GPX4 degradation contributes to fluoride-induced neuronal ferroptosis and cognitive impairment via mtROS-chaperone-mediated autophagy.

Ferroptosis is a newly recognized type of programmed cell death that is implicated in the pathophysiological process of neurological disorders. Our previous studies have revealed that exposure to high concentrations of fluoride for long periods of time induces hippocampal neural injury and cognitive deficits. However, whether ferroptosis is involved in fluoride-induced neuronal death and the underlying mechanism remain unknown. In this study, the results indicated that exposure to high fluoride triggered ferroptosis in SH-SY5Y cells and in the hippocampus of mice. Fluoride exposure accelerated the lysosomal degradation of GPX4 and led to neuronal ferroptosis, while GPX4 overexpression protected SH-SY5Y cells against fluoride-induced neurotoxicity. Intriguingly, the enhanced chaperone-mediated autophagy (CMA) induced by fluoride stimulation was responsible for GPX4 degradation because the inhibition of CMA activity by LAMP2A knockdown effectively prevented fluoride-induced GPX4 loss. Furthermore, mitochondrial ROS (mtROS) accumulation caused by fluoride contributed to CMA activation-mediated GPX4 degradation and subsequent neuronal ferroptosis. Notably, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or the ROS scavenger N-acetyl-L-cysteine (NAC) alleviated fluoride-evoked hippocampal neuronal death and synaptic injury as well as cognitive deficits in mice. The present studies indicates that ferroptosis is a novel mechanism of fluoride-induced neurotoxicity and that chronic fluoride exposure facilitates GPX4 degradation via mtROS chaperone-mediated autophagy, leading to neuronal ferroptosis and cognitive impairment.

Methylmercury induces inflammatory response and autophagy in microglia through the activation of NLRP3 inflammasome.

Methylmercury (MeHg) is a global environmental pollutant with neurotoxicity, which can easily crosses the blood-brain barrier and cause irreversible damage to the human central nervous system (CNS). CNS inflammation and autophagy are known to be involved in the pathology of neurodegenerative diseases. Meanwhile, MeHg has the potential to induce microglia-mediated neuroinflammation as well as autophagy. This study aims to further explore the exact molecular mechanism of MeHg neurotoxicity. We conducted in vitro studies using BV2 microglial cell from the central nervous system of mice. The role of inflammation and autophagy in the damage of BV2 cells induced by MeHg was determined by detecting cell viability, cell morphology and structure, reactive oxygen species (ROS), antioxidant function, inflammatory factors, autophagosomes, inflammation and autophagy-related proteins. We further investigated the relationship between the inflammatory response and autophagy induced by MeHg by inhibiting them separately. The results indicated that MeHg could invade cells, change cell structure, activate NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and autophagosome, release a large amount of inflammatory factors and trigger the inflammatory response and autophagy. It was also found that MeHg could disrupt the antioxidant function of cells. In addition, the inhibition of NLRP3 inflammasome alleviated both cellular inflammation and autophagy, while inhibition of autophagy increased cellular inflammation. Our current research suggests that MeHg might induce BV2 cytotoxicity through inflammatory response and autophagy, which may be mediated by the NLRP3 inflammasome activated by oxidative stress.

Metformin modulates autophagic pathway in renal fibrosis induced by carbon tetrachloride in adult male albino rats.

BACKGROUNDS: Chronic kidney disease (CKD) is a global public health problem. All progressive chronic kidney disease (CKD) is characterized by tubulointerstitial fibrosis. Exposure to high concentrations of carbon tetrachloride (including vapor) can destroy the kidneys. Autophagy played an important role in maintaining the homeostasis of organs. Impaired autophagy was frequently associated with renal damage and fibrosis. Recent data suggests that metformin protects against a variety of kidney disorders. AIM: To investigate the protective role of metformin on carbon tetrachloride induced renal damage via autophagy pathway. MATERIALS AND METHODS: Forty adult male albino rats were divided into four equal groups (10 rats, each); Group 1: control group. Group 2: olive oil group received olive oil 1.5 mg/kg twice weekly S.C for 12 weeks. Group 3: The ccl4 group, the rats were received ccl4 1.5 mg/kg twice weekly S.C for 12 weeks. Group 4: CCL4 and Metformin group received concomitant treatment of CCL4, 1.5 mg/kg twice weekly S.C and 100 mg/kg/day Metformin orally for 12 weeks. After sacrifice, kidneys were taken from all animal groups and processed for light and electron microscopy, immunological studies and biochemical tests. Statistical analysis was done. RESULTS: Administration of ccl4 resulted in histopathological changes in the kidney tissue in the form of areas of tissue destruction, inflammatory cell infiltration, congestion and fibrosis. Ultrastructurally, irregular thickening of GBM was observed. Improvement was noticed with concomitant treatment of ccl4 with metformin. CONCLUSION: Metformin administration can modulate histological and biochemical effects in the renal tissue induced by of ccl4.

Drug repurposing-based nanoplatform via modulating autophagy to enhance chemo-phototherapy against colorectal cancer.

Multi-modal combination therapy is regarded as a promising approach to cancer treatment. Combining chemotherapy and phototherapy is an essential multi-modal combination therapy endeavor. Ivermectin (IVM) is a potent antiparasitic agent identified as having potential antitumor properties. However, the fact that it induces protective autophagy while killing tumor cells poses a challenge to its further application. IR780 iodide (IR780) is a near-infrared (NIR) dye with outstanding photothermal therapy (PTT) and photodynamic therapy (PDT) effects. However, the hydrophobicity, instability, and low tumor uptake of IR780 limit its clinical applications. Here, we have structurally modified IR780 with hydroxychloroquine, an autophagy inhibitor, to synthesize a novel compound H780. H780 and IVM can form H780-IVM nanoparticles (H-I NPs) via self-assembly. Using hyaluronic acid (HA) to modify the H-I NPs, a novel nano-delivery system HA/H780-IVM nanoparticles (HA/H-I NPs) was synthesized for chemotherapy-phototherapy of colorectal cancer (CRC). Under NIR laser irradiation, HA/H-I NPs effectively overcame the limitations of IR780 and IVM and exhibited potent cytotoxicity. In vitro and in vivo experiment results showed that HA/H-I NPs exhibited excellent anti-CRC effects. Therefore, our study provides a novel strategy for CRC treatment that could enhance chemo-phototherapy by modulating autophagy.

[Effect of Shikonin on Autophagy and Apoptosis of Human Promyelocytic Leukemia Cells].

OBJECTIVE: To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism. METHODS: Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins. RESULTS: Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05). CONCLUSION: Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.

[Inhibition of Autophagy Augments the Anticancer Activity of KPT-330 in Mantle Cell Lymphoma Cells].

OBJECTIVE: To investigate the influence of novel CRM1 inhibitor KPT-330 on the autophagy of mantle cell lymphoma (MCL) cells, and effect of KPT-330 on the prolifiration of MCL cells in the presence or absence of autophagy inhibitor. METHODS: CCK-8 assay was used to detect the effect of KPT-330 on MCL cell lines Z-138， Jeko-1， Granta-519， Rec-1. The effect of KPT-330 on autophagy features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope and detecting protein expression of LC3B-II assessed by Western blot. Further combined application of lysosomal inhibitor Chloroquine (CQ) was used to observe its effect on the increase of LC3B-Ⅱ caused by KPT-330. CalcuSyn 2.0 software was used to detected the Combination index (CI) of KPT-330 combined with CQ. RESULTS: The proliferation of MCL cell lines （Z-138, Jeko-1, Grant-519, Rec-1） could be inhibited by KPT-330 in a dose-dependent manner (r =0.930, 0.946, 0.691, 0.968 respectively). The number of acidic vesicular organelles (AVO) and the expression of LC3B-II were increased in KPT-330 treated Jeko-1 and Granta-519 cells in a dose-dependent manner (r Jeko-1=0.993, r Granta-519=0.971). LC3B-II protein amounts still increased upon KPT-330 treatment with the existence of lysosomal inhibitor CQ in Jeko-1 and Granta-519 cells, which was higher than CQ or KPT-330 single drug group. The combination of KPT-330 and CQ produced the synergistic effects on cells proliferation inhibition with CalcuSyn 2.0 analysis. CONCLUSION: KPT-330 can inhibit MCL cell proliferation and induce autophagy. KPT-330 combined with autophagy inhibitor CQ could produce synergistic anti MCL effects, providing experimental basis for clinical combination therapy.

Phenylbutyric acid inhibits hypoxia-induced trophoblast apoptosis and autophagy in preeclampsia via the PERK/ATF-4/CHOP pathway.

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.

[Research Progress on the Role of Berberine in Hematologic Malignancies and Its Related Mechanisms --Review].

Berberine, a traditional Chinese medicine, is an isoquinoline alkaloid extracted from the rhizome of Coptis chinensis. It has anti-inflammatory and antidiarrheal effects and is commonly used in the treatment of infections and gastrointestinal diseases. In recent years, studies have found that berberine can play a wide range of anti-cancer effects in the treatment of leukemia, lymphoma, multiple myeloma, etc. In hematologic malignancies, berberine can induce autophagy, promote apoptosis, regulate cell cycle, inhibit inflammatory response, cause oxidative damage to cancer cells and interact with miRNA to inhibit the proliferation, migration and colony formation of cancer cells. This paper will review the role and related mechanisms of berberine in hematological malignancies.

Copper enhances aggregational toxicity of mutant huntingtin in a Drosophila model of Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder with clinical presentations of moderate to severe cognitive, motor, and psychiatric disturbances. HD is caused by the trinucleotide repeat expansion of CAG of the huntingtin (HTT) gene. The mutant HTT protein containing pathological polyglutamine (polyQ) extension is prone to misfolding and aggregation in the brain. It has previously been observed that copper and iron concentrations are increased in the striata of post-mortem human HD brains. Although it has been shown that the accumulation of mutant HTT protein can interact with copper, the underlying HD progressive phenotypes due to copper overload remains elusive. Here, in a Drosophila model of HD, we showed that copper induces dose-dependent aggregational toxicity and enhancement of Htt-induced neurodegeneration. Specifically, we found that copper increases mutant Htt aggregation, enhances the accumulation of Thioflavin S positive ß-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases ß-amyloid aggregates in the HD pathological model. These findings reveal a direct role of copper in potentiating mutant Htt protein-induced aggregational toxicity, and further indicate the potential impact of environmental copper exposure in the disease onset and progression of HD.

Maternal PFOS exposure in mice induces hepatic lipid accumulation and inflammation in adult female offspring: Involvement of microbiome-gut-liver axis and autophagy.

Perfluorooctane sulfonates (PFOS) are the persistent organic pollutants. In the present study, 0, 0.3, or 3-mg/kg PFOS were administered to pregnant mice from GD 11 to GD 18. The histopathology of liver and intestine, serum and hepatic lipid levels, lipid metabolism related genes, and gut microbiota were examined in adult female offspring. The results suggested that maternal PFOS exposure increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and induced F4/80+ macrophage infiltration in adult female offspring, in addition to the elevation of TNF-α and IL-1ß mRNA levels in low-dose and high-dose groups, respectively. Furthermore, maternal exposure to PFOS increased serum triglyceride (TG) and hepatic total cholesterol (TC) levels, which was associated with the alteration of the process of fatty acid transport and ß-oxidation, TG synthesis and transport, cholesterol synthesis and excretion in the liver. The AMPK/mTOR/autophagy signaling was also inhibited in the liver of adult female offspring. Moreover, changes in gut microbiota were also related to lipid metabolism, especially for the Desulfovibrio, Ligilactobacillus, Enterorhabdus, HT002 and Peptococcaceae_unclassified. Additionally, maternal exposure to PFOS decreased mRNA expressions of the tight junction protein and AB+ goblet cells in the colon, while increasing the overproduction of lipopolysaccharides (LPS) and F4/80+ macrophage infiltration. Collectively, maternal PFOS exposure induced liver lipid accumulation and inflammation, which strongly correlated with the disruption of the gut-liver axis and autophagy in adult female offspring, highlighting the persistent adverse effects in offspring exposed to PFOS.

Induction of autophagy-dependent and caspase- and microtubule-acetylation-independent cell death by phytochemical-stabilized gold nanopolygons in colorectal adenocarcinoma cells.

Collective functionalization of the phytochemicals of medicinal herbs on nanoparticles is emerging as a potential cancer therapeutic strategy. This study presents the facile synthesis of surface-functionalized gold nanoparticles using Bacopa monnieri (Brahmi; Bm) phytochemicals and their therapeutically relevant mechanism of action in the colorectal cancer cell line, HT29. The nanoparticles were characterized using UV-visible spectroscopy, TEM-EDAX, zeta potential analysis, TGA, FTIR and 1H NMR spectroscopy, and HR-LC-MS. The particles (Bm-GNPs) were of polygonal shape and were stable against aggregation. They entered the target cells and inhibited the viability and clonogenicity of the cells with eight times more antiproliferative efficacy (25 ± 1.5 µg mL-1) than Bm extract (Bm-EX). In vitro studies revealed that Bm-GNPs bind tubulin (a protein crucial in cell division and a target of anticancer drugs) and disrupt its helical structure without grossly altering its tertiary conformation. Like other antitubulin agents, Bm-GNPs induced G2/M arrest and ultimately killed the cells, as confirmed using flow cytometry analyses. ZVAD-FMK-mediated global pan-caspase inhibition and the apparent absence of cleaved caspase-3 in treated cells indicated that the death did not involve the classic apoptosis pathway. Cellular ultrastructure analyses, western immunoblots, and in situ immunofluorescence visualization of cellular microtubules revealed microtubule-acetylation-independent induction of autophagy as the facilitator of cell death. Together, the data indicate strong antiproliferative efficacy and a possible mechanism of action for these designer nanoparticles. Bm-GNPs, therefore, merit further investigations, including preclinical evaluations, for their therapeutic potential as inducers of non-apoptotic cell death.

Selective Targeting of Regulated Rhabdomyosarcoma Cells by Trinuclear Ruthenium(II)-Arene Complexes.

The use of benzimidazole-based trinuclear ruthenium(II)-arene complexes (1-3) to selectively target the rare cancer rhabdomyosarcoma is reported. Preliminary cytotoxic evaluations of the ruthenium complexes in an eight-cancer cell line panel revealed enhanced, selective cytotoxicity toward rhabdomyosarcoma cells (RMS). The trinuclear complex 1 was noted to show superior short- and long-term cytotoxicity in RMS cell lines and enhanced selectivity relative to cisplatin. Remarkably, 1 inhibits the migration of metastatic RMS cells and maintains superior activity in a 3D multicellular spheroid model in comparison to that of the clinically used cisplatin. Mechanistic insights reveal that 1 effectively induces genomic DNA damage, initiates autophagy, and prompts the intrinsic and extrinsic apoptotic pathways in RMS cells. To the best of our knowledge, 1 is the first trinuclear ruthenium(II) arene complex to selectively kill RMS cells in 2D and 3D cell cultures.

Cycloplatinated (II) Complex Based on Isoquinoline Alkaloid Elicits Ferritinophagy-Dependent Ferroptosis in Triple-Negative Breast Cancer Cells.

The development and optimization of metal-based anticancer drugs with novel cytotoxic mechanisms have emerged as key strategies to overcome chemotherapeutic resistance and side effects. Agents that simultaneously induce ferroptosis and autophagic death have received extensive attention as potential modalities for cancer therapy. However, only a limited set of drugs or treatment modalities can synergistically induce ferroptosis and autophagic tumor cell death. In this work, we designed and synthesized four new cycloplatinated (II) complexes harboring an isoquinoline alkaloid C∧N ligand. On screening the in vitro activity of these agents, we found that Pt-3 exhibited greater selectivity of cytotoxicity, decreased resistance factors, and improved anticancer activity compared to cisplatin. Furthermore, Pt-3, which we demonstrate can initiate potent ferritinophagy-dependent ferroptosis, exhibits less toxic and better therapeutic activity than cisplatin in vivo. Our results identify Pt-3 as a promising candidate or paradigm for further drug development in cancer treatment.

Quercetin and tanshinone prevent mitochondria from oxidation and autophagy to inhibit KGN cell apoptosis through the SIRT1/SIRT3-FOXO3a axis.

Granulosa cells are somatic cells located inside follicles that play a crucial role in the growth and development of follicles. Quercetin and tanshinone are two key monomers in traditional Chinese medicine that have antioxidant and anti-aging properties. The KGN cell apoptosis model caused by triptolide (TP) was employed in this work to investigate granulosa cell death and medication rescue. Quercetin and tanshinone therapy suppressed KGN cell death and oxidation while also regulating the expression of critical apoptosis and oxidation-related markers such as B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Further research revealed that the effects of Quercetin and Tanshinone were accomplished via deacetylation of FOXO3A in the cytoplasm and mitochondria via the SIRT1/SIRT3-FOXO3a axis. In summary, Quercetin and tanshinone protect KGN cells from apoptosis by reducing mitochondrial apoptosis and oxidation via the SIRT1/SIRT3-FOXO3a axis.

Astragaloside IV inhibits idiopathic pulmonary fibrosis through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway.

As the main active ingredient of Astragalus, Astragaloside IV (AS-IV) can ameliorate pulmonary fibrosis. In this experiment, we studied how AS-IV reduces idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) or TGF-ß1 was treated in mice or alveolar epithelial cells to mimic IPF in vivo as well as in vitro. ASV-IV alleviated levels of inflammatory cytokines and fibrosis markers in IPF model. Through detection of autophagy-related genes, ASV-IV was observed to induce autophagy in IPF. Besides, ASV-IV inhibited miR-21 expression in IPF models, and overexpression of miR-21 could reverse the protective potential of ASV-IV on IPF. PTEN was targeted by miR-21 and was up-regulated by ASV-IV in IPF models. In addition, levels of inflammatory cytokines and fibrosis markers, autophagy, as well as the PI3K/AKT/mTOR pathway regulated by ASV-IV could be neutralized after treatment with autophagy inhibitors, miR-21 mimics, or si-PTEN. Our study demonstrates that ASV-IV inhibits IPF through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway, suggesting that ASV-IV could be acted to be a promising therapeutic method for IPF.

Se-methylselenocysteine ameliorates mitochondrial function by targeting both mitophagy and autophagy in the mouse model of Alzheimer's disease.

Background: Alzheimer's disease (AD) exerts tremendous pressure on families and society due to its unknown etiology and lack of effective treatment options. Our previous study had shown that Se-methylselenocysteine (SMC) improved the cognition and synaptic plasticity of triple-transgenic AD (3 × Tg-AD) mice and alleviated the related pathological indicators. We are dedicated to investigating the therapeutic effects and molecular mechanisms of SMC on mitochondrial function in 3 × Tg-AD mice. Methods: Transmission electron microscopy (TEM), western blotting (WB), mitochondrial membrane potential (ΔΨm), mitochondrial swelling test, and mitochondrial oxygen consumption test were used to evaluate the mitochondrial morphology and function. Mitophagy flux and autophagy flux were assessed with immunofluorescence, TEM and WB. The Morris water maze test was applied to detect the behavioral ability of mice. Results: The destroyed mitochondrial morphology and function were repaired by SMC through ameliorating mitochondrial energy metabolism, mitochondrial biogenesis and mitochondrial fusion/fission balance in 3 × Tg-AD mice. In addition, SMC ameliorated mitochondria by activating mitophagy flux via the BNIP3/NIX pathway and triggering autophagy flux by suppressing the Ras/Raf/MEK/ERK/mTOR pathway. SMC remarkably increased the cognitive ability of AD mice. Conclusions: This research indicated that SMC might exert its therapeutic effect by protecting mitochondria in 3 × Tg-AD mice.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Emodin activates autophagy to suppress oxidative stress and pyroptosis via mTOR-ULK1 signaling pathway and promotes multi-territory perforator flap survival.

BACKGROUND: Multi-territory perforator flap reconstruction has been proven effective in treating large skin and soft tissue defects in clinical settings. However, in view of that the multi-territory perforator flap is prone to partial postoperative necrosis, increasing its survival is the key to the success of reconstruction. In this study, we aimed to clarify the effect of emodin on multi-territory perforator flap survival. METHODS: Flap survival was assessed by viability area analysis, infrared laser imaging detector, HE staining, immunohistochemistry, and angiography. Western blotting, immunofluorescence assays, and real-time fluorescent quantitative PCR were performed to detect the indicators of oxidative stress, pyroptosis and autophagy. RESULTS: After emodin treatment, the multi-territory perforator flap showed a significantly increased survival rate, which was shown to be closely related to the inhibition of oxidative stress and pyroptosis and enhanced autophagy. Meanwhile, the use of autophagy inhibitor 3 MA was found to reverse the inhibitory effects of emodin on oxidative stress and pyroptosis and weaken the improving effect of emodin on flap survival, suggesting that autophagy plays a critical role in emodin-treated flaps. Interestingly, our mechanistic investigations revealed that the positive effect of emodin on multi-territory perforator flap was attributed to the mTOR-ULK1 signaling pathway activation. CONCLUSIONS: Emodin can inhibit oxidative stress and pyroptosis by activating autophagy via the mTOR-ULK1 pathway, thereby improving the multi-territory perforator flap survival.

Ganoderic acid A suppresses autophagy by regulating the circFLNA/miR-486-3p/CYP1A1/XRCC1 axis to strengthen the sensitivity of lung cancer cells to cisplatin.

OBJECTIVES: Reportedly, ganoderic acid A (GA-A) increases the sensitivity of hepatocellular carcinoma cells to cisplatin (DDP) chemotherapy. Therefore, this study aims to fathom the influence of GA-A on lung cancer cells. METHODS: After the construction of A549/DDP cells through exposure to DDP, the effects of GA-A on A549 and A549/DDP cells were revealed by cellular functional assays, western blot and quantitative reverse transcription PCR (qRT-PCR). The DDP-resistant lung cancer tumor was established in vivo, followed by further validation of the mechanism of GA-A. RESULTS: GA-A suppressed the viability, migration, and invasion while downregulating Beclin and autophagy marker LC3II/LC3I levels and upregulating P62 levels in A549 and A549/DDP cells. These effects were reversed by circFLNA overexpression. Also, GA-A reinforced the sensitivity of A549/DDP cells to DDP, elevated the apoptosis and regulated the circFLNA/miR-486-3p/cytochrome P450 family 1 subfamily A member 1 (CYP1A1)/X-ray repair cross-complementing 1 (XRCC1) axis. The reversal effects of circFLNA overexpression on GA-A-induced viability and apoptosis of A549/DDP cells could all be counteracted in the presence of 3MA. GA-A inhibited lung cancer tumor growth and blocked autophagy. CONCLUSION: GA-A suppresses autophagy by regulating the circFLNA/miR-486-3p/CYP1A1/XRCC1 axis to strengthen the sensitivity of lung cancer cells to DDP.