Molecular characterization of STEAP3 in lung squamous cell carcinoma: Regulating EGFR to affect cell proliferation and ferroptosis.

Six-transmembrane epithelial antigen of the prostate 3 (STEAP3) has been reported to play a regulatory role in various types of cancers. However, its involvement in lung squamous cell carcinoma (LUSC) remains understudied. Here, we aimed to explore the biological functions and underlying mechanisms of STEAP3 in LUSC. Intersection genes associated with LUSC and ferroptosis were analyzed using the Venn method, STRING, GEPIA and UALCAN databases. The expression of STEAP3 was detected by qPCR and western blotting assay. Cell proliferation and viability were determined using the cell counting kit-8 assay and EDU staining. Oxidative stress and lipid peroxidation were measured by corresponding kits and DCFH-DA staining. Ferroptosis was evaluated by Phen Green SK and Western blot assay. The correlation between STEAP3 and EGFR was predicted by the TIMER and starBase database. Co-immunoprecipitation was conducted to verify the binding of STEAP3 and EGFR. The data demonstrated a significant upregulation of STEAP3 expression in LUSC cell lines. Silencing of STEAP3 suppressed H2170 cell viability and proliferation while promoting oxidative stress and lipid peroxidation through increased levels of MDA and ROS, as well as inhibited SOD activity. In addition, knockdown of STEAP3 induced ferroptosis through the regulation of ferroptosis-related proteins. Moreover, the binding between STEAP3 and EGFR was predicted and confirmed in LUSC. EGFR overexpression reversed the effects of STEAP3 silencing on H2170 cell viability, proliferation, oxidative stress, and ferroptosis. To summarize, the inhibition of STEAP3/EGFR may serve as a promising therapeutic target for LUSC treatment, as it can suppress LUSC proliferation and promote lipid peroxidation and ferroptosis.

Susceptibility of cervical cancer to dihydroartemisinin-induced ferritinophagy-dependent ferroptosis.

The clinical therapeutics of cervical cancer is limited due to the drug resistance and metastasis of tumor. As a novel target for antitumor therapy, ferroptosis is deemed to be more susceptible for those cancer cells with resistance to apoptosis and chemotherapy. Dihydroartemisinin (DHA), the primary active metabolites of artemisinin and its derivatives, has exhibited a variety of anticancer properties with low toxicity. However, the role of DHA and ferroptosis in cervical cancer remained unclear. Here, we showed that DHA could time-dependently and dose-dependently inhibit the proliferation of cervical cancer cells, which could be alleviated by the inhibitors of ferroptosis rather than apoptosis. Further investigation confirmed that DHA treatment initiated ferroptosis, as evidenced by the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA) and liquid peroxidation (LPO) levels and simultaneously depletion of glutathione peroxidase 4 (GPX4) and glutathione (GSH). Moreover, nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy was also induced by DHA leading to subsequent increases of intracellular labile iron pool (LIP), exacerbated the Fenton reaction resulting in excessive ROS production, and enhanced cervical cancer ferroptosis. Among them, we unexpectedly found that heme oxygenase-1 (HO-1) played an antioxidant role in DHA-induced cell death. In addition, the results of synergy analysis showed that the combination of DHA and doxorubicin (DOX) emerged a highly synergistic lethal effect for cervical cancer cells, which was related also to ferroptosis. Overall, our data revealed the molecular mechanisms that DHA triggered ferritinophagy-dependent ferroptosis and sensitized to DOX in cervical cancer, which may provide novel avenues for future therapy development.

Downregulation of TNFAIP1 alleviates OGD/R­induced neuronal damage by suppressing Nrf2/GPX4­mediated ferroptosis.

TNFα-induced protein 1 (TNFAIP1) serve a role in neurovascular disease. However, the potential role and molecular mechanism of TNFAIP1 in cerebral ischemia-reperfusion (I/R) remains elusive. In the present study, reverse transcription-quantitative PCR and western blotting were used to assess TNFAIP1 mRNA and protein expression levels in PC12 cells. Furthermore, using Cell Counting Kit-8, flow cytometry and western blotting, cell viability and apoptosis were evaluated. Oxidative stress was evaluated using DCFH-DA staining and ELISA was used for assessment of inflammatory factors. Expression of components in the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and ferroptosis were assessed using western blotting analysis and an iron assay kit. TNFAIP1 expression was significantly upregulated in oxygen glucose deprivation and reperfusion (OGD/R)-injured PC12 cells. However, knocking down TNFAIP1 expression restored PC12 cell viability and decreased apoptosis following OGD/R-challenge. Furthermore, TNFAIP1 silencing significantly suppressed OGD/R-induced oxidative stress and inflammatory damage in PC12 cells. TNFAIP1 knockdown inhibited ferroptosis via activation of the Nrf2 signaling pathway in OGD/R-injured PC12 cells. Erastin treatment reversed the beneficial effects of TNFAIP1 knockdown on PC12 cell viability, apoptosis alleviation, oxidative stress and inflammation following OGD/R treatment. These results suggested that TNFAIP1 knockdown could alleviate OGD/R-induced neuronal cell damage by suppressing Nrf2-mediated ferroptosis, which might lay the foundation for the investigation of targeted-therapy for cerebral I/R injury in clinic.

Canagliflozin mitigates ferroptosis and improves myocardial oxidative stress in mice with diabetic cardiomyopathy.

Canagliflozin (Cana), an anti-diabetes drug belongs to sodium-glucose cotransporter 2 inhibitor, is gaining interest because of its extra cardiovascular benefits. Ferroptosis is a new mode of cell death, which can promote the occurrence of diabetic cardiomyopathy (DCM). Whether Cana can alleviate DCM by inhibiting ferroptosis is the focus of this study. Here, we induced DCM models in diabetic C57BL6 mice and treated with Cana. Meanwhile, in order to exclude its hypoglycemic effect, the high glucose model in H9C2 cells were established. In the in vivo study, we observed that Cana could effectively alleviate the damage of cardiac function in DCM mice, including the increasing of lactate dehydrogenase (LDH) and cardiac troponin I (cTnI), the alleviating of myocardial fiber breakage, inflammation, collagen fiber deposition and mitochondrial structural disorder. We evaluated reactive oxygen species (ROS) levels by DCFH-DA and BODIPY 581/591 C11, in vitro Cana reduced ROS and lipid ROS in H9C2 cells induced by high glucose. Meanwhile, JC-1 fluorochrome assay showed that the decreased mitochondrial membrane potential (MMP) was increased by Cana. Furthermore, the inhibitory effects of Cana on myocardial oxidative stress and ferroptosis were verified in vivo and in vitro by protein carbonyl (PCO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH). As a key inducer of ferroptosis, the deposition of total iron and Fe2+ can be inhibited by Cana both in vivo and in vitro. In addition, western blot results indicated that the expression of ferritin heavy-chain (FTN-H) was down-regulated, and cystine-glutamate antiporter (xCT) was up-regulated by Cana in DCM mice and cells, suggesting that Cana inhibit ferroptosis by balancing cardiac iron homeostasis and promoting the system Xc-/GSH/GPX4 axis in DCM. These findings underscore the fact that ferroptosis plays an important role in the development and progression of DCM and targeting ferroptosis may be a novel strategy for prevention and treatment. In conclusion, Cana may exert some of its cardiovascular benefits by attenuating ferroptosis.

Therapeutic Effect and Mechanism of Negative Pressure Wound Therapy with Huoxue Shengji Decoction Instillation for Chronic Skin Ulcers.

Background: Negative pressure wound therapy (NPWT) with instillation (NPWTi) is a new treatment for chronic skin ulcers (CSUs), but the choice of perfusate is still investigated. The clinical application of Huoxue Shengji (HXSJ) decoction has been proved to promote the formation of granulation. The formation of fresh granulation, angiogenesis, and proliferation of vascular endothelial cells are closely related. The purpose of this study was to observe the clinical efficacy of NWPT with HXSJ decoction instillation in the treatment of CSUs and to explore the potential mechanism by which HXSJ decoction promotes proliferation of vascular endothelial cells at the cellular level. Methods: In the clinical study, the random number table was used to divide the patients into three groups (patients were numbered by visit time and assigned a random number and grouped by the remainder after the random number was divided by 3, and when the number of patients in one group reached 20, the enrolment of this group is stopped), including NPWT combined with HXSJ decoction instillation (group A), NPWT combined with normal saline instillation (group B), and NPWT (group C). Related indexes were examined, including the wound cavity volume, bacterial culture, histopathology examination, time periods of debridement, repair methods, and the time of ulcer healing. In the basic research, the effect of HXSJ decoction on the proliferation of HUVECs was analysed by CCK-8 assay and RT-PCR and western blot were used to quantify the VEGF and VEGFR-2 expression in the relevant signalling pathway. Results: There was no significant difference in the improvement rate of invasive cavity volume (P > 0.05) between groups A and B, but a significant difference was observed between groups A and C (P < 0.05). There was no significant difference in microbial reduction among groups (all P > 0.05). Histopathological examination showed that the microvascular count in group A was significantly higher than that in groups B and C (both P < 0.01) and there was no statistical difference between groups B and C (P > 0.05). There were no significant differences in the number of invasive lesions and repair methods among the groups (all P > 0.05). The healing time of group A was significantly faster than those of groups B and C (compared to group B, P < 0.05; compared to group C, P < 0.01), and there was no statistical difference between groups B and C (P > 0.05). In the cellular experiments, concentration screening was performed and 125 µg/mL HXSJ decoction showed the most significant effect on the proliferation of HUVECs and also enhanced the expression of VEGF and VEGFR-2. Conclusion: HXSJ decoction can enhance the expression of VEGF and VEGFR-2 and promote the proliferation of HUVECs. Treatment with NWPT with HXSJ decoction instillation can further reduce the wound cavity volume; meanwhile, it can promote blood vessel formation in ulcer wounds, thus accelerating the healing of CSUs.

WetA and VosA are distinct regulators of conidiation capacity, conidial quality, and biological control potential of a fungal insect pathogen.

Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana, a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA, two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, ΔwetA and ΔvosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of ΔwetA showed more defective features than those of ΔvosA, including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of ΔvosA than of ΔwetA. As a result, conidial sensitivity to cell wall perturbation was elevated in ΔwetA but unaffected in ΔvosA, which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H2O2 or the fungicide carbendazim. Moreover, ΔwetA lost more virulence to Galleria mellonella larvae than ΔvosA. All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana.

Beauveria bassiana, a filamentous entomopathogen, has five distinct superoxide dismutases (SODs), including cytosolic and mitochondrial MnSODs (Sod2/3) which have proved contributing primarily to intracelluar SOD activity and additively to antioxidation and virulence. Here we characterized cytosolic Cu/ZnSOD (Sod1), mitochondrial FeSOD (Sod4) and cell wall-anchored Cu/ZnSOD (Sod5). The latter two are unexplored despite existence in many filamentous fungi, and their subcellular localization was well confirmed with specifically stained cells expressing Sod4::eGFP or Sod5::eGFP fusion. Total SOD activity decreased by ∼15% in Δsod1 but increased by 11-20% in three sod4 knockdown mutants (Δsod4 was lethal) when co-cultivated with menadone and H2O2. Surprisingly, total catalase activity decreased much more in the sod4 mutants (69-75%) than in Δsod1 (27-33%) under normal and oxidative conditions. However, Δsod5 showed little change in either SOD or catalase activity. Transcript levels of SOD partners and five catalases also changed more dramatically in the sod4 mutants than in Δsod1 and Δsod5. As a consequence of global effect, intracellular ROS levels induced by both oxidants were higher in Δsod1 than in the sod4 mutants and Δsod5. All the mutants were differentially more sensitive to the two oxidants and UV-A/UV-B irradiations and less virulent to Galleria mellonella larvae but not responsive to high osmolarity, cell wall stress and high temperature. Taken together with previously characterized Sod2 and Sod3, our results provide full insight into the SOD family, unveiling the interactions of each SOD with other partners and catalases in the antioxidant reaction associated with the fungal biocontrol potential.