Hepatocellular carcinoma (HCC) is one of the paramount causes of cancer-related death worldwide. Despite recent advances have been made in clinical treatments of HCC, the general prognosis of patients remains poor. Therefore, it is imperative to develop a less toxic and more effective therapeutic strategy. Currently, series of cellular, molecular, and pharmacological experimental approaches were utilized to address the unrecognized characteristics of disulfiram (DSF), pursuing the goal of repurposing DSF for cancer therapy. We found that DSF/Cu selectively exerted an efficient cytotoxic effect on HCC cell lines, and potently inhibited migration, invasion, and angiogenesis of HCC cells. Importantly, we confirmed that DSF/Cu could intensively impair mitochondrial homeostasis, increase free iron pool, enhance lipid peroxidation, and eventually result in ferroptotic cell death. Of note, a compensatory elevation of NRF2 accompanies the process of ferroptosis, and contributes to the resistance to DSF/Cu. Mechanically, we found that DSF/Cu dramatically activated the phosphorylation of p62, which facilitates competitive binding of Keap1, thus prolonging the half-life of NRF2. Notably, inhibition of NRF2 expression via RNA interference or pharmacological inhibitors significantly facilitated the accumulation of lipid peroxidation, and rendered HCC cells more sensitive to DSF/Cu induced ferroptosis. Conversely, fostering NRF2 expression was capable of ameliorating the cell death activated by DSF/Cu. Additionally, DSF/Cu could strengthen the cytotoxicity of sorafenib, and arrest tumor growth both in vitro and in vivo, by simultaneously inhibiting the signal pathway of NRF2 and MAPK kinase. In summary, these results provide experimental evidence that inhibition of the compensatory NRF2 elevation strengthens HCC cells more vulnerable to DSF/Cu induced ferroptosis, which facilitates the synergistic cytotoxicity of DSF/Cu and sorafenib.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with limited treatment options. Cisplatin (DDP) is used as a mainstay of chemotherapeutic agents in combination with other drugs or radiotherapy for PDAC therapy. However, DDP exhibits severe side-effects that can lead to discontinuation of therapy, and the acquired drug resistance of tumor cells presents serious clinical obstacles. Therefore, it is imperative to develop a more effective and less toxic therapeutic strategy. We and others have previously discovered that dihydroartemisinin (DHA) represents a safe and promising therapeutic agent to preferentially induce cancer cell ferroptosis. In the present study, we find that DHA could intensively strengthen the cytotoxicity of DDP and significantly reduce its effective concentrations both in vitro and in vivo. Combination of DHA and DDP synergistically inhibits the proliferation and induces DNA damage of PDAC cells. Mechanically, the combinative treatment impairs mitochondrial homeostasis, characterized by destroyed mitochondrial morphology, decreased respiratory capacity, reduced ATP production, and accumulated mitochondria-derived ROS. Further studies show that ferroptosis contributes to the cytotoxic effects in PDAC cells under the challenge of DHA and DDP, together with catastrophic accumulation of free iron and unrestricted lipid peroxidation. Moreover, pharmacologic depleting of the free iron reservoir or reconstituted expression of FTH contributes to the tolerance of DHA/DDP-induced ferroptosis, while iron addition accelerates the ferroptotic cell death. In summary, these results provide experimental evidence that DHA acts synergistically with DDP and renders PDAC cells vulnerable to ferroptosis, which may act as a promising therapeutic strategy.

Axonal degeneration is a common pathological feature in many acute and chronic neurological diseases such as spinal cord injury (SCI). SARM1 (sterile alpha and TIR motif-containing 1), the fifth TLR (Toll-like receptor) adaptor, has diverse functions in the immune and nervous systems, and recently has been identified as a key mediator of Wallerian degeneration (WD). However, the detailed functions of SARM1 after SCI still remain unclear.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the fourth leading cause of cancer-related death globally, which is characterized by complicated pathophysiology, high recurrence rate, and poor prognosis. Our previous study has demonstrated that disulfiram (DSF)/Cu could be repurposed for the treatment of HCC by inducing ferroptosis. However, the effectiveness of DSF/Cu may be compromised by compensatory mechanisms that weaken its sensitivity. The mechanisms underlying these compensatory responses are currently unknown. Herein, we found DSF/Cu induces endoplasmic reticulum stress with disrupted ER structures, increased Ca²⁺ level and activated expression of ATF4. Further studies verified that DSF/Cu induces both ferroptosis and cuproptosis, accompanied by the depletion of GSH, elevation of lipid peroxides, and compensatory increase of xCT. Comparing ferroptosis and cuproptosis, it is interesting to note that GSH acts at the crossing point of the regulation network and therefore, we hypothesized that compensatory elevation of xCT may be a key aspect of the therapeutic target. Mechanically, knockdown of ATF4 facilitated the DSF/Cu-induced cell death and exacerbated the generation of lipid peroxides under the challenge of DSF/Cu. However, ATF4 knockdown was unable to block the compensatory elevation of xCT and the GSH reduction. Notably, we found that DSF/Cu induced the accumulation of ubiquitinated proteins, promoted the half-life of xCT protein, and dramatically dampened the ubiquitination-proteasome mediated degradation of xCT. Moreover, both pharmacologically and genetically suppressing xCT exacerbated DSF/Cu-induced cell death. In conclusion, the current work provides an in-depth study of the mechanism of DSF/Cu-induced cell death and describes a framework for the further understanding of the crosstalk between ferroptosis and cuproptosis. Inhibiting the compensatory increase of xCT renders HCC cells more susceptible to DSF/Cu, which may provide a promising synergistic strategy to sensitize tumor therapy and overcome drug resistance, as it activates different programmed cell death.

Background: CDGSH iron sulfur domain 2 (CISD2) is an iron–sulfur protein with a [2Fe–2S] cluster, which is critical for cell proliferation and iron homeostasis. It has been demonstrated that aberrant expression of CISD2 is associated with the progression of multiple cancers. However, the underlying mechanism of CISD2 in regulating tumorigenesis remains obscure. Methods: Bioinformatics strategies were used to investigate the protein interaction network and functional annotation of CISD2. In the functional experiment, cell viability was measured by CCK-8 kit. The levels of cellular reactive oxygen species (ROS), intracellular free iron, lipid peroxides, and lysosomal activity were determined by DCF-DA, RPA, C11-BODIPY, and cathepsin B staining, respectively. The glutathione (GSH) content was determined using a GSH assay kit. Results: We showed that knockdown of CISD2 significantly accelerated the Erastin-induced ferroptotic cell death with excess lipid peroxidation, GSH exhaustion, and iron accumulation, while overexpression of CISD2 hindered the sensitivity to Erastin. Further assays via confocal microscopy and western blot exhibited that CISD2 knockdown markedly enhanced the lysosomal activity, and activated ferritinophagy under the exposure of Erastin. Pharmacological inhibition of lysosomal function could inhibit the degradation of ferritin heavy chain (FTH), and attenuate the phenotypes of ferroptosis, such as accelerated iron accumulation and lipid peroxidation. Notably, we found that Erastin-induced compensatory elevation of nuclear factor erythroid 2-related factor 2 (NRF2) could be eliminated in CISD2 depletion cells. Mechanically, CISD2 knockdown promoted the degradation of autophagy adaptor p62 and resulted in an increased binding affinity of Keap1 with NRF2, thus leading to the increased ubiquitination and subsequent degradation of NRF2. Enforced expression of NRF2 reversed the sensitivity of shCISD2 cells to ferroptosis both in vitro and in vivo. Conversely, enforced expression of Keap1 exacerbated the degradation of NRF2, reduced the transcriptional expression of FTH and heme oxygenase 1 (HO-1), increased the oxidative damage, and thus further facilitated ferroptosis. Conclusion: Taken together, our current results illustrated two parallel mechanisms involved in the shCISD2-mediated ferroptosis. One was that shCISD2 enhanced the accumulation of free iron via ferritinophagy-dependent ferritin turnover; the other was that CISD2 depletion induced the inhibition of the p62–Keap1–NRF2 pathway, which resulted in oxidative stress and ferroptosis.