DSpace community: 生命科學研究所

探討細胞內氧化還原水平對外泌體生成之影響;Investigation of the effects of intracellular redox status on exosome biogenesis

title: 探討細胞內氧化還原水平對外泌體生成之影響;Investigation of the effects of intracellular redox status on exosome biogenesis abstract: 細胞外囊泡（extracellular vesicles， EVs）是細胞間訊息傳遞的重要媒介，其中外泌體主要源自內體系統，經由內體膜向內出芽形成腔內囊泡，進一步形成多泡體（multivesicular ibodies， MVBs）。MVB 後續可與溶酶體融合並進行降解，亦可與細胞膜融合而將外泌體釋放至細胞外。此過程涉及 ESCRT 相關貨物分選、Rab GTPase 介導的囊泡運輸、細胞膜錨定及膜融合等多項調控機制。已有研究指出，細胞內氧化壓力的增加可能影響內體成熟、溶酶體功能及 EV 釋放，但降低細胞內氧化水平是否直接與抑制或促進外泌體生成有影響，目前仍然未知。因此，本研究以 A375 人類黑色素瘤細胞及 WJMSC 臍帶間質幹細胞為研究模型，探討不同抗氧化處理所造成的細胞內氧化狀態改變，是否會影響外泌體生合成、MVB 命運及 EV 釋放。研究中分析細胞內及小型細胞外囊泡( small extracellular vesicles ， sEVs) 中之外泌體與 MVB 相關蛋白，包括 CD63、CD81、TSG101、Syn-1、Rab27a、Rab7、WDR4 及 LAMP1，並搭配奈米粒子追蹤分析（nanoparticle tracking analysis， NTA），評估不同處理對 sEVs 生成與釋放的影響。研究結果顯示，細胞內氧化水平與外泌體生成之間並非單一線性之關係，而具有細胞類型及抗氧化物種類依賴性。在 A375 細胞中，抗氧化處理可造成多種外泌體或 MVB 相關蛋白表現改變，顯示腫瘤細胞可能隨細胞內氧化狀態變化，重新調節內體貨物分選機制、MVB 生成、囊泡運輸及外泌體釋放路徑。相較之下，在 WJMSC 細胞中，Compound A 與 Compound Compound .ß 雖皆可降低細胞內氧化水平，卻對 sEV 釋放產生不同影響。Compound A 處理後，儘管部分細胞內 MVB 或溶酶體相關蛋白表現下降，單次NTA檢測中sEV 數量增加，顯示其可能促進 MVB 與細胞膜融合或提高囊泡釋放效率，而非單純增加 MVB 相關蛋白表現。另一方面，Compound ß 處理後雖使部分細胞內囊泡運輸相關蛋白上升，但外泌體標誌蛋白表現及單次NTA檢驗結果均顯示 sEV 釋放下降，；細胞內 Rab7 與 LAMP1 顯著增加，同時 Rab27a 與 WDR4 亦顯著增加，顯示分泌與溶酶體相關路徑可能同時受到調控，因此現有結果尚不足以將 MVB 命運簡化為單一降解方向。推測此處理可能改變 MVB 的命運，使其較傾向進入溶酶體降解途徑，而非與細胞膜融合並釋放至細胞外。綜合而言，本研究顯示，抗氧化物對外泌體生成及 sEV 釋放的影響，無法僅以降低細胞內氧化水平作為單一且唯一解釋，而應同時考量其對內體成熟、MVB 生成、貨物分選、囊泡運輸、溶酶體功能及膜融合效率的整體調控。不同細胞來源對氧化還原狀態變化具有不同反應，顯示腫瘤細胞與間質幹細胞可能採取不同的內體—溶酶體路徑調控策略。本研究支持細胞內氧化狀態參與外泌體生合成調控的觀點，並進一步指出，不同抗氧化物可能透過改變 MVB 命運選擇與 sEV 釋放效率，影響細胞間訊息傳遞。;Extracellular vesicles (EVs) serve as pivotal conduits for intercellular communication. Among distinct EV subpopulations， exosomes predominantly originate from the endosomal pathway via inward budding of the endosomal membrane， resulting in the generation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Subsequently， MVBs face alternative physiological fates: undergoing lysosomal fusion for degradation， or docking and fusing with the plasma membrane to liberate exosomes into the extracellular milieu. This delicate machinery is governed by an intricate regulatory network encompassing ESCRT-dependent cargo sorting， Rab GTPase-mediated vesicular trafficking， and specialized membrane tethering and fusion events. Although elevated intracellular oxidative stress has been implicated in modulating endosomal maturation， lysosomal clearance， and EV secretion， whether attenuating intracellular redox status directly promotes or suppresses exosome biogenesis remains poorly defined. To elucidate whether variations in intracellular redox states exert a direct influence on exosome biogenesis， MVB fate determination， and sEV release， we employed human melanoma A375 cells and Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) subjected to distinct antioxidant interventions. Exosome- and MVB-associated molecular signatures—including CD63， CD81， TSG101， Syntenin-1 (Syn-1)， Rab27a， Rab7， WDR4， and LAMP1—were systematically profiled in both cellular lysates and isolated small extracellular vesicles (sEVs)， accompanied by nanoparticle tracking analysis (NTA) to quantify physical secretion kinetics. Our findings reveal a non-linear relationship between intracellular oxidative levels and exosome biogenesis， characterized by pronounced cell-type specificity and antioxidant dependency. In A375 cells， antioxidant treatment dynamically recalibrated the expressionsignatures of exosomal and MVB-associated proteins， indicating that malignant cells reconfigure cargo sorting， MVB generation， and exocytotic pathways in response to redox fluctuations. Conversely， in WJMSCs， Compound A and Compound ß triggered divergent sEV secretion profiles despite achieving comparable efficacy in scavenging intracellular reactive species. Notably， Compound A administration selectively enhanced sEV release kinetics—as reflected by elevated particle counts—despite the downregulation of specific intracellular MVB/lysosomal proteins， suggesting that Compound A facilitates plasma membrane fusion efficiency rather than merely driving vesicle synthesis. In stark contrast， while Compound ß upregulated specific trafficking-related intracellular machinery， both molecular markers and NTA metrics converged on a net reduction in sEV secretion. Crucially， the marked upregulation of Rab7 and LAMP1， alongside elevated Rab27a and WDR4 levels， implies a concurrent activation of both secretory and degradative machinery. Rather than adhering to a simplified exocytotic route， MVBs under Compound $\beta$ treatment are preferentially shunted toward lysosomal degradation over plasma membrane exocytosis. Collectively， this study demonstrates that antioxidant-mediated modulation of exosome biogenesis extends beyond a generic reduction in oxidative load. Instead， it reflects an integrated recalibration of endosomal maturation， cargo sorting， vesicular trafficking， and membrane fusion kinetics. By demonstrating that distinct cell types and antioxidant moieties differentially govern MVB fate selection， these findings establish intracellular redox status as a crucial molecular rheostat in extracellular vesicle biology.

全氟辛烷磺酸PFOS及短鏈替代物FBSA對順鉑抗藥性泌尿上皮癌細胞惡性表徵;EMT及發炎反應之影響研究 Effects of PFOS and the Short-Chain Alternative FBSA on Malignant Phenotypes， Epithelial Mesenchymal Transition， and Inflammatory Responses in Cisplatin-Resistant Urothelial Carcinoma Cells

title: 全氟辛烷磺酸PFOS及短鏈替代物FBSA對順鉑抗藥性泌尿上皮癌細胞惡性表徵;EMT及發炎反應之影響研究 Effects of PFOS and the Short-Chain Alternative FBSA on Malignant Phenotypes， Epithelial Mesenchymal Transition， and Inflammatory Responses in Cisplatin-Resistant Urothelial Carcinoma Cells abstract: 本研究旨探討全氟及多氟烷基物質（per- and polyfluoroalkyl substances， PFAS）在促進化療抗藥性泌尿上皮癌（urothelial carcinoma， UC）惡性進展中的作用及其潛在分子機制。PFAS為廣泛存在於環境中的持久性污染物，具有高度穩定性與生物累積性，近年研究指出其暴露與多種癌症風險上升相關。然而，目前對於 PFAS 是否能進一步促進已具化療抗藥性之腫瘤細胞惡化，仍缺乏系統性研究。本研究聚焦於長期低劑量PFAS暴露對抗藥性腫瘤行為之影響，並比較長鏈代表物質全氟辛烷磺酸（PFOS）與短鏈替代物全氟丁烷磺酸（FBSA）之作用差異。此實驗以人類膀胱癌細胞株 J82 及其經持續順鉑處理建立之抗藥性衍生株 J82 CR0.65 為實驗模型。相較於親代細胞，J82-CR0.65 細胞呈現顯著提升之遷移能力與基質金屬蛋白酶（matrix metalloproteinases， MMPs）表現，顯示其具備較強之惡性特徵。在長期低劑量PFOS暴露條件下，兩種細胞株皆出現細胞存活率與集落形成能力上升，其中以抗藥性細胞 J82-CR0.65 的變化更為顯著，顯示抗藥性腫瘤細胞對 PFAS 誘導之惡性表型具有較高敏感性。進一步分析顯示，PFOS 暴露可隨時間促進上皮–間質轉換（epithelial–mesenchymal transition， EMT）及細胞外基質（extracellular matrix， ECM）重塑，並伴隨 MMP-2、MMP-7及MMP-9等分子表現上調。此外，在親代細胞中，短期（7天）PFOS暴露已可誘導EMT相關蛋白（如N-cadherin、Snail 及 fibronectin）表現增加，並使其 MMP 表現趨近於抗藥性細胞之型態；而在抗藥性細胞中，長期（約30天）PFOS暴露則主要誘導發炎相關訊號分子，包括IL-1β、IL-18及KRAS之上升。在抗藥性細胞中，上述發炎與EMT變化並未伴隨顯著之活性氧（reactive oxygen species， ROS）變化，顯示PFOS可能透過非典型氧化壓力途徑，促進EMT與發炎訊號之交互作用（EMT–inflammatory axis），進而驅動腫瘤惡性進展。此外，短鏈PFAS替代物FBSA 亦呈現類似效應，包括誘導 EMT 相關分子、MMP 表現及趨化激素（如CXCL5、CXCL9）轉錄上升，顯示即使為替代性PFAS，仍可能保留其促腫瘤進展之潛在能力。綜合以上結果，本研究指出長期PFAS暴露可透過協同活化EMT、細胞外基質重塑及發炎訊號途徑，促進順鉑抗藥性泌尿上皮癌細胞之惡性進展，且抗藥性細胞對環境污染物具有較高反應性。本研究不僅補足PFAS於抗藥性腫瘤進展中的角色，也進一步強調環境暴露因子在腫瘤惡化過程中的重要性，對未來癌症治療策略與環境風險評估具有潛在意義。;This study aimed to investigate the role of per- and polyfluoroalkyl substances (PFAS) in promoting malignant progression in chemoresistant urothelial carcinoma (UC) and to elucidate the underlying molecular mechanisms. PFAS are persistent environmental pollutants characterized by high chemical stability and bioaccumulation， and increasing evidence has linked their exposure to elevated cancer risk. However， whether PFAS further exacerbate malignant phenotypes in chemoresistant tumor cells remains largely unclear. Therefore， this study focused on the effects of long-term， low-dose PFAS exposure on chemoresistant cancer cells， with a comparative analysis of the long-chain compound perfluorooctane sulfonate (PFOS) and the short-chain alternative perfluorobutane sulfonate (FBSA). Human bladder cancer J82 cells and their cisplatin-resistant derivative， J82-CR0.65， were used as experimental models. Compared with parental cells， J82-CR0.65 cells exhibited enhanced migratory capacity and elevated expression of matrix metalloproteinases (MMPs)， indicating a more aggressive phenotype. Under long-term， low-dose PFOS exposure， both cell lines showed increased cell viability and clonogenic potential， with more pronounced effects observed in the resistant cells， suggesting that chemoresistant cells are more susceptible to PFAS-induced malignant progression.Further analyses revealed that PFOS exposure promoted epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling in a time-dependent manner， accompanied by upregulation of MMP-2， MMP-7， and MMP-9. In parental J82 cells， short-term PFOS exposure (7 days) induced EMT-related markers， including N-cadherin， Snail， and fibronectin， and shifted the MMP expression profile toward that observed in resistant cells. In contrast， prolonged PFOS exposure (approximately 30 days) in J82-CR0.65 cells predominantly activated inflammatory signaling pathways， as evidenced by increased expression of IL-1β， IL-18， and KRAS. Notably， these inflammatory and EMT-associated changes occurred without significant alterations in intracellular reactive oxygen species (ROS)， suggesting that PFOS may promote malignant progression through a noncanonical mechanism involving crosstalk between EMT and inflammatory signaling (EMT–inflammatory axis)， rather than through classical oxidative stress pathways. The short-chain PFAS alternative FBSA elicited comparable effects， including induction of EMT markers， MMP expression， and chemokine transcription (e.g.， CXCL5 and CXCL9)， indicating that replacement PFAS may retain pro-tumorigenic potential. In conclusion， long-term PFAS exposure promotes malignant progression in cisplatin-resistant urothelial carcinoma through coordinated activation of EMT， extracellular matrix remodeling， and inflammatory signaling pathways. These findings suggest that chemoresistant tumor cells exhibit heightened responsiveness to environmental stressors such as PFAS， and highlight the potential contribution of environmental exposure to tumor progression and therapeutic resistance.

兒科癌症亞型中 MYCN/AURKA 依賴性的表徵及利用變構 AURKA 抑制劑進行聯合標靶治療的研究;Characterization of MYCN/AURKA Dependency in Pediatric Cancer Subtypes for Co-Targeting with an Allosteric AURKA Inhibitor

title: 兒科癌症亞型中 MYCN/AURKA 依賴性的表徵及利用變構 AURKA 抑制劑進行聯合標靶治療的研究;Characterization of MYCN/AURKA Dependency in Pediatric Cancer Subtypes for Co-Targeting with an Allosteric AURKA Inhibitor abstract: 透過基因擴增導致的 MYCN 過度活化是高風險神經母細胞瘤的核心特徵，儘管接受了密集治療，該疾病的臨床預後依然不佳。在MYCN擴增的腫瘤中，極光激酶 A（Aurora kinase A， AURKA）可透過蛋白質交互作用穩定 MYCN，進而促進惡性腫瘤進展。在本研究中，資料庫分析顯示，同時高表達MYCN與AURKA的神經母細胞瘤患者，其存活預後顯著差於僅單獨高表達任一基因的腫瘤患者。鑑於兒童惡性腫瘤通常是由胚胎谱系特性而非累積突變所驅動，本研究將兒童癌症世代分析擴展至尋找其他發育相關的兒童腫瘤時，鑑定出了一組伴隨MYCN與AURKA同時高表達的軟組織肉瘤；其中，在尤文氏肉瘤（Ewing’s sarcoma）的共同高表達與整體存活率呈現強烈的負相關。在神經母細胞瘤與尤文氏肉瘤細胞株中，MYCN的表達水平與對AURKA 異位調節抑制劑（包括alisertib和6K465）的敏感性呈正相關。為評估其臨床轉譯潛力，本研究在 SK-N-BE(2) CDX（細胞株來源的異種移植）模型中，比較了alisertib與 DBPR728（6K465的前體藥物）的抗腫瘤療效。結果顯示，每週給藥一次300 mg/kg 的 DBPR728，比每日給藥一次50 mg/kg 的alisertib 能產生更持久的腫瘤抑制效果。為了尋找此類MYCN/AURKA雙重抑制劑敏感性相關的預測性基因特徵，本研究利用有限的細胞毒性與轉錄組圖譜，透過彈性網路（Elastic Net）k-fold重複交叉驗證開發了預測模型。將這些模型應用於癌症細胞株百科全書（CCLE）與 St. Jude 資料庫時發現，藥物敏感性與參與神經相關途徑的基因具有相關性。這些結果為兒童癌症患者的分層提供了潛在依據。總體而言，這些發現將MYCN與AURKA的共同活化定義為一種治療脆弱性，為進一步研發針對MYCN與AURKA高表達之兒童癌症的靶向藥物提供了有力支持。;Excessive activation of MYCN via gene amplification is a defining feature of high-risk neuroblastoma， which continues to have poor clinical outcomes despite intensive therapy. In MYCN-amplified tumors， Aurora kinase A (AURKA) contributes to promote malignancies by stabilizing MYCN via protein-protein interactions. In this study， database analyses revealed that neuroblastoma co-overexpressing MYCN and AURKA exhibit significantly worst survival outcomes compared with tumors overexpressing either one gene. Given that pediatric malignancies are frequently driven by embryonic lineage identities rather than accumulated mutations， expanding pediatric cancer cohort analysis to identify other developmentally related pediatric tumor identified a subset of soft-tissue sarcoma with concurrent MYCN and AURKA co-overexpression， among which Ewing’s sarcoma showed a strong negative correlation between co-overexpression and overall survival. Across neuroblastoma and Ewing’s sarcoma cell lines， MYCN expression levels positively correlated with sensitivity to AURKA allosteric inhibitors， including alisertib and 6K465. To evaluate translational potential， the antitumor efficacy of alisertib and DBPR728 (the prodrug of 6K465) in the SK-N-BE(2) CDX model were compared. DBPR728 administered at 300 mg/kg once weekly produced more durable tumor suppression than alisertib at 50 mg/kg once daily. To identify predictive gene signatures associated with sensitivity to AURKA allosteric inhibitors， predictive models using Elastic Net k-fold repetitive cross-validation were developed on a limited set of cytotoxicity and transcriptomic profiles. Applying these models to Cancer Cell Line Encyclopedia (CCLE) and St. Jude datasets revealed that sensitivity correlates with genes involved in the neuronal-related pathways. These results suggest a potential basis for pediatric cancer patient stratification. Collectively， these findings define coactivation of MYCN and AURKA as a therapeutic vulnerability， warranting further development of AURKA-targeting agents for pediatric cancers.

Targeted Attenuation of an ER-Associated Degradation (ERAD) Pathway Enhances Mitochondrial Genome Quality Control in Yeast;Targeted Attenuation of an ER-Associated Degradation (ERAD) Pathway Enhances Mitochondrial Genome Quality Control in Yeast

title: Targeted Attenuation of an ER-Associated Degradation (ERAD) Pathway Enhances Mitochondrial Genome Quality Control in Yeast;Targeted Attenuation of an ER-Associated Degradation (ERAD) Pathway Enhances Mitochondrial Genome Quality Control in Yeast abstract: 線粒體是重要的細胞器，負責調節細胞能量代謝、生物合成和訊號傳導，其功能高度依賴於線粒體DNA（mtDNA）的完整性。在酵母菌（Saccharomyces cerevisiae）中，失去mtDNA會導致呼吸缺陷的小型表型，提供了一個強健的模型來研究線粒體基因組的穩定性和品質控制。雖然線粒體的品質控制主要由線粒體內部的機制調節，但越來越多的證據表明，線粒體外的蛋白質穩定性途徑也可能影響線粒體的穩態。內質網相關降解（ERAD）途徑是一個核心的蛋白質品質控制系統，負責識別和降解內質網中的錯誤摺疊蛋白。雖然ERAD在維持內質網蛋白質穩態方面已有較完整的研究，但其對線粒體基因組穩定性的影響仍不清楚。本研究旨在探討破壞ERAD組分是否會影響酵母（Saccharomyces cerevisiae）的線粒體品質控制和線粒體DNA穩定性。線粒體基因組的不穩定性是使用小型頻率測試在溴化乙錠誘導的壓力條件下評估的。生成了一系列針對 ERAD-L、ERAD-M 和 ERAD-C 組件的基因刪除株。總體而言，ERAD 並不作為線粒體基因組穩定性的統一調控因子。它對線粒體品質控制的貢獻具有途徑特異性，且大多是間接的，如通過泛素穩態、蛋白質穩定性平衡和應激反應信號介導。這些研究結果突顯了線粒體基因組完整性主要由內在線粒體機制維持，並能緩衝與 ER 相關降解途徑的干擾。;Mitochondria are essential organelles that regulate cellular energy metabolism， biosynthesis， and signaling， and their function critically depend on the integrity of mitochondrial DNA (mtDNA). In Saccharomyces cerevisiae， loss of mtDNA results in respiratory-deficient petite phenotypes， providing a robust model to investigate mitochondrial genome stability and quality control. While mitochondrial quality control is largely governed by intra-mitochondrial mechanisms， increasing evidence suggests that extramitochondrial proteostasis pathways may also influence mitochondrial homeostasis. The endoplasmic reticulum-associated degradation (ERAD) pathway is a central protein quality control system responsible for the recognition and degradation of misfolded protein in endoplasmic reticulum. Although ERAD is well characterized in maintaining ER proteostasis， its contribution in mitochondrial DNA quality control (mtDNA QC) is remained poorly defined. This study aims to investigate whether disruption of ERAD components affects mitochondrial DNA quality control (mtDNA QC) in Saccharomyces cerevisiae. Mitochondrial DNA quality control (mtDNA QC) was assessed using petite frequency assays under ethidium bromide-induced stress conditions. A series of gene deletion strains targeting ERAD-L， ERAD-M and ERAD-C components were generated. Overall， ERAD does not acts as a uniform regulator of mitochondrial genome stability. Its contribution to mitochondrial DNA quality control (mtDNA QC) is pathway substrate-specific and largely indirectly， like mediated through ubiquitin homeostasis， proteostasis balance， and stress-response signaling. These findings highlight that mitochondrial genome integrity is primarily maintained by intrinsic mitochondrial mechanisms buffered against disruption of ER-associated degradation pathways.