Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Combination of erastin and celastrol induced NSCLC cell death in vitro . (A) HCC827, A549 and H1299 cells were treated with erastin at the indicated concentrations for 24 h, and cell growth was assayed by a CCK‐8 assay. (B) HCC827, A549 and H1299 cells were treated with celastrol at the indicated concentrations for 24 h, and cell growth was assessed by a CCK‐8 assay. (C) HCC827, A549 and H1299 cells were treated with indicated combinations of concentrations of erastin and celastrol for 24 h. Cell viability was measured by a CCK‐8 assay. (D) Cell death was measured by a PI assay using flow cytometry. (E) Combination index (CI) analyses were performed to determine synergy using the Calcusyn software. (F) Representative phase‐contrast microscopic images of HCC827 cells treated with either erastin or celastrol or their combination for 24 h. Most of the cells became smaller in size and round in shape after cotreatment with celastrol and erastin for 24 h. Scale bars, 200 µ m . (G) Cell morphology was examined by quantitative holographic phase microscopy. The scale bar indicates the cell height. Scale bars, 200 µ m . (H) Representative images of colony formation of HCC827 cells after exposure to either erastin or celastrol, or in combination for 24 h. The mean ± SD is shown, n = 3. Statistical significance was determined using two‐tailed unpaired Student’s t ‐tests. *** P < 0.001. n.s., not significant.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Software, Microscopy, Two Tailed Test

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Combination index (CI) values calculated for erastin and celastrol synergy experiment.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques:

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Combination of erastin and celastrol failed to induce apoptosis or ferroptosis. (A) HCC827 cells were treated with erastin and celastrol for 24 h and analyzed using PI/Annexin V‐FITC flow cytometry. (B) HCC827 cells were cotreated with erastin and celastrol in the absence or presence of ferroptosis inhibitor (Fer‐1) or apoptosis inhibitor (Z‐VAD‐FMK) for 24 h. Cell viability was assessed by a CCK‐8 assay. (C) Cleavage of caspase‐3 and expression of Bax and Bcl‐2 were detected by western blotting. (D) Lipid ROS generation was assessed by flow cytometry using C11‐BODIPY. HCC827 cells were treated with either erastin or celastrol or their combination for 24 h. (E) Cellular iron levels were assessed by flow cytometry using Calcein‐AM. HCC827 cells were treated as described in D. (F) HCC827 cells were cotreated with celastrol and erastin in the absence or presence of FAC, DFO, or DFX for 24 h. Cell viability was measured by a CCK‐8 assay (G) Western blot analysis showed that erastin and celastrol cotreatment up‐regulated the expression of TFR1, FPN1, FTH, and FTL in HCC827 cells. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way ANOVA with Tukey’s post hoc test. * P < 0.05, *** P < 0.001. n.s., not significant.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

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

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Cotreatment with celastrol and erastin induced ROS‐dependent autophagy. (A) HCC827 cells were cotreated with celastrol and erastin for 24 h. Cells were stained with DCFH‐DA, and the fluorescence intensity was examined by flow cytometry. (B) HCC827 cells were cotreated with erastin and celastrol in the presence or absence of NAC (0.5 m m ) for 24 h, and cell viability and cell death were measured by CCK‐8 and PI assays. (C) Representative images of LC3II puncta formation after treatment with either erastin or celastrol or their combination for 24 h. Scale bars, 10 µ m . (D) Autophagy‐related proteins, including ATG5, ATG7, p62, Beclin‐1, and LC3, were detected by western blotting after cotreatment with celastrol and erastin for 24 h. (E) HCC827 cells were cotreated with erastin and celastrol in the presence or absence of NAC for 24 h. Autophagy‐related proteins, including ATG5, ATG7, p62, Beclin‐1, and LC3‐I/II, were detected by western blotting. (F) HCC827 cells were treated as described in E. Representative images of LC3‐II puncta formation are presented. Scale bars, 10 µ m . (G) Western blotting demonstrated the knockdown of ATG5 or ATG7 prevented up‐regulation of autophagy‐related genes. (H) Representative images of LC3‐II puncta formation in SCR, ATG5‐KD, and ATG7‐KD cells. All the indicated cells were treated with erastin and celastrol for 24 h. Scale bars, 10 µ m . (I) CCK‐8 and PI assays showing that ATG5 or ATG7 knockdown mitigated cell death induced by cotreatment with celastrol and erastin. (J) ATG5 knockout in HCC827 cells. (K, L) ATG5 knockout substantially alleviated erastin‐ and celastrol‐induced cell death. (M) Representative images of colony formation of SCR and ATG5‐KO cells after erastin and celastrol cotreatment. (O) Western blotting showing overexpression of ATG5 enhanced LC3‐II up‐regulation. (P) Overexpression of ATG5 potentiated cell death induced by cotreatment with celastrol and erastin. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way or two‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Staining, Fluorescence, Flow Cytometry, CCK-8 Assay, Western Blot, Knockdown, Knock-Out, Over Expression

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Cotreatment with celastrol and erastin induced mitochondrial dysfunction in HCC827 cells. (A) Mitochondrial ROS generation was assessed using flow cytometry using the MitoSOX™ Red mitochondrial superoxide indicator. (B) Mitochondrial membrane potential was determined after treatment. Cells were stained with a Rhodamine‐123 probe and analyzed by flow cytometry. (C) Mitochondrial mass was assessed by flow cytometry after staining with the MitoTracker Green probe. (D) Mitochondrial copy number was examined by real‐time PCR. (E) TEM was used to indicate the mitochondrial morphology. Intact cristae and normal morphological characteristics were observed in HCC827 cells. Swollen mitochondria with fractured cristae appeared in celastrol‐ and erastin‐treated cells. M, mitochondria. Scale bars, 0.5 µ m . (F) Flow cytometry to distinguish populations with normal membrane potential (healthy mitochondria) from those with reduced membrane potential (damaged mitochondria) among HCC827 cell populations. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Flow Cytometry, Membrane, Staining, Real-time Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Cotreatment with celastrol and erastin induced PINK1/Parkin‐dependent mitophagy. (A) Colocalization of p62 and TOM20 was visualized under confocal microscopy. Representative fluorescent images of cells immunostained with p62 and TOM20 after erastin and celastrol cotreatment for 24 h are shown. The yellow dots in the merge panel indicated the activation of mitophagy. Scale bars, 10 µ m . (B) Western blotting was used to measure the protein levels of PINK1 and Parkin in HCC827 cells treated with celastrol and erastin for 24 h. (C) HCC827 cells were cotreated with erastin and celastrol in the presence or absence of NAC for 24 h. The expression levels of PINK1 and Parkin were detected by western blotting. (D) Western blotting analysis of PINK1 and Parkin in the cytosolic and mitochondrial fractions of HCC827 cells treated with erastin and celastrol for 24 h. (E) Western blotting analysis of the effect of PINK1 knockdown on the expression of Parkin. (F) Colocalization of p62 and TOM20 was reduced in PINK1‐KD cells. Representative fluorescent images of cells immunostained with p62 and TOM20 after cotreatment with celastrol and erastin for 24 h are shown. Scale bars, 10 µ m . (G) Ubiquitination levels of protein in the cytosolic and mitochondrial fractions in SCR and PINK1‐KD cells were determined by western blotting. (H, I) Cell viability and cell death were measured by CCK‐8 and PI assays. The mean ± SD is shown, n = 3. Statistical significance was determined using two‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Confocal Microscopy, Activation Assay, Western Blot, Expressing, Knockdown, Ubiquitin Proteomics, CCK-8 Assay

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Cotreatment with celastrol and erastin induced DRP1‐mediated mitochondrial fission. (A) Representative fluorescence photographs of mitochondria following erastin and celastrol cotreatment for 24 h. Mitochondrial morphology was analyzed by TOM20 staining. Scale bars, 10 µ m . (B) Western blotting analysis of mitochondrial fission proteins (DRP1, OPA1, FIS1, and MFF) and mitochondrial fusion proteins (MFN1 and MFN2) in HCC827 cells after cotreatment with celastrol and erastin for 24 h. (C) The cytosolic and mitochondrial proteins were separately extracted. The expression levels of DRP1, FIS1, and OPA1 were evaluated by western blotting. TOM20 and HSP90 were used as the loading controls for mitochondria and cytosol, respectively. (D) Cotreatment enhanced the interaction between DRP1 and FIS1. HCC827 cells were treated as described in A. (E) SCR and DRP1‐KD cells were subjected to subcellular fractionation for the isolation of cytosol and mitochondria for western blotting. (F) Ubiquitination levels of protein in the cytosolic and mitochondrial fractions in SCR and DRP1‐KD cells were determined by western blotting analysis. (G, H) Knockdown of DRP1‐attenuated cell death induced by erastin and celastrol cotreatment. Cell viability and cell death were measured by CCK‐8 and PI assays (I) HCC827 cells were cotreated with erastin and celastrol in the presence or absence of Mdivi‐1 (20 µ m ) for 24 h. The mitochondrial morphology was analyzed by TOM20 staining. Scale bars, 10 µ m . (J) HCC827 cells were treated as described in H, and the expression of mitochondrial fission proteins (DRP1 and OPA1) and mitophagy proteins (PINK1 and Parkin) was examined by western blotting. (K) Cell viability and cell death were measured by CCK‐8 and PI assays. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way or two‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Fluorescence, Staining, Western Blot, Expressing, Fractionation, Isolation, Ubiquitin Proteomics, Knockdown, CCK-8 Assay

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Activation of p38 promotes mitochondrial fission induced by cotreatment with celastrol and erastin. (A) Western blotting showed that phosphorylation of p38 and DRP1 was increased by cotreatment with celastrol and erastin. (B) Cotreatment with celastrol and erastin enhanced the interaction between DRP1 and p38. Complexes coimmunoprecipitated using antiserum against DRP1 were blotted with p38 or DRP1 antibodies. (C) Inhibition of p38 alleviated mitochondrial fission. The expression of DRP1, OPA1, and FIS1 was examined by western blotting. (D) CCK‐8 assay demonstrated that inhibition of p38 by SB203580 or SB202190 further exacerbated the cell death induced by cotreatment with celastrol and erastin. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Expressing, CCK-8 Assay

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: HSF1 activation and HSP expression are associated with the resistance to cotreatment with celastrol and erastin. (A) Protein levels of HSP and HSF1 in cells exposed to erastin or/and celastrol for 24 h. (B) Western blotting showed that knockdown of HSF1 with shRNA inhibited up‐regulation of HSPs. (C) Inhibition of HSF1 by KRIBB11 reduced the up‐regulation of HSP expressions. The phosphorylation of HSF1 and protein levels of HSP was investigated by western blotting. (D) Knockdown of HSF1 promoted cell death induced by cotreatment with celastrol and erastin in HCC827 cells. Cell death was measured by CCK‐8 and PI assays (E) CCK‐8 and PI assays demonstrated that inhibition of HSF1 by KRIBB11 or triptolide exacerbated the cell death induced by cotreatment with celastrol and erastin. (F) Erastin and celastrol cotreatment significantly increased HSF1 phosphorylation at S326 and HSF1 nuclear translocation. Immunofluorescence staining using antibodies against p‐HSF1 S326 and HSF1 was observed using a confocal microscope. Scale bars, 10 µ m . (G) Nuclear translocation of HSF1 is enhanced by cotreatment with celastrol and erastin. HCC827 cells were fractionated into cytoplasmic and nuclear proteins. Lamin B1 and HSP90 were used as nuclear and cytoplasmic markers, respectively. (H) Cross‐linking experiment showed that cotreatment with celastrol and erastin promoted HSF1 trimer formation. (I) HSF1, BRG1, and Pol II recruitment to the HSP70 promoter was enhanced by cotreatment with celastrol and erastin. SCR and HSF1‐KD HCC827 cells were treated as described in A, and ChIP‐qPCR analyses were performed using HSF1, BRG1 and Pol II antibodies. The mean ± SD is shown, n = 3. Statistical significance was determined using one‐way or two‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, shRNA, Inhibition, Phospho-proteomics, CCK-8 Assay, Translocation Assay, Immunofluorescence, Staining, Microscopy, ChIP-qPCR

Journal: Molecular Oncology

Article Title: Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS–mitochondrial fission–mitophagy axis

doi: 10.1002/1878-0261.12936

Figure Lengend Snippet: Antitumor activity of erastin and celastrol cotreatment on xenograft models of NSCLC. (A) Nude mice were injected subcutaneously with indicated HCC827 cells. Images of isolated tumors from nude mice treated with single‐agent erastin (5 mg·kg −1 ), celastrol (1 mg·kg −1 ), and their combination. Tumor volumes (B) and weights (C) of HCC827 xenografts in each treatment group are shown. (D) Tumor lysates were subjected to western blotting for HSF1, HSP27, LC3, and Parkin. (E) H&E staining tissue images obtained from major organs of erastin or/and celastrol‐treated mice for the in vivo toxicity evaluation. Scale bar, 100 µ m . (F) HSF1 knockdown HCC827 cells were more sensitive to erastin and celastrol cotreatment in vivo . Representative images of subcutaneous tumors in mice inoculated with stable HSF1 knockdown HCC827 cells compared with the control group. Tumor volumes (G) and weights (H) of subcutaneous tumors in mice inoculated with stable HSF1 knockdown or SCR HCC827 cells. (I) Western blotting was performed to examine HSF1, HSP27, LC3, and Parkin protein expression in subcutaneous tumors. (J) Proposed mechanism of celastrol‐ and erastin‐induced cell death in NSCLC. The mean ± SD is shown, n = 6. Statistical significance was determined using one‐way or two‐way ANOVA with Tukey’s post hoc test. *** P < 0.001.

Article Snippet: Erastin (S7242), celastrol (S1290), PD98059 (S1177), Z‐VAD‐FMK (S7023), Ferrostatin‐1 (S1177), KRIBB11 (S8402), and NAC (S1623) were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activity Assay, Injection, Isolation, Western Blot, Staining, In Vivo, Knockdown, Control, Expressing