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Browsing by Author "Han, Bumsoo"
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Item Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells(Elsevier B.V., 2014-01-24) Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo; Department of Biomedical Engineering, School of Engineering and TechnologyRecent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.Item New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types(Elsevier, 2024) Gampala, Silpa; Moon, Hye-ran; Wireman, Randall; Peil, Jacqueline; Kiran, Sonia; Mitchell, Dana K.; Brewster, Kylee; Mang, Henry; Masters, Andi; Bach, Christine; Smith-Kinnamen, Whitney; Doud, Emma H.; Rai, Ratan; Mosley, Amber L.; Quinney, Sara K.; Clapp, D. Wade; Hamdouchi, Chafiq; Wikel, James; Zhang, Chi; Han, Bumsoo; Georgiadis, Millie M.; Kelley, Mark R.; Fishel, Melissa L.; Pediatrics, School of MedicineAP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.Item Recapitulation of complex transport and action of drugs at tumor microenvironment using tumor-microenvironment-on-chip(Elsevier, 2016-09-28) Han, Bumsoo; Qu, Chunjing; Park, Kinam; Konieczny, Stephen F.; Korc, Murray; Medicine, School of MedicineTargeted delivery aims to selectively distribute drugs to targeted tumor tissue but not to healthy tissue. This can address many of clinical challenges by maximizing the efficacy but minimizing the toxicity of anti-cancer drugs. However, complex tumor microenvironment poses various barriers hindering the transport of drugs and drug delivery systems. New tumor models that allow for the systematic study of these complex environments are highly desired to provide reliable test beds to develop drug delivery systems for targeted delivery. Recently, research efforts have yielded new in vitro tumor models, the so called tumor-microenvironment-on-chip, that recapitulate certain characteristics of the tumor microenvironment. These new models show benefits over other conventional tumor models, and have the potential to accelerate drug discovery and enable precision medicines. However, further research is warranted to overcome their limitations and to properly interpret the data obtained from these models. In this article, key features of the in vivo tumor microenvironment that are relevant to drug transport processes for targeted delivery was discussed, and the current status and challenges for developing in vitro transport model systems was reviewed.Item Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target(BMC, 2021-08-10) Gampala, Silpa; Shah, Fenil; Lu, Xiaoyu; Moon, Hye-ran; Babb, Olivia; Umesh Ganesh, Nikkitha; Sandusky, George; Hulsey, Emily; Armstrong, Lee; Mosely, Amber L.; Han, Bumsoo; Ivan, Mircea; Yeh, Jing-Ruey Joanna; Kelley, Mark R.; Zhang, Chi; Fishel, Melissa L.; Pediatrics, School of MedicineBackground: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.Item Subcellular domain-dependent molecular hierarchy of SFK and FAK in mechanotransduction and cytokine signaling(Springer NPG, 2017-08-22) Wan, Qiaoqiao; TruongVo, ThucNhi; Steele, Hannah E.; Ozcelikkale, Altug; Han, Bumsoo; Wang, Yingxiao; Oh, Junghwan; Yokota, Hiroki; Na, Sungsoo; Biomedical Engineering, School of Engineering and TechnologyFocal adhesion kinase (FAK) and Src family kinases (SFK) are known to play critical roles in mechanotransduction and other crucial cell functions. Recent reports indicate that they reside in different microdomains of the plasma membrane. However, little is known about their subcellular domain-dependent roles and responses to extracellular stimuli. Here, we employed fluorescence resonance energy transfer (FRET)-based biosensors in conjunction with collagen-coupled agarose gels to detect subcellular activities of SFK and FAK in three-dimensional (3D) settings. We observed that SFK and FAK in the lipid rafts and nonrafts are differently regulated by fluid flow and pro-inflammatory cytokines. Inhibition of FAK in the lipid rafts blocked SFK response to fluid flow, while inhibition of SFK in the non-rafts blocked FAK activation by the cytokines. Ex-vivo FRET imaging of mouse cartilage explants showed that intermediate level of interstitial fluid flow selectively decreased cytokine-induced SFK/FAK activation. These findings suggest that SFK and FAK exert distinctive molecular hierarchy depending on their subcellular location and extracellular stimuli.