Pub Date : 2025-11-01DOI: 10.1016/j.bbagen.2025.130878
Weichen Zhan , Xiaowei Ding , Zhongrui Cui , Yizhuo Wu , Yiwen Gu , Hanxiao Cheng , Xinxin Ge , Yun Wang , Jiangyun Luo , Bing Xiao
Despite significant advancements in liposome-mediated transfection technology over the past decades, achieving optimal transfection efficiency with lipoplex remains challenging in certain primary cells, such as vascular smooth muscle cells, endothelial cells, and suspension cells. Here, we present an innovative approach to significantly enhance Lipofectamine-based transient transfection efficiency in hard-to-transfect cells by applying elevated cyclic hydrostatic pressure (CHP). The plasmids encoding the enhanced green fluorescent protein (EGFP) were transfected using Lipofectamine 3000 reagent, and the transfection efficiency was evaluated by Western blot or flow cytometry. Our results demonstrate that CHP (0.0083 Hz, 0–100 mmHg) significantly enhanced the transfection efficiency of lipoplex in primary human aortic smooth muscle cells (HASMCs) and other difficult-to-transfect cell types. Mechanistic studies revealed that the enhancement of liposome-mediated transfection by CHP was dependent on the activation of clathrin-dependent endocytic pathways. Importantly, this mechanical stimulation did not affect the proliferative or migratory capacities of HASMCs, despite the identification of significantly modulated proteins (5.8 % of the total proteome) by proteomic analysis. This study establishes a novel, safe strategy to enhance lipoplex-mediated nucleic acid delivery in challenging-to-transfect cell types.
{"title":"Elevated cyclic hydrostatic pressure enhances the transfection activity of lipoplexes by activating clathrin-mediated endocytosis","authors":"Weichen Zhan , Xiaowei Ding , Zhongrui Cui , Yizhuo Wu , Yiwen Gu , Hanxiao Cheng , Xinxin Ge , Yun Wang , Jiangyun Luo , Bing Xiao","doi":"10.1016/j.bbagen.2025.130878","DOIUrl":"10.1016/j.bbagen.2025.130878","url":null,"abstract":"<div><div>Despite significant advancements in liposome-mediated transfection technology over the past decades, achieving optimal transfection efficiency with lipoplex remains challenging in certain primary cells, such as vascular smooth muscle cells, endothelial cells, and suspension cells. Here, we present an innovative approach to significantly enhance Lipofectamine-based transient transfection efficiency in hard-to-transfect cells by applying elevated cyclic hydrostatic pressure (CHP). The plasmids encoding the enhanced green fluorescent protein (EGFP) were transfected using Lipofectamine 3000 reagent, and the transfection efficiency was evaluated by Western blot or flow cytometry. Our results demonstrate that CHP (0.0083 Hz, 0–100 mmHg) significantly enhanced the transfection efficiency of lipoplex in primary human aortic smooth muscle cells (HASMCs) and other difficult-to-transfect cell types. Mechanistic studies revealed that the enhancement of liposome-mediated transfection by CHP was dependent on the activation of clathrin-dependent endocytic pathways. Importantly, this mechanical stimulation did not affect the proliferative or migratory capacities of HASMCs, despite the identification of significantly modulated proteins (5.8 % of the total proteome) by proteomic analysis. This study establishes a novel, safe strategy to enhance lipoplex-mediated nucleic acid delivery in challenging-to-transfect cell types.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130878"},"PeriodicalIF":2.2,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145437017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-01DOI: 10.1016/j.bbagen.2025.130875
Xiyu Mei , Chuang Ke , Ziyun Gao , Fan Yang , Zhenlin Huang , Bin Lu , Lili Ji
Diabetes mellitus (DM) is associated with a poor prognosis of aggressive breast cancer. Vascular dysfunction is commonly found during the development of both cancer and diabetes. We previously reported that the disruption of vascular endothelial phenotype induced by tumor necrosis factor-α (TNFα) accelerated the trans-endothelial metastasis of triple-negative breast cancer (TNBC). Herein, we explored the role of vascular endothelial cells in diabetes-induced TNBC metastasis. Both type 2 DM (T2DM) and type 1 DM (T1DM) enhanced the metastasis of TNBC in vivo. T2DM increased the expression of endothelial phenotype vascular endothelial cadherin (VE-cadherin), platelet-endothelial cell adhesion molecule (PECAM-1/CD31), and mesenchymal markers including vimentin and fibroblast specific protein-1 (FSP-1/S100A4) in tumor vessels. T1DM increased the expression of vimentin and FSP-1, but suppressed the expression of VE-cadherin in tumor vessels. Hyperglycemia elevated the production of TNFα in vivo and in vitro. TNFα reduced the trans-endothelial electrical resistance (TEER) value of both human mammary microvascular endothelial cells (HMMECs) and human umbilical vein endothelial cells (HUVECs). Expressions of vimentin and α-smooth muscle actin (α-SMA) were also increased in TNFα-treated both HMMECs and HUVECs. The number of trans-endothelial migrated MDA-MB-231 cells through TNFα-treated HMMECs or HUVECs monolayer was elevated. Moreover, glioma-associated oncogene 1 (Gli-1) was remarkably accumulated in the nucleus of TNFα-stimulated HMMECs and DM-induced tumor vessels. Both Gli-1 siRNA and GANT61 (an inhibitor of Gli-1) could abrogate the increased TNBC trans-endothelial migration through TNFα-treated ECs. We demonstrated that DM might promote TNBC metastasis via activating the TNFα/Gli-1 axis initiated vascular endothelial mesenchymal-like phenotype.
{"title":"Hyperglycemia accelerated the metastasis of triple-negative breast cancer via promoting TNFα/Gli-1 axis in endothelial cells","authors":"Xiyu Mei , Chuang Ke , Ziyun Gao , Fan Yang , Zhenlin Huang , Bin Lu , Lili Ji","doi":"10.1016/j.bbagen.2025.130875","DOIUrl":"10.1016/j.bbagen.2025.130875","url":null,"abstract":"<div><div>Diabetes mellitus (DM) is associated with a poor prognosis of aggressive breast cancer. Vascular dysfunction is commonly found during the development of both cancer and diabetes. We previously reported that the disruption of vascular endothelial phenotype induced by tumor necrosis factor-<em>α</em> (TNF<em>α</em>) accelerated the trans-endothelial metastasis of triple-negative breast cancer (TNBC). Herein, we explored the role of vascular endothelial cells in diabetes-induced TNBC metastasis<em>.</em> Both type 2 DM (T2DM) and type 1 DM (T1DM) enhanced the metastasis of TNBC <em>in vivo</em>. T2DM increased the expression of endothelial phenotype vascular endothelial cadherin (VE-cadherin), platelet-endothelial cell adhesion molecule (PECAM-1/CD31), and mesenchymal markers including vimentin and fibroblast specific protein-1 (FSP-1/S100A4) in tumor vessels. T1DM increased the expression of vimentin and FSP-1, but suppressed the expression of VE-cadherin in tumor vessels. Hyperglycemia elevated the production of TNF<em>α in vivo</em> and <em>in vitro</em>. TNF<em>α</em> reduced the trans-endothelial electrical resistance (TEER) value of both human mammary microvascular endothelial cells (HMMECs) and human umbilical vein endothelial cells (HUVECs). Expressions of vimentin and <em>α</em>-smooth muscle actin (<em>α</em>-SMA) were also increased in TNF<em>α</em>-treated both HMMECs and HUVECs. The number of trans-endothelial migrated MDA-MB-231 cells through TNF<em>α</em>-treated HMMECs or HUVECs monolayer was elevated. Moreover, glioma-associated oncogene 1 (Gli-1) was remarkably accumulated in the nucleus of TNF<em>α</em>-stimulated HMMECs and DM-induced tumor vessels. Both Gli-1 siRNA and GANT61 (an inhibitor of Gli-1) could abrogate the increased TNBC trans-endothelial migration through TNF<em>α</em>-treated ECs. We demonstrated that DM might promote TNBC metastasis via activating the TNF<em>α</em>/Gli-1 axis initiated vascular endothelial mesenchymal-like phenotype.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130875"},"PeriodicalIF":2.2,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145437054","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-29DOI: 10.1016/j.bbagen.2025.130874
Marcela Andrade-Soares , Mayra Alves , Clara Rodrigues-Ferreira , Jarlene A. Lopes , Thuany Crisóstomo , Gloria Costa-Sarmento , Christina M. Takiya , Amaury Pereira-Acácio , Adalberto Vieyra
Acute kidney injury (AKI) induced by ischemia-reperfusion (I/R) remains a significant clinical challenge due to its rapid progression, limited therapeutic options, and high morbidity. Mitochondrial dysfunction is a critical component of AKI pathogenesis, contributing to oxidative stress, impaired bioenergetics, and tissue injury. Extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) have emerged as potential candidates for organ protection through the modulation of inflammatory and oxidative pathways. This study evaluated the effects of EV secreted by hypoxia-preconditioned adipose-derived MSC on mitochondrial function in a rat model of I/R-induced AKI. Wistar rats were assigned to four groups: SHAM, I/R, SHAM + EV, and I/R + EV. Hypoxia-preconditioned EV (2 × 109) or vehicle were administered subcapsularly 1 h prior to bilateral renal artery clamping (45 min ischemia, 1 h reperfusion). Histological analyses demonstrated that EV treatment effectively prevented tubular injury, inflammatory infiltration, and preserved renal architecture. EV enhanced Nrf2 nuclear translocation, upregulated HO-1 expression, and stabilized antioxidant defenses. Furthermore, EV preserved mitochondrial membrane potential, respiratory control ratio, ATP synthesis, and the abundance of electron transport chain complexes I, III, and IV, although complex II remained vulnerable. Proton leak responses were unaffected. These results demonstrate that hypoxia-preconditioned MSC-derived EV exert rapid protective effects on renal mitochondria and redox homeostasis during early reperfusion, offering a promising therapeutic strategy for AKI prevention in clinical scenarios such as transplantation and major cardiovascular surgeries. Further studies are needed to characterize the cargo of EV and their long-term outcomes.
{"title":"Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells preserve mitochondrial functions and redox homeostasis in ischemia–reperfusion-induced acute kidney injury","authors":"Marcela Andrade-Soares , Mayra Alves , Clara Rodrigues-Ferreira , Jarlene A. Lopes , Thuany Crisóstomo , Gloria Costa-Sarmento , Christina M. Takiya , Amaury Pereira-Acácio , Adalberto Vieyra","doi":"10.1016/j.bbagen.2025.130874","DOIUrl":"10.1016/j.bbagen.2025.130874","url":null,"abstract":"<div><div>Acute kidney injury (AKI) induced by ischemia-reperfusion (I/R) remains a significant clinical challenge due to its rapid progression, limited therapeutic options, and high morbidity. Mitochondrial dysfunction is a critical component of AKI pathogenesis, contributing to oxidative stress, impaired bioenergetics, and tissue injury. Extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) have emerged as potential candidates for organ protection through the modulation of inflammatory and oxidative pathways. This study evaluated the effects of EV secreted by hypoxia-preconditioned adipose-derived MSC on mitochondrial function in a rat model of I/R-induced AKI. Wistar rats were assigned to four groups: SHAM, I/R, SHAM + EV, and I/R + EV. Hypoxia-preconditioned EV (2 × 10<sup>9</sup>) or vehicle were administered subcapsularly 1 h prior to bilateral renal artery clamping (45 min ischemia, 1 h reperfusion). Histological analyses demonstrated that EV treatment effectively prevented tubular injury, inflammatory infiltration, and preserved renal architecture. EV enhanced Nrf2 nuclear translocation, upregulated HO-1 expression, and stabilized antioxidant defenses. Furthermore, EV preserved mitochondrial membrane potential, respiratory control ratio, ATP synthesis, and the abundance of electron transport chain complexes I, III, and IV, although complex II remained vulnerable. Proton leak responses were unaffected. These results demonstrate that hypoxia-preconditioned MSC-derived EV exert rapid protective effects on renal mitochondria and redox homeostasis during early reperfusion, offering a promising therapeutic strategy for AKI prevention in clinical scenarios such as transplantation and major cardiovascular surgeries. Further studies are needed to characterize the cargo of EV and their long-term outcomes.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130874"},"PeriodicalIF":2.2,"publicationDate":"2025-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145420862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-25DOI: 10.1016/j.bbagen.2025.130871
Zhiyong Peng , Xiaojuan Cao , Hejun Gao , Cuiling Li , Jinhan Zhang , Youtan Liu
Sepsis, a severe inflammatory response to infection, is characterized by complex and rapidly evolving pathophysiology with high mortality. Mitochondrial DNA (mtDNA) in exosomes is a key damage-associated molecular pattern implicated in sepsis; however, its exact role and mechanisms are unclear. This study investigates how exosome-derived mtDNA induces mitochondrial dysfunction via protein kinase C delta (PKCδ), leading to endothelial barrier disruption and the progression of sepsis. Our analysis revealed significantly elevated levels of the mtDNA markers ND2 and D-loop in serum exosomes from sepsis patients compared to healthy controls. These elevated exosomal mtDNA levels correlated with disease severity and showed a positive association with lung injury markers, including SRAGE, SP-D, and CC16. In vitro experiments demonstrated that both isolated mtDNA and exosomes significantly impaired mitochondrial membrane potential, increased reactive oxygen species (ROS) levels, and reduced the oxygen consumption rate (OCR), suggesting the induction of mitochondrial dysfunction. Moreover, mtDNA promoted endothelial cell damage and increased permeability via PKCδ. Crucially, PKCδ knockdown markedly restored mtDNA-induced mitochondrial dysfunction and cellular permeability damage. In conclusion, Exosome-derived mtDNA triggers mitochondrial dysfunction and endothelial barrier disruption via PKCδ, promoting sepsis progression, suggesting potential therapeutic targets.
{"title":"Exosome-derived mtDNA disrupts endothelial barrier integrity and accelerates sepsis progression by inducing mitochondrial dysfunction through the PKCδ gene","authors":"Zhiyong Peng , Xiaojuan Cao , Hejun Gao , Cuiling Li , Jinhan Zhang , Youtan Liu","doi":"10.1016/j.bbagen.2025.130871","DOIUrl":"10.1016/j.bbagen.2025.130871","url":null,"abstract":"<div><div>Sepsis, a severe inflammatory response to infection, is characterized by complex and rapidly evolving pathophysiology with high mortality. Mitochondrial DNA (mtDNA) in exosomes is a key damage-associated molecular pattern implicated in sepsis; however, its exact role and mechanisms are unclear. This study investigates how exosome-derived mtDNA induces mitochondrial dysfunction via protein kinase C delta (PKCδ), leading to endothelial barrier disruption and the progression of sepsis. Our analysis revealed significantly elevated levels of the mtDNA markers ND2 and D-loop in serum exosomes from sepsis patients compared to healthy controls. These elevated exosomal mtDNA levels correlated with disease severity and showed a positive association with lung injury markers, including SRAGE, SP-D, and CC16. In vitro experiments demonstrated that both isolated mtDNA and exosomes significantly impaired mitochondrial membrane potential, increased reactive oxygen species (ROS) levels, and reduced the oxygen consumption rate (OCR), suggesting the induction of mitochondrial dysfunction. Moreover, mtDNA promoted endothelial cell damage and increased permeability via PKCδ. Crucially, PKCδ knockdown markedly restored mtDNA-induced mitochondrial dysfunction and cellular permeability damage. In conclusion, Exosome-derived mtDNA triggers mitochondrial dysfunction and endothelial barrier disruption via PKCδ, promoting sepsis progression, suggesting potential therapeutic targets.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130871"},"PeriodicalIF":2.2,"publicationDate":"2025-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145428824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-24DOI: 10.1016/j.bbagen.2025.130867
Morgan C. Marsh , Shawn C. Owen
Split protein systems, including split fluorescent proteins and split enzymes, have predominantly been applied to monitor protein-protein interactions but have recently gained popularity as components in induced complementation diagnostic platforms. Many of these systems are limited by self-affinity which limits the dynamics or causes a false-positive high background signal. To overcome these challenges, it is necessary to either modify the interacting residues of established split fragments or by exploring new split-sites. Using the computational modeling tool SPELL to identify stable candidates, we developed five unique split sites for TEM-1 β-lactamase to compare structure and activity versus the original split site. We monitored structural changes through MMS IR spectroscopy as well as functional activity of each split enzyme. Expected structural changes of each fragment and changes in self-affinity driven complementation were observed. By utilizing specific binding proteins attached to the split enzyme, we monitored enzyme activity with forced complementation when bound to the target compared to auto-complementation when the enzymes were auto-complexed in solution. Of the five new split sites, one maintained high binding-mediated complementation activity with significant reduction in background auto-complementation. Changing the split site is a relatively simple approach to reduce background activity while maintaining on-target activity. The ability to tune split-systems has the potential to enable more sensitive and complex studies that previously were limited by the high background from self-affinity.
{"title":"Selection and evaluation of new sites for splitting beta-lactamase to modulate auto-complementation enzyme activity","authors":"Morgan C. Marsh , Shawn C. Owen","doi":"10.1016/j.bbagen.2025.130867","DOIUrl":"10.1016/j.bbagen.2025.130867","url":null,"abstract":"<div><div>Split protein systems, including split fluorescent proteins and split enzymes, have predominantly been applied to monitor protein-protein interactions but have recently gained popularity as components in induced complementation diagnostic platforms. Many of these systems are limited by self-affinity which limits the dynamics or causes a false-positive high background signal. To overcome these challenges, it is necessary to either modify the interacting residues of established split fragments or by exploring new split-sites. Using the computational modeling tool SPELL to identify stable candidates, we developed five unique split sites for TEM-1 β-lactamase to compare structure and activity versus the original split site. We monitored structural changes through MMS IR spectroscopy as well as functional activity of each split enzyme. Expected structural changes of each fragment and changes in self-affinity driven complementation were observed. By utilizing specific binding proteins attached to the split enzyme, we monitored enzyme activity with forced complementation when bound to the target compared to auto-complementation when the enzymes were auto-complexed in solution. Of the five new split sites, one maintained high binding-mediated complementation activity with significant reduction in background auto-complementation. Changing the split site is a relatively simple approach to reduce background activity while maintaining on-target activity. The ability to tune split-systems has the potential to enable more sensitive and complex studies that previously were limited by the high background from self-affinity.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130867"},"PeriodicalIF":2.2,"publicationDate":"2025-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145428825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The recent introduction of “cuproptosis” into the oncological lexicon has opened up new horizons for cancer therapy, yet our understanding of how this process operates in lung adenocarcinoma (LUAD) is still limited. In this work we have investigated the core genetic factors and provided theoretical support for the application of cuproptosis in the treatment of LUAD. Through bioinformatics analysis, we found that FDX1 was significantly downregulated in LUAD and highly correlated with cuproptosis markers. Therefore, we chose FDX1 as the research object.
Methods
Biochemical and bioinformatic analyses were assessed FDX1 and CEBPA expression in LUAD. The binding relationship between CEBPA and FDX1 was validated via CHIP and dual-luciferase assays. FDX1 expression in LUAD cells was evaluated by qRT-PCR and Western blot. The impact of FDX1 overexpression on LUAD progression was examined using CCK-8 and Transwell assays. Experiments involving CCK-8, copper ion measurement, cuproptosis-related protein detection, and DLAT immunofluorescence confirmed the successful LUAD cuproptosis model.
Results
FDX1 was downregulated in LUAD tissues and cells, and its overexpression inhibited LUAD cell migration, invasion, and proliferation. Cuproptosis significantly reduced LUAD cell viability and the protein levels of Lipoy-DLAT, DLAT, and FDX1, while increasing HSP70 expression, DLAT aggregation, and intracellular copper ion levels. CEBPA, a transcriptional activator of FDX1, positively correlated with and bound to it. Overexpressed FDX1 enhanced cuproptosis in LUAD cells, an effect partially reversible by CEBPA suppression.
Conclusion
These analyses were performed to construct a proposed cellular model of cuproptosis in LUAD.
{"title":"The CEBPA/FDX1 axis elevates sensitivity to cuproptosis in lung adenocarcinoma cells","authors":"Yongsheng Zhao , Renyan Zheng , Kexin Luo , Haiyang Zhao , Wanping Xiang","doi":"10.1016/j.bbagen.2025.130872","DOIUrl":"10.1016/j.bbagen.2025.130872","url":null,"abstract":"<div><h3>Background</h3><div>The recent introduction of “cuproptosis” into the oncological lexicon has opened up new horizons for cancer therapy, yet our understanding of how this process operates in lung adenocarcinoma (LUAD) is still limited. In this work we have investigated the core genetic factors and provided theoretical support for the application of cuproptosis in the treatment of LUAD. Through bioinformatics analysis, we found that FDX1 was significantly downregulated in LUAD and highly correlated with cuproptosis markers. Therefore, we chose FDX1 as the research object.</div></div><div><h3>Methods</h3><div>Biochemical and bioinformatic analyses were assessed FDX1 and CEBPA expression in LUAD. The binding relationship between CEBPA and FDX1 was validated <em>via</em> CHIP and dual-luciferase assays. FDX1 expression in LUAD cells was evaluated by qRT-PCR and Western blot. The impact of FDX1 overexpression on LUAD progression was examined using CCK-8 and Transwell assays. Experiments involving CCK-8, copper ion measurement, cuproptosis-related protein detection, and DLAT immunofluorescence confirmed the successful LUAD cuproptosis model.</div></div><div><h3>Results</h3><div>FDX1 was downregulated in LUAD tissues and cells, and its overexpression inhibited LUAD cell migration, invasion, and proliferation. Cuproptosis significantly reduced LUAD cell viability and the protein levels of Lipoy-DLAT, DLAT, and FDX1, while increasing HSP70 expression, DLAT aggregation, and intracellular copper ion levels. CEBPA, a transcriptional activator of FDX1, positively correlated with and bound to it. Overexpressed FDX1 enhanced cuproptosis in LUAD cells, an effect partially reversible by CEBPA suppression.</div></div><div><h3>Conclusion</h3><div>These analyses were performed to construct a proposed cellular model of cuproptosis in LUAD.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1870 1","pages":"Article 130872"},"PeriodicalIF":2.2,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Radiation therapy for cancer takes advantage of the higher sensitivity of tumor cells to radiation compared to normal tissue, but some cancers, such as glioblastoma (GBM) and malignant melanoma, acquire radiation resistance (radioresistance), rendering treatment ineffective. Radioresistance is characterized by strong activation of DNA repair mechanisms in response DNA damage induced by radiation, together with possession of malignant property such as enhanced invasiveness and metastasis, though the molecular mechanisms involved remain to be fully established. Here, we show that P2X7 receptor-specific inhibitors suppress the γ-irradiation-induced DNA damage response (DDR) and enhance cell death of A172 GBM cells. In contrast, ATP, a P2X7 receptor ligand, promotes the DDR and suppresses cell death. Irradiation immediately induced ATP release from cells, and P2X7 receptor inhibitor suppressed the release of ATP. Furthermore, P2X7 receptor inhibitors suppress the release of high mobility group box 1 (HMGB1), which is known to promote cancer cell migration. Inhibitors of the receptor for advanced glycation end products (RAGE) also suppress ATP-induced cell motility, indicating that the P2X7-HMGB1-RAGE pathway contributes to radiation-induced malignant transformation. These data indicate firstly that the P2X7 receptor promotes the γ-irradiation-induced DDR, leading to increased resistance of GBM cells to γ-radiation-induced death, and secondly that the P2X7 receptor and extracellular ATP may be involved in the γ-irradiation-induced acquisition of malignant property such as cytoskeletal changes and enhanced motility in GBM cells.
{"title":"P2X7 receptor contributes to DNA damage repair and acquisition of malignant phenotypes in irradiated human glioblastoma cells","authors":"Hiromu Seki , Kazuki Kitabatake , Fumiaki Uchiumi , Sei-ichi Tanuma , Mitsutoshi Tsukimoto","doi":"10.1016/j.bbagen.2025.130873","DOIUrl":"10.1016/j.bbagen.2025.130873","url":null,"abstract":"<div><div>Radiation therapy for cancer takes advantage of the higher sensitivity of tumor cells to radiation compared to normal tissue, but some cancers, such as glioblastoma (GBM) and malignant melanoma, acquire radiation resistance (radioresistance), rendering treatment ineffective. Radioresistance is characterized by strong activation of DNA repair mechanisms in response DNA damage induced by radiation, together with possession of malignant property such as enhanced invasiveness and metastasis, though the molecular mechanisms involved remain to be fully established. Here, we show that P2X7 receptor-specific inhibitors suppress the γ-irradiation-induced DNA damage response (DDR) and enhance cell death of A172 GBM cells. In contrast, ATP, a P2X7 receptor ligand, promotes the DDR and suppresses cell death. Irradiation immediately induced ATP release from cells, and P2X7 receptor inhibitor suppressed the release of ATP. Furthermore, P2X7 receptor inhibitors suppress the release of high mobility group box 1 (HMGB1), which is known to promote cancer cell migration. Inhibitors of the receptor for advanced glycation end products (RAGE) also suppress ATP-induced cell motility, indicating that the P2X7-HMGB1-RAGE pathway contributes to radiation-induced malignant transformation. These data indicate firstly that the P2X7 receptor promotes the γ-irradiation-induced DDR, leading to increased resistance of GBM cells to γ-radiation-induced death, and secondly that the P2X7 receptor and extracellular ATP may be involved in the γ-irradiation-induced acquisition of malignant property such as cytoskeletal changes and enhanced motility in GBM cells.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 12","pages":"Article 130873"},"PeriodicalIF":2.2,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-16DOI: 10.1016/j.bbagen.2025.130870
Jia Jing Cai , Yi Lin Shen , Jia Lin , Jun Tao Ren , Yan Zhi Yi , Xue Cheng Li , Ke Xin Jia , Jun Yi Liu , Guo Ming Su , Yong Yan Song , Qi Wei Guo , Ding Zhi Fang
The metabolic syndrome-associated gene (MSAG) encodes a polypeptide of MSAG composed of 110 amino acids and was observed to responds to high glucose conditions. However, scant studies have been documented since it was first reported in 2009. The function of MSAG and its underlying mechanisms remain unclear. To explore them, antibodies against MSAG are needed. Therefore, the current study aimed to generate a polyclonal antibody against MSAG and assess its applications. The pET-28a-mMSAG vector was constructed for prokaryotic expression. The recombinant MSAG was obtained and identified with a coverage of 100 %. Subsequently, a polyclonal antibody against MSAG was prepared by rabbit immunization with an antibody titer exceeding 1:128,000 as determined by ELISA. The prepared antibody was validated using MSAG knockdown mouse and HepG2 cells. The mRNA and protein levels of MSAG were lower in MSAG knockdown mouse livers and HepG2 cells. Additionally, MSAG mRNA and protein levels in mouse hepatic tissues were significantly increased following the intervention with a high carbohydrate diet as evaluated by RT-qPCR and western blotting using the prepared antibody. This reproducibly demonstrated that MSAG was involved in the regulation of glucose metabolism. Taken together, the rabbit anti-MSAG polyclonal antibody has been successfully generated in the current study, which lays the foundation for future studies to explore the functions of MSAG and its underlying mechanisms.
{"title":"Development of a polyclonal antibody against the protein encoded by the metabolic syndrome-associated gene for dissecting its function and underlying mechanism","authors":"Jia Jing Cai , Yi Lin Shen , Jia Lin , Jun Tao Ren , Yan Zhi Yi , Xue Cheng Li , Ke Xin Jia , Jun Yi Liu , Guo Ming Su , Yong Yan Song , Qi Wei Guo , Ding Zhi Fang","doi":"10.1016/j.bbagen.2025.130870","DOIUrl":"10.1016/j.bbagen.2025.130870","url":null,"abstract":"<div><div>The metabolic syndrome-associated gene (<em>MSAG</em>) encodes a polypeptide of MSAG composed of 110 amino acids and was observed to responds to high glucose conditions. However, scant studies have been documented since it was first reported in 2009. The function of MSAG and its underlying mechanisms remain unclear. To explore them, antibodies against MSAG are needed. Therefore, the current study aimed to generate a polyclonal antibody against MSAG and assess its applications. The pET-28a-m<em>MSAG</em> vector was constructed for prokaryotic expression. The recombinant MSAG was obtained and identified with a coverage of 100 %. Subsequently, a polyclonal antibody against MSAG was prepared by rabbit immunization with an antibody titer exceeding 1:128,000 as determined by ELISA. The prepared antibody was validated using <em>MSAG</em> knockdown mouse and HepG2 cells. The mRNA and protein levels of <em>MSAG</em> were lower in <em>MSAG</em> knockdown mouse livers and HepG2 cells. Additionally, <em>MSAG</em> mRNA and protein levels in mouse hepatic tissues were significantly increased following the intervention with a high carbohydrate diet as evaluated by RT-qPCR and western blotting using the prepared antibody. This reproducibly demonstrated that MSAG was involved in the regulation of glucose metabolism. Taken together, the rabbit anti-MSAG polyclonal antibody has been successfully generated in the current study, which lays the foundation for future studies to explore the functions of MSAG and its underlying mechanisms.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 12","pages":"Article 130870"},"PeriodicalIF":2.2,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-11DOI: 10.1016/j.bbagen.2025.130868
Zenab Shahzad , Ramish Rafay , Saeed Khan , Amir Kazory , Nancy D. Denslow , Abdel A. Alli
The mechanism underlying calcium oxalate (CaOx) stone formation remains unclear, although recurrent kidney stone formation has been linked to injury of kidney tubular cells in response to their exposure to higher oxalate and CaOx crystals. We hypothesized that CaOx induces the externalization and membrane enrichment of the negatively charged phospholipid phosphatidylserine (PS), while simultaneously enhancing the expression of calcium-binding proteins as a protective mechanism against cell death in inner medullary collecting duct (IMCD) cells. Annexin A2, a calcium-dependent phospholipid-binding protein, may play a protective role in CaOx stone formation. Mouse IMCD (mIMCD3) cells were treated with CaOx before performing a targeted lipidomic analysis of membrane fractions. Additionally, mIMCD3 cells were challenged with methyl-beta-cyclodextrin (MBCD) PS liposomes, Annexin A2 was evaluated by Western blotting and densitometric analysis. Lipidomic analysis revealed an enrichment of multiple PS lipid species in membrane fractions of mIMCD3 cells treated with CaOx. Western blotting and densitometric analysis further demonstrated a significant increase in Annexin A2 protein expression in the membrane fractions of mIMCD3 cells treated with MBCD PS liposomes. CaOx and MBCD PS liposomes were found to differentially affect membrane fluidity in mIMCD3 cells. Importantly, CaOx treatment caused an increase in ACSL4 and caspase 9 protein expression, indicating initiation of ferroptosis and apoptosis, respectively. The siRNA mediated knockdown of annexin A2 further augmented the upregulation of caspase 9 after CaOx treatment. Collectively, these findings suggest that Annexin A2, a calcium-binding and plasma membrane repair protein, is upregulated in response to CaOx-induced PS enrichment in mIMCD3 cells.
{"title":"Calcium oxalate alters lipid profiles and annexin A2 enrichment in membrane fractions of murine inner medullary collecting duct cells","authors":"Zenab Shahzad , Ramish Rafay , Saeed Khan , Amir Kazory , Nancy D. Denslow , Abdel A. Alli","doi":"10.1016/j.bbagen.2025.130868","DOIUrl":"10.1016/j.bbagen.2025.130868","url":null,"abstract":"<div><div>The mechanism underlying calcium oxalate (CaOx) stone formation remains unclear, although recurrent kidney stone formation has been linked to injury of kidney tubular cells in response to their exposure to higher oxalate and CaOx crystals. We hypothesized that CaOx induces the externalization and membrane enrichment of the negatively charged phospholipid phosphatidylserine (PS), while simultaneously enhancing the expression of calcium-binding proteins as a protective mechanism against cell death in inner medullary collecting duct (IMCD) cells. Annexin A2, a calcium-dependent phospholipid-binding protein, may play a protective role in CaOx stone formation. Mouse IMCD (mIMCD3) cells were treated with CaOx before performing a targeted lipidomic analysis of membrane fractions. Additionally, mIMCD3 cells were challenged with methyl-beta-cyclodextrin (MBCD) PS liposomes, Annexin A2 was evaluated by Western blotting and densitometric analysis. Lipidomic analysis revealed an enrichment of multiple PS lipid species in membrane fractions of mIMCD3 cells treated with CaOx. Western blotting and densitometric analysis further demonstrated a significant increase in Annexin A2 protein expression in the membrane fractions of mIMCD3 cells treated with MBCD PS liposomes. CaOx and MBCD PS liposomes were found to differentially affect membrane fluidity in mIMCD3 cells. Importantly, CaOx treatment caused an increase in ACSL4 and caspase 9 protein expression, indicating initiation of ferroptosis and apoptosis, respectively. The siRNA mediated knockdown of annexin A2 further augmented the upregulation of caspase 9 after CaOx treatment. Collectively, these findings suggest that Annexin A2, a calcium-binding and plasma membrane repair protein, is upregulated in response to CaOx-induced PS enrichment in mIMCD3 cells.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 12","pages":"Article 130868"},"PeriodicalIF":2.2,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-10DOI: 10.1016/j.bbagen.2025.130869
M. Davigo , F. Caiment , M. Verheijen , F.J. van Schooten , M. van Herwijnen , A. Mommers , A. Opperhuizen , R. Talhout , A.H.V. Remels
Introduction
IQOS is a Heated Tobacco Product marketed as reduced-risk alternative to cigarettes. This is based on limited industry-independent toxicological evidence.
Methods
Cigarette Smoke Extract (CSE) and IQOS extract (IQOSE) were generated by puffing mainstream emissions in PBS. Nicotine levels were quantified via liquid chromatography-mass spectrometry (LC-MS). Subsequently, undifferentiated Primary human Bronchial Epithelial Cells (PBECs) from three donors were exposed (24 h) to 1 %CSE, 1 %IQOSE, 3 %IQOSE (v/v), or nicotine concentrations (1.5, 6 or 18 μg/mL), representative of the levels present in the extracts. Transcriptomic alterations were assessed via RNA sequencing, and specific mRNA alterations were validated at the protein level by Western blot.
Results
IQOSE contained about 4-fold more nicotine than CSE. Nicotine did not significantly affect PBECs transcriptome. Substantial differences in the amount of differentially expressed genes (DEGs) were observed in response to CSE and IQOSE between donors. However, in all donors, 3 %IQOSE exposure downregulated pathways involved in cell cycle progress. In addition, both mRNA and protein levels of molecules controlling autophagy and mitophagy increased in response to IQOSE in one donor, and the disease Chronic Obstructive Pulmonary Disease (COPD) was enriched in cells from two donors upon exposure to CSE or IQOSE. In the most responsive donor, pathways associated with ER-stress and DNA replication were deregulated.
Conclusions
Despite significant inter-donor variability, IQOSE induced specific gene signatures associated with pathways known to be involved in (smoking-related) lung diseases in all donors. Collectively, these findings provide novel insights into the molecular toxicity of IQOS mainstream emissions in human bronchial epithelial cells.
{"title":"Alterations in the transcriptome of human primary bronchial epithelial cells exposed to emissions from a heated tobacco product","authors":"M. Davigo , F. Caiment , M. Verheijen , F.J. van Schooten , M. van Herwijnen , A. Mommers , A. Opperhuizen , R. Talhout , A.H.V. Remels","doi":"10.1016/j.bbagen.2025.130869","DOIUrl":"10.1016/j.bbagen.2025.130869","url":null,"abstract":"<div><h3>Introduction</h3><div>IQOS is a Heated Tobacco Product marketed as reduced-risk alternative to cigarettes. This is based on limited industry-independent toxicological evidence.</div></div><div><h3>Methods</h3><div>Cigarette Smoke Extract (CSE) and IQOS extract (IQOSE) were generated by puffing mainstream emissions in PBS. Nicotine levels were quantified <em>via</em> liquid chromatography-mass spectrometry (LC-MS). Subsequently, undifferentiated Primary human Bronchial Epithelial Cells (PBECs) from three donors were exposed (24 h) to 1 %CSE, 1 %IQOSE, 3 %IQOSE (<em>v</em>/v), or nicotine concentrations (1.5, 6 or 18 μg/mL), representative of the levels present in the extracts. Transcriptomic alterations were assessed <em>via</em> RNA sequencing, and specific mRNA alterations were validated at the protein level by Western blot.</div></div><div><h3>Results</h3><div>IQOSE contained about 4-fold more nicotine than CSE. Nicotine did not significantly affect PBECs transcriptome. Substantial differences in the amount of differentially expressed genes (DEGs) were observed in response to CSE and IQOSE between donors. However, in all donors, 3 %IQOSE exposure downregulated pathways involved in cell cycle progress. In addition, both mRNA and protein levels of molecules controlling autophagy and mitophagy increased in response to IQOSE in one donor, and the disease Chronic Obstructive Pulmonary Disease (COPD) was enriched in cells from two donors upon exposure to CSE or IQOSE. In the most responsive donor, pathways associated with ER-stress and DNA replication were deregulated.</div></div><div><h3>Conclusions</h3><div>Despite significant inter-donor variability, IQOSE induced specific gene signatures associated with pathways known to be involved in (smoking-related) lung diseases in all donors. Collectively, these findings provide novel insights into the molecular toxicity of IQOS mainstream emissions in human bronchial epithelial cells.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 12","pages":"Article 130869"},"PeriodicalIF":2.2,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145273567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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