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.

SNX14 inhibits autophagy via the PI3K/AKT/mTOR signaling cascade in breast cancer cells.

BACKGROUND: Sorting nexin 14 (SNX14) is a member of the sorting junction protein family. Its specific roles in cancer development remain unclear. Therefore, in this study, we aimed to determine the effects and underlying mechanisms of SNX14 on autophagy of breast cancer cells to aid in the therapeutic treatment of breast cancer. METHODS: In this study, we performed in vitro experiments to determine the effect of SNX14 on breast cancer cell growth. Moreover, we used an MCF7 breast cancer tumor-bearing mouse model to confirm the effect of SNX14 on tumor cell growth in vivo. We also performed western blotting and quantitative polymerase chain reaction to identify the mechanism by which SNX14 affects breast cancer MCF7 cells. RESULTS: We found that SNX14 regulated the onset and progression of breast cancer by promoting the proliferation and inhibiting the autophagy of MCF7 breast cancer cells. In vivo experiments further confirmed that SNX14 knockdown inhibited the tumorigenicity and inhibited the growth of tumor cells in tumor tissues of nude mice. In addition, western blotting analysis revealed that SNX14 modulate the autophagy of MCF7 breast cancer cells via the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin kinase signaling pathway. CONCLUSION: Our findings indicate that SNX14 is an essential tumor-promoting factor in the development of breast cancer.

[Elesclomol-Cu Induces Cuproptosis in Human Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the effects of elesclomol-Cu (ES-Cu) on the proliferation and cuproptosis of human acute myeloid leukemia (AML) cells. METHODS: The effects of ES-Cu on the proliferation of AML cells and the AML cells pre-treated with ammonium tetrathiomolybdate (TTM) were examined by CCK-8 assay. The Calcein/PI kit was used to detected the changes in activity and cytotoxicity of AML cells induced by ES-Cu. Flow cytometry and Cytation3 fully automated cell imaging multifunctional detection system were used to analyze DCFH-DA fluorescence intensity, so as to determine the level of reactive oxygen species (ROS). The GSH and GSSG detection kits were used to measure the intracellular GSH content. Western blot was used to detected the expression of cuproptosis-related proteins ATP7B, FDX1, DLAT and DPYD. RESULTS: ES-Cu inhibited the proliferation of Kasumi-1 and HL-60 cells in a concentration-dependent manner (r Kasumi-1=-0.99， r HL-60=-0.98). As the concentration of ES-Cu increased, the level of intracellular ROS also increased (P <0.01-0.001). TTM could significantly reverse the inhibitory effect of ES-Cu on cell proliferation and its promoting effect on ROS. With the increase of ES-Cu concentration, the content of GSH was decreased (r =-0.98), and Western blot showed that the protein expressions of ATP7B, FDX1, DLAT and DPYD were significantly reduced (P <0.05). CONCLUSION: ES-Cu can induce cuproptosis in AML cells, which provides a new idea for the treatment of AML.

Regulation of NCOA4-mediated iron recycling ameliorates paraquat-induced lung injury by inhibiting ferroptosis.

Paraquat (PQ) is an irreplaceable insecticide in many countries for the advantage of fast-acting and broad-spectrum. However, PQ was classified as the most prevailing poisoning substance for suicide with no specific antidote. Therefore, it is imperative to develop more effective therapeutic agents for the treatment of PQ poisoning. In the present study, both the RNA-Seq and the application of various cell death inhibitors reflected that ferroptosis exerts a crucial regulatory role in PQ poisoning. Moreover, we found PQ strengthens lipid peroxidation as evidenced by different experimental approaches. Of note, pretreatment of iron chelation agent DFO could ameliorate the ferroptotic cell death and alleviate the ferroptosis-related events. Mechanistically, PQ treatment intensively impaired mitochondrial homeostasis, enhanced phosphorylation of AMPK, accelerated the autophagy flux and triggered the activation of Nuclear receptor coactivator 4-ferritin heavy chain (NCOA4-FTH) axis. Importantly, the activation of autophagy was observed prior to the degradation of ferritin, and inhibition of autophagy could inhibit the accumulation of iron caused by the ferritinophagy process. Genetic and pharmacological inhibition of ferritinophagy could alleviate the lethal oxidative events, and rescue the ferroptotic cell death. Excitingly, in the mouse models of PQ poisoning, both the administration of DFO and adeno-associated virus-mediated FTH overexpression significantly reduced PQ-induced ferroptosis and improved the pathological characteristics of pulmonary fibrosis. In summary, the current work provides an in-depth study on the mechanism of PQ intoxication, describes a framework for the further understanding of ferroptosis in PQ-associated biological processes, and demonstrates modulation of iron metabolism may act as a promising therapeutic agent for the management of PQ toxicity.

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.

The potential of targeting cuproptosis in the treatment of kidney renal clear cell carcinoma.

Renal cell carcinoma (RCC) is one of the top ten malignancies and tumor-related causes of death worldwide. The most common histologic subtype is kidney renal clear cell carcinoma (KIRC), accounting for approximately 75% of all RCC cases. Early resection is considered the basic treatment for patients with KIRC. However, approximately 30% of these patients experience recurrence post-operation. Cuproptosis, an autonomous mechanism for controlling cell death, encompasses various molecular mechanisms and multiple cellular metabolic pathways. These pathways mainly include copper metabolic signaling pathways, mitochondrial metabolism signaling pathways, and lipoic acid pathway signaling pathways. Recent evidence shows that cuproptosis is identified as a key cell death modality that plays a meaningful role in tumor progression. However, there is no published systematic review that summarizes the correlation between cuproptosis and KIRC, despite the fact that investigations on cuproptosis and the pathogenesis of KIRC have increased in past years. Researchers have discovered that exogenous copper infusion accelerates the dysfunction of mitochondrial dysfunction and suppresses KIRC cells by inducing cuproptosis. The levels of tricarboxylic acid cycle proteins, lipoic acid protein, copper, and ferredoxin 1 (FDX1) were dysregulated in KIRC cells, and the prognosis of patients with high FDX1 expression is better than that of patients with low expression. Cuproptosis played an indispensable role in the regulation of tumor microenvironment features, tumor progression, and long-term prognosis of KIRC. In this review, we summarized the systemic and cellular metabolic processes of copper and the copper-related signaling pathways, highlighting the potential targets related to cuproptosis for KIRC treatment.

Systematic review and meta-analysis of the curative effects and safety of endoscopic sinus surgery in children with chronic sinusitis with nasal polyps.

Background: At present, the surgical treatment of sinusitis with nasal polyps has made great progress, but its recurrence rate is still high. Therefore, this time, meta-analysis is used to study the therapeutic effect of endoscopic sinus surgery on children with chronic sinusitis with nasal polyps, analyze its effectiveness and safety, and provide theoretical basis for clinical treatment. Methods: Boolean logic searching was adopted to retrieve articles from various databases, including PubMed, Medline, and Chinese National Knowledge Infrastructure (CNKI), published from the establishment of the database to December 30, 2021. The following search terms were used: "endoscopic sinus surgery", "antrochoanal polyps", "chronic sinusitis with nasal polyps", and "nasal polyps". Comparative studies on traditional surgical treatment and endoscopic sinus surgery were also screened out. Review Manager was utilized for meta-analysis. Results: A total of 9 references were included in the study, and most of them were low risk bias (medium and high quality). Meta-analysis showed that there was no statistical heterogeneity between the control group and the experimental group (Chi2=0.03, I2=0%, P=0.98). According to the fixed effect model analysis, the number of patients with polyp formation in the experimental group was significantly less than that in the control group (Z=2.65, P=0.008). Compared with the control group, there is no statistical heterogeneity in the postoperative recurrence (Chi2=1.59, I2=0%, P=0.45). According to the analysis of fixed effect model, the postoperative recurrence in the experimental group is significantly less than that in the control group (Z=2.92, OR =2.78, 95% CI: 1.40-5.52, P=0.004). Compared with the control group, the results of postoperative visual analogue scale (VAS) were statistically different (Chi2=12.63, I2=84%, P=0.002). According to the random effect model analysis, the VAS score of the experimental group was significantly lower than that of the control group (Z=18.06, MD =4.51, 95% CI: 3.96-5.05, P<0.00001). Discussion: Endoscopic sinus surgery could reduce the postoperative recurrence and pain of patients, and showed high curative effects and safety in the treatment of children with chronic sinusitis with nasal polyps.

Antineoplastic prescription among patients with colorectal cancer in eight major cities of China, 2015-2019: an observational retrospective database analysis.

OBJECTIVES: It is unclear what is driving rising colorectal cancer (CRC) treatment costs in China, whether an adjustment in drug prices changes use and total cost. This study aims to estimate trends in drug use, prescribing patterns and spending for antineoplastic drug therapies for CRC in major cities of China. METHODS: Information from 128 811 antineoplastic drug prescriptions in CRC was retrospectively collected from the Hospital Prescription Analysis Cooperative Project. The prescriptions extracted included demographic information of patients, the generic name and the price of antineoplastic drugs. The Mann-Kendall and Cochran-Armitage trend test was used to estimate the trends of antineoplastic agent usage. RESULTS: The number of antineoplastic prescriptions ranged from 18 966 in 2015 to 34 219 in 2019. Among the prescriptions collected in this study, the annual cost of antineoplastic drugs increased by 117.2%, and average prescription cost increased by 20%. Throughout the study period, the most prescribed antineoplastic drugs were capecitabine, oxaliplatin, fluorouracil and irinotecan, representing 49%, 27%, 21% and 9% of (per cent of visits (PV)). The PV of bevacizumab and cetuximab increased by 494% and 338% (from 1.8% and 1.3% in 2015 to 10.7% and 5.7% in 2019). In prescribing patterns of antineoplastic agents, monotherapy gradually decreased, while combination therapy, especially three-drug combination, increased significantly from 1.35% to 7.31%. CONCLUSION: This study estimated recent trends of antineoplastic drug use and expenditure for Chinese patients with CRC. These results would inform CRC treatment decisions, including health insurance negotiation, precision therapy access, allocation of research funding and evaluation of the financial burden of CRC drug treatment.

DKK3 attenuates JNK and AP-1 induced inflammation via Kremen-1 and DVL-1 in mice following intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is the most devastating stroke subtype, with a poor prognosis and few proven treatments. Neuroinflammation is associated with ICH-induced brain injury and unfavorable outcomes. There is growing evidence that Dickkopf (DKK) 3 plays a key role in the adaptive anti-inflammatory and neuroprotective responses following intracerebral hemorrhage. This study aimed to evaluate the protective effects of DKK3 against brain edema and neuroinflammation in a mice model of ICH. METHODS: Male, adult CD1 mice were subjected to sham or ICH surgery using a collagenase injection model. ICH animals received either recombinant DKK3, Kremen-1 siRNA, or DVL-1 siRNA. The neurobehavioral deficits were evaluated at 24 h, 72 h, and 28 days after ICH induction. Western blot and immunofluorescence were employed to examine the expression and localization of DKK3, Kremen-1, Dishevelled-1 (DVL-1), c-JUN N-terminal kinase (JNK), Activator protein-1 (AP-1), cleaved caspase-1, NF-κB, and IL-1ß in the brain. RESULTS: The expression of endogenous DKK3 and DVL-1 was transiently decreased after ICH compared to that in the sham group. Compared to the mice of ICH, exogenous rDKK3 administration reduced the brain water content and affected the neurological functions in ICH mice. Moreover, DKK3 was colocalized with Kremen-1 in microglia. Using a Kremen-1 or DVL-1 siRNA-induced in vivo knockdown approach, we demonstrated that the effects of DKK3 against ICH were mediated, at least partly, by the Kremen-1 and DVL-1 pathways. CONCLUSIONS: DKK3 improves the neurological outcomes, potentially by decreasing JNK/AP-1-mediated inflammation, thereby ameliorating the short- and long-term sequelae after ICH.

Macrophage stimulating protein preserves blood brain barrier integrity after intracerebral hemorrhage through recepteur d'origine nantais dependent GAB1/Src/ß-catenin pathway activation in a mouse model.

Blood brain barrier (BBB) disruption is an important contributor to brain edema and neurological deficits following intracerebral hemorrhage (ICH). Macrophage stimulating protein (MSP) is a hepatocyte growth factor-like protein that mediates its functions via activating receptor tyrosine kinase recepteur d'origine nantais (RON). Grb2-associated binder 1 (GAB1) is a docking protein that mediates downstream receptor signal transduction pathways. This study aimed to evaluate the role of MSP and RON activated signaling pathway in preserving BBB integrity after collagenase-induced ICH. ICH mice received recombinant human MSP (rhMSP) or rhMSP combined with siRNA knockdown of RON or GAB1. rhMSP was administered by intranasal route 1 h after ICH. Brain edema, neurobehavior, BBB tight junction protein expression, and BBB permeability were evaluated. The expression of endogenous MSP and p-RON was decreased after ICH. Exogenous rhMSP administration reduced brain edema, neurological deficits, BBB permeability, and increased the expression of tight junction proteins in ICH mice. rhMSP administration increased the expression of p-RON, p-GAB1, p-Src, nuclear ß-catenin, and tight junction proteins after ICH. These effects were reversed with RON and GAB1 siRNA. We conclude that MSP activation of RON preserved BBB integrity via GAB-1/Src/ß-catenin pathway, thereby reducing brain edema and neurological deficits after ICH in mice.

Adropin preserves the blood-brain barrier through a Notch1/Hes1 pathway after intracerebral hemorrhage in mice.

Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood-brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase-induced ICH was investigated in mice. At 1-h post-ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N-cadherin expression and reducing extravasation of albumin. Moreover, in vivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway.

Development and validation of a rapid LC-MS/MS method to quantify letrozole in human plasma and its application to therapeutic drug monitoring.

A selective, rapid, and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed and validated for the determination of letrozole (LTZ) in human plasma, using anastrozole as internal standard (IS). Sample preparation was performed by one-step protein precipitation with methanol. The analyte and IS were chromatographed on a reversed-phase YMC-ODS-C18 column (2.0 × 100 mm i.d., 3 µm) with a flow rate of 0.3 mL/min. The mobile phase consisted of water containing 0.1% formic acid (v/v) and methanol containing 0.1% formic acid (v/v). The mass spectrometer was operated in selected reaction monitoring mode through electrospray ionization ion mode using the transitions of m/z 286.2 → 217.1 for LTZ and m/z 294.1 → 225.1 for IS, respectively. The method was validated for selectivity, linearity, lower limit of quantitation, precision, accuracy, matrix effects and stability in accordance with the US Food and Drug Administration guidelines. Linear calibration curves were 1.0-60.0 ng/mL. Intra- and inter-batch precision (CV) for LTZ were <9.34%, and the accuracy ranged from 97.43 to 105.17%. This method was successfully used for the analysis of samples from patients treated with LTZ in the dose of 2.5 mg/day. It might be suitable for therapeutic drug monitoring of these patients and contribute to predict the risk of adverse reactions.

Cardioprotective effects of Glycyrrhiza uralensis extract against doxorubicin-induced toxicity.

The purpose of the present study was to investigate the cardioprotective effects of Glycyrrhiza uralensis extract (GUE) against doxorubicin (DOX)-induced cardiotoxicity. Imprinting control region (ICR) mice were treated with saline, DOX (20 mg/kg intraperitoneal [ip] for once), GUE (100 mg/kg intragastric [ig] for 8 days), co-treatments with DOX and GUE (100 mg/kg ig for 8 days), and amifostine (100 mg/kg intravenous [iv] for once), respectively. Serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB), glutathione peroxidase (GSH-P(X)) activity, and glutathione (GSH) level in heart tissue were measured. Histopathologic analysis of heart tissue was also performed. Treatment with GUE significantly protected the mice from DOX-induced cardiotoxicity, indicated by decreased levels of serum LDH and CK-MB, improved heart morphology and increased GSH-P(X) activity and GSH level. Additionally, GUE did not compromise the tumor-inhibitory effect of DOX. In conclusion, our studies imply the potentially clinical application of GUE to overcome the cardiotoxicity of doxorubicin.

[Discovering active components from traditional Chinese medicine by component-knockout approach].

OBJECTIVE: A component-knockout approach was established to discover active components from traditional Chinese medicine. METHOD: According to the principle of gene knockout technique, an experiment workflow for component-knockout method was developed, which is distinct from the bio-guided screening method. The differences of therapeutic efficacies between different combinations of individual components were analyzed by some statistical methods including Analysis of Variance (ANOVA), whilst a set of criterias were established to assess active components. By comparing the difference of drug efficacy between the original formulae and the mixture being knockout certain component, the active components can be identified. RESULT: The presented component-knockout method was applied to discover the active components of Shenmai formulae for the synergistic effects on the cyclophosphamide chemotherapy for S180 tumor-bearing mice. The results indicated that panoxadiol, a type of ginsenosides, were the effective components of Shenmai formulae. CONCLUSION: A new method for identifying effective components from Chinese medicinal formulae was developed and successfully applied to discover the active components of Shenmai Formulae, which possesses the synergistic actions towards chemotherapy process.