA-CU prevents doxorubicin-induced NF-kB activation. ( A ) FACS analysis of p65 NF-κB phosphorylation in WT-874 cells treated with 5 µM CU-CPT22 or PLGA-CU and/or 1 µM LIPO-DOXO or doxorubicin for 48 h, or left untreated. The individual values and means ± SEM of mean fluorescence intensity (MFI) from three independent experiments are reported, as well as representative FACS histograms where light grey histograms show the signal obtained with an isotype control antibody, dark grey histograms with the specific monoclonal antibody. ( B and C ) Representative Western blot analysis of phosphorylated and total p65 NF-κB in 4T1 and MDA-MB-231 cells treated as in ( A ). The graphs report the individual values and means ± SEM of the fold increase in NF-κB phosphorylation in treated samples as compared to control cells cultured in medium from three independent experiments. The black dotted line indicates the baseline activation level of control (medium) cells. ( D and E ) ELISA of IL-6 and TGF-β released into the supernatant of 4T1 cells cultured for 48 h as described in ( A ). ( F and G ) Representative images of tumorspheres (Magnification 10X), and graph showing sphere-generating ability (number of tumorspheres generated every 10 3 plated cells) of 4T1 cells cultured for 5 days in tumorsphere-forming conditions in the absence or presence of IL-6 (10 ng/mL) or PGN-SA (200 µg/mL). ( H ) Means ± SEM of the percentage of Sca1 + cells in 4T1 tumorspheres treated as in ( F ). ( I ) MTT assays on 4T1 cells treated for 48 h with IL-6 (10 ng/mL) or PGN-SA (200 µg/mL). The black dotted line represents the viability of the control condition, normalized to 100%. Deviations above or below this line indicate an increase or decrease in cell viability relative to the control. In all panels, N = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA.

Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000] (ammonium salt) (DPPE-PEG2000-methoxy), (1,2-dioleoyl-sn-glycero-3-poshosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (DOPE-CF) and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids Inc. CU-CPT22 was purchased from Tocris Bioscience.

Techniques: Activation Assay, Phospho-proteomics, Fluorescence, Control, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Generated

Journal: International Journal of Nanomedicine

Article Title: Dual-Action Theranostic Nanoparticles Delivering Toll-Like Receptor 2 Inhibitors and Chemotherapy Target Breast Cancer Cells and the Tumor Microenvironment

doi: 10.2147/IJN.S556284

Figure Lengend Snippet: PLGA-CU impairs angiogenesis. ( A ) Individual values and means ± SEM of the % of CD31 + cells and ( B ) representative CD31 IHC staining of tumors. Magnification X20. ( C – E ) ELISA analysis of VEGF released into the supernatant by 4T1, MDA-MB-231 or Bend.3 cells treated with PLGA-CU and/or LIPO-DOXO or free CU-CPT22 or doxorubicin for 48 h or left untreated. The graphs show individual values and means ± SEM of VEGF concentration from three independent experiments. ( F ) bEnd.3 cells were treated as in E, then apoptosis was analyzed. The graph shows individual values and means ± SEM of the % of Annexin-V + from three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001, ****P < 0.0001, Two-Way ( A ) or One-Way ANOVA.

Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2000-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-col)-2000] (ammonium salt) (DPPE-PEG2000-methoxy), (1,2-dioleoyl-sn-glycero-3-poshosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (DOPE-CF) and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar Lipids Inc. CU-CPT22 was purchased from Tocris Bioscience.

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Concentration Assay