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.

A Type VI Secretion System Facilitates Fitness, Homeostasis, and Competitive Advantages for Environmental Adaptability and Efficient Nicotine Biodegradation.

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or microniche. It is known that bacteria can benefit from the type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with antimicrobial and anti-ROS activities. Wild-type strain JY-Q grew better than the AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated TseN (type VI secretion system effector for nicotine tolerance), homologs of which were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+ TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, the T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage, in addition to the well-studied T6SS-dependent antimicrobial activities.IMPORTANCE Mixtures of various pollutants and the coexistence of numerous species of organisms are usually found in adverse environments. Concerning biodegradation of nitrogen-heterocyclic contaminants, the scientific community has commonly focused on screening functional enzymes that transform pollutants into intermediates of attenuated toxicity or for primary metabolism. Here, we identified dual roles of the T6SS effector TseN in Pseudomonas sp. strain JY-Q, which is capable of degrading nicotine. The T6SS in strain JY-Q is able to deliver TseN to kill competitors and provide a growth advantage by a contact-independent pattern. TseN could monitor the intracellular NAD+ level by its hydrolase activity, causing cytotoxicity in competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by the immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated the nicotine tolerance of JY-Q. We therefore reveal a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.

The protective function of miR-378 in the ischemia-reperfusion injury during renal transplantation and subsequent interstitial fibrosis of the renal allograft.

Survival time of kidney transplant patients is primarily threatened by chronic allograft dysfunction (CAD), whose typical feature is interstitial fibrosis and tubular atrophy (IF/TA). CAD could be caused by ischemia-reperfusion injury (IRI) during renal transplantation. MiR-378 is correlated with multiple kidney diseases and implicated in CAD. To clarify the underlying mechanism of miR-378 on renal allograft, we utilize renal unilateral IRI mice and H/R NRK52E cells. To evaluate the acute tubular damage, we determined the apoptotic rate by TUNEL assay and examined mice kidney sections of H&E staining 1 day after IRI. To assess the chronic renal interstitial inflammation and fibrosis, we detected the infiltration rates of CD45R+ leukocytes and Ly6b+ neutrophils by immunohistochemistry, examined mice kidney sections of picrosirius staining and measured the mRNA level of Col1a1 14 days after IRI. To investigate the H/R injury of NRK52E cells, MTT assay was performed to detect cell viability, TUNEL assay was performed to determine apoptotic rate and luciferase reporter assay was carried out to demonstrate the potential target of miR-378. Moreover, we determined the levels of miR-378 of renal allograft biopsies in healthy patients and patients diagnosed with IF/TA. We found agomir-378 treatment significantly reduced the apoptotic rate and tubular damage scores assessed by H&E staining 1 day after IRI. Agomir-378 treatment also decreased infiltration rates of both CD45R+ leukocytes and Ly6b+ neutrophils and fibrosis examined by picrosirius staining and by the mRNA level of Col1a1 14 days after IRI. Experiments in vitro revealed that miR-378 increased cell viability and decreased apoptotic rate of NRK52E cells subjected to H/R. Additionally, luciferase reporter assay confirmed that caspase 3 was targeted by miR-378 directly. Furthermore, we found the levels of miR-378 were significantly lower in renal allografts of patients with IF/TA than those of healthy controls. Taken together, we have found that miR-378 has potential protective effects on renal allografts to prevent IRI during kidney transplantation and following IF/TA of renal allografts.

Comparative Genomics Reveals Specific Genetic Architectures in Nicotine Metabolism of Pseudomonas sp. JY-Q.

Microbial degradation of nicotine is an important process to control nicotine residues in the aqueous environment. In this study, a high active nicotine degradation strain named Pseudomonas sp. JY-Q was isolated from tobacco waste extract (TWE). This strain could completely degrade 5.0 g l-1 nicotine in 24 h under optimal culture conditions, and it showed some tolerance even at higher concentrations (10.0 g l-1) of nicotine. The complete genome of JY-Q was sequenced to understand the mechanism by which JY-Q could degrade nicotine and tolerate such high nicotine concentrations. Comparative genomic analysis indicated that JY-Q degrades nicotine through putative novel mechanisms. Two candidate gene cluster duplications located separately at distant loci were predicted to be responsible for nicotine degradation. These two nicotine (Nic) degradation-related loci (AA098_21325-AA098_21340, AA098_03885-AA098_03900) exhibit nearly completely consistent gene organization and component synteny. The nicotinic acid (NA) degradation gene cluster (AA098_17770-AA098_17790) and Nic-like clusters were both predicted to be flanked by mobile genetic elements (MGE). Furthermore, we analyzed the regions of genomic plasticity (RGP) in the JY-Q strain and found a dynamic genome carrying a type VI secretion system (T6SS) that promotes nicotine metabolism and tolerance based on transcriptomics and used in silico methods to identify the T6SS effector protein. Thus, a novel nicotine degradation mechanism was elucidated for Pseudomonas sp. JY-Q, suggesting its potential application in the bioremediation of nicotine-contaminated environments, such as TWEs.

Multivariate Boosting for Integrative Analysis of High-Dimensional Cancer Genomic Data.

In this paper, we propose a novel multivariate component-wise boosting method for fitting multivariate response regression models under the high-dimension, low sample size setting. Our method is motivated by modeling the association among different biological molecules based on multiple types of high-dimensional genomic data. Particularly, we are interested in two applications: studying the influence of DNA copy number alterations on RNA transcript levels and investigating the association between DNA methylation and gene expression. For this purpose, we model the dependence of the RNA expression levels on DNA copy number alterations and the dependence of gene expression on DNA methylation through multivariate regression models and utilize boosting-type method to handle the high dimensionality as well as model the possible nonlinear associations. The performance of the proposed method is demonstrated through simulation studies. Finally, our multivariate boosting method is applied to two breast cancer studies.

Effects of alkaloid sinomenine on levels of IFN-γ, IL-1ß, TNF-α and IL-6 in a rat renal allograft model.

AIMS: To evaluate the immunosuppressive efficacy of alkaloid sinomenine (SIN) and the synergistic effects in combination with cyclosporin A (CsA) in acute rejection after rat renal allograft. MATERIALS & METHODS: Animals were treated with saline in group 1, SIN (30 mg/kg/d) in group 2, CsA (2.5 mg/kg/d) in group 3 and SIN (30 mg/kg/d) + CsA(2.5 mg/kg/d) in group 4. Another 12 syngeneic renal transplantation animals were treated with saline as control. Survival time is observed. The levels of serum creatinine (Scr) and blood urea nitrogen (Bun) were detected; the secretion of IFN-γ, IL-1ß, TNF-α and IL-6 were detected by ELISA. The kidneys were fixed to perform histological staining. RESULTS: The mean survival time was 8.00 ± 2.10 days in group 1, 10.67 ± 1.21 days in group 2, 11.00 ± 1.41 days in group 3 and 19.67 ± 2.80 days in group 4, while all the recipients survived more than 180 days in the control group. The 24-h urinary volume and urinary time of the other three groups were increased significantly compared with group 1. The levels of Scr and Bun, levels of IFN-γ, IL-1ß, TNF-α and IL-6 were significantly higher in group 1 than that in the other three groups; there were significant differences between group 4 and group 2 or 3. CONCLUSION: Our study showed that SIN had immunosupression effects in rat renal allograft models, it also had a synergistic effect in combination with CsA, which provided a new immunosuppressant for clinical application.