GSH exhaustion via inhibition of xCT-GSH-GPX4 pathway synergistically enhanced DSF/Cu-induced cuproptosis in myelodysplastic syndromes.

The clinical application of the therapeutic approach in myelodysplastic syndromes (MDS) remains an insurmountable challenge for the high propensity for progressing to acute myeloid leukemia and predominantly affecting elderly individuals. Thus, the discovery of molecular mechanisms underlying the regulatory network of different programmed cell death holds great promise for the identification of therapeutic targets and provides insights into new therapeutic avenues. Herein, we found that disulfiram/copper (DSF/Cu) significantly repressed the cell viability, increased reactive oxygen species (ROS) accumulation, destroyed mitochondrial morphology, and altered oxygen consumption rate. Further studies verified that DSF/Cu induces cuproptosis, as evidenced by the depletion of glutathione (GSH), aggregation of lipoylated DLAT, and induced loss of Fe-S cluster-containing proteins, which could be rescued by tetrathiomolybdate and knockdown of ferredoxin 1 (FDX1). Additionally, GSH contributed to the tolerance of DSF/Cu-mediated cuproptosis, while pharmacological chelation of GSH triggered ROS accumulation and sensitized cell death. The xCT-GSH-GPX4 axis is the ideal downstream component of ferroptosis that exerts a powerful protective mechanism. Notably, classical xCT inhibitors were capable of leading to the catastrophic accumulation of ROS and exerting synergistic cell death, while xCT overexpression restored these phenomena. Simvastatin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase, has beneficial effects in repurposing for inhibiting GPX4. Similarly, the combination treatment of DSF/Cu and simvastatin dramatically decreased the expression of GPX4 and Fe-S proteins, ultimately accelerating cell death. Moreover, we identified that the combination treatment of DSF/Cu and simvastatin also had a synergistic antitumor effect in the MDS mouse model, with the reduced GPX4, increased COX-2 and accumulated lipid peroxides. Overall, our study provided insight into developing a novel synergistic strategy to sensitize MDS therapy by targeting ferroptosis and cuproptosis.

Inhibiting the compensatory elevation of xCT collaborates with disulfiram/copper-induced GSH consumption for cascade ferroptosis and cuproptosis.

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.

Functional deficiency of succinate dehydrogenase promotes tumorigenesis and development of clear cell renal cell carcinoma through weakening of ferroptosis.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinomas, with high mortality and poor prognoses worldwide. Succinate dehydrogenase (SDH) consists of four nuclear-encoded subunits and it is the only complex involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Previous studies have shown decreased SDH activity in ccRCC. However, the role and underlying molecular mechanisms of SDH in ccRCC initiation and development remain unclear. In the present study, pan-cancer analysis of SDH gene expression was analyzed and the relationship between SDH gene expression and clinicopathological parameters was assessed using different databases. cBioPortal, UACLAN, and Tumor Immune Estimation Resource (TIMER) were subsequently utilized to analyze genetic alterations, methylation, and immune cell infiltration of SDH genes in ccRCC patients. We found SDHs were significantly downregulated in ccRCC tissues and correlated with ccRCC progression. Increased methylation and high SDH promoter mutation rates may be the cause of reduced expression of SDHs in ccRCC. Moreover, the interaction network showed that SDH genes were correlated with ferroptosis-related genes. We further demonstrated that SDH inhibition dampened oxidative phosphorylation, reduced ferroptotic events, and restored ferroptotic cell death, characterized by eliminated mitochondrial ROS levels, decreased cellular ROS and diminished peroxide accumulation. Collectively, this study provides new insights into the regulatory role of SDH in the carcinogenesis and progression of ccRCC, introducing a potential target for advanced antitumor therapy through ferroptosis.

CDC25C is a prognostic biomarker and correlated with mitochondrial homeostasis in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a common digestive tract malignant tumor with an extremely poor prognosis. The survival and prognosis may significantly improve if it is diagnosed early. Therefore, identifying biomarkers for early diagnosis is still considered a great clinical challenge in PAAD. Cell Division Cycle 25C (CDC25C), a cardinal cell cycle regulatory protein, directly mediates the G2/M phase and is intimately implicated in tumor development. In the current study, we aim to explore the possible functions of CDC25C and determine the potential role of CDC25C in the early diagnosis and prognosis of PAAD. Expression analysis indicated that CDC25C was overexpressed in PAAD . In addition, survival analysis revealed a strong correlation between the enhanced expression of CDC25C and poor survival in PAAD. Furthermore, pathway analysis showed that CDC25C is related to TP53 signaling pathways, glutathione metabolism, and glycolysis. Mechanically, our in vitro experiments verified that CDC25C was capable of promoting cell viability and proliferation. CDC25C inhibition increases the accumulation of ROS, inhibits mitochondrial respiration, suppresses glycolysis metabolism and reduces GSH levels. To summarize, CDC25C may be involved in energy metabolism by maintaining mitochondrial homeostasis. Our results suggested that CDC25C is a potential biological marker and promising therapeutic target of PAAD.

Significance of Common Blood Test Indexes in the Diagnosis and Prognosis of Multiple Myeloma.

BACKGROUND: The goal of this study is to explore the clinical value of routine tests in multiple myeloma (MM). METHODS: A total of 179 MM patients, newly diagnosed in our hospital from January 2010 to December 2018 (case group), as well as 352 cases of healthy individuals (control group) were evaluated. Albumin (Alb), globulin (Glb), albumin/globulin (A/G), creatinine (Cr), calcium (Ca), hemoglobin (Hb), lactate dehydrogenase (LDH), platelet count (Plt), and platelet distribution width (PDW) were compared between the analyzed groups. Respective tests were screened by forward selection. Thereafter, screened out indicators were identified through logistic regression analysis. Risk prediction nomogram, area under curve (AUC), calibration, decision curve analysis (DCA), and clinical impact curve (CIC) were further performed. At the same time, routine test indicators of MM patients for stage and subtype diagnosis, were compared. A correlation analysis between these test indicators and respective disease stages was performed. High stage group and low stage groups were subsequently compared to define the predictive value of single and combined indicators of disease severity. RESULTS: Except for Ca, the difference between the case and control groups for all other blood indicators was statistically significant (p < 0.05). Moreover, the difference in positive rate(s) was statistically significant (p < 0.05). The receiver operating characteristic (ROC) curve of Alb, Hb, and PDW harbored robust discrimination (AUC = 0.960) and appropriate calibration. The DCA and CIC showed that the resulting nomogram had a superior net benefit in predicting MM. Among all indicators, only LDH was statistically reduced in MM patients at ISS stages I, II, and III (p < 0.05). Interestingly, the ISS stage of respective MM patients was positively correlated with Cr (τ = 0.392), while it was negatively correlated with Hb (τ = -0.364). Alb, Glo, A/G, and Hb were significantly distinct between heavy chain (IgG, IgA) and LC, while few significant differences were found between the ISS stages. Lastly, the AUC (0.828) for Cr was greater than that for all other single and combined indicators. CONCLUSIONS: The effective application of major indicators measured in routine blood tests can provide important clues for the diagnosis and prognosis of MM.

FTH promotes the proliferation and renders the HCC cells specifically resist to ferroptosis by maintaining iron homeostasis.

BACKGROUND: Ferroptosis is a newly identified type of programmed cell death, which preferentially targets iron-rich cancer cells such as hepatocellular carcinoma (HCC). Ferritin heavy chain (FTH) is a major iron storing nanocage to store redox-inactive iron, and harbors ferroxidase activity to prevent the iron-mediated production of ROS. Our previous studies have demonstrated that FTH acts as a protective role to increase the cellular resistance to ferroptosis. However, the specific role of FTH in the development of HCC and ferroptosis resistance remains unclear. METHODS: The indicated databases were used for bioinformatics analysis. The abilities of cell proliferation, migration were measured by cell proliferation assay, transwell assay and wound healing assay. The levels of reactive oxygen species (ROS), lipid peroxide, free iron, mitochondrial superoxide, mitochondrial morphology and mitochondrial membrane potential (MMP) were determined by DCF-DA, C11-BODIPY, mitoSOX, mitoTracker, JC-10 and TMRM staining, respectively. The mitochondrial oxygen consumption rate was monitored by the Seahorse XF24 Analyzer. RESULTS: The pan-cancer analysis was performed and showed that FTH expression is upregulated in multiple cancers, such as LIHC, CHOL, HNSC, compared to corresponding normal tissues. In addition, the level of serum ferritin is positively associated with the progression of hepatitis, cirrhosis liver and hepatocellular carcinoma. Further investigation shed light on the strong correlation between FTH expression and tumor grades, cancer stages and prognosis of HCC. Importantly, the proteins interaction network elucidated that FTH is involved in iron homeostasis maintenance and lysosomal-dependent degradation. Enforced expression of FTH accelerates proliferation, migration and endows HCC cells specifically resistant to ferroptosis, but does not protect against cell death caused by cytotoxic compounds like oxaliplatin, irinotecan, and adriamycin. Mechanically, FTH reconstituted cells exhibit diminished peroxides accumulation, reduce mitochondrial ROS level, attenuate the impaired mitochondrial respiratory and rescue the mitochondrial homeostasis. Notably, FTH expression boosts tumorigenic potential in vivo with increased PCNA staining and lesser lipid peroxides generation. CONCLUSION: These results provide new insights that FTH acts as an oncogene in the carcinogenesis and progression of HCC, and is hopeful to be a potential target for therapeutic intervention through ferroptosis.

TEOA Promotes Autophagic Cell Death via ROS-Mediated Inhibition of mTOR/p70S6k Signaling Pathway in Pancreatic Cancer Cells.

Pancreatic cancer is a common malignant tumor with high mortality, and novel therapeutic options have focused on ameliorating its poor prognosis. TEOA, a traditional Chinese herbal medicine, exhibits anti-inflammatory and anti-cancer activities. Our recent study has shown that TEOA inhibits proliferation and induces DNA damage in diffuse large B-cell lymphoma cells by activating the ROS-mediated p38 MAPK pathway. However, its effects on pancreatic cancer cells remain unknown. In the present study, we evaluated the effects of TEOA on the proliferation, migration of pancreatic cancer cells and explored the possible underlying mechanism of action. We found that TEOA significantly inhibited the proliferation and migration of pancreatic cancer cells in a time- and dose-dependent manner. Mechanistically, TEOA significantly induced mitochondrial dysfunction in PANC1 and SW1990 cells, as evidenced by the collapse of the mitochondrial membrane potential, exhausted ATP level, and excessive accumulation of intracellular ROS. Notably, our further experiments showed that TEOA induced autophagic cell death in pancreatic ductal adenocarcinoma cells by inactivating the ROS-dependent mTOR/p70S6k signaling pathway. More importantly, both pharmacological or genetic blocking of the autophagic flux signal could partly restore the cytotoxicity of TEOA, whereas activation of autophagy by rapamycin or EBSS induced starvation facilitated the cytotoxicity of TEOA. Concomitantly, N-acetylcysteine, a ROS scavenger, abolished the inhibition of the mTOR signaling pathway, thus preventing autophagy and restoring cell viability. Taken together, our results reveal that TEOA can lead to ROS-dependent autophagic cell death of pancreatic cancer cells by inducing mitochondrial dysfunction, which might be a promising therapeutic agent for pancreatic cancer.

CISD3 inhibition drives cystine-deprivation induced ferroptosis.

Ferroptosis, a new form of programmed cell death, not only promotes the pathological process of various human diseases, but also regulates cancer progression. Current perspectives on the underlying mechanisms remain largely unknown. Herein, we report a member of the NEET protein family, CISD3, exerts a regulatory role in cancer progression and ferroptosis both in vivo and in vitro. Pan-cancer analysis from TCGA reveals that expression of CISD3 is generally elevated in various human cancers which are consequently associated with a higher hazard ratio and poorer overall survival. Moreover, knockdown of CISD3 significantly accelerates lipid peroxidation and accentuates free iron accumulation triggered by Xc- inhibition or cystine-deprivation, thus causing ferroptotic cell death. Conversely, ectopic expression of the shRNA-resistant form of CISD3 (CISD3res) efficiently ameliorates the ferroptotic cell death. Mechanistically, CISD3 depletion presents a metabolic reprogramming toward glutaminolysis, which is required for the fuel of mitochondrial oxidative phosphorylation. Both the inhibitors of glutaminolysis and the ETC process were capable of blocking the lipid peroxidation and ferroptotic cell death in the shCISD3 cells. Besides, genetic and pharmacological activation of mitophagy can rescue the CISD3 knockdown-induced ferroptosis by eliminating the damaged mitochondria. Noteworthily, GPX4 acts downstream of CISD3 mediated ferroptosis, which fails to reverse the homeostasis of mitochondria. Collectively, the present work provides novel insights into the regulatory role of CISD3 in ferroptotic cell death and presents a potential target for advanced antitumor activity through ferroptosis.

Overcoming the compensatory elevation of NRF2 renders hepatocellular carcinoma cells more vulnerable to disulfiram/copper-induced ferroptosis.

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.

DHA exhibits synergistic therapeutic efficacy with cisplatin to induce ferroptosis in pancreatic ductal adenocarcinoma via modulation of iron metabolism.

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.

Identification of Frataxin as a regulator of ferroptosis.

Ferroptosis is a newly discovered form of non-apoptotic regulated cell death and is characterized by iron-dependent and lipid peroxidation. Due to the enhanced dependence on iron in cancer cells, induction of ferroptosis is becoming a promising therapeutic strategy. However, the precise underlying molecular mechanism and regulation process of ferroptosis remains largely unknown. In the present study, we demonstrate that the protein Frataxin (FXN) is a key regulator of ferroptosis by modulating iron homeostasis and mitochondrial function. Suppression of FXN expression specifically repressed the proliferation, destroyed mitochondrial morphology, impeded Fe-S cluster assembly and activated iron starvation stress. Moreover, suppression of FXN expression significantly enhanced erastin-induced cell death through accelerating free iron accumulation, lipid peroxidation and resulted in dramatic mitochondria morphological damage including enhanced fragmentation and vanished cristae. In addition, this type of cell death was confirmed to be ferroptosis, since it could be pharmacologically restored by ferroptotic inhibitor Fer-1 or GSH, but not by inhibitors of apoptosis, necrosis. Vice versa, enforced expression of FXN blocked iron starvation response and erastin-induced ferroptosis. More importantly, pharmacological or genetic blocking the signal of iron starvation could completely restore the resistance to ferroptosis in FXN knockdown cells and xenograft graft in vivo. This paper suggests that FXN is a novel ferroptosis modulator, as well as a potential provided target to improve the antitumor activity based on ferroptosis.

Downregulation of CISD2 Has Prognostic Value in Non-Small Cell Lung Cancer and Inhibits the Tumorigenesis by Inducing Mitochondrial Dysfunction.

CISD2, a NEET protein that coordinates 2Fe-2S clusters through its CDGSH domain, is critical for normal development and iron homeostasis. CISD2 plays an important role in Fe-S cluster transfer and promotes cancer proliferation. However, its specific role in the development of non-small cell lung cancer (NSCLC) remains unclear. Bioinformatics of pan-cancer analysis from The Cancer Genome Atlas show that CISD2 has an aberrant expression in most types of human cancers. Moreover, CISD2 expression is associated with a higher hazard ratio and exhibits significantly poorer overall survival in lung adenocarcinoma (LUAD), uveal melanoma, head and neck squamous cell carcinoma, brain lower grade glioma, kidney chromophobe, and liver hepatocellular carcinoma. Further investigation revealed that CISD2 is highly expressed in LUAD and LUSC, which is associated with clinical pathological stages. In addition, survival data collected from GSE31210 and GSE13213, two datasets from the NCBI Gene Expression Omnibus, also confirmed that high CISD2 expression is associated with unfavorable survival in patients with LUAD. A cell-based assay indicated that the knockdown of CISD2 inhibited proliferation, invasion, and migration in A549 cells. Additionally, CISD2 knockdown accelerated the accumulation of cellular and mitochondrial reactive oxygen species, destroying the mitochondrial morphology and function. Moreover, CISD2 inhibition activated the iron starvation response, thus, accelerating iron accumulation in A549 cells. Pretreatment with DFO, the iron chelator, blocked mitochondrial dysfunction in CISD2-knockdown cells. Collectively, the present study provides novel insights into the regulatory role of CISD2 in NSCLC and presents a potential target to improve antitumor activity based on oxidative stress.