Browsing by Subject "Hydrolases"

Now showing 1 - 9 of 9
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    BCL-XL Protects ASS1-Deficient Cancers from Arginine Starvation-Induced Apoptosis
    (American Association for Cancer Research, 2025) Panda, Prashanta Kumar; Paschoalini Mafra, Ana Carolina; Bastos, Alliny C. S.; Cao, Li; Bonet, Maria Serra; Brashears, Caitlyn B.; Chen, Ethan Yang; Benedict-Hamilton, Heather M.; Ehrhardt, William; Bomalaski, John; Dehner, Carina; Rogers, Leonard C.; Oyama, Toshinao; Van Tine, Brian A.; Pathology and Laboratory Medicine, School of Medicine
    Purpose: Argininosuccinate synthetase 1 (ASS1) silencing in carcinomas and sarcomas leads to a dependence on extracellular arginine for survival. Arginine deprivation therapies, such as PEGylated arginine deiminase (ADI-PEG20), have shown limited effectiveness, which may be due to underlying mechanisms that inhibit apoptosis. Experimental design: The effects of ADI-PEG20 on cell-cycle regulation, apoptosis, and BCL-XL-mediated survival pathways in ASS1-deficient cancer cells were determined. The mechanism of cell death protection was determined by assessing caspase and PARP cleavage, CDK2 activity, MCL1 expression, and the interactions among BCL-XL, BAX, and BAK. In vitro synergy was determined, and in vivo efficacy was modeled. Results: Treatment with ADI-PEG20 led to reduced CDK2 activity and inhibited cell-cycle progression but did not induce significant cell death. BCL-XL was found to bind to BAX and BAK, preventing the initiation of apoptosis despite arginine starvation. Inhibition of BCL-XL allowed proapoptotic BAX and BAK to initiate the intrinsic apoptosis pathway, leading to increased cell death. This was found to be synergistic in vitro and efficacious in combination in vivo. Conclusions: The study identifies BCL-XL as a key factor limiting the efficacy of arginine starvation therapies. Combining BCL-XL inhibitors with arginine deprivation strategies may overcome this resistance and enhance therapeutic outcomes. These findings provide a strong preclinical rationale for testing this combination approach in phase 1 clinical trials for ASS1-deficient cancers.
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    Mechanisms of Cytoskeletal Dysregulation in the Kidney Proximal Tubule During ATP Depletion and Ischemia
    (2009-08) Zhang, Hao; Atkinson, Simon J.; Harrington, Maureen A.; Marrs, James A.; Quilliam, Lawrence A.
    Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.
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    The role of acid sphingomyelinase in autophagy
    (2014-07-11) Justice, Matthew Jose; Petrache, Irina; Blum, Janice Sherry, 1957-; Wek, Ronald C.
    Autophagy is a conserved cellular process that involves sequestration and degradation of cytosolic contents. The cell can engulf autophagic cargo (lipids, long-lived proteins, protein aggregates, and pathogens) through a double bound membrane called an autophagosome that fuses with a lysosome where hydrolases then degrade these contents. This process is one of the main defenses against starvation and is imperative for newborns at birth. Research on this process has increased exponentially in the last decade since its discovery almost a half a century ago. It has been found that autophagy is an important process in many diseases, continues to be at the forefront of research, and is clearly not fully understood. Our preliminary cell culture data in endothelial and epithelial cells show that a blockade of the de novo ceramide synthesis pathway, during treatment with an autophagy stimulus (cigarette smoke extract exposure), does not result in any reduction in autophagy or autophagic flux. Conversely, when acid sphingomyelinase (ASM) is pharmacologically inhibited, which prevents the generation of ceramide from sphingomyelin in an acidic environment, a profound increase in autophagy is observed. In this work, we hypothesize that (ASM) is an endogenous inhibitor of autophagy. ASM has two forms, a secreted form and a lysosomal form. N-terminal processing in the Golgi determines its cellular fate. In the lysosomal form, the phosphodiesterase is bound in the lysosomal membrane. The pharmacological inhibition mechanism is to release ASM from the membrane and allow other hydrolases to actively degrade the enzyme which, in turn, decreases the activity of ASM. This suggests that either the activity of ASM is a regulator of autophagy or that the presence of ASM, activity aside, is required for the lysosomal nutrient sensing machinery (LYNUS) to function properly. Here, we show that ASM is, in fact, an endogenous inhibitor of autophagy in vitro. The phosphorylation status of P70 S6k, a downstream effector of mammalian target of rapamycin (mTOR), which is part of the LYNUS, shows that dissociation of ASM from the membrane regulates mTOR and disturbs the LYNUS in such a manner as to signal autophagy.
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    Role of Rap1 in Angiogenesis and Tumor Invasion
    (2009-08) Yan, Jingliang; Quilliam, Lawrence A.; Atkinson, Simon J.; Ingram, David A., Jr.; Pavalko, Fredrick M.; Shou, Weinian; Yoder, Mervin C.
    Rap1a and Rap1b are two closely related members of the Ras family of small GTPases. Despite their high sequence similarity, the two proteins serve non-redundant functions in cells and organs. Rap1a plays critical roles during mouse development, and both Rap1a and Rap1b are required for angiogenesis. In glioblastoma cells, however, Rap1b plays a more unique role in tumor cell invasion. Loss of rap1a in mice resulted in 40% embryonic lethality, and caused cardiac defects in mouse embryos and cardiac hypertrophy in adult mice. These phenotypes, distinct from those of the rap1b knockout mice, suggest differential roles of the two GTPases during mouse development. Angiogenesis, the formation of new blood vessels by endothelial cells, is impaired by the loss of rap1. Blood vessel growth into FGF2-containing Matrigel plugs was absent from rap1a-/- mice and aortic rings derived from rap1a-/- mice failed to sprout primitive endothelial tubes in response to FGF2 when embedded in Matrigel. Knocking down of either rap1a or rap1b in human micro-vascular endothelial cells (HMVECs) confirmed that Rap1 plays key roles in endothelial cell function. The knockdown of rap1a or 1b resulted in decreased adhesion to extracellular matrices and impaired cell migration. Rap1 deficient endothelial cells failed to form 3-D tubular structures when plated on Matrigel in vitro. The activation of ERK, p38, and Rac, important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either Rap1 protein was depleted. In U373 human glioblastoma multiforme cells, depletion of rap1b, but not rap1a drastically reduced tumor cell invasion by decreasing the activity of secreted matrix metalloproteinase 2 (MMP2). The adhesion of cells to the extracellular matrices collagen or fibronectin, but not to vitronectin, was decreased upon rap1b depletion. However, a mild increase in proliferation associated with elevation in ERK1/2, p38, Akt and ribosomal S6 protein activation was observed in cells depleted of either rap1a or rap1b. When an MEK1/2 inhibitor U0126 was used, the phosphorylation of p38, Akt and S6 were decreased, however, to various levels, suggesting complex regulatory pathways mediate Rap1 action in glioblastoma cells.
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    Step-growth thiol-ene photopolymerization to form degradable, cytocompatible and multi-structural hydrogels
    (2014-01-17) Shih, Han; Lin, Chien-Chi; Xie, Dong; Bottino, Marco
    Hydrogels prepared from photopolymerization have been used for a variety of tissue engineering and controlled release applications. Polymeric biomaterials with high cytocompatibility, versatile degradation behaviors, and diverse material properties are particularly useful in studying cell fate processes. In recent years, step-growth thiol-ene photochemistry has been utilized to form cytocompatible hydrogels for tissue engineering applications. This radical-mediated gelation scheme utilizes norbornene functionalized multi-arm poly(ethylene glycol) (PEGNB) as the macromer and di-thiol containing molecules as the crosslinkers to form chemically crosslinked hydrogels. While the gelation mechanism was well-described in the literature, the network properties and degradation behaviors of these hydrogels have not been fully characterized. In addition, existing thiol-ene photopolymerizations often used type I photoinitiators in conjunction with an ultraviolet (UV) light source to initiate gelation. The use of cleavage type initiators and UV light often raises biosafety concerns. The first objective of this thesis was to understand the gelation and degradation properties of thiol-ene hydrogels. In this regard, two types of step-growth hydrogels were compared, namely thiol-ene hydrogels and Michael-type addition hydrogels. Between these two step-growth gel systems, it was found that thiol-ene click reactions formed hydrogels with higher crosslinking efficiency. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEGNB macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, it was found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation rate of thiol-ene hydrogels, different macromer contents and peptide crosslinkers with different amino acid sequences were used. A chymotrypsin-sensitive peptide was also used as part of the hydrogel crosslinkers to render thiol-ene hydrogels enzymatically degradable. The second objective of this thesis was to develop a visible light-mediated thiol-ene hydrogelation scheme using a type II photoinitiator, eosin-Y, as the only photoinitiator. This approach eliminates the incorporation of potentially cytotoxic co-initiator and co-monomer that are typically used with a type II initiator. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, it was found that the visible light-mediated thiol-ene hydrogels were highly cytocompatible for human mesenchymal stem cells (hMSCs) and pancreatic MIN6 beta-cells. It was also found that eosin-Y could be repeatedly excited for preparing step-growth hydrogels with multilayer structures. This new gelation chemistry may have great utilities in controlled release of multiple sensitive growth factors and encapsulation of multiple cell types for tissue regeneration.