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.
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 upregulated 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
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.Keywords Breast cancer . Nitric oxide . p53 . Reactive oxygen species (ROS) . Glycolysis and apoptosis . Aerobic and anaerobic metabolisms
During the late 19th century, a number of dye types were used in early microscopic investigations of simple plant and tissue materials (Cook 1997). These included (1) hematoxylin and eosin (H&E) used separately, (2) picric acid and carmine used concurrently, and, finally, (3) the combination of H&E (Cook 1997;Gill 2010). The H&E staining method evolved rapidly into a traditional staining standard used for morphological study within histology and cytology settings throughout the world (Cook 1997;Shi et al. 2011;Gill 2010). This standard staining technique is well known within the histopathology and cytopathology communities. The hematoxylin stains nuclei, nucleic acids, and other cellular components (such as keratohyalin granules) blue in color, while eosin normally stains both extracellular and cytoplasmic proteins red. As such, H&E is particularly useful in identifying morphological changes valuable in the diagnosis of cancer and other disease states. There are many H&E protocols available, with selection of the most appropriate being subject to the needs and setting of the investigation (Sullivan-Brown et al. 2011 AbstractElectrocautery and directed energy devices (DEDs)
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