Using NMR Spectroscopy To Measure Protein Binding Capacity on Gold Nanoparticles.

A simple one-dimensional 1H NMR experiment that quantifies protein bound to gold nanoparticles has been developed for upper-division biochemistry and physical chemistry students. This laboratory experiment teaches the basics of NMR techniques, which is a highly effective tool in protein studies and supports students to understand the concepts of NMR spectroscopy and nanoparticle-protein interactions. Understanding the interactions of gold nanoparticles (AuNPs) with biological macromolecules is becoming increasingly important as interest in the clinical use of nanoparticles has been on the rise. Applications in drug delivery, biosensing, diagnostics, and enhanced imaging are all tangible possibilities with a better understanding of AuNP-protein interactions. The ability to use AuNPs as biosensors for drug delivery methods in cellular uptake is dependent on the amount of protein that is able to bind to the surface of the nanoparticle. This laboratory experiment solidifies concepts such as quantitative NMR spectroscopy while reinforcing precision laboratory titrations. Students learn how 1H proton NMR spectra can be used to measure free protein in solution and protein bound to AuNPs. A simple formula is used to determine the binding capacity of the nanoparticle. This analysis helps students to understand the impact of nanoparticle-protein interactions, and it allows them to conceptualize macromolecular binding using NMR spectroscopy.

Loss of LGR5 through Therapy-induced Downregulation or Gene Ablation Is Associated with Resistance and Enhanced MET-STAT3 Signaling in Colorectal Cancer Cells.

Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.

Dual mTORC1/2 inhibition compromises cell defenses against exogenous stress potentiating Obatoclax-induced cytotoxicity in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy and have a dismal 4-year event-free survival (EFS) of 37%. We have previously shown that mTOR activation contributes to AT/RT's aggressive growth and poor survival. Targeting the mTOR pathway with the dual mTORC1/2 inhibitor TAK-228 slows tumor growth and extends survival in mice bearing orthotopic xenografts. However, responses are primarily cytostatic with limited durability. The aim of this study is to understand the impact of mTOR inhibitors on AT/RT signaling pathways and design a rational combination therapy to drive a more durable response to this promising therapy. We performed RNASeq, gene expression studies, and protein analyses to identify pathways disrupted by TAK-228. We find that TAK-228 decreases the expression of the transcription factor NRF2 and compromises AT/RT cellular defenses against oxidative stress and apoptosis. The BH3 mimetic, Obatoclax, is a potent inducer of oxidative stress and apoptosis in AT/RT. These complementary mechanisms of action drive extensive synergies between TAK-228 and Obatoclax slowing AT/RT cell growth and inducing apoptosis and cell death. Combination therapy activates the integrative stress response as determined by increased expression of phosphorylated EIF2α, ATF4, and CHOP, and disrupts the protective NOXA.MCL-1.BIM axis, forcing stressed cells to undergo apoptosis. Combination therapy is well tolerated in mice bearing orthotopic xenografts of AT/RT, slows tumor growth, and extends median overall survival. This novel combination therapy could be added to standard upfront therapies or used as a salvage therapy for relapsed disease to improve outcomes in AT/RT.