Acquired resistance to chemotherapeutic drugs is the primary cause of treatment failure in the clinic. While multiple factors contribute to this resistance, increased expression of ABC transporters—such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins—play significant roles in the development of resistance to various chemotherapeutics. We found that Erastin, a ferroptosis inducer, was significantly cytotoxic to NCI/ADR-RES, a P-gp-expressing human ovarian cancer cell line. Here, we examined the effects of both Erastin and RSL3 (Ras-Selected Ligand 3) on reversing Adriamycin resistance in these cell lines. Our results show that Erastin significantly enhanced Adriamycin uptake in NCI/ADR-RES cells without affecting sensitive cells. Furthermore, we observed that Erastin enhanced Adriamycin cytotoxicity in a time-dependent manner. The selective iNOS inhibitor, 1400W, reduced both uptake and cytotoxicity of Adriamycin in P-gp-expressing NCI/ADR-RES cells only. These findings were also confirmed in a BCRP-expressing human breast cancer cell line (MCF-7/MXR), which was selected for resistance to Mitoxantrone. Both Erastin and RSL3 were found to be cytotoxic to MCF-7/MXR cells. Erastin significantly enhanced the uptake of Hoechst dye, a well-characterized BCRP substrate, sensitizing MCF-7/MXR cells to Topotecan. The effect of Erastin was inhibited by 1400W, indicating that iNOS is involved in Erastin-mediated enhancement of Topotecan cytotoxicity. RSL3 also significantly increased Topotecan cytotoxicity. Our findings—demonstrating increased cytotoxicity of Adriamycin and Topotecan in P-gp- and BCRP-expressing cells—suggest that ferroptosis inducers may be highly valuable in combination with other chemotherapeutics to manage patients’ cancer burden in the clinical setting.

Erastin (ER) induces cell death through the formation of reactive oxygen species (ROS), resulting in ferroptosis. Ferroptosis is characterized by an accumulation of ROS within the cell, leading to an iron-dependent oxidative damage-mediated cell death. ER-induced ferroptosis may have potential as an alternative for ovarian cancers that have become resistant due to the presence of Ras mutation or multi-drug resistance1 (MDR1) gene expression. We used K-Ras mutant human ovarian tumor OVCAR-8 and NCI/ADR-RES, P-glycoprotein-expressing cells, to study the mechanisms of ER-induced cell death. We used these cell lines as NCI/ADR-RES cells also overexpresses superoxide dismutase, catalase, glutathione peroxidase, and transferase compared to OVCAR-8 cells, leading to the detoxification of reactive oxygen species. We found that ER was similarly cytotoxic to both cells. Ferrostatin, an inhibitor of ferroptosis, reduced ER cytotoxicity. In contrast, RSL3 (RAS-Selective Ligand3), an inducer of ferroptosis, markedly enhanced ER cytotoxicity in both cells. More ROS was detected in OVCAR-8 cells than NCI/ADR-RES cells, causing more malondialdehyde (MDA) formation in OVCAR-8 cells than in NCI/ADR-RES cells. RSL3, which was more cytotoxic to NCI/ADR-RES cells, significantly enhanced MDA formation in both cells, suggesting that glutathione peroxidase 4 (GPX4) was involved in ER-mediated ferroptosis. ER treatment modulated several ferroptosis-related genes (e.g., CHAC1, GSR, and HMOX1/OX1) in both cells. Our study indicates that ER-induced ferroptotic cell death may be mediated similarly in both NCI/ADR-RES and OVCAR-8 cells. Additionally, our results indicate that ER is not a substrate of P-gp and that combinations of ER and RSL3 may hold promise as more effective treatment routes for ovarian cancers, including those that are resistant to other current therapeutic agents.

Ferroptosis has emerged as a promising therapeutic strategy for drug-resistant cancers; however, the molecular mechanisms governing ferroptosis susceptibility in breast cancer remain poorly defined. Here, we have investigated distinct ferroptosis resistance mechanisms in parental MCF-7 breast cancer cells and their BCRP-overexpressing derivative, MCF-7/MXR. MCF-7/MXR cells displayed robust erastin (ER)-induced ferroptosis characterized by extensive lipid peroxidation, ROS accumulation, and suppression of the xCT–GSH–GPX4 axis. In contrast, MCF-7 cells were resistant to ER, exhibiting minimal lipid damage despite measurable ER-induced oxidative stress. We found that this resistance is mediated not by the canonical GPX4 pathway, but by a potent compensatory antioxidant system centered on the FSP1–CoQ10–NADPH axis. Pharmacological inhibition of FSP1 strongly sensitized MCF-7 cells to ER, with minimal effects in MXR cells. ER differentially regulated ferroptosis-associated genes, downregulating GPX4, and SLC7A11 in MXR cells but upregulating the GPX4 pathway in MCF-7 cells. Additionally, ER downregulated FSP1 and NQO1 in MCF-7 cells without affecting their expressions in MXR cells. This mechanistic divergence highlights that ferroptosis resistance in breast cancer is context-dependent and mediated by pathway-specific antioxidant programs. Co-targeting FSP1 and GPX4 therefore represents a rational strategy to overcome ferroptosis resistance in MCF-7-like breast cancers.

Detrimental side effects of drugs like doxorubicin, which can cause cardiotoxicity, pose barriers for preventing cancer progression, or treating cancer early through molecular interception. Extracellular vesicles (EVs) are valued for their potential as biomarkers of human health, chemical and molecular carcinogenesis, and therapeutics to treat disease at the cellular level. EVs are released both during normal growth and in response to toxicity and cellular death, playing key roles in cellular communication. Consequently, EVs may hold promise as precision biomarkers and therapeutics to prevent or offset damaging off-target effects of chemotherapeutics. EVs have promise as biomarkers of impending cardiotoxicity induced by chemotherapies and as cardioprotective therapeutic agents. However, EVs can also mediate cardiotoxic cues, depending on the identity and past events of their parent cells. Understanding how EVs mediate signaling is critical toward implementing EVs as therapeutic agents to mitigate cardiotoxic effects of chemotherapies. For example, it remains unclear how mixtures of EV populations from cells exposed to toxins or undergoing different stages of cell death contribute to signaling across cardiac tissues. Here, we present our perspective on the outlook of EVs as future clinical tools to mitigate chemotherapy-induced cardiotoxicity, both as biomarkers of impending cardiotoxicity and as cardioprotective agents. Also, we discuss how heterogeneous mixtures of EVs and transient exposures to toxicants may add complexity to predicting outcomes of exogenously applied EVs. Elucidating how EV cargo and signaling properties change during dynamic cellular events may aid precision prevention of cardiotoxicity in anticancer treatments and development of safer chemotherapeutics.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.

Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and high-risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. Particularly, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, where many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed association with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.