Kim M. Elseth

Synthesis and Biological Analysis of Thiotetra(ethylene glycol) monomethyl Ether-Functionalized Porphyrazines: Cellular Uptake and Toxicity Studies

The porphyrazines (pzs), a class of porphyrin analogues, are being investigated for their potential use as tumor imaging/therapeutic agents. We here examine six peripherally-functionalized M[pz(AnB4-n)] pzs with n=4, 3, or 2 (in a trans conformation) and M = H2 or Zn, where A is an [S((CH2)2O)4Me]2 unit and B is a fused β,β′-diisopropyloxybenzo group. Cell viability/proliferation assays and fluorescence microscopy were carried out in both tumor and normal cells. Dark toxicity studies disclosed that four of the compounds exhibited toxicity in both normal and tumor cells; one was nontoxic in both normal and tumor cells, and one was selectively toxic to normal cells. Additionally, three of the pzs showed enhanced photo-induced toxicity with these effects in some cases being observed at treatment concentrations of up to ten-fold lower than that needed for a response in Photofrin. All six compounds were preferentially absorbed by tumor cells, suggesting that they have potential as in vitro diagnostic agents and as aids in the isolation and purification of aberrant cells from pathological specimens. In particular, two promising diagnostic candidates have been identified as part of this work.

Part III. Molecular changes induced by high nitric oxide adaptation in human breast cancer cell line BT-20 (BT-20-HNO): a switch from aerobic to anaerobic metabolism

Nutrient deprivation and reactive oxygen species (ROS) play an important role in breast cancer mitochondrial adaptation. Adaptations to these conditions allow cells to survive in the stressful microenvironment of the tumor bed. This study is directed at defining the consequences of High Nitric Oxide (HNO) exposure to mitochondria in human breast cancer cells. The breast cancer cell line BT-20 (parent) was adapted to HNO as previously reported, resulting in the BT-20-HNO cell line. Both cell lines were analyzed by a variety of methods including MTT, LDH leakage assay, DNA sequencing, and Western blot analysis. The LDH assay and the gene chip data showed that BT-20-HNO was more prone to use the glycolytic pathway than the parent cell line. The BT-20-HNO cells were also more resistant to the apoptotic inducing agent salinomycin, which suggests that p53 may be mutated in these cells. Polymerase chain reaction (PCR) followed by DNA sequencing of the p53 gene showed that it was, in fact, mutated at the DNA-binding site (L194F). Western blot analysis showed that p53 was significantly upregulated in these cells. These results suggest that free radicals, such as nitric oxide (NO), pressure human breast tumor cells to acquire an aggressive phenotype and resistance to apoptosis. These data collectively provide a mechanism by which the dysregulation of ROS in the mitochondria of breast cancer cells can result in DNA damage.

Gastroesophageal Reflux Disease (GERD): Is There More to the Story?

Gastroesophageal reflux disease (GERD) affects both men and women worldwide, with the most common symptom of GERD being frequent heartburn. If left untreated, more serious diseases including esophagitis and/or esophageal cancer may result. GERD has been commonly held to be the result of gastric acid refluxing into the esophagus. Recent work, however, has shown that there are acid‐producing cells in the upper aerodigestive tract. In addition, acid‐producing bacteria located within the upper gastrointestinal tract and oral cavity may also be a contributing factor in the onset of GERD. Proton pump inhibitors (PPIs) are commonly prescribed for treating GERD; these drugs are designed to stop the production of gastric acid by shutting down the H+/K+‐ATPase enzyme located in parietal cells. PPI treatment is systemic and therefore significantly different than traditional antacids. Although a popular treatment choice, PPIs exhibit substantial interpatient variability and commonly fail to provide a complete cure to the disease. Recent studies have shown that H+/K+‐ATPases are expressed in tissues outside the stomach, and the effects of PPIs in these nongastric tissues have not been fully explored. Likewise, acid‐producing bacteria containing proton pumps are present in both the oral cavity and esophagus, and PPI use may also adversely affect these bacteria. The use of PPI therapy is further complicated by the two philosophical approaches to treating this disease: to treat only symptoms or to treat continuously. The latter approach frequently results in unwanted side effects which may be due to the PPIs acting on nongastric tissues or the microbes which colonize the upper aerodigestive tract.

Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines

There is a lack of understanding of the casual mechanisms behind the observation that some breast adenocarcinomas have identical morphology and comparatively different cellular growth behavior. This is exemplified by a differential response to radiation, chemotherapy, and other biological intervention therapies. Elevated concentrations of the free radical nitric oxide (NO), coupled with the up-regulated enzyme nitric oxide synthase (NOS) which produces NO, are activities which impact tumor growth. Previously, we adapted four human breast cancer cell lines: BT-20, Hs578T, T-47D, and MCF-7 to elevated concentrations of nitric oxide (or high NO [HNO]). This was accomplished by exposing the cell lines to increasing levels of an NO donor over time. Significantly, the HNO cell lines grew faster than did each respective (“PARENT”) cell line even in the absence of NO donor-supplemented media. This was evident despite each “parent” being morphologically equivalent to the HNO adapted cell line. Herein, we characterize the HNO cells and their biological attributes against those of the parent cells. Pairs of HNO/parent cell lines were then analyzed using a number of key cellular activity criteria including: cell cycle distribution, DNA ploidy, response to DNA damage, UV radiation response, X-ray radiation response, and the expression of significant cellular enzymes. Other key enzyme activities studied were NOS, p53, and glutathione S-transferase-pi (GST-pi) expression. HNO cells were typified by a far more aggressive pattern of growth and resistance to various treatments than the corresponding parent cells. This was evidenced by a higher S-phase percentage, variable radioresistance, and up-regulated GST-pi and p53. Taken collectively, this data provides evidence that cancer cells subjected to HNO concentrations become resistant to free radicals such as NO via up-regulated cellular defense mechanisms, including p53 and GST-pi. The adaptation to NO may explain how tumor cells acquire a more aggressive tumor phenotype.

Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines

The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC); however, the role NO plays in tumor pathophysiology is still not well understood. We, herein, report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted (“parent”) cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate that the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis.

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated (“parent”) cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide

Previously, we demonstrated that A549, a human lung cancer cell line, could be adapted to the free radical nitric oxide (NO●). NO● is known to be over expressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serve as a useful model system to study the biology of NO●. To see if tumor cells can similarly be adapted to any free radical with the same outcome, herein we successfully adapted A549 cells to high levels of hydrogen peroxide (HHP). A549-HHP, the resulting cell line, was more resistant and grew better then the parent cell line, and showed the following characteristics: (1) resistance to hydrogen peroxide, (2) resistance to NO●, (3) growth with and without hydrogen peroxide, and (4) resistance to doxorubicin. Gene chip analysis was used to determine the global gene expression changes between A549-parent and A549-HHP and revealed significant changes in the expression of over 1,700 genes. This gene profile was markedly different from that obtained from the A549-HNO cell line. The mitochondrial DNA content of the A549-HHP line determined by quantitative PCR favored a change for a more anaerobic metabolic profile. Our findings suggest that any free radical can induce resistance to other free radicals; this is especially important given that radiation therapy and many chemotherapeutic agents exert their effect via free radicals. Utilizing this model system to better understand the role of free radicals in tumor biology will help to develop new therapeutic approaches to treat lung cancer.

Application of Immunohistochemical Staining to Detect Antigen Destruction as a Measure of Tissue Damage

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, “antigen destruction immunohistochemistry” (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Part I-mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways.

Our previous studies demonstrate that A549, a human lung adenocarcinoma line, could be adapted to the free radical nitric oxide (NO●). NO● has been shown to be overexpressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serves as a useful model system to study the role of NO● in tumor biology. It is well known that DNA damage response (DDR) is altered in cancer cells and NO● is known to cause DNA damage. Modulations in molecular mechanisms involved in DNA damage response in A549-HNO cells can provide better insights into the enhanced growth behavior of these cells. Thus, here, we carried out a series of time course experiments by treating A549 and A549-HNO cells with NO● donor and examining levels of proteins involved in the DDR pathway. We observed induced expression of key components of DDR pathway in A549-HNO cells. The HNO cells showed sustained expression of key proteins involved in both nonhomologous end joining (NHEJ) and homologous recombination pathways, whereas parent cells only expressed low levels of NHEJ pathway proteins. Further with prolonged NO● exposure, ATR, Chk1, and p53 were activated and upregulated in HNO cells. Activation of p53 results in inhibition of apoptosis through induced Mcl1 expression. It also leads to cell cycle modulation. Interestingly, several reports show that cancer stem cells have enhanced expression of proteins involved in DNA damage response and also activated an antiapoptotic response. Our results here suggest that our HNO adapted A549 cells have increased activation of DNA damage response pathway proteins which can lead to better DNA repair function. Enhanced DDR leads to activation of antiapoptosis response and modulation in the cell cycle which may lead to better survival of these cells under harsh conditions. Thus, our present investigation further supports the hypothesis that HNO exposure leads to survival of these cells.

Part II—mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints

Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1–S and G2–M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1–S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher than those of the parent cells. This suggests an initial bypass of cell cycle checkpoints as p21CIP1 can inhibit the activity of all cyclin/Cdk complexes. p21CIP1 is also known to inhibit p53-induced apoptosis. This could be important during later phases of the cell cycle to allow time for repair of damaged DNA and thus better survival of HNO cells.