Kim L. O’Neill

The role of oxidative stress in prostate cancer.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are not only byproducts of normal cellular metabolism, but also play important roles in cell signaling. However, when the levels of ROS and RNS increase, cells are exposed to oxidative stresses, which activate a variety of mechanisms to allow them to cope with these changes. Studies have shown that oxidative stress conditions play an important role in both the initiation and the progression of prostate cancer by regulating molecules such as DNA, enhancers, transcription factors, and cell cycle regulators. Other studies have shown that antioxidants, molecules that protect cells against oxidative stress, play a role in prevention of prostate cancer. This review summarizes the effects of oxidative stress on the development of prostate cancer and explores the potential of ROS regulators as preventatives for prostate cancer.

Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A 2 during early phase of apoptosis

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A(2). The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43 degrees C). The appearance of membrane susceptibility to secretory phospholipase A(2) was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A(2), live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA(2) was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA(2) is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.

Virion disruption by ozone-mediated reactive oxygen species

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Reversed-phase liquid chromatography of proteins and peptides using multimodal copolymer-encapsulated silica

Multimodal copolymer-encapsulated particles for liquid chromatography were prepared by bonding 1-octadecene and unsaturated carboxylic acids on silica particles (5 microm diameter, 300 A pores) for liquid chromatography of proteins. These multimodal copolymer-encapsulated particles can provide both hydrophobic and hydrogen bonding interactions with polar compounds. The chromatographic performance of these multimodal copolymer-encapsulated particles for peptide and protein separations was evaluated under reversed-phase conditions. Compared with typical C8-bonded silica, polymer-encapsulated particles were more stable in acidic mobile phases and provided better recoveries, especially for large proteins (Mr>0.5 x 10(6)). Totally hydrophobic polymer-encapsulated particles were found to produce broad peaks for proteins, and significant improvements were observed by introducing hydrophilic groups (-COOH) onto the polymer-encapsulated surface to form a multimodal phase. For the reversed-phase liquid chromatography of peptides and proteins, improved selectivity and increased solute retention were found using the multimodal polymer-encapsulated particles. More peaks were resolved for the separation of complex peptide mixtures such as protein digests using the multimodal polymer-encapsulated particles as compared to totally hydrophobic polymer-encapsulated particles.

Macrophage Polarization and Its Role in Cancer

The immune system plays an important role in the development of, and progression of cancer. Macrophages exhibit a variety of responses according to varying stimuli, and express different functions depending upon the microenvironment surrounding them. Macrophages can be pro-inflammatory (M1) or anti-inflammatory (M2). Research studies have shown that infiltration of macrophages can account for >50% of the tumor mass in some cancers, aid in metastasis by inducing angiogenesis, and signify a poor prognosis. Macrophages that migrate to the tumor site, remain there, and aid in angiogenesis and metastasis are termed tumor associated macrophages (TAMs) and are thought to express an M2 phenotype. This review will examine the polarization states of macrophages, their functions and role in cancer, their activation pathways and metabolism, and potential approaches to cancer immunotherapies using macrophages.

DNA damage caused by inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate, on Burkitts lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage at concentrations of ammonium nitrate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m(2), 400 J/m(2), 600 J/m(2), 850 J/m(2)), as measured by the comet assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 cells (p<0.001). Apoptosis was shown in Raji and HepG2 cells when exposed to high concentrations of ammonium nitrate (200 μg/ml and 400 μg/ml) for 1h in samples without ultraviolet exposure, as assessed by the comet assay. However, the level of apoptosis greatly diminished after ultraviolet exposure at these concentrations. Over a 24h period, at intervals of 1, 4, 8, 12, 18, and 24h, we also observed that ammonium nitrate decreased viability in Raji and HepG2 cell lines and inhibited cell growth. Ammonium sulfate-induced DNA damage was minimal in both cell lines, but there remained a significant difference (p<0.05) between the ultraviolet radiation treated and negative control samples. These results indicate that the inorganic particulate compound, ammonium nitrate, induced DNA strand breaks at all concentrations, and indications of apoptosis at high concentrations in Raji and HepG2 cells, with ultraviolet radiation preventing apoptosis at high concentrations. We hypothesize that ultraviolet radiation may inhibit an essential cellular mechanism, possibly involving p53, thereby explaining this phenomenon. Further studies are necessary to characterize the roles of apoptosis inhibition induced by DNA damage caused by inorganic particulate matter.

A review of HPRT and its emerging role in cancer

Hypoxanthine guanine phosphoribosyltransferase (HPRT) is a common salvage housekeeping gene with a historically important role in cancer as a mutational biomarker. As an established and well-known human reporter gene for the evaluation of mutational frequency corresponding to cancer development, HPRT is most commonly used to evaluate cancer risk within individuals and determine potential carcinogens. In addition to its use as a reporter gene, HPRT also has important functionality in the body in relation to purine regulation as demonstrated by Lesch–Nyhan patients whose lack of functional HPRT leads to significant purine overproduction and further neural complications. This regulatory role, in addition to an established connection between other salvage enzymes and cancer development, points to HPRT as an emerging influence in cancer. Recent work has shown that not only is the enzyme upregulated within malignant tumors, it also has significant surface localization within some cancer cells. With this is mind, HPRT has the potential to become a significant biomarker not only for the characterization of cancer, but also for its potential treatment.

Examination of Hypoxanthine Guanine Phosphoribosyltransferase as a biomarker for colorectal cancer patients

ABSTRACT The aim of this study is to investigate these enzymes as possible biomarkers in two colorectal cancer cell lines: HT29, SW480, SW620, and Colo205. With 1,168,929 individuals currently diagnosed with colorectal cancer in the United States, there remains a need to find biomarkers to improve diagnosis and expand treatment options for patients. Due to their role in proliferation and cell cycle regulation, we hypothesized an increase in salvage pathway enzyme (APRT, DCK, and HPRT) expression and possible presentation within colon cancer cells. Enzyme surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. General protein expression was evaluated utilizing immunohistochemistry and Western blot analysis. While we found no statistically significant presence of either APRT or DCK on the membranes of SW620, Colo205, and HT29 cells, but found significant expression of HPRT on the surface of HT29, SW480, and SW620 cells. The average population fluorescence increased by 28%, 58%, and 40% in HT29, SW620, and SW480 cells, respectively, when compared to isotype controls. Confocal microscopy images revealed direct overlap between SW620 cells stained with a membrane dye and anti-HPRT antibody, indicating co-localization on the plasma membrane. In addition, cells treated with gold labelled HPRT antibody experienced significant changes in gold weight percentage on both SW620 and HT29 cells when compared to isotype controls. When evaluating expression within normal tissue, there was insignificant levels of HPRT binding. These data collectively suggest that HPRT may be a possible biomarker target for the identification and treatment of colorectal cancer.

The expansion of targetable biomarkers for CAR T cell therapy

Biomarkers are an integral part of cancer management due to their use in risk assessment, screening, differential diagnosis, prognosis, prediction of response to treatment, and monitoring progress of disease. Recently, with the advent of Chimeric Antigen Receptor (CAR) T cell therapy, a new category of targetable biomarkers has emerged. These biomarkers are associated with the surface of malignant cells and serve as targets for directing cytotoxic T cells. The first biomarker target used for CAR T cell therapy was CD19, a B cell marker expressed highly on malignant B cells. With the success of CD19, the last decade has shown an explosion of new targetable biomarkers on a range of human malignancies. These surface targets have made it possible to provide directed, specific therapy that reduces healthy tissue destruction and preserves the patient’s immune system during treatment. As of May 2018, there are over 100 clinical trials underway that target over 25 different surface biomarkers in almost every human tissue. This expansion has led to not only promising results in terms of patient outcome, but has also led to an exponential growth in the investigation of new biomarkers that could potentially be utilized in CAR T cell therapy for treating patients. In this review, we discuss the biomarkers currently under investigation and point out several promising biomarkers in the preclinical stage of development that may be useful as targets.

TK1 Membrane Expression May Play a Role in the Invasion Potential of A549 Lung Cancer Cells

Thymidine kinase 1 (TK1) is a well-studied cancer biomarker. It is commonly found upregulated in the serum of cancer patients, and its levels correlate with stage and grade, disease progression, and prognosis. It has recently been reported that TK1 localizes on the plasma cell membrane of hematological and solid malignancies, and not on the membrane of normal healthy cells, and while on the membrane, TK1 has enzymatic activity. However, the function of TK1 on the surface membrane is not well understood. Here, we hypothesize that it may have a role in tumor invasion and migration. It has been shown that TK1 expression levels positively correlate with epithelia to mesenchymal transition (EMT) markers in patients with breast cancer as they progress from HER2+ to triple negative breast cancer. In this study, we silenced TK1 expression by siRNA and show that TK1’s membrane expression is significantly downregulated at 60 hours post transfection. Using a Matrigel-based quantitative invasion assay, we measured cell invasion potential in cells either expressing or lacking TK1 on their membrane and found that cells that lack TK1 on their membrane exhibit decreased invasion potential. These results suggest that TK1’s presence on the membrane may play a role in invasion and cell migration in cancer.