LC3 Protein Research Articles

Licochalcone B, a Natural Autophagic Agent for Alleviating Oxidative Stress-Induced Cell Death in Neuronal Cells and Caenorhabditis elegans Models.

Autophagy has been implicated in the regulation of neuroinflammation and neurodegenerative disorders. Licochalcone B (LCB), a chalcone from Glycyrrhiza inflata, has been reported to have anti-cancer, anti-oxidation and anti-β–amyloid fibrillation effects; however, its effect in autophagy remain un-investigated. In the current study, the potential neuro-protective role of LCB in terms of its anti-oxidative, anti-apoptotic, and autophagic properties upon oxidative stress-induced damage in neuronal cells was investigated. With the production of reactive oxygen species (ROS) as a hallmark of neuroinflammation and neurodegeneration, hydrogen peroxide (H2O2) was adopted to stimulate ROS-induced cell apoptosis in PC-12 cells. Our findings revealed that LCB reduced cell cytotoxicity and apoptosis of PC-12 cells upon H2O2-stimulation. Furthermore, LCB increased the level of the apoptosis-associated proteins caspase-3 and cleaved caspase-3 in H2O2-induced cells. LCB effectively attenuated the level of oxidative stress markers such as MDA, SOD, and ROS in H2O2-induced cells. Most importantly, LCB was confirmed to possess its anti-apoptotic effects in H2O2-induced cells through the induction of ATG7-dependent autophagy and the SIRT1/AMPK signaling pathway. As a novel autophagic inducer, LCB increased the level of autophagy-related proteins LC3–II and decreased p62 in both neuronal cells and Caenorhabditis elegans (C. elegans) models. These results suggested that LCB has potential neuroprotective effects on oxidative damage models via multiple protective pharmacological mechanisms.

LncRNA NEAT1 induces autophagy through the miR-128-3p/ADAM28 axis to suppress apoptosis of nonsmall-cell lung cancer.

This study aimed to identify the molecular mechanism underlying NEAT1 regulation of non-small-cell lung cancer (NSCLC) autophagy and apoptosis. The expression levels of NEAT1, miR-128-3p, and ADAM28 in NSCLC tissues and cells were examined using qRT-PCR. The relationships between NEAT1, miR-128-3p, and ADAM28 expression levels and prognosis of NSCLC patients were investigated using the Kaplan-Meier analysis method. The interactions between NEAT1, miR-128-3p, and ADAM28 were confirmed by dual-luciferase reporter assay or FISH assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry assays, respectively. Apoptosis and autophagy-related proteins Bax, cleaved caspase-3, cleaved caspase-9, Bcl-2, LC3, Beclin-1, and p62 were analyzed by western blotting. Finally, an in vivo NSCLC mouse xenograft model was established to confirm the in vitro data. We showed NEAT1 and ADAM28 expression levels were upregulated, while the miR-128-3p level was downregulated in NSCLC tissues and cells. NSCLC patients with high NEAT1 expression levels, low miR-128-3p levels, or high ADAM28 levels had significantly reduced overall survival times. Silencing of NEAT1 inhibited NSCLC cell autophagy and promoted apoptosis by sponging miR-128-3p. MiR-128-3p directly targeted ADAM28 and suppressed ADAM28 expression, which led to deactivation of the JAK2/STAT3 signaling pathway. Furthermore, ADAM28 overexpression attenuated miR-128-3p overexpression-induced apoptosis and autophagy inhibition in NSCLC by increasing the phosphorylation of JAK2 and STAT3. NEAT1 depletion or miR-128-3p overexpression inhibited autophagy and promoted apoptosis in vivo by suppressing ADAM28. In other word, silencing NEAT1 inhibited autophagy and then promoted NSCLC cell apoptosis by deactivating the JAK2/STAT3 signaling pathway through regulation of miR-128-3p/ADAM28 axis.

The Reduced Graphene Oxide (rGO) Induces Apoptosis, Autophagy and Cell Cycle Arrest in Breast Cancer Cells.

Reduced graphene oxide (rGO) has already been reported as a potential cytostatic agent in various cancers. However, the mechanisms underlying rGO’s cytotoxicity are still insufficiently understood. Thus, the aim of the study was to investigate the molecular and cellular effects of rGO in breast cancer. Given this, two cell lines, MDA-MB-231 and ZR-75-1, were analyzed using MTT test, flow cytometry and Western blot assay. Incubation with rGO resulted in a multitude of effects, including the stimulation of autophagy, cell cycle arrest and, finally, the apoptotic death of cancer cells. Notably, rGO had minimal effect on normal human fibroblasts. Apoptosis in cancer cells was accompanied by decreased mitochondrial membrane potential, the deregulated expression of mitochondrial proteins and the activation of caspase 9 and caspase 3, suggesting that rGO predominantly induced apoptosis via intrinsic pathway. The analysis of LC3 protein expression revealed that rGO also caused autophagy in breast cancer cells. Moreover, rGO treatment resulted in cell cycle arrest, which was accompanied by deregulated p21 expression. Altogether, rGO seems to have multidirectional cytostatic and cytotoxic effects in breast cancer cells, making it a promising agent worthy of further investigation.

OPN inhibits autophagy through CD44, integrin and the MAPK pathway in osteoarthritic chondrocytes.

BackgroundTo investigate whether osteopontin (OPN) affects autophagy in human osteoarthritic chondrocytes and determine the roles of CD44, αvβ3 integrin and the Mitogen-activated protein kinase (MAPK) pathway in this progress.MethodsFirst, we compared the autophagy levels in the human osteoarthritis (OA) and normal cartilage, then, we cultured human OA chondrocytes in vitro and treated cells with recombinant human OPN (rhOPN) to determine autophagy changes. Next, the anti-CD44 and anti-CD51/61 monoclonal antibodies (Abs) or isotype IgG were used to determine the possible role of CD44 and αvβ3 integrin; subsequently, an inhibitor of the ERK MAPK pathway was used to investigate the role of ERK MAPK. Western blotting was used to measure the Beclin1, LC3 II and MAPK proteins expressions, mRFP-GFP-LC3 confocal imaging and transmission electron microscopy were also used to detect the autophagy levels. Cell Counting Kit-8 (CCK-8) was used to assay the proliferation and activity of chondrocytes.ResultsThe LC3 protein was greatly decreased in OA cartilage compared to normal cartilage, and OPN suppressed the autophagy activity in chondrocytes in vitro. Blocking experiments with anti-CD44 and anti-CD51/61 Abs indicated that OPN could suppress the expression of LC3II and Beclin1 through αvβ3 integrin and CD44. Our results also indicated that the ratio of p-ERK/ERK but not p-P38/P38 and p-JNK/JNK was increased after the rhOPN treatment. The ERK inhibitor inhibited the activity of OPN in the suppression of autophagy, and the CCK-8 results showed that rhOPN could promote chondrocyte proliferation.ConclusionOPN inhibited chondrocyte autophagy through CD44 and αvβ3 integrin receptors and via the ERK MAPK signaling pathway.

LIPOPOLYSACCHARIDE INHIBITS AUTOPHAGY IN HUMAN DERMAL FIBROBLASTS IN VITRO

Objective: Fibroblasts are mesenchymal cells of dermal origin that provide structural integrity for connective tissue by producing the extracellular matrix proteins. They are the dominant component of the tumor microenvironment and are then referred to as cancer-associated fibroblasts (CAFs). CAFs play an important role in cancer cell growth. Autophagy is an intracellular self-degradative process that balances cell energy sources and regulates tissue homeostasis. Autophagy is now considered a critical process in skin health and skin cancer. The objective of this study is to investigate the effect of lipopolysaccharide (LPS) on the autophagy mechanism in human dermal fibroblasts (HDFs) in vitro. Methods: HDFs were incubated with 0.1, 0.5, 1 and 5 μg/mL of LPS for 24 h. The viability of cells was determined using WST-1 assay. The protein expressions of beclin-1, p62, LC3-I and LC3-II were analyzed by Western blotting. Results: The viability of HDFs incubated with 0.1, 0.5, 1 and 5 μg/mL of LPS did not show any significant differences compared to untreated cells. Beclin-1 protein expression was upregulated with all LPS concentrations. The protein expression of p62 was also induced with all LPS concentrations. Furthermore, the ratio of LC3-II/LC3-I protein expression was increased with all LPS concentrations. Conclusion: Our results demonstrate that LPS inhibits autophagy in HDFs through the upregulation of p62 and LC3 protein expression. These findings suggest the relationship between inflammation and autophagy in skin cells and might contribute to understanding the key role of autophagy in the development of skin cancer.

Targeting microglial autophagic degradation of the NLRP3 inflammasome for identification of thonningianin A in Alzheimer’s disease

BackgroundNLRP3 inflammasome-mediated neuroinflammation plays a critical role in the pathogenesis and development of Alzheimer’s disease (AD). Microglial autophagic degradation not only decreases the deposits of extracellular Aβ fibrils but also inhibits the activation of NRLP3 inflammasome. Here, we aimed to identify the potent autophagy enhancers from Penthorum chinense Pursh (PCP) that alleviate the pathology of AD via inhibiting the NLRP3 inflammasome.MethodsAt first, autophagic activity-guided isolation was performed to identify the autophagy enhancers in PCP. Secondly, the autophagy effect was monitored by detecting LC3 protein expression using Western blotting and the average number of GFP-LC3 puncta per microglial cell using confocal microscopy. Then, the activation of NLRP3 inflammasome was measured by detecting the protein expression and transfected fluorescence intensity of NLRP3, ASC, and caspase-1, as well as the secretion of proinflammatory cytokines. Finally, the behavioral performance was evaluated by measuring the paralysis in C. elegans, and the cognitive function was tested by Morris water maze (MWM) in APP/PS1 mice.ResultsFour ellagitannin flavonoids, including pinocembrin-7-O-[4″,6″-hexahydroxydiphenoyl]-glucoside (PHG), pinocembrin-7-O-[3″-O-galloyl-4″,6″-hexahydroxydiphenoyl]-glucoside (PGHG), thonningianin A (TA), and thonningianin B (TB), were identified to be autophagy enhancers in PCP. Among these, TA exhibited the strongest autophagy induction effect, and the mechanistic study demonstrated that TA activated autophagy via the AMPK/ULK1 and Raf/MEK/ERK signaling pathways. In addition, TA effectively promoted the autophagic degradation of NLRP3 inflammasome in Aβ(1–42)-induced microglial cells and ameliorated neuronal damage via autophagy induction. In vivo, TA activated autophagy and improved behavioral symptoms in C. elegans. Furthermore, TA might penetrate the blood-brain barrier and could improve cognitive function and ameliorate the Aβ pathology and the NLRP3 inflammasome-mediated neuroinflammation via the AMPK/ULK1 and Raf/MEK/ERK signaling pathways in APP/PS1 mice.ConclusionWe identified TA as a potent microglial autophagy enhancer in PCP that promotes the autophagic degradation of the NLRP3 inflammasome to alleviate the pathology of AD via the AMPK/ULK1 and Raf/MEK/ERK signaling pathways, which provides novel insights for TA in the treatment of AD.

Activating Hypoxia-Inducible Factor-1α Reduces Myocardial Ischemia-Reperfusion Injury in Mice Through Hexokinase II

To verify that HIF-1α/HKII pathway is the key mechanism to alleviate myocardial ischemiareperfusion (IR) injury in aged mice through HIF-1α and HKII inhibitors. A mouse I/R model was carried out in young and old C57BL/6 mice for 60 min and reperfusion for 120 min. Mice were injected intraperitoneally with AAV-9 virus to introduce HIF-1α 24 h before ischemia. After 2 h of reperfusion, the mitochondrial ultrastructure, ATP content, membrane potential, and protein expression of HIF-1α, LC3, Bax, Bcl-2, Caspese-9, Caspase-3, and Cyt-3 were detected. After 24 h, the myocardial infarction area and cardiac ability were evaluated. Young mice and old mice have different protective effects after acute ischemia/reperfusion injury. After the introduction of HIF-1α by AAV-9 virus, the expression of the downstream target gene HKII can be up-regulated. At the same time, it reduces the expression of key proteins LC3, Bax, Caspese-9, and Caspase-3. Stabilize the membrane potential, ultimately reduce the area of myocardial infarction and improve heart function. Young mice and old mice have different protective effects after acute ischemia/reperfusion injury. The I/R in the older is caused by HIF-1α. HIF-1α inhibits the opening of mPTP by up-regulating HKII, stabilizes mitochondrial membrane potential, protects mitochondrial integrity, and reduces myocardial ischemia-reperfusion damage to the myocardium of elderly mice.

Dapagliflozin Improves Diabetic Cardiomyopathy by Modulating the Akt/mTOR Signaling Pathway.

Background Dapagliflozin can significantly improve heart failure, and Cx43 is one of the molecular mechanisms of heart failure. This study investigated the effect of dapagliflozin on Cx43 and Akt/mTOR signaling pathway in ventricular myocytes. Methods A rat model of type 2 diabetes mellitus was established by high-fat diet combined with streptozotocin, and the animals were treated randomly with dapagliflozin. The morphological changes of the myocardium were observed by hematoxylin eosin staining, and the expression and distribution of Cx43 in ventricular myocytes were detected by immunohistochemistry. And Western blot determined the expressions of Cx43, Akt, mTOR, p62, and LC3 proteins in rat myocardium. Results Compared with the normal control group, the heart rate of diabetic rats decreased significantly (p < 0.05), QRS wave of ECG widened, and QT interval prolonged (p < 0.05). Dapagliflozin treatment in diabetic rats resulted in improvements in these ECG indexes (p < 0.05) with early administration group obtaining greater efficacy than the late administration group (p < 0.05). In the normal control group, the cardiomyocytes were arranged orderly, and the expression of Cx43 was dense, uniform, and regular, which was higher than that in the intercalated disc. In the diabetic control model group, the cardiomyocytes were enlarged and presented disorderly with detection of Cx43 in the cytoplasm. Early use of dapagliflozin better improved these myocardial tissue lesions. Of note, as diabetic rats exhibited decreased expression of Cx43, Akt, and mTOR (p < 0.05), increased p62 expression (p < 0.05), and decreased LC3-II/I ratio (p < 0.05), administration of dapagliflozin partially reversed the expression of the above proteins (p < 0.05) with greater improvement in the early administration group compared with the late administration group (p < 0.05). Conclusions Dapagliflozin increases the expression of Cx43 in cardiomyocytes of diabetic rats and thereby alleviates heart failure partly through regulating the Akt/mTOR signaling pathway.

Grape seed proanthocyanidin extract induces apoptotic and autophagic cell death in rheumatoid arthritis fibroblast-like synoviocytes.

In this study, we aimed to evaluate the association between grape seed proanthocyanidin extract (GSPE) and rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLSs) and to investigate whether GSPE induces cell death in RA-FLSs. The FLSs were isolated from RA synovial tissues. Cell viability and cell cycle staging were analyzed using a hemocytometer and flow cytometry. Caspase 3 and poly (ADP-ribose) polymerase (PARP) proteins were analyzed using Western blotting with z-VAD-fmk. Protein LC3 and polyubiquitin-binding protein p62 that were degraded by autophagy were evaluated using Western blotting with 3-methyladenine and chloroquine. Reactive oxygen species (ROS) were also evaluated. When RA-FLSs were treated with GSPE, cell viability decreased, the number of cells in sub-G1 and G2/M phases increased, and the expression of pro-PARP and pro-caspase 3 proteins decreased in a concentration-dependent manner. This result was offset, when the cells were co-treated with the pan-caspase inhibitor z-VAD-fmk. The reduced cell viability, increased expression of LC3-II protein, and reduced expression of p62 protein with GSPE treatment were offset, when RA-FLSs were co-treated with GSPE and autophagy inhibitors 3-methyladenine and chloroquine. The level of ROS in RA-FLSs treated with GSPE was significantly lower than treatment with N-acetyl-cysteine, a ROS inhibitor. Our study results show that GSPE induces apoptotic and autophagic cell death and inhibites reactive oxygen species in RA-FLSs.

Caryophyllene Oxide Induces Ferritinophagy by Regulating the NCOA4/FTH1/LC3 Pathway in Hepatocellular Carcinoma.

Ferritinophagy is associated with tumor occurrence, development, and therapy effects. Ferritinophagy and ferroptosis are regulated by iron metabolism and are closely connected. LC3 protein is a key protein in autophagy. Following the binding of NCOA4 to FTH1, it links to LC3Ⅱ in lysosomes, a symbol of ferritinophagy. A ferritinophagy’s inducer is likely to open new avenues for anticancer medication research and development. In this study, we discovered that caryophyllene oxide has a substantial inhibitory effect on HCCLM3 and HUH7 cells, by regulating the level of cellular oxidative stress, and the levels of autophagy and iron metabolism in HCCLM3 and HUH7 cells, leading to a ferritinophagy-related phenomenon. Furthermore, the results of T-AOC, DPPH free radical scavenging rate, and hydroxyl radical inhibition indicated that caryophyllene oxide can inhibit cell anti-oxidation. The examination of the ferritinophagy-related process revealed that caryophyllene oxide promotes the production and accumulation of intracellular reactive oxygen species and lipid peroxidation. NCOA4, FTH1, and LC3Ⅱ were found to be targeted regulators of caryophyllene oxide. Caryophyllene oxide regulated NCOA4, LC3 Ⅱ, and FTH1 to promote ferritinophagy. In vivo, we discovered that caryophyllene oxide can lower tumor volume, significantly improve NCOA4 and LC3 protein levels in tumor tissue, and raise Fe2+ and malondialdehyde levels in serum. The compound can also reduce NRF2, GPX4, HO-1, and FTH1 expression levels. The reduction in the expression levels of NRF2, GPX4, HO-1, and FTH1 by caryophyllene oxide also inhibited GSH and hydroxyl radical’s inhibitory capacities in serum, and promoted iron deposition in tumor tissue resulting in the inhibition of tumor growth. In summary, our study revealed that caryophyllene oxide mostly kills liver cancer cells through ferritinophagy-mediated ferroptosis mechanisms. In conclusion, caryophyllene oxide may be used as a ferritinophagy activator in the field of antitumor drug research and development.

Induction of Autophagy by Ursolic Acid Promotes the Elimination of Trypanosoma cruzi Amastigotes From Macrophages and Cardiac Cells.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an infectious illness endemic to Latin America and still lacks an effective treatment for the chronic stage. In a previous study in our laboratory, we established the protective role of host autophagy in vivo during T. cruzi infection in mice and proposed this process as one of the mechanisms involved in the innate immune response against this parasite. In the search for an autophagy inducer that increases the anti-T. cruzi response in the host, we found ursolic acid (UA), a natural pentacyclic triterpene with many biological actions including autophagy induction. The aim of this work was to study the effect of UA on T. cruzi infection in vitro in the late infection stage, when the nests of intracellular parasites are forming, in both macrophages and cardiac cells. To test this effect, the cells were infected with T. cruzi for 24 h and then treated with UA (5–10 µM). The data showed that UA significantly decreased the number of amastigotes found in infected cells in comparison with non-treated cells. UA also induced the autophagy response in both macrophages and cardiac cells under the studied conditions, and the inhibition of this pathway during UA treatment restored the level of infection. Interestingly, LC3 protein, the main marker of autophagy, was recruited around amastigotes and the acidic probe LysoTracker localized with them, two key features of xenophagy. A direct cytotoxic effect of UA was also found on trypomastigotes of T. cruzi, whereas epimastigotes and amastigotes displayed more resistance to this drug at the studied concentrations. Taken together, these data showed that this natural compound reduces T. cruzi infection in the later stages by promoting parasite damage through the induction of autophagy. This action, in addition to the effect of this compound on trypomastigotes, points to UA as an interesting lead for Chagas disease treatment in the future.

Lysosomal dysfunction is associated with NLRP3 inflammasome activation in chronic unpredictable mild stress-induced depressive mice

NLRP3 inflammasome pathway-mediated inflammatory response is closely associated with depression. Increasing attention has been recently paid to the links between autophagy and depression, however, the relationship between autophagy and NLRP3 inflammasome in depressive behavior remain poorly understood. In the present study, the potential roles of autophagy-lysosome pathway in NLRP3 inflammasome regulation were investigated both in vivo (chronic unpredictable mild stress (CUMS)-induced depressive mouse model) and in vitro (LPS-induced cellular model) model. It demonstrated that CUMS induces depressive-like behaviors in mice, accompanied by increased expression of NLRP3 inflammasome and inflammatory responses. Meanwhile, it promoted the autophagosome marker LC3 and autophagic adapter protein p62 accumulation, accompanied by the decrease of lysosomal cathepsins B and D expression in the prefrontal cortex of mice. Notably, a significant colocalization of NLRP3 and LC3 in CUMS mice by immunofluorescence co-staining were observed. For the in vitro study, disrupting the lysosomal function with Baf A1 significantly increased the LPS-induced NLRP3 inflammasome accumulation and pro-inflammatory factors (IL-1β and IL-18) production in BV2 cells. Collectively, our results suggested that the autophagic process is related to NLRP3 inflammasome activation, and dysfunctional lysosome in autophagy-lysosomal pathway may retard NLRP3 inflammasome degradation, facilitating the production of pro-inflammatory factors, thereby contributing to depressive behavior in CUMS mice.

Effects of captopril on glucose metabolism and autophagy in liver and muscle from mice with type 1 diabetes and diet-induced obesity

Impaired metabolic functions underlie the pathophysiology of diabetes and obesity. The renin-angiotensin system (RAS) is one pathway related to the pathophysiology of both diseases. RAS activation in metabolically active tissues exerts pro-inflammatory effects via angiotensin II (Ang II), linked to dysfunction in cellular processes such as autophagy, which is associated with obesity and diabetes. Here, we determined whether RAS is involved in metabolic dysregulations in a Type 1 Diabetes (T1D) mouse model, treated with captopril, and in an obesity mouse model (Agt-Tg) that overexpresses angiotensinogen (Agt) in adipose tissue. T1D mice had lower plasma leptin, resistin and higher non-esterified fatty acids (NEFA) compared to wild type (Wt) mice, even under captopril treatment. Further, mRNA levels for Agt, At1, Insr, and Beclin1 were upregulated in muscle and liver of T1D mice with captopril compared to Wt. Moreover, autophagy markers LC3 and p62 proteins were decreased, regardless of captopril treatment in the liver from T1D mice. In obese Wt mice, captopril increased muscle Irs1 gene levels. Further, captopril reduced mRNA levels of At1, Insr, Ampk, Beclin1, Atg12, and Lc3 in the liver from both Wt and Agt-Tg mice, while Agt, At1, Insr, and Atg12 expression was reduced in Agt-Tg mice without captopril treatment. Irs1 expression was decreased in the liver from obese Wt mice treated with captopril. Our results suggest that captopril treatment upregulates components of RAS, insulin signaling, and autophagy in both muscle and liver, indicating potential utility of captopril in targeting both insulin sensitivity and autophagy in diabetes and obesity.

Astragalus-Scorpion Drug Pair Inhibits the Development of Prostate Cancer by Regulating GDPD4-2/PI3K/AKT/mTOR Pathway and Autophagy.

Objective: Prostate cancer (PCa) is an epithelial malignancy of the prostate that currently lacks effective treatment. Traditional Chinese medicine (TCM) can play an anticancer role through regulating the immune system, anti-tumor angiogenesis, regulating tumor cell apoptosis, autophagy dysfunction, and other mechanisms. This study attempted to explore the active ingredients and potential mechanism of action of the Astragalus–Scorpion (A–S) drug pair in PCa, in order to provide new insights into the treatment of PCa. Methods: Network pharmacology was used to analyze the A–S drug pair and PCa targets. Bioinformatics analysis was used to analyze the LncRNAs with significant differences in PCa. The expression of LC3 protein was detected by immunofluorescence. CCK8 was used to detect cell proliferation. The expressions of GDPD4-2, AC144450.1, LINC01513, AC004009.2, AL096869.1, AP005210.1, and BX119924.1 were detected by RT-qPCR. The expression of the PI3K/AKT/mTOR pathway and autophagy-related proteins were detected by western blot. LC-MS/MS was used to identify the active components of Astragalus and Scorpion. Results: A–S drug pair and PCa have a total of 163 targets, which were mainly related to the prostate cancer and PI3K/AKT pathways. A–S drug pair inhibited the formation of PCa, promoted the expression of LC3Ⅱ and Beclin1 proteins, and inhibited the expression of P62 and PI3K–AKT pathway proteins in PCa mice. Astragaloside IV and polypeptide extract from scorpion venom (PESV) were identified as the main active components of the A–S drug pair. GDPD4-2 was involved in the treatment of PCa by Astragaloside IV-PESV. Silencing GDPD4-2 reversed the therapeutic effects of Astragaloside IV-PESV by regulating the PI3K/AKT/mTOR pathway. Conclusion: Astragaloside IV-PESV is the main active components of A–S drug pair treated PCa by regulating the GDPD4-2/PI3K–AKT/mTOR pathway and autophagy.

Abstract 2996: Pre-activation of autophagy impacts PARP inhibitor sensitivity of prostate cancer cells

Abstract Introduction: Advanced prostate cancer (APC) is one of the most common causes of cancer-related mortality in males largely related to the development of resistance to hormonal and chemotherapeutic based regimens. Recently, the PARP inhibitor (PARPi) olaparib was shown to improve progression-free and overall survival in patients with APC although resistance remains a barrier to cure. Therapeutic resistance in cancers can be mediated by autophagy, a cellular recycling process. Here we addressed the role of autophagy in the development of olaparib resistance in APC. Methods: Autophagy levels and olaparib sensitivity were evaluated in prostate cancer (PC) cell lines (LNCaP, C4-2B and PC-3) by western blots and clonogenic assays, respectively. We introduced a double-tagged (mCherry-GFP) LC3 protein in cells to monitor autophagic flux by confocal microscopy and evaluated the temporal importance of autophagy activation by rapamycin on olaparib sensitivity. We measured cell proliferation (Incucyte), cell cycle (flow cytometry) and DNA repair (microscopy). Finally, we confirmed the role of autophagy in olaparib sensitivity using CRISPR/Cas9 Atg16L1 knockout cell lines. Results: We found a correlation between the basal level of autophagy and olaparib sensitivity in PC cell lines. PC-3 cells were olaparib resistant and had a higher basal level of autophagy compared to LNCaP and C4-2B cells, which were PARPi sensitive. When autophagy was pre-activated before olaparib treatment, PC cell lines became more resistant by increasing homologous recombination activity. This autophagy-mediated resistance was partially regulated by a balance between SQSTM1 and FLNA protein levels in the nucleus. Inhibition of autophagy or post-activated autophagy prevented development of PARPi resistance. Conclusion: The autophagic flux is associated with PARPi resistance in PC cell lines by regulating DNA repair. Inhibiting autophagy may lead to new strategies to increase PARPi efficacy. Understanding the role of autophagy in PC progression may lead to strategies that prevent the development of PARPi resistance. Citation Format: Maxime Cahuzac, Benjamin Péant, Hubert Fleury, Anne-Marie Mes-Masson, Fred Saad. Pre-activation of autophagy impacts PARP inhibitor sensitivity of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2996.

Hepatocyte nuclear factor 1A suppresses innate immune response by inducing degradation of TBK1 to inhibit steatohepatitis

Non-alcoholic steatohepatitis (NASH), a progressive form of non-alcoholic fatty liver disease (NAFLD), is characterised by chronic liver inflammation, which can further progress into complications such as liver cirrhosis and NASH-associated hepatocellular carcinoma (HCC) and therefore has become a growing health problem worldwide. The type I interferon (IFN) signaling pathway plays a pivotal role in chronic inflammation; however, the molecular mechanisms underlying NAFLD/NASH from the perspective of innate immune response has not yet been fully explored. In this study, we elucidated the mechanisms of how innate immune response modulates NAFLD/NASH pathogenesis, and demonstrated that hepatocyte nuclear factor-1alpha (HNF1A) was suppressed and the type I IFN production pathway was activated in liver tissues of patients with NAFLD/NASH. Further experiments suggested that HNF1A negatively regulates the TBK1-IRF3 signaling pathway by promoting autophagic degradation of phosphorylated-TBK1, which constrains IFN production, thereby inhibiting the activation of type I IFN signaling. Mechanistically, HNF1A interacts with the phagophore membrane protein LC3 through its LIR-docking sites, and mutations of LIRs (LIR2, LIR3, LIR4, and LIRs) block the HNF1A-LC3 interaction. In addition, HNF1A was identified not only as a novel autophagic cargo receptor but also to specifically induce K33-linked ubiquitin chains on TBK1 at Lys670, thereby resulting in autophagic degradation of TBK1. Collectively, our study illustrates the crucial function of the HNF1A-TBK1 signaling axis in NAFLD/NASH pathogenesis via cross-talk between autophagy and innate immunity.

Effect of starvation and refeeding on reactive oxygen species, autophagy and oxidative stress in Chinese perch (Siniperca chuatsi) muscle growth.

In skeletal muscle, autophagy regulates the development and growth of muscle fibres and maintains the normal muscle metabolism. Under starvation and refeeding conditions, the effect of reactive oxygen species (ROS) levels on skeletal muscle autophagy is still unclear, although the excessive accumulation of ROS has been shown to increase autophagy in cells. The purpose of this study was to explore the effects of starvation and diet after starvation on the autophagy of adult Chinese perch muscle, and to determine the level of ROS in the muscle. We performed zero (Normal control), three and seven starvation treatments on adult Chinese perch, and returned to normal feeding for 3 days after starvation for 7 days. In the muscles of the adult Chinese perch muscle after 3 days of starvation, the autophagy marker protein LC3 and the number of autophagosomes remained basically the same as in the normal feeding situation. However, on starvation for 7 days, the mitochondrial autophagy was sensitive and the number of autophagosomes increased, but the antioxidant-related molecules (malondialdehyde, catalase, glutathione S-transferase, glutathione and anti-superoxide anion) decreased and the accumulation of ROS was obvious. In addition, the extended starvation time also increased the level of LC3 protein. However, by refeeding after starvation this nutritional stress resulted in a decrease in ROS levels and a partial restoration of antioxidant enzyme activity. Our data show that in the adult Chinese perch muscle, starvation could reduce the antioxidant activity through the accumulation of ROS, and that the number of autophagosomes continues to increase. Refeeding after starvation could effectively compensate for the level of ROS, and restore the mRNA abundance of antioxidant genes and the activity of antioxidant enzymes to reduce autophagy and improve feed efficiency. Further research should optimize starvation conditions to reduce autophagy in muscles and maintain normal muscle metabolism.

Therapeutic Effect and Mechanism of Cinnamyl Alcohol on Myocardial Ischemia-Reperfusion Injury.

Objective To investigate the effect of CA on autophagy and its molecular mechanism after myocardial ischemia/reperfusion injury (MI/RI). Methods The MI/RI model was established by the ligation of the left anterior descending coronary artery with ischemia and reperfusion. In vitro cell models were established using hypoxia/reoxygenation. Western blot was used to determine the expression levels of beclin-1, P62, and LC3 II. The expression levels of IL-1β, IL-6, TNFα, and apoptosis-related genes Bax, Cyt-c, and Bcl-2 were detected by qRT-PCR. Cell activity was detected by CCK-8. Apoptosis was detected by TUNEL staining. Results Beclin-1, P62, and LC3 II protein expression and LC3 II/LC3 I level were significantly increased after myocardial ischemia-reperfusion injury. Compared with model group, CA downregulated beclin-1, P62, and LC3 II protein expression and LC3 II/LC3 I level in the myocardium. The results of cell-level experiments showed that CA inhibited the autophagy response of the cardiomyocytes induced by hypoxia-reperfusion injury. Mechanism studies showed that CA targeted the inhibition of ATG12. Knocking down ATG12 reduces the production of inflammatory cytokines induced by H/R. The knockdown of ATG12 also reduced apoptosis and injury of the myocardial cells. Conclusion Myocardial ischemia-reperfusion can enhance autophagy response and promote apoptosis. CA plays a protective role in myocardium by targeting ATG12, thereby inhibiting autophagy and improving myocardial cell apoptosis.

Tanshinone IIA exerts autophagic cell death through down-regulation of β-catenin in renal cell carcinoma cells

Renal cell carcinoma (RCC), also called kidney cancer, is one of the most common malignancies worldwide, including the United States and China. Because of the characteristics of RCC that are both insidious and largely insensitive to chemo-radiation, the incidence and mortality of RCC are increasing every year. However, there are few studies describing anti-cancer effects of the natural compounds on RCC as compared to other cancers. Here, we analyzed the anti-neoplastic impact of Tanshinone IIA (TSN) on RCC cells. We noted that TSN increased the expression of LC3 proteins while having little effect on PARP and Alix protein expression. We found that TSN up-regulated the expression of autophagy-related proteins such as Atg7 and Beclin-1. Moreover, TSN promoted the formation of autophagic vacuoles such as autophagosomes and autolysosomes. However, treatment with 3-Methyladenine (3-MA) or Chloroquine (CQ), slightly decreased the ability of TSN to induce autophagy, but still autophagy occurred. In addition, TSN inhibited translocation of β-catenin into the nucleus, and β-catenin deletion and TSN treatment in RCC increased the expression of LC3 protein. Overall, our findings indicate that TSN can exert significant anti-tumor effects through down-regulation of β-catenin to induce autophagic cell death.

The Multidirectional Effect of Azelastine Hydrochloride on Cervical Cancer Cells.

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15–90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15–25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45–90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future.