Mitochondria-mediated Apoptosis Research Articles

Fraxetin reduces endometriotic lesions through activation of ER stress, induction of mitochondria-mediated apoptosis, and generation of ROS

BackgroundFraxetin, a phytochemical obtained from Fraxinus rhynchophylla, is well known for its anti-inflammatory and anti-fibrotic properties. However, fraxetin regulates the progression of endometriosis, which is a benign reproductive disease that results in low quality of life and infertility. Hypothesis/purposeWe hypothesized that fraxetin may have therapeutic effects on endometriosis and aimed to elucidate the underlying mechanisms of mitochondrial function and tiRNA regulation. Study designEndometriotic animal models and cells (End1/E6E7 and VK2/E6E7) were used to identify the mode of action of fraxetin. MethodsAn auto-implanted endometriosis animal model was established and the effects of fraxetin on lesion size reduction were analyzed. Cell-based assays including proliferation, cell cycle, migration, apoptosis, mitochondrial function, calcium efflux, and reactive oxygen species (ROS) were performed. Moreover, fraxetin signal transduction was demonstrated by western blotting and qPCR analyses. ResultsFraxetin inhibited proliferation and migration by inactivating the P38/JNK/ERK mitogen-activated protein kinase (MAPK) and AKT/S6 pathways. Fraxetin dissipates mitochondrial membrane potential, downregulates oxidative phosphorylation (OXPHOS), and disrupts redox and calcium homeostasis. Moreover, it triggered endoplasmic reticulum stress and intrinsic apoptosis. Furthermore, we elucidated the functional role of tiRNAHisGTG in endometriosis by transfection with its inhibitor. Finally, we established an endometriosis mouse model and verified endometriotic lesion regression and downregulation of adhesion molecules with inflammation. ConclusionThis study suggests that fraxetin is a novel therapeutic agent that targets mitochondria and tiRNAs. This is the first study to demonstrate the mechanisms of tiRNAHisGTG with mitochondrial function and cell fates and can be applied as a non-hormonal method against the progression of endometriosis.

Evaluating the cytotoxicity mechanism of the cell-penetrating peptide TP10 on Jurkat cells

TP10, a classic cell-penetrating peptide, shows a high degree of similarity to AMPs in structure. Although TP10 has been widely used in drug delivery, the mechanism underlying its cytotoxicity is yet to be elucidated. Herein, we explored the cell-killing mechanism of TP10 against human leukemia Jurkat cells. TP10 induced necrosis in Jurkat cells via rapid disruption of cell membranes, particularly at high concentrations. Although mitochondria in Jurkat cells were damaged by TP10, mitochondria-mediated apoptosis did not occur, possibly due to intracellular ATP depletion. Necroptosis in TP10-treated Jurkat cells became an alternative route of apoptosis. Our results demonstrate that necrosis and necroptosis rather than apoptosis are involved in the cell-killing mechanism of TP10, which contributes to the understanding of its toxicity.

Premna puberula root petroleum ether extract inhibits proliferation, migration and invasion, and induces apoptosis through mitochondrial pathway in non-small cell lung cancer A549 cells

Premna puberula, a medicine food homologous plant, is utilized in the preparation of cold tofu and as traditional Chinese medicine. This study aimed to identify the chemical components of P. puberula root petroleum ether extract (PEE) and clarify its anticancer properties and associated mechanisms. The MTT results of the four extracts (petroleum ether extract, ethyl acetate extract, n-butanol extract, and water extract) from P. puberula root showed that the PEE had good cytotoxic activity against A549 cells (IC50 = 38.01 ± 3.36 μg/mL) and low toxicity to normal cells (IC50 = 76.85 ± 3.18 μg/mL). The GC-FID/MS analysis revealed fifty compounds, which made up 98.6 % of the total PEE. Further antitumor assay demonstrated that PEE induced cell cycle arrest in the S phase by up-regulating p21 and cyclin E2 expression, thereby inhibiting the proliferation of A549 cells. Simultaneously, it induces apoptosis through the mitochondrial-mediated apoptosis pathway, which significantly up-regulated the ratio of Bax/Bcl-2, down-regulated mitochondrial membrane potential (ΔΨm), promoted the release of Cyt c, activated caspase-9 and caspase-3, thereby resulting in PARP cleavage. It prevented the migration and invasion of A549 cells by reducing the expression of MMP-2 and N-cadherin. In conclusion, P. puberula root PEE can be a new source of antitumor agents and has significant anticancer activity in vitro.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Modulation of PI3K/Akt/GSK3β signaling cascade through G protein-coupled receptor 55 (GPR55) activation: Prenatal lysophosphatidylinositol attenuates valproic acid-induced synaptic abnormalities and mitochondrial dysfunction

AimsDysregulation of PI3K/Akt/GSK3β signaling has been implicated in various neurological disorders, including autism spectrum disorder (ASD). G protein-coupled receptor 55 (GPR55) has recently emerged as a potential regulator of this signaling cascade. This study explores the intricate modulation of the PI3K/Akt/GSK3β signaling cascade via GPR55 activation and its potential therapeutic implications in the context of autism-associated neuronal impairments. Main methodsValproic acid (VPA) was administered on embryonic day 12 (E12) to induce ASD, and lysophosphatidylinositol (LPI), a GPR55 agonist, was used prenatally to modulate the receptor activity. Golgi-cox staining was performed to observe neuronal morphology, and Hematoxylin and eosin (H and E) staining was carried out to quantify damaged neurons. Enzyme-linked immunosorbent assay (ELISA) was implemented to identify molecular mediators involved in neuroprotection. Key findingsPrenatal VPA exposure resulted in significant abnormalities in synaptic development, which were further evidenced by impairments in social interaction and cognitive function. When LPI was administered, most of the synaptic abnormalities were alleviated, as reflected by higher neuron and dendritic spine count. LPI treatment also reduced cytoplasmic cytochrome c concentration and related neuronal cell death. Mechanistically, GPR55 activation by LPI increases the expression of phospho-Akt and phospho-GSK3β, leading to the activation of this signaling in the process of rescuing synaptic abnormalities and mitochondria-mediated neuronal apoptosis. SignificanceThe observed therapeutic effects of GPR55 activation shed light on its significance as a prospective target for ameliorating mitochondrial dysfunction and dendritic spine loss, offering novel prospects for developing targeted interventions to alleviate the neuropathological causes of ASD.

Silver(I) Bromide Phosphines Induce Mitochondrial-Mediated Apoptosis in Malignant Human Colorectal Cells.

Due to its emerging resistance to current therapies, colon cancer remains one of the most difficult types of cancer to treat. Silver, a non-invasive metal, is well-known for its antimicrobial and anti-cancer properties. Two novel silver(I) phosphine complexes, [silver(I) diphenyl-2-pyridylphosphine]Br (1) and [silver(I) is 4-(dimethylamino)phenyldiphenylphosphine]Br (2), were synthesized and characterized by elemental analysis, infrared spectroscopy, and nuclear magnetic resonance (1H, 13C, 31P). To assess the complexes' potentials as antiproliferative agents, experiments were conducted on human colorectal cancer cells (HT-29) in vitro. The evaluation involved the analysis of morphological changes, the performance of an alamarBlue® proliferation assay, and the undertaking of flow cytometric analyses to detect mitochondrial alterations. Complex 1 displayed superior selectivity and significant inhibitory effects on malignant HT-29 cells while exhibiting minimal toxicity towards two non-malignant HEK-293 and MRHF cells. Moreover, after 24 h of treatment, complex 1 (IC50, 7.49 µM) demonstrated higher efficacy in inhibiting cell proliferation compared with complex 2 (IC50, 21.75 µM) and CDDP (IC50, 200.96 µM). Flow cytometric studies indicated that complex 1 induced regulated cell death, likely through mitochondrial-mediated apoptosis. Treatment with complex 1 induced morphological changes indicative of apoptosis, which includes membrane blebbing, PS externalization, increased levels of reactive oxygen species (ROS) and mitochondrial membrane depolarization (ΔΨm). These observations suggest that complex 1 targets the mitochondria and holds promise as a novel metal-based anti-cancer therapeutic for the selective treatment of colorectal cancer.

Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury

Ethnopharmacological relevanceErigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368–1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of “Zuo tan you huan.” In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. Aim of the studyIn this study, a metabonomics study based on ultraperformance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. Materials and methodsThis study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. ResultsErigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP–kynurequinolinic acid and succinylornithine–urea cycle–NO metabolic pathways. ConclusionsThis paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.

A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer.

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

Effects of Alginic Acid on the Porcine Granulosa Cells and Maturation of Porcine Oocytes.

Alginic acid (AA) from brown algae is a marine organic compound. There is extensive use of AA in the food industry and healthcare, suggesting a high probability of AA exposure. The present study investigates the effects of AA on porcine ovarian granulosa cells (GCs) and oocytes to explore its mechanism in female reproduction because of its adverse effects on reproduction. The study adds 20 µM AA to the porcine primary ovarian GCs medium and porcine oocyte in vitro maturation (IVM) medium. Estrogen and progesterone levels are downregulated in GCs. Reactive oxygen species are excessive, and the antioxidant capacity declines. Then mitochondria-mediated apoptosis pathway is involved in GCs apoptosis. In addition, scores of autophagosomes are found in the experimental cells. Furthermore, AA significantly inhibits the proliferation of GCs around cumulus-oocyte complexes (COCs) accompanied by abnormal spindle assembly, chromosome arrangement disorder, and aberrant cortical granules distribution in oocytes, leading to a decreased oocyte maturation rate. These findings suggest that 20 µM AA is toxic to sow reproduction by interfering with estrogen production, oxidative stress, mitochondria-mediated apoptosis, autophagy in GCs of sows, and oocyte maturation.

Inhibition of autophagy can promote the apoptosis of bladder cancer cells induced by SC66 through the endoplasmic reticulum stress pathway

Bladder cancer is among the ten most prevalent cancer types worldwide, and its prognosis has not improved significantly in the past three decades because of cognitive limitations in the molecular mechanisms that drive the malignant progression of bladder cancer. Therefore, there is an urgent need to identify new therapeutic drugs or molecular targets to improve the prognosis of patients with bladder cancer. SC66, a novel allosteric inhibitor of AKT, has recently been reported to exert potent anticancer effects on various cancer cells. However, the mechanisms underlying its anticancer effects in bladder cancer remain largely unknown. Consequently, this study aimed to conduct a series of molecular and cellular biology experiments to verify the anticancer effect and potential mechanism of action of SC66 in bladder cancer in vitro. A xenograft tumor model was established to confirm its anticancer role in vivo. Our results showed that SC66 inhibited cell proliferation, triggered mitochondria-mediated apoptosis, and initiated autophagy in bladder cancer cells dose-dependently. In addition, our results suggested that SC66-caused apoptosis and autophagy were endoplasmic reticulum stress-dependent. Interestingly, the activation of autophagy can partially protect bladder cancer cells from apoptosis under endoplasmic reticulum stress induced by SC66 treatment. This study shows that SC66 exerts its anticancer impact on bladder cancer by inhibiting cell proliferation and inducing apoptosis. It also reveals that inhibiting autophagy can increase the cytotoxic effects of SC66 in bladder cancer. Overall, this is the first study on the anticancer effect of SC66 mediated by the endoplasmic reticulum stress pathway and the first report on the AKT-independent anticancer mechanism of SC66 in bladder cancer. Conclusively, exploring the relationship between apoptosis, autophagy, and endoplasmic reticulum stress induced by SC66 indicates that SC66 is a promising novel agent for patients with bladder cancer.

Synthetic azo-dye, Tartrazine induces neurodevelopmental toxicity via mitochondria-mediated apoptosis in zebrafish embryos

Tartrazine (TZ), or E 102 or C Yellow, is a commonly used azo dye in the food and dyeing industries. Its excessive usage beyond permissible levels threatens human health and the aquatic environment. While previous studies have reported adverse effects such as mutagenicity, carcinogenicity, and reproductive toxicity. Our study aimed to comprehensively evaluate the developmental neurotoxicity of TZ exposure via biochemical and behavioral examinations and explored the underlying mechanism via gene expression analyses. TZ at an environmentally relevant concentration (50 mg/L) significantly induces oxidative stress, altered antioxidant (SOD, CAT and GSH) response, triggered cellular damage (MDA and LDH), and induced neuro-biochemical changes (AChE and NO). Gene expression analyses revealed broad disruptions in genes associated with antioxidant defense (sod1, cat, and gstp1), mitochondrial dysfunction (mfn2, opa1, and fis1),evoked inflammatory response (nfkb, tnfa, and il1b), apoptosis activation (bcl2, bax, and p53), and neural development (bdnf, mbp, and syn2a). Behavioral analysis indicated altered thigmotaxis, touch response, and locomotion depending on the concentration of TZ exposure. Remarkably, the observed effective concentrations were consistent with the permitted levels in food products, highlighting the neurodevelopmental effects of TZ at environmentally relevant concentrations. These findings provide valuable insights into the underlying molecular mechanisms, particularly the role of mitochondria-mediated apoptosis, contributing to TZ-induced neurodevelopmental disorders in vivo.

Synergistic effects of MFG-E8 and whey protein on mitigating d-galactose-induced sarcopenia through PI3K/AKT/PGC-1α and MAPK/ERK signaling pathways

Milk fat globule epidermal growth factor 8 (MFG-E8) and whey protein have emerged as promising bionutrient supplements for enhancing skeletal muscle mass and function. In the present study, aging-related sarcopenia rat model was employed to elucidate the effects of the combined administration of MFG-E8 and whey protein on the catabolism and anabolism of gastrocnemius protein. Combined intervention led to notable enhancements in the antioxidative stress status and mitochondrial biogenesis capacity of gastrocnemius muscle fibers in the aging rats, concomitant with a significant inhibition of lipid accumulation. Moreover, the synergistic effect of MFG-E8 and whey protein was found to exert modulatory effects on key signaling pathways, including PI3K/Akt/PGC-1α pathway and MAPK/ERK signaling pathways in the gastrocnemius muscle of the aging rats. Specifically, this combined intervention was observed to promote mitochondrial biogenesis and regulate the expression of protein anabolism and catabolism-related regulators, thereby facilitating the alleviation of mitochondrial oxidative stress and enhancing biogenesis in gastrocnemius tissues. The findings of our study provide compelling evidence for the potential of MFG-E8 as a promising dietary supplement with antisarcopenic properties to ameliorate muscle protein metabolism disorders and mitigate mitochondrial-mediated myoblast apoptosis induced by oxidative stress.

Coleus vettiveroides ethanolic root extract induces cytotoxicity by intrinsic apoptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) contributes to more than 80% of all primary cancers globally and ranks fourth in cancer-related deaths, due to the lack of an effective, definite therapeutic drug. Coleus vettiveroides (CV) has been usedin Indian traditional medicine to treat diabetes, liver ailments, skin diseases, leukoderma, and leprosy. This study investigates the anticancer effect of CV ethanolic root extract in HepG2 cells. HepG2 cells were treated with CV extract, and its cytotoxicity was analyzed by MTT assay. AO/EB staining, propidium iodide staining, DCFH-DA assay, phalloidine staining, flow cytometry, and qPCR studies were performed for ROS expression, apoptosis and cell cycle analysis. The phytochemical analysis confirmed the presence of quercetin and galangin in CV root extract. The results showed that CV inhibited the proliferation of HepG2 cells, with altered cellular and nuclear morphology. CV was also found to increase intracellular ROS levels and oxidative stress markers in HepG2 cells. CV significantly altered the actin microfilament distribution in HepG2 cells and caused cell cycle arrest at the sub G0 -G1 phase. CV also induced mitochondria-mediated apoptosis, as evidenced by increased expression of p53, Bax, cytochrome C, Apaf-1, PARP, caspase-3 and caspase-9, and downregulated Bcl-2 expression. Therefore, CV exerts its anticancer effect by inducing mitochondrial dysfunction, oxidative stress, cytoskeletal disorganization, cell cycle arrest, and mitochondria-mediated apoptosis, and it could be a potent therapeutic option for HCC.

Targeted inhibition of mTOR by BML-275 induces mitochondrial-mediated apoptosis and autophagy in prostate cancer

Prostate cancer (PCa) is the most frequently diagnosed cancer among men and the second leading cause of death in Western countries. Clinically, screening drugs and develop developing new therapeutics to treat PCa is of great significance. In this study, BML-275 was demonstrated to exert potent antitumor effects in PCa by antagonizing mTOR activity. In cultured PCa cells, BML-275 treatment reduced the expression levels of c-Myc and survivin, promoted the activation of p53, and thereby induced p21/cyclin D1/CDK4/6-dependent cell cycle G1/S arrest. As a result, BML-275 inhibited cellular proliferation and induced mitochondrial-mediated apoptosis. In addition, BML-275 treatment triggered autophagy. Interestingly, EACC-mediated suppression of autophagy did not affect BML-275-induced proliferation and apoptosis. Nude mouse tumorigenic experiments also confirmed that BML-275 inhibited PCa growth, induced PCa cell apoptosis and autophagy. Mechanistically, the activities of PI3K/AKT and AMPK pathways were downregulated by BML-275 treatment in vitro and in vivo. Importantly, mTOR, a common downstream negative protein of PI3K/AKT and AMPK signaling, was induced to inactivate, which may be associated with the induction of apoptosis and autophagy. The pharmacological activation of mTOR by MHY1485 abolished the induction of apoptosis and autophagy of BML-275. Molecular docking results showed that BML-275 can bind to the FKRP12-rapamycin binding site on mTOR protein, and thereby may have the same inhibitory activity on mTOR as rapamycin. Thus, these findings indicated that BML-275 induces mitochondrial-mediated apoptosis and autophagy in PCa by targeting mTOR inhibition. BML-275 may be a potential candidate for the treatment of PCa.

Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate

BackgroundBenign prostatic hyperplasia (BPH) is a common disease in elderly men, mainly resulted from an imbalance between cell proliferation and death. Glutathione peroxidase 3 (GPX3) was one of the differentially expressed genes in BPH identified by transcriptome sequencing of 5 hyperplastic and 3 normal prostate specimens, which had not been elucidated in the prostate. This study aimed to ascertain the mechanism of GPX3 involved in cell proliferation, apoptosis, autophagy and ferroptosis in BPH.MethodsHuman prostate tissues, GPX3 silencing and overexpression prostate cell (BPH-1 and WPMY-1) models and testosterone-induced rat BPH (T-BPH) model were utilized. The qRT-PCR, CCK8 assay, flow cytometry, Western blotting, immunofluorescence, hematoxylin and eosin, masson’s trichrome, immunohistochemical staining and transmission electron microscopy analysis were performed during in vivo and in vitro experiments.ResultsOur study indicated that GPX3 was localized both in the stroma and epithelium of prostate, and down-regulated in BPH samples. Overexpression of GPX3 inhibited AMPK and activated ERK1/2 pathway, thereby inducing mitochondria-dependent apoptosis and G0/G1 phase arrest, which could be significantly reversed by MEK1/2 inhibitor U0126 preconditioning. Moreover, overexpression of GPX3 further exerted anti-autophagy by inhibiting AMPK/m-TOR and up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4, mitochondrial GPX4 and cytoplasmic GPX4) to antagonize autophagy-related ferroptosis. Consistently, GPX3 deficiency generated opposite changes in both cell lines. Finally, T-BPH rat model was treated with GPX3 indirect agonist troglitazone (TRO) or GPX4 inhibitor RAS-selective lethal 3 (RSL3) or TRO plus RSL3. These treatments produced significant atrophy of the prostate and related molecular changes were similar to our in vitro observations.ConclusionsOur novel data manifested that GPX3, which was capable of inducing apoptosis via AMPK/ERK1/2 pathway and antagonizing autophagy-related ferroptosis through AMPK/m-TOR signalling, was a promising therapeutic target for BPH in the future.

Lobetyolin inhibits cell proliferation and induces cell apoptosis by downregulating ASCT2 in gastric cancer.

Gastric cancer (GC) is a heterogeneous disease and is the fifth most common cancer worldwide. Lobetyolin, as a bioactive ingredient extracted from Codonopsis pilosula (Franch.) Nannf., has been reported to exert anti-tumor effects in several cancer types. This study was aimed to investigate the role of lobetyolin in GC and the associated mechanism. MKN-45 and MKN-28 cells were incubated with concentrations of lobetyolin for 24h. The viability and survival of GC cells were evaluated by performing MTT assay. Glutamine uptake, Adenosine Triphosphate, reactive oxygen species (ROS), and glutathione levels were measured by corresponding kits. Apoptosis and mitochondrial membrane potential of GC cells were determined by flow cytometry. Alanine, serine, cysteine-preferring transporter 2 (ASCT2) and the AKT/GSK3β/c-Myc pathway protein levels were examined by western blotting. Xenograft model and immunohistochemical staining were used to evaluate the pharmacological effects of lobetyolin in mice in vivo. We found that lobetyolin treatment suppressed the proliferative capacity of both MKN-45 and MKN-28 cells in a concentration-dependent manner. Lobetyolin reduced the uptake of glutamine and downregulated the expression levels of ASCT2 in GC cells and xenograft tumors. Lobetyolin effectively restrained the growth of tumors in vivo. In addition, lobetyolin induced the accumulation of ROS to attenuate mitochondria-mediated apoptosis via downregulation of ASCT2 expression. Lobetyolin promoted the phosphorylation of c-Myc and suppressed the phosphorylation of GSK3β and AKT in both MKN-45 and MKN-28 cells. The level of total Nrf2 protein was reduced after lobetyolin treatment. Overall, lobetyolin exerts anti-cancer effects by repressing cell proliferation and inducing cell apoptosis via downregulation of ASCT2 in GC.

Anti-rheumatoid arthritis effects of total saponins from Rhizoma Panacis Majoris on adjuvant-induced arthritis in rats and rheumatoid arthritis fibroblast-like synoviocytes

BackgroundTotal saponins from Rhizoma Panacis Majoris (RPMTG) showed significant antitumour activity in our previous studies. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) with tumour-like characteristics have received attention as a therapeutic target for RA. However, the potential effect and mechanism of action of RPMTG against RA-FLS remain unclear. ObjectiveThe study investigated the therapeutic effect of RPMTG on adjuvant-induced arthritis (AIA) in rats, and the regulation effect and underlying mechanism on apoptosis, autophagy of RA-FLS. MethodsThe therapeutic effect of RPMTG was determined by the symptoms and signs of AIA rats. The production of inflammatory cytokines was detected by ELISA. Histopathological change of the ankle and synovial tissues were detected by HE staining. Flow cytometry, Hoechst 33342/PI staining, MDC staining, and TEM were used to determine the effects of RPMTG on apoptosis and autophagy. Western blotting was applied to detect the expression levels of proteins. ResultsIn AIA rats, RPMTG treatment ameliorated paw swelling, and arthritis score, restored synovial histopathological changes, inhibited the expression of IL-6 and IL-1β, exhibiting its potent anti-arthritis effect. In vitro, RPMTG depressed the proliferation of RA-FLS, arrested cell cycle in G0/G1 phase, and induced mitochondria-mediated apoptosis. Moreover, RPMTG significantly inhibited the autophagy in vivo and in vitro, proved by decreasing the expression of autophagy-related indicators (LC3II/LC3I, Beclin-1). Mechanistically, the study demonstrated that the activation of p38 MAPK and PI3K/Akt/mTOR pathways was mainly involved in the therapeutic effects of RPMTG. Interestingly, the effect of RPMTG on apoptosis was reversed after Rapamycin treatment, which preliminarily demonstrated that the inhibitory effect of RPMTG on autophagy was beneficial to the effect on inducing apoptosis. The regulation effect of RPMTG concurrently on apoptosis and autophagy revealed its unique advantages in RA treatment. ConclusionRPMTG showed potent therapeutic effects on AIA rats and induced apoptosis, inhibited autophagy mainly through activating the p38 MAPK and PI3K/Akt/mTOR pathways in RA-FLS.

Co-exposure to molybdenum and cadmium evokes necroptosis and decreases apoptosis in duck myocardium

Superfluous molybdenum (Mo) and cadmium (Cd) in the environment are detrimental to organisms through their accumulation. The NF-κB/TNF-α axis plays a vital part in regulating necroptosis and apoptosis. However, the impacts of Mo and/or Cd on myocardium injury in ducks and the function of NF-κB/TNF-α axis are not clear in the process. In this research, ducks exposed to different dosages of Mo and/or Cd were applied as the study object. The findings substantiated that the accumulation of Mo and/or Cd caused elements imbalance and necroptosis in myocardial tissue. As p-NF-κB/TNF-α expression up-regulated, RIPK1/RIPK3/p-MLKL expression significantly increased in all treatment groups, while the expression of c-caspase-8/3 markedly decreased. Moreover, apoptosis rate obviously decreased in Cd treated groups and clearly elevated in Mo group. Mitochondria-mediated apoptosis was activated by excessive Mo and inhibited by Mo + Cd, but Cd exposure alone had little effect on it. Collectively, our research confirmed that Mo and/or Cd evoked necroptosis via NF-κB/TNF-α axis, and decreased death receptor-mediated apoptosis in duck myocardium, the impacts of Mo and/or Cd on mitochondrial-mediated apoptosis were different. These results are significant for studying toxicology of Mo and/or Cd and preserving the ecosystem.

Mitochondria-mediated apoptosis and endoplasmic reticulum stress are involved in the toxicity induced by copper in the porcine spleen.

Copper (Cu) is one of the common heavy metal pollutants in the environment, and its toxic mechanisms have been extensively studied. However, the immunotoxicity induced by Cu remains rarely reported, and the effects of Cu on endoplasmic reticulum stress and mitochondria-mediated apoptosis have been little studied in the spleen. In this study, pigs were fed with different contents of Cu (10, 125, and 250mg/kg Cu) for 80days to establish a toxicity model. The results showed the Cu exposure triggered endoplasmic reticulum stress in the spleen, as evidenced by increased mRNA and protein levels of GRP94, GRP78, CHOP, XBP1, ATF6, and JNK; the positive rate of GRP78 increased by immunofluorescence analysis. Additionally, mitochondrial fission and fusion homeostasis were disrupted, the expression levels of mitochondrial dynamics-related genes Mfn1, Mfn2, and OPA1 decreased, DRP1 increased, and the positive rate of Mfn1 decreased by immunofluorescence analysis. Furthermore, Cu exposure could induce apoptosis, as demonstrated by the increased expression level of related proteins and genes Bak, Bax, Caspase-3, P53, and Cytc. In conclusion, these results suggest chronic Cu exposure can lead to endoplasmic reticulum stress and imbalance in mitochondrial dynamics and induced apoptosis of pig spleen, and these results provided new insights into the underlying mechanism of Cu exposure caused splenic toxicity, which has public health implications where humans and animals are exposed to copper contamination.

Endogenous PTEN acts as the key determinant for mTOR inhibitor sensitivity by inducing the stress-sensitized PTEN-mediated death axis in KSHV-associated malignant cells.

As a part of viral cancer evolution, KSHV-infected human endothelial cells exert a unique transcriptional program via upregulated mTORC1 signaling. This event makes them sensitive to mTOR inhibitors. Master transcriptional regulator PTEN acts as the prime regulator of mTOR and determining factor for mTOR inhibitory drug resistance and sensitivity. PTEN is post-translationally modified in KSHV-associated cell lines and infected tissues. Our current study is an attempt to understand the functional role of upstream modulator PTEN in determining the sensitivity of mTOR inhibitors against KSHV-infected cells in an in vitro stress-responsive model. Our analysis shows that, despite phosphorylation, endogenous levels of intact PTEN in different KSHV-infected cells compared to normal and non-infected cells are quite high. Genetic overexpression of intact PTEN showed functional integrity of this gene in the infected cells in terms of induction of a synchronized cell death process via cell cycle regulation and mitochondria-mediated apoptosis. PTEN overexpression enhanced the mTOR inhibitory drug activity, the silencing of which hampers the process against KSHV-infected cells. Additionally, we have shown that endogenous PTEN acts as a stress balancer molecule inside KSHV-infected cells and can induce stress-sensitized death program post mTOR inhibitor treatment, lined up in the ATM-chk2-p53 axis. Moreover, autophagic regulation was found as a major regulator in mTOR inhibitor-induced PTEN-mediated death axis from our study. The current work critically intersected the PTEN-mediated stress balancing mechanism where autophagy has been utilized as a part of the KSHV stress management system and is specifically fitted and switched toward autophagy-mediated apoptosis directing toward a therapeutic perspective.