Browsing by Subject "Reactive oxygen species"

The ability of photocatalytic nanoparticles (NPs) to produce reactive oxygen species (ROS) has inspired research into several new applications and technologies, including water purification, contaminant remediation, and self-cleaning surface coatings. As a result, NPs continue to be incorporated into a wide variety of increasingly complex products. With the increased use of NPs and nano-enabled products and their subsequent disposal, NPs will make their way into the environment. Currently, many unanswered questions remain concerning how changes to the NP surface chemistry that occur in natural waters will impact reactivity. This work seeks to investigate potential influences on photoreactivity – specifically the impact of functionalization, the influence of anions, and interactions with biological objects - so that ROS generation in natural aquatic environments may be better understood.

To this aim, titanium dioxide nanoparticles (TiO2) and fullerene nanoparticles (FNPs) were studied in terms of their reactive endpoints: ROS generation measured through the use of fluorescent or spectroscopic probe compounds, virus and bacterial inactivation, and contaminant degradation. Physical characterization of NPs included light scattering, electron microscopy and electrophoretic mobility. These systematic investigations into the effect of functionalization, sorption, and aggregation on NP aggregate structure, size, and reactivity improve our understanding of trends that impact nanoparticle reactivity.

Engineered functionalization of FNPs was shown to impact NP aggregation, ROS generation, and viral affinity. Fullerene cage derivatization can lead to a greater affinity for the aqueous phase, smaller mean aggregate size, and a more open aggregate structure, favoring greater rates of ROS production. At the same time however, fullerene derivatization also decreases the 1O2 quantum yield and may either increase or decrease the affinity for a biological surface. These results suggest that the biological impact of fullerenes will be influenced by changes in the type of surface functionalization and extent of cage derivatization, potentially increasing the ROS generation rate and facilitating closer association with biological targets.

Investigations into anion sorption onto the surface of TiO2 indicate that reactivity will be strongly influenced by the waters they are introduced into. The type and concentration of anion impacted both aggregate state and reactivity to varying degrees. Specific interactions due to inner sphere ligand exchange with phosphate and carbonate have been shown to stabilize NPs. As a result, waters containing chloride or nitrate may have little impact on inherent reactivity but will reduce NP transport via aggregation, while waters containing even low levels of phosphate and carbonate may decrease “acute” reactivity but stabilize NPs such that their lifetime in the water column is increased.

Finally, ROS delivery in a multicomponent system was studied under the paradigm of pesticide degradation. The presence of bacteria or chlorpyrifos in solution significantly decreased bulk ROS measurements, with almost no OH detected when both were present. However, the presence of bacteria had no observable impact on the rate of chlorpyrifos degradation, nor chlorpyrifos on bacterial inactivation. These results imply that investigating reactivity in simplified systems may significantly over or underestimate photocatalytic efficiency in realistic environments, depending on the surface affinity of a given target.

This dissertation demonstrates that the reactivity of a system is largely determined by NP surface chemistry. Altering the NP surface, either intentionally or incidentally, produces significant changes in reactivity and aggregate characteristics. Additionally, the photocatalytic impact of the ROS generated by a NP depends on the characteristics of potential targets as well as on the characteristics of the NP itself. These are complicating factors, and the myriad potential exposure conditions, endpoints, and environmental systems to be considered for even a single NP highlight the need for functional assays that employ environmentally relevant conditions if risk assessments for engineered NPs are to be made in a timely fashion so as not to be outpaced by, or impede, technological advances.

Apoptotic dysregulation is a hallmark of cancer cells. The inability of cancer cells to undergo apoptosis may lead to therapeutic resistance. Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression and ErbB2 activation. ErbB-targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. Little is known about the molecular mechanisms of therapeutic resistance in IBC cells. We hypothesized that apoptotic dysregulation leads to therapeutic resistance of IBC cells to therapeutic agents, including ErbB-targeting agents. To determine whether apoptotic dysregulation and changes in anti-apoptotic proteins leads to resistance of IBC cells to therapeutic agents, we performed a variety of in vitro-based studies using agents that are used in the clinic to treat IBC patients. The sensitivity of both ErbB2 overexpressing and ErbB1 activated IBC cells to various therapeutic agents was evaluated using various cell death and apoptosis assays, and anti-apoptotic protein expression post-treatment was determined using western blot analysis. The overarching theme observed was that x-linked inhibitor of apoptosis protein (XIAP) expression inversely correlated with sensitivity of cells to therapeutic agents with various mechanisms of action, including TNF-related apoptosis inducing ligand (TRAIL), doxorubicin, cisplatin, paclitaxel, and two ErbB-targeting agents: trastuzumab and a lapatinib-analog (GW583340). Moreover, there was a specific and marked overexpression of XIAP in cells with de novo resistance to trastuzumab and with acquired resistance to GW583340. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. Stable XIAP overexpression using a lentiviral system reversed sensitivity to therapeutic agents (TRAIL and GW583340) in parental IBC cells. Moreover, XIAP downregulation in cells resistant to therapeutic agents (TRAIL, trastuzumab, and GW583340) resulted in decreased viability and increased apoptosis, demonstrating that XIAP is required for survival of cells with resistance to these agents. A novel mechanism of GW583340 oxidative stress-induced mediated apoptosis was identified, and resistant cells had increased antioxidant expression and capability. Interesting, inhibition of XIAP function overcame this increase in antioxidant potential, demonstrating a new function for XIAP in oxidative stress-induced apoptosis. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to therapeutic agents.

Imbalance between oxygen consumption and supply often makes tumors hypoxic (Bristow and Hill 2008). Tumor hypoxia is significantly correlated with aggressive tumor growth, ineffective response to radiation and chemotherapy, and as a result, poor patient prognosis. Hyperthermia (HT) is a strong adjuvant treatment to overcome these challenges of tumor hypoxia because it causes tumor reoxygenation at temperatures lower than 43ºC (Song, Park, and Griffin 2001). However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we determine that 1 hour HT activates hypoxia-inducible factor-1 (HIF-1) and its downstream targets, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) in tumors. Consistent with HIF-1 activation and upregulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption rates. As a result, tumor hypoxia is reduced after HT suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Since HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. Mechanistically, we demonstrate that NADPH oxidase-mediated reactive oxygen species (ROS) production upregulates HIF-1 after HT. Further, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 (NOX1) through the ERK pathway.

A major research effort at Duke focuses on combinations of HT and doxorubicin in the treatment of locally advanced breast and other cancers. Thus, we investigated whether there are HIF-1 responses to doxorubicin treatment. We reveal that doxorubicin also activates HIF-1. Unlike HT, doxorubicin-induced HIF-1 promotes persistent tumor angiogenesis. We also reveal that the signal transducer and activator of transcription 1 (STAT1)/inducible nitric oxide synthase (iNOS) pathway causes HIF-1α accumulation in an oxygen-independent manner. We show that activated STAT1 upregulates iNOS expression and promotes nitric oxide (NO) production in tumor cells resulting in HIF-1α stabilization. We further determine that both iNOS inhibitor, 1400W and STAT1 inhibitor, epigallocatechin-3-gallate (EGCG) significantly decrease intracellular NO production and suppress doxorubicin-induced normoxic HIF-1α accumulation.

HIF-1 is often considered a promising therapeutic target because of its role in tumor progression (Semenza 2003) and therapy resistance (Moeller et al. 2004). However, our findings suggest that HIF-1 plays a pleiotropic role in response to HT and chemotherapy. Therefore, to preferentially take advantage of HT-induced HIF-1 activation and also to suppress its deleterious effects induced by chemotherapy or as we have previously reported, by radiation (Moeller et al. 2004), HIF-1 inhibition needs to be carefully regulated in a time-sensitive manner to achieve optimal therapeutic effects.