erastin and Carcinoma--Non-Small-Cell-Lung

Despite recent progress in non-small-cell lung cancer (NSCLC) treatment, treatment outcomes remain poor, mainly because of treatment resistance or toxicity. Erastin is a ferroptosis inducer that has shown promising cytotoxic effects in various types of cancers, including NSCLC. Celastrol is a triterpene extracted from the Tripterygium wilfordii that exhibits potential anticancer activity. However, the side effects of celastrol are severe and limit its clinical application. Combination therapy is a promising strategy to overcome the compensatory mechanisms and unwanted off-target effects. In the present study, we found that erastin synergized with celastrol to induce cell death at nontoxic concentrations. The combined treatment with celastrol and erastin significantly increased reactive oxygen species (ROS) generation, disrupted mitochondrial membrane potential, and promoted mitochondrial fission. Furthermore, cotreatment with erastin and celastrol initiated ATG5/ATG7-dependent autophagy, PINK1/Parkin-dependent mitophagy, and the expression of heat shock proteins (HSPs) in an HSF1-dependent manner. HSF1 knockdown further enhanced cell death in vitro and inhibited tumor growth in vivo. Our findings indicate that the combination of celastrol with erastin may represent a novel therapeutic regimen for patients with NSCLC and warrants further clinical evaluation.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Female; Ferroptosis; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mitochondrial Dynamics; Mitophagy; Pentacyclic Triterpenes; Piperazines; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Growing evidence confirms that ferroptosis plays an important role in tumor growth inhibition. However, some non-small-cell lung cancer (NSCLC) cell lines are less sensitive to erastin-induced ferroptotic cell death. Elucidating the mechanism of resistance of cancer cells to erastin-induced ferroptosis and increasing the sensitivity of cancer cells to erastin need to be addressed. In our experiment, erastin and acetaminophen (APAP) cotreatment inhibited NSCLC cell viability and promoted ferroptosis and apoptosis, accompanied with attenuation of glutathione and ectopic increases in lipid peroxides. Erastin and APAP promoted NSCLC cell death by regulating nucleus translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); and the ferroptosis induced by erastin and APAP was abrogated by bardoxolone methyl (BM) with less generation of reactive oxygen species and malondialdehyde. As a downstream gene of Nrf2, heme oxygenase-1 expression decreased significantly with the cotreatment of erastin and APAP, which could be rescued by BM. In vivo experiment showed that the combination of erastin and APAP had a synergic therapeutic effect on xenograft of lung cancer. In short, the present study develops a new effective treatment for NSCLC by synergizing erastin and APAP to induce ferroptosis.

Topics: Acetaminophen; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Drug Synergism; Ferroptosis; Gene Expression Regulation, Neoplastic; Heme Oxygenase-1; Humans; Lipid Peroxides; Malondialdehyde; Mice; NF-E2-Related Factor 2; Oleanolic Acid; Piperazines; Signal Transduction; Xenograft Model Antitumor Assays

Although ferroptosis has been recognized as a novel antitumoral treatment, high expression of nuclear factor erythroid 2-related factor 2 (NRF2) has been reported to be an antioxidant transcript factor that protects malignant cells from ferroptosis. Previous findings indicated that metallothionein 1D pseudogene (MT1DP), a long noncoding RNA (lncRNA), functioned to aggravate oxidative stress by repressing antioxidation. Here we aimed at assessing whether MT1DP could regulate erastin-induced ferroptosis on non-small cell lung cancer (NSCLC) and elucidating the mechanism. We found that ectopic expression of MT1DP sensitized A549 and H1299 cells to erastin-induced ferroptosis through downregulation of NRF2; in addition, ectopic MT1DP upregulated malondialdehyde (MDA) and reactive oxygen species (ROS) levels, increased intracellular ferrous iron concentration, and reduced glutathione (GSH) levels in cancer cells exposed to erastin, whereas downregulation of MT1DP showed the opposite effect. RNA pulldown assay and dual-luciferase reporter assay confirmed that MT1DP modulated the expression of NRF2 via stabilizing miR-365a-3p. As low solubility of erastin limits its efficient application, we further prepared folate (FA)-modified liposome (FA-LP) nanoparticles for targeted co-delivery of erastin and MT1DP to enhance the bioavailability and the efficiency of the drug/gene combination. Erastin/MT1DP@FA-LPs (E/M@FA-LPs) sensitized erastin-induced ferroptosis with decreased cellular GSH levels and elevated lipid ROS. In vivo analysis showed that E/M@FA-LPs had a favorable therapeutic effect on lung cancer xenografts. In short, our findings identify a novel strategy to elevate erastin-induced ferroptosis in NSCLCs acting through the MT1DP/miR-365a-3p/NRF2 axis.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Ferroptosis; Humans; Liposomes; Lung Neoplasms; Mice; NF-E2-Related Factor 2; Piperazines; Transfection

Ferroptosis is a new mode of regulated cell death, which is completely distinct from other cell death modes based on morphological, biochemical, and genetic criteria. This study evaluated the therapeutic role of ferroptosis in classic chemotherapy drugs, including the underlying mechanism.. Cell viabilitywas detected by using the methylthiazoltetrazlium dye uptake method. RNAiwas used to knockout iron-responsive element binding protein 2, and polymerase chain reaction, western blot was used to evaluate the efficiency. Intracellular reduced glutathione level and glutathione peroxidases activitywere determined by related assay kit. Intracellularreactive oxygen species levelswere determined by flowcytometry. Electron microscopywas used to observe ultrastructure changes in cell.. Among five chemotherapeutic drugs screened in this study, cisplatin was found to be an inducer for both ferroptosis and apoptosis in A549 and HCT116 cells. The depletion of reduced glutathione caused by cisplatin and the inactivation of glutathione peroxidase played the vital role in the underlying mechanism. Besides, combination therapy of cisplatin and erastin showed significant synergistic effect on their anti-tumor activity.. Ferroptosis had great potential to become a new approach in anti-tumor therapies and make up for some classic drugs, which open up a new way for their utility in clinic.

Topics: A549 Cells; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Death; Cisplatin; Colorectal Neoplasms; Fibrosarcoma; Glutathione Peroxidase; HCT116 Cells; Humans; Lung Neoplasms; Neoplasms; Piperazines

The tumour‑suppressor protein p53 is a key regulator of multiple cellular processes and exerts its tumour‑suppressor function by inducing apoptotic cell death. However, emerging evidence indicates that p53 is also involved in inducing ferroptosis, which is a unique iron‑dependent form of non‑apoptotic cell death triggered by the RAS‑selective lethal small molecule erastin. Previous studies have shown that erastin exposure induces increased ROS accumulation and oxidative stress. In the present study, we incubated A549 cells with erastin and detected ROS accumulation. Semi‑quantitative western blotting was performed to analyse the effect of the induced ROS on p53 activity. To determine how ROS activate p53, NAC, an ROS scavenger, and KU‑55933, an ATM kinase inhibitor, were employed to co‑incubate with erastin, followed by western blot analysis. Either p53 or SLC7A11 siRNA was introduced into A549 cells to silence the target‑gene expression, followed by ROS detection to illustrate the regulatory role of ROS‑activated p53 on its target gene SLC7A11. Annexin V‑FITC/PI staining was performed to detect the induction of apoptotic cell death by erastin exposure. To further assess the effects of erastin treatment on cellular proliferation, EdU staining and cell cycle flow cytometric analysis were performed. Erastin exposure upregulated and activated p53 and thus, transcriptionally activated its downstream target genes, including p21 and Bax, in lung cancer A549 cells dependent on erastin‑induced ROS. Subsequently, activated p53 by erastin treatment suppressed SLC7A11 and induced ROS accumulation, indicating the potential feedback loop between p53 and erastin‑induced ROS. By employing the caspase inhibitor Z‑VAD‑FMK, it was revealed that erastin‑induced p53 contributed to both ferroptotic and apoptotic cell death and inhibited cell proliferation via arresting the cell cycle at G1 phase. Collectively, these results indicated that p53 may contribute to the cytotoxic and cytostatic effects associated with establishing a feedback loop with ROS induced by erastin.

Topics: Amino Acid Transport System y+; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Piperazines; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53