Autophagy In Cells Research Articles

PCSK9 induces endothelial cell autophagy by regulating the PI3K/ATK pathway in atherosclerotic coronary heart disease.

Atherosclerosis is a chronic inflammatory disease of the arteries, and its pathogenesis is related to endothelial dysfunction. It has been found that the protein convertase subtilin/kexin9 type (PCSK9) plays an important role in AS, but its specific mechanism is still unclear. In this study, we first cultured human umbilical vein endothelial cells (HUVECs) with 50 or 100μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 hours to establish a coronary atherosclerosis cell model. The results showed that ox-LDL induced HUVEC injury and autophagy and upregulated PCSK9 protein expression in HUVECs in a concentration-dependent manner. Silencing PCSK9 expression with siRNA inhibited ox-LDL-induced HUVEC endothelial dysfunction, inhibited the release of inflammatory factors, promoted HUVEC proliferation and inhibited apoptosis. In addition, ox-LDL increased the expression of LC3B-I and LC3B-II and decreased the expression of p62. However, these processes are reversed by sh-PCSK9. In addition, sh-PCSK9 can inhibit PI3K, AKT and mTOR phosphorylation and promote autophagy. Taken together, our research shows that silencing PCSK9 inhibits the PI3K/ATK/mTOR pathway to activate ox-LDL-induced autophagy in vascular endothelial cells, alleviating endothelial cell injury and inflammation.

VD3/VDR attenuates bisphenol A-induced impairment in mouse Leydig cells via regulation of autophagy.

Bisphenol A (BPA) is an endocrine disruptor with reproductive toxicity. Further, 1,25-dihydroxyvitamin D3 (VD3) plays an important role in male reproduction by binding vitamin D receptor (VDR) and mediating the pleiotropic biological actions that include spermatogenesis. However, whether VD3/VDR regulates the effect of BPA on Leydig cells (LCs) injury remains unknown. This study aimed to explore the effects of VD on BPA-induced cytotoxicity in mouse LCs. Hereby, LCs treated with BPA, VD3, or both were subjected to the assays of cell apoptosis, proliferation, autophagy, and levels of target proteins. This study unveiled that cell viability was dose-dependently reduced after exposure to BPA. BPA treatment significantly inhibited LC proliferation, induced apoptosis, and also downregulated VDR expression. By jointly analyzing transcriptome data and Comparative Toxicogenomics Database (CTD) data, autophagy signaling pathways related to testicular development and male reproduction were screened out. Therefore, the autophagy phenomenon of cells was further detected. The results showed that BPA treatment could activate cell autophagy, Vdr-/- inhibits cell autophagy, and active VD3 does not have a significant effect on the autophagy of normal LCs. After VD3 and BPA were used in combination, the autophagy of cells was further enhanced, and VD3 could alleviate BPA-induced damage of LCs. In conclusion, this study found that supplementing VD3 could eliminate the inhibition of BPA on VDR expression, further enhance LCs autophagy effect, and alleviate the inhibition of LCs proliferation and induction of apoptosis by BPA, playing a protective role in cells. The research results will provide valuable strategies to alleviate BPA-induced reproductive toxicity.

Palmitic acid impairs human and mouse placental function by inhibiting trophoblast autophagy through induction of acyl-coenzyme A-binding protein (ACBP) upregulation.

Can exposure to palmitic acid (PA), a common saturated fatty acid, modulate autophagy in both human and mouse trophoblast cells through the regulation of acyl-coenzyme A-binding protein (ACBP)? PA exposure before and during pregnancy impairs placental development through mechanisms involving placental autophagy and ACBP expression. High-fat diets, including PA, have been implicated in adverse effects on human placental and fetal development. Despite this recognition, the precise molecular mechanisms underlying these effects are not fully understood. Extravillous trophoblast (EVT) cell line HTR-8/SVneo and human trophoblast stem cell (hTSC)-derived EVT (hTSCs-EVT) were exposed to PA or vehicle control for 24 h. Female wild-type C57BL/6 mice were divided into PA and control groups (n = 10 per group) and subjected to a 12-week dietary intervention. Afterward, they were mated with male wild-type C57BL/6 mice and euthanized on Day 14 of gestation. Female ACBPflox/flox mice were also randomly assigned to control and PA-exposed groups (each with 10 mice), undergoing the same dietary intervention and mating with ACBPflox/floxELF5-Cre male mice, followed by euthanasia on Day 14 of gestation. The study assessed the effects of PA on mouse embryonic development and placental autophagy. Additionally, the role of ACBP in the pathogenesis of PA-induced placental toxicity was investigated. The findings were validated using real-time PCR, Western blot, immunofluorescence, transmission electron microscopy, and shRNA knockdown approaches. Exposure to PA-upregulated ACBP expression in both human HTR-8/SVneo cells and hTSCs-EVT, as well as in mouse placenta. PA exposure also induced autophagic dysfunction in HTR-8/SVneo cells, hTSCs-EVT, and mouse placenta. Through studies on ACBP placental conditional knockout mice and ACBP knockdown human trophoblast cells, it was revealed that reduced ACBP expression led to trophoblast malfunction and affected the expression of autophagy-related proteins LC3B-II and P62, thereby impacting embryonic development. Conversely, ACBP knockdown partially mitigated PA-induced impairment of placental trophoblast autophagy, observed both in vitro in human trophoblast cells and in vivo in mice. N/A. Primary EVT cells from early pregnancy are fragile, limiting research use. Maintaining their viability is tough, affecting data reliability. The study lacks depth to explore PA diet cessation effects after 12 weeks. Without follow-up, understanding postdiet impacts on pregnancy stages is incomplete. Placental abnormalities linked to elevated PA diet in embryos lack confirmation due to absence of control groups. Clarifying if issues stem solely from PA exposure is difficult without proper controls. Consuming a high-fat diet before and during pregnancy may result in complications or challenges in successfully carrying the pregnancy to term. It suggests that such dietary habits can have detrimental effects on the health of both the mother and the developing fetus. This work was supported in part by the National Natural Science Foundation of China (82171664, 82301909) and the Natural Science Foundation of Chongqing Municipality of China (CSTB2022NS·CQ-LZX0062, cstc2019jcyj-msxmX0749, and cstc2021jcyj-msxmX0236). The authors declare that they have no conflict of interest. N/A.

SLC34A2 promotes cell proliferation by activating STX17-mediated autophagy in esophageal squamous cell carcinoma.

Solute carrier family 34 member 2 (SLC34A2) has been implicated in the development of various malignancies. However, the clinical significance and underlying molecular mechanisms of SLC34A2 in esophageal squamous cell carcinoma (ESCC) remain elusive. Western blotting, quantitative real-time PCR and immunohistochemistry were utilized to evaluate the expression levels of SLC34A2 mRNA/protein in ESCC cell lines or tissues. Kaplan-Meier curves were employed for survival analysis. CCK-8, colony formation, EdU and xenograft tumor model assays were conducted to determine the impact of SLC34A2 on ESCC cell proliferation. Cell cycle was examined using flow cytometry. RNA-sequencing and enrichment analysis were carried out to explore the potential signaling pathways. The autophagic flux was evaluated by western blotting, mRFP-GFP-LC3 reporter system and transmission electron microscopy. Immunoprecipitation and mass spectrometry were utilized for identification of potential SLC34A2-interacting proteins. Cycloheximide (CHX) chase and ubiquitination assays were conducted to test the protein stability. The expression of SLC34A2 was significantly upregulated in ESCC and correlated with unfavorable clinicopathologic characteristics particularly the Ki-67 labeling index and poor prognosis of ESCC patients. Overexpression of SLC34A2 promoted ESCC cell proliferation, while silencing SLC34A2 had the opposite effect. Moreover, SLC34A2 induced autophagy to promote ESCC cell proliferation, whereas inhibition of autophagy suppressed the proliferation of ESCC cells. Further studies showed that SLC34A2 interacted with an autophagy-related protein STX17 to promote autophagy and proliferation of ESCC cells by inhibiting the ubiquitination and degradation of STX17. These findings indicate that SLC34A2 may serve as a prognostic biomarker for ESCC.

Gypenoside XLIX Activates the Sirt1/Nrf2 Signaling Pathway to Inhibit NLRP3 Inflammasome Activation to Alleviate Septic Acute Lung Injury.

Currently, treatment options for acute lung injury (ALI) are limited. Gypenoside XLIX (Gyp-XLIX) is known for its anti-inflammatory properties, but there is a lack of extensive research on its effects against ALI. This study induced ALI in mice through cecal ligation and puncture surgery and investigated the biological activity and potential mechanisms of Gypenoside XLIX (40mg/kg) by intraperitoneal injection. The in vitro ALI model was established using mouse lung epithelial (MLE-12) cells stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Various methods, including Hematoxylin and Eosin (H&E) staining, biochemical assay kits, Quantitative Polymerase Chain Reaction (qPCR) analysis, Western blotting, Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay, immunofluorescence, and flow cytometry, were employed for this research. The results indicated that pretreatment with Gypenoside XLIX significantly alleviated pathological damage in mouse lung tissues and reduced the expression levels of inflammatory factors. Additionally, Gypenoside XLIX inhibited ROS levels and NLRP3 inflammasome, possibly mediated by the Sirt1/Nrf2 signaling pathway. Moreover, Gypenoside XLIX significantly inhibited sepsis-induced lung cell apoptosis and excessive autophagy of mitochondria. Specifically, it suppressed mitochondrial pathway apoptosis and the Pink1/Parkin pathway of mitochondrial autophagy. These findings reveal the multifaceted effects of Gypenoside XLIX in anti-inflammatory, antioxidative, and inhibition of cell apoptosis and autophagy. This provides strong support for its therapeutic potential in sepsis-related lung injuries.

Phenformin activates ER stress to promote autophagic cell death via NIBAN1 and DDIT4 in oral squamous cell carcinoma independent of AMPK

The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

Repair effect of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles on ovarian injury induced by cisplatin.

Small extracellular vesicles (sEVs) secreted by human umbilical cord have therapeutic effects on various degenerative diseases. However, the characteristics and potential functions of human umbilical cord mesenchymal stem cells (huMSCs)-derived sEVs, especially the role of premature ovarian failure (POF), are poorly understood. Here, we isolated and characterized huMSCs and their sEVs. huMSCs highly expressed CD73, CD90, and CD105. huMSC-sEVs showed typical exosomal features, highly expressing CD9, TSG101, and CD63. It was shown that huMSC-sEVs could be taken up by granulosa cells (GCs) and damaged ovarian tissue, which increased the levels of hormone secretion and reduced GCs apoptosis. We further confirmed that the levels of follicle-stimulating hormone in rat serum decreased dramatically, while the levels of estrogen (E2）and anti-mullerian hormone (AMH) increased significantly with the treatment of huMSC-sEVs. Meanwhile, huMSC-sEVs treatment greatly reduced cell apoptosis and autophagy, while increased the phosphorylation levels of p-PI3K and p-Akt. Therefore, treatment with huMSC-sEVs significantly inhibited GCs apoptosis, improved ovarian morphology, promoted follicular development, inhibited follicular over-atresia, and improved ovarian reserve capacity in POF rats. Our study verified that activation of PI3K/Akt signaling pathway and regulation of cellular autophagy, thus reducing GCs death, are the mechanisms by which huMSC-sEVs restore ovarian tissue function.

Targeting programmed cell death in inflammatory bowel disease through natural products: New insights from molecular mechanisms to targeted therapies.

Inflammatory bowel disease (IBD) is an autoimmune disorder primarily characterized by intestinal inflammation and recurrent ulceration, leading to a compromised intestinal barrier and inflammatory infiltration. This disorder's pathogenesis is mainly attributed to extensive damage or death of intestinal epithelial cells, along with abnormal activation or impaired death regulation of immune cells and the release of various inflammatory factors, which contribute to the inflammatory environment in the intestines. Thus, maintaining intestinal homeostasis hinges on balancing the survival and functionality of various cell types. Programmed cell death (PCD) pathways, including apoptosis, pyroptosis, autophagy, ferroptosis, necroptosis, and neutrophil extracellular traps, are integral in the pathogenesis of IBD by mediating the death of intestinal epithelial and immune cells. Natural products derived from plants, fruits, and vegetables have shown potential in regulating PCD, offering preventive and therapeutic avenues for IBD. This article reviews the role of natural products in IBD treatment by focusing on targeting PCD pathways, opening new avenues for clinical IBD management.

Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer.

Appendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line. PTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37°C and 42°C with and without bromelain pretreatment. From October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs. Bromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius)

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

New insights into resveratrol attenuates hepatotoxicity in emamectin benzoate-exposed grass carp (Ctenopharyngodon idella) via NO system/NF-κB signaling pathway

Emamectin benzoate (EMB) is extensively used as a crop protection agent. Overuse of EMB poses a serious threat to the quality of water and non-target organisms in the environment. Resveratrol (RES) is a natural phytoalexin with the function of anti-oxidation and anti-inflammation. Nonetheless, it is unclear whether EMB affects the expression of cytokines and induces autophagy, apoptosis, and necroptosis of hepatocytes (L8824 cell) in grass carp (Ctenopharyngodon idella), and whether RES has an attenuate function in this process. Therefore, we established the L8824 cells model of EMB exposure and treated it with RES. The results showed that compared with the control (CON) group, EMB exposure significantly increased the nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, and the expression of iNOS and phosphorylated nuclear factor kappa B (p-NF-κB) (P < 0.05). In addition, compared with the CON group, the results of flow cytometry and dansylcadaverine (MDC) staining showed a significant increase in apoptosis and autophagy in the EMB-exposed group (P < 0.05) with the activation of the B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax)/cysteine-aspartic acid protease 3 (Caspase-3)/cysteine-aspartic acid protease 9 (Caspase-9) pathway and microtubule-associated protein light chain 3 (LC3)/sequestosome 1 (p62)/Beclin1 pathway. EMB exposure significantly increased the mRNA and protein expression of receptor-interacting protein 1 (RIPK1)/receptor-interacting protein 3 (RIPK3)/mixed the lineage kinase domain-like (MLKL) pathway (P < 0.05). Moreover, EMB exposure significantly increased the expression of genes related to immunity (immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin D (IgD), and antimicrobial peptide-related genes expression including β-defensin and hepcidin) (P < 0.05). The addition of RES significantly diminished autophagy, apoptosis, necroptosis, and immunity-related gene expression by inhibiting iNOS activity, NO content, and the protein expression of iNOS and p-NF-κB. In conclusion, RES attenuated autophagy, apoptosis, and necroptosis in EMB-exposed L8824 cells via suppression of the NO system/NF-κB signaling pathway.

A review: targeting UBR5 domains to mediate emerging roles and mechanisms: chance or necessity?

Ubiquitinases are known to catalyze ubiquitin chains on target proteins to regulate various physiological functions like cell proliferation, autophagy, apoptosis, and cell cycle progression. As a member of E3 ligase, ubiquitin protein ligase E3 component n-recognin 5 (UBR5) belongs to the HECT E3 ligase and has been reported to be correlated with various pathophysiological processes. In this review, we give a comprehensive insight into the structure and function of UBR5. We discuss the specific domains of UBR5 and explore their biological functions separately. Furthermore, we describe the involvement of UBR5 in different pathophysiological conditions, including immune response, virus infection, DNA damage response and protein quality control. Moreover, we provide a thorough summary of the important roles and regulatory mechanisms of UBR5 in cancers and other diseases. On the whole, investigating the domains and functions of UBR5, elucidating the underlying mechanisms of UBR5 with various substrates in detail may provide new theoretical basis for the treatment of diseases, including cancers, which could improve future studies to construct novel UBR5-targeted therapy strategies.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids

BackgroundInduced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.MethodsDifferentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.ResultsWe observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.ConclusionOur data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Mechanisms of persistent hemolysis-induced middle kidney injury in grass carp (Ctenopharyngodon idella)

Infection-induced hemolysis results in intravascular hemolysis, which releases hemoglobin (Hb) into the tissues. Free Hb exhibits cytotoxic, oxidative, and pro-inflammatory effects, leading to systemic inflammation, vascular constriction dysfunction, thrombosis, and proliferative vascular lesions. Currently, the impact of intravascular hemolysis on the middle kidney in fish is unclear. Here, the injection of phenylhydrazine (PHZ) was used to establish a persistent hemolysis model in grass carp. The determination results revealed that the PHZ-induced hemolysis caused conspicuous tissue damage in the kidneys of grass carp, increased the levels of Cr in the serum and the expression indicators of kidney injury-related genes in the middle kidney. Prussian blue staining indicated that PHZ-induced hemolysis significantly increased the deposition of iron ions in the kidneys of grass carp, and activated the expression levels of iron metabolism-related genes. The results of oxidative damage-related experiments indicate that under PHZ treatment, the activity of middle kidney cells decreases, and the production of oxidative damage markers malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) increases, simultaneously inhibiting the activity of antioxidant enzymes and upregulating the transcription levels of antioxidant enzyme-related genes. Additionally, the analysis of inflammatory factors revealed a significant upregulation of genes associated with inflammation induced by PHZ-induced hemolysis. The transcriptome analysis was performed to further explore the molecular regulatory effects of hemolysis on tissues, the analysis revealed the treatment of PHZ activated various of programmed cell death (PCD) pathways, including ferroptosis, apoptosis, and autophagy. In summary, this study found that sustained hemolysis in fish results in Hb and iron ion deposition in middle kidney, promoting oxidative damage, ultimately inducing various forms of PCD.

Abstract PO2-28-03: E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis

Abstract Electronic cigarettes (E-cigs) have emerged as a significant public health concern due to their increasing popularity. These products have been associated with various harmful effects on human health. In the United States, breast cancer is the most diagnosed cancer among women, and its recurrence and metastasis are primary contributors to morbidity and mortality. Two critical factors in cancer progression are Epithelial-mesenchymal transition (EMT) and Cancer stem-like cells (CSCs), both of which play pivotal roles in metastasis. As a result, targeting the signaling pathways involved in these processes has become a promising avenue for cancer therapy. Autophagy, a cellular process, has dual roles in cancer development depending on the cancer type and stage. Given these considerations, our hypothesis centers on disrupting the EMT-mediated metastatic cascade in breast cancer by targeting autophagy in Cancer Stem Cells (CSCs). To investigate this hypothesis, we conducted experiments using mouse breast epithelial cells (HC11) and Triple-negative mouse breast cancer cells (4T1) exposed to E-cig aerosol at the air-liquid interphase (ALI) for 1 hr followed by a 24 hr recovery period. Our findings indicate that exposure to E-cig aerosols led to a decrease in epithelial markers (E-Cadherin and β-catenin) and an increase in mesenchymal markers (N-Cadherin and Vimentin) in HC11 cells. This suggests that normal cells may acquire characteristics associated with solitary migration. Furthermore, we observed significant changes in the levels of autophagy-related proteins (LC3B, Beclin-1, ATG12, and ATG16) and upregulation of stem cell markers (OCT4, SOX2, NANOG, KLF4, and c-MYC) in response to E-cig aerosol exposure, both in HC11 and 4T1 cells. Additionally, these alterations extended to changes in the expression of genes associated with the Transforming Growth Factor-beta (TGF-β) signaling pathway, known to play a potential role in promoting EMT. Our results provide a deeper understanding of the interplay between EMT, CSCs, and the autophagy process in the context of ALI exposure in normal and cancer cells. This underscores the need for further research into the shared molecular mechanisms of these three processes to identify common therapeutic targets that can simultaneously affect them. Citation Format: Shilpa Thota, Rizwana Begum, Naveen Chintalaramulu, Biplov Sapkota, Abhishek Pandit. E-Cigarette Aerosol Exposure: Unraveling the Nexus of Autophagy, Cancer Stem Cells, and EMT in Breast Cancer Metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-03.

The mechanism of high mobility group box-1 protein and its bidirectional regulation in tumors.

High-mobility group box-1 protein (HMGB1) is a nonhistone chromatin-related protein widely found in eukaryotic cells. It is involved in the transcription, replication, and repair of DNA to maintain nuclear homeostasis. It participates in cell growth, differentiation, and signal transduction. Recent studies showed that HMGB1 has a bidirectional regulatory effect on tumors by regulating TLR4/MYD88/NF-κB and RAGE/AMPK/mTOR signaling pathways. On the one hand, it is highly expressed in a variety of tumors, promoting tumor proliferation and invasion, while on the other hand, it induces autophagy and apoptosis of tumor cells and stimulates tumor-infiltrating lymphocytes to produce an anti-tumor immune response. At present, HMGB1 could be used as a target to regulate the drug resistance and prognostication in cancer. Clinical applications of HMGB1 in cancer need further in-depth studies.

Abstract PO3-14-12: SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients

Abstract Background: Several prospective, randomized clinical trials showed that the CDK4/6 inhibitor palbociclib in combination with either letrozole or fulvestrant significantly improves progression-free survival (PFS) in patients with ER+/HER2- metastatic breast cancer (MBC). However, in some cases, there is development of endocrine resistance and ultimate disease progression. Molecular analysis performed in tissue specimens collected in the PALOMA-3 study, comparing fulvestrant and Palbociclib versus fulvestrant placebo, demonstrated that one of the genes associated with an increased clinical benefit is SIRT3 (Sirtuin 3) which is thought to control mitochondrial integrity and metabolism (Cristofanilli M. et al., Lancet Oncol. 2016). This study aimed to evaluate in preclinical models the predictive role of SIRT3 in ER+/HER2- breast tumors. Methods: Using lentiviruses we generated MCF-7 and T47D cells stably expressing either control shRNA (sh ctr) or shRNA targeting SIRT3 (shSIRT3). With these models, we studied cell viability, tumor growth, apoptosis, and autophagy in MCF-7 cells treated with fulvestrant and palbociclib as a single treatment or in combination, these either alone or in combination with shSIRT3 (knockdown). Nude mice were used for our in vivo studies, and sh ctr or shSIRT3 MCF7 cells were injected. Results: We showed in vitro that the response to palbociclib was significantly associated with levels of SIRT3 expressionas high or low in ER+ cells (p&amp;lt; 0.0001). Furthermore, we found that irreversible cell growth inhibition mediates the beneficial effect of palbociclib in SIRT3 high expressing cellscompared to low expression (p&amp;lt; 0.001).Then, we evaluated the mechanistic activity of SIRT3. We showed thatSIRT3-low expression cells induced enhanced sensitivity to palbociclib by targeting the reactive oxygen species (ROS)/autophagy axis:(i) SIRT3 regulated the mitochondrial homeostasis (e.g. glucose, lactate, pyruvate, succinates), (ii) ROS levels where lower in sh crt comparing and shSIRT3 in treated and untreated cells (p&amp;lt; 0.001), (iii) in terms of senescence,a higher level of positive cells for β-gal activity was found (p≤0.05) and (iv) for autophagy, our western blot analysis showed cleaved LC3 proteins. Our in vivo studies showed that SIRT3 regulated treatment response to palbociclib with a significant decrease in tumor weight and tumor volume (p&amp;lt; 0.01) and importantly, after immunohistochemistry analysis, we validated the role of SIRT3 in regulating the proteins involved in the autophagy/senescence balance, was mainly in epithelial cells compared to stromal cells. Conclusion: Our preclinical studies demonstrated that elevated SIRT3 expression levels sensitizes ER+/HER2- breast tumors to palbociclib treatment. This study suggested that SIRT3 expression could represent a potential predictive biomarker in HR+ MBC patients treated with Palbociclib. Citation Format: Maroua Manai, Ghada Sahraoui, Raoudha Doghri, Lorenzo Gerratana, Paolo D’Amico, Youbin Zhang, Jeannine Donahue, Ami Shah, Carolina Reduzzi, Wenan Qiang, Massimo Cristofanilli. SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-12.

Abstract PO2-28-01: Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer

Abstract Caveolin-1 (Cav-1) is a lipid raft protein with a dual role, serving as a tumor suppressor and a promoter. Studies have unveiled Cav-1’s significance in breast cancer progression, impacting various aspects, including cell proliferation, apoptosis, autophagy, invasion, migration, and metastasis. Previously, our research demonstrated that the knockout (KO) of Cav-1 from epithelial cells reduced lung metastasis originating from primary tumors. In this study, we sought to investigate whether Cav-1 KO in epithelial cells increases sensitivity to doxorubicin (Dox) using both in vitro and in vivo studies. In our in vitro study, we observed reduced cell numbers in doxorubicin-treated Cav-1 KO cells compared to Dox-treated 4T1 cells (wild type). Flow cytometry-based cell cycle assay showed a significant increase in G2M phase arrest in Dox-treated Cav-1 KO cells compared with wild-type, confirming reduced cell growth and proliferation. To validate the increased sensitivity to Dox in vivo, we used a syngeneic tumor model using Cav-1 KO and 4T1 wild-type cells injected into the mammary fat of female Balb/c mice intraperitoneally. Subsequently, after one week of tumor cell injection, both groups received Dox at a dose of 8 mg/kg body weight/week for 3 weeks (a cumulative dose of 24 mg/kg body weight). Primary tumor sizes exhibited a significant reduction in Dox-treated mice, and this was further reduced in Cav-1 KO mice compared to the wild type. To quantify lung metastasis, we explanted the mice’s lungs and plated dissociated single cells in complete RPMI media supplemented with 6-thioguanine (6-GT). The number of 6-TG-resistant 4T1 metastatic cells was significantly lower in the lungs' suspension of Cav-1 KO mice treated with Dox compared to wild-type Dox-treated mice. Our findings confirm that knockout of Cav-1 increased the sensitivity of Dox treatment and inhibited breast cancer lung metastasis in Balb/c mice. Our ongoing analysis of the molecular pathways seeks to elucidate the underlying mechanism. Citation Format: Abhishek Pandit, Dhirendra Singh, Rashmi Pathak, Shilpa Thota, Joseph Francis. Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-01.

Mitochondria-targeting metallodrugs for cancer therapy: perspectives from cell death modes.

Mitochondria, recognized as the cellular powerhouses, are indispensable organelles responsible for crucial cellular processes, such as energy metabolism, material synthesis, and signaling transduction. Their intricate involvement in a broad spectrum of diseases, particularly cancer, has propelled the exploration of mitochondria-targeting treatment as a promising strategy for cancer therapy. Since the groundbreaking discovery of cisplatin, the trajectory of research on the development of metal complexes have been marked by continuous advancement, giving rise to a diverse array of metallodrugs characterized by variations in ligand types, metal center properties, and oxidation states. By specifically targeting mitochondria, these metallodrugs exhibit the remarkable ability to elicit various programmed cell death pathways, encompassing apoptosis, autophagy, and ferroptosis. This review primarily focuses on recent developments in transition metal-based mitochondria-targeting agents, offering a comprehensive exploration of their capacity to induce distinct cell death modes. The aim is not only to disseminate knowledge but also to stimulate an active field of research toward new clinical applications and novel anticancer mechanisms.

Myelin basic protein antagonizes the SARS-CoV-2 protein ORF3a-induced autophagy inhibition

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS.