Evita G. Weagel

A novel method for predicting antioxidant activity based on amino acid structure.

Epidemiological studies show a positive correlation between oxidative stress and chronic disease development such as heart disease and cancer. While several antioxidant compounds with varying physical and chemical characteristics are able to reduce oxidative stress in biological systems, relatively few studies have been performed to examine the structural characteristics that produce potent antioxidants. We examined 20 essential and non-essential amino acids using the ORAC assay and used a simplest-case amino acid model to gather data to make predictions regarding the antioxidant activity of non-amino acid compounds; we also tested our findings on chalcone and nitrone data from the current literature. We observed that the sp(2)-hybridized carbons were the most consistent predictors of antioxidant activity in all groups. Valence electron to carbon ratio and length of conjugated double bond groups also emerged as important structural characteristics. Further testing may help to elucidate more accurate trends, as well as nonlinear relationships.

Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

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.

Biomarker analysis and clinical relevance of TK1 on the cell membrane of Burkitt’s lymphoma and acute lymphoblastic leukemia

TK1 is an enzyme involved in DNA synthesis and repair. TK1 is usually found elevated in cancer patients’ serum, which makes it a useful tumor proliferation biomarker that strongly correlates with cancer stage, metastatic capabilities, and recurrence risk. In this study, we show that TK1 is upregulated and localizes on the plasma membrane of Burkitt’s lymphoma, acute promyelocytic leukemia, T cell leukemia, and acute lymphoblastic leukemia (ALL). Using flow cytometry, we confirmed that TK1 localizes on the surface of Raji, HL60, and Jurkat cell lines and on ALL clinical samples. Using fluorescent microscopy, we found a strong association of TK1 with the plasma membrane in Raji, HL60, and Jurkat cell lines. These findings were also confirmed by scanning electron microscopy. Our study also shows that this phenomenon does not occur on normal resting or proliferating lymphocytes. In addition, we show that membrane TK1 is found in all oligomeric forms ranging from monomer to tetramer and exhibits enzymatic activity. These findings suggest TK1 as a possible target for immunotherapy with the potential to be utilized in the treatment of hematological cancers.

Pharmacologic immunosuppression of mononuclear phagocyte phagocytosis by caffeine

Caffeine is the most widely used neurostimulant in the world. There is considerable debate on its effect on immune cells as it has been shown to antagonize adenosine receptors (ARs), which mediate an anti‐inflammatory switch in activated immune cells. A second target is phosphodiesterase, where it acts as an inhibitor. If the primary effect of caffeine on mononuclear phagocytes were to antagonize ARs we would expect cells exposed to caffeine to have a prolonged proinflammatory response. The aim of this study was to investigate the effects and mechanism of action of caffeine in mononuclear phagocytes. Human mononuclear phagocytes were separated from whole blood and pretreated with protein kinase A inhibitor (PKA) and then exposed to micromolar physiological concentrations of caffeine. Phagocytosis and phagocytosis exhaustion were quantified using flow cytometry. Treatments were analyzed and compared to controls, using a beta regression controlling for factors of age, gender, caffeine intake, and exercise. We found that caffeine suppresses phagocytosis at micromolar physiological concentrations. This suppression was prevented when mononuclear phagocytes were pretreated with PKA inhibitor, suggesting that caffeines phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an anti‐inflammatory response. Additionally, these effects are altered by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in mononuclear phagocyte activation. These results demonstrate that caffeine may be acting as a phosphodiesterase inhibitor and suppressing phagocytosis in mononuclear phagocytes by promoting an anti‐inflammatory response.

Abstract 2149: Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620

Colorectal cancer is one of the most common cancers in the world, affecting nearly 1.2 million people in the United States alone. The aim of this study is to investigate the salvage pathway enzyme HPRT as a possible biomarker in two colorectal cancer cell lines: HT29 and SW620. HPRT is a transferase in the purine salvage pathway that functions primarily by catalyzing the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Because of its role in proliferation and cell cycle regulation, we hypothesized an increase in HPRT expression within cancer cells, which could potentially lead to presentation on the surface of the cell. HPRT surface localization was assessed utilizing confocal microscopy, flow cytometry, and scanning electron microscopy. These techniques allowed us to visualize HPRT on the plasma membrane and quantify expression. There was statistically significant expression of HPRT on the surface of both HT29 and SW620 cells with a 28% and 58% fluorescent shift in the population, respectively. Confocal microscopy images revealed overlap between cells stained with a membrane dye and anti-HPRT FITC antibody, which indicates a direct relationship between HPRT and the plasma membrane of SW620 cells. To visualize the location of HPRT on the plasma membrane, anti-HPRT antibodies were labelled with gold and protein presence was quantified using an electron microscope, which measured the gold elemental weight percentage of each sample. When exposed to anti-HPRT antibody, the gold weight percentage of the samples significantly increased with 12.4% gold in SW620 cells and 11.2% gold in HT29 cells, indicating significant HPRT surface presence. The observed localization of HPRT to the plasma membrane was variable between the two different cancer cell lines. SW620 cells, which are faster growing and more aggressive, had significantly higher HPRT presentation that HT29 cells. These data collectively suggest that HPRT may be a possible biomarker target for the identification and possible treatment of colorectal cancer cells. Citation Format: Evita G. Weagel, Michelle H. Townsend, Michael D. Anderson, Edwin J. Velazquez, K Scott Weber, Richard A. Robison, Kim L. O9Neill. Unusual expression of HPRT on the surface of the colorectal cancer cell lines HT29 and SW620 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149. doi:10.1158/1538-7445.AM2017-2149

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.

Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies

BackgroundLung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1’s potential as a tumor biomarker and immunotherapeutic target and clinical relevance.MethodsWe assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1’s clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients.ResultsWe found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1’s localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients.ConclusionsOur results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer.

Abstract 1949: Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma

The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt’s B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt’s Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt’s Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt’s lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt’s B-cell lymphoma pediatric patients. Citation Format: Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O9Neill. Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2017-1949

Abstract 1239: The expression of salvage pathway enzymes in non-small cell lung cancer cells

The aim of this study is to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in lung cancer cells to determine if they could serve as biomarkers for lung cancer diagnosis and potential treatment. In both men and women, lung cancer is the most lethal cancer in the world, and accounts for more than 30% of cancer-related deaths. We chose to evaluate the salvage pathway enzymes due to an established relationship between the serum biomarker Thymidine Kinase 1 (TK1) and lung cancer. Two non-small cell lung carcinoma cell lines were utilized for this analysis (NCI-H460 and A549) along with cancer tissue and healthy tissue from 27 lung squamous carcinoma patients. The surface localization of these enzymes was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry (IHC). Throughout our investigation, we found no significant expression of DCK or APRT on the surface of non-small cell lung cancer cells, but found a significant presence of HPRT on the plasma membrane of both NCI-H460 and A549 cells. The average population florescence of cells treated with HPRT antibodies increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison to controls. To ensure expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and EDAX. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (p-value 0.012) in comparison to isotype controls. Finally, the general upregulation of the protein was observed in patient tissue samples, with approximately 44% of lung cancer tissues showing significant HPRT upregulation when compared to healthy tissue samples. This differential upregulation shows an altered HPRT expression within some patients, which may help explain the presence of this presumed cytosolic enzyme on the surface of lung cancer cells. These results strongly indicate a unique relationship between cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of non-small cell lung cancers. Citation Format: Michelle H. Townsend, Evita G. Weagel, Michael D. Anderson, Edwin Velazquez, Richard A. Robison, Kim L. O9Neill. The expression of salvage pathway enzymes in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1239. doi:10.1158/1538-7445.AM2017-1239