A quantitative analysis of chemotherapy-induced reactive oxidative species using genetically encoded sensors and generators
Author(s)Huang, Beijing Kara
Massachusetts Institute of Technology. Department of Biological Engineering.
Hadley D. Sikes.
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Recent advances in chemotherapeutic development have targeted vital mechanisms that ensure survival of cancer cells; these include the ability to evade immune surveillance, undergo metabolic adaptations and form a defense mechanism against oxidative stress. Cancer cells often possess higher endogenous levels of reactive oxidative species (ROS) compared to normal cells due to the cumulative effects from genomic instability, inflammation and oncogene activation, and they become increasingly reliant on the cell antioxidant network to prevent this elevated oxidative stress from becoming toxic. Thus, chemotherapeutics that inhibit the antioxidant network and thereby elevating ROS are thought to be promising candidates that can selectively eliminate tumor cells. Despite the promises of these molecules, chemotherapeutics modulating ROS levels have mostly fallen short of their projected impact. We believe that a thorough understanding of the quantity, location and duration of ROS generation needed to cause tumor cell toxicity, will be important for understanding the mechanism of current successful chemotherapeutics and designing future ROS-based drug candidates. In this thesis, we explored the use of genetically encoded sensors and generators of ROS, H₂O₂ in particular, to answer these important redox biology questions. We began by developing a deeper understanding of how to use these protein-based peroxide sensors in a quantitative manner. We created a technique quantifying intracellular peroxide levels by converting the fluorescence signal outputs from these sensors into more meaningful intracellular concentrations. This was accomplished via a combination of kinetic modeling, biochemical measurements and image analysis techniques. We also explored the cell to cell heterogeneity in sensor response to H₂O₂ stimulation, and found that the intracellular expression level of the sensor is correlated with the ratio-metric response of the probe. Further kinetic modeling analysis showed that the slow recycling step of activated sensor was responsible for the correlation. In the second part of the thesis, we used these sensors in combination with enzymatic generators that can produce H202 endogenously in a kinetically controlled manner. These tools allowed us to quantitatively determine that there are two toxicity thresholds, a total accumulation of H₂O₂ and intracellular concentration, that are needed for H₂O₂ -mediated cell death. We also applied these tools to investigate the mechanism of two ROS-based chemotherapeutics, phenethyl isocyanate (PEITC) and piperlongumine. We found that depletion of the glutathione antioxidant by these drugs was unimportant to the toxicity mechanism, and the amount of oxidative stress generated by these compounds was not enough to induce significant toxicity by itself. The final part of the thesis involves technology development for a next generation enzymatic ROS generator. We explored the use of P450-BM3, an enzyme that can generate superoxide and hydrogen peroxide through a reaction that requires only NADPH and oxygen. While this reaction in the wild type protein is slow, it can be engineered to have much higher catalytic rates. We demonstrated through various protein engineering approaches that we could create P450-BM3 proteins with enhanced generation of H₂O₂. We also were able to express correctly folded, active enzymes inside mammalian cells that utilize intracellular NADPH and oxygen to produce H₂O₂.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering.
Massachusetts Institute of Technology