Proteins as biomarkers for tobacco-induced lung cancer development and chemoprevention
Open Access
- Author:
- Bortner Jr, James
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 20, 2011
- Committee Members:
- Dr Karam El Bayoumy, Dissertation Advisor/Co-Advisor
Karam E El Bayoumy, Committee Chair/Co-Chair
Arunangshu Das, Committee Member
Gail Lynn Matters, Committee Member
David Spencer Phelps, Committee Member
John Peter Richie Jr., Committee Member
Cara Lynne Schengrund, Committee Member - Keywords:
- biomarkers
proteomics
2D-DIGE
iTRAQ
NNK
mouse model
cigarette smoking
plasma
NSCLC
adenocarcinoma
chemoprevention
selenium - Abstract:
- Background: As discussed in Chapter 1, lung cancer continues to be the most common cancer diagnosed in the world and is the leading cause of cancer-related death in both men and women in the United States. The non-small cell lung cancer (NSCLC) subtype adenocarcinoma is the leading histological type clinically diagnosed in the United States, accounting for over 30% of all cases. The primary risk factor associated with lung cancer is related to cigarette smoking; approximately 87% of lung cancer deaths in the United States are attributed to cigarette smoking. Existing approaches using conventional treatment strategies, including surgery, radiation therapy, and chemotherapy, have generated minimally significant results for lung cancer survival; 5-year survival for all stages combined is 16%. Furthermore, the disease is often diagnosed at advanced stages of development, beyond the benefit of current treatment. Therefore, alternative approaches in the management of lung cancer, such as early detection and prevention, are urgently needed. In the present investigation we embark upon two strategies with promising clinical implications: identification of (informative) molecular biomarkers related to the development of tobacco-induced lung cancer and the discovery of novel chemopreventive agents. Hypotheses: Current clinical protein biomarkers for detection of lung cancer lack sensitivity and specificity. The proteomics field can contribute greatly to the understanding of mechanisms in cancer progression and treatment response. Therefore, in Chapter 2 we hypothesized that by using proteomic approaches in a well-defined preclinical mouse lung tumorigenesis model induced using the tobacco carcinogen NNK, protein biomarkers could be obtained to provide an accurate view of molecular alterations during lung cancer development. Such biomarkers can provide utility for early detection, but also in the validation of chemoprevention strategies. Smokers are at risk for a variety of health concerns including lung cancer. Non-invasive biological fluids such as blood plasma contains molecular profiles related to current health status. Therefore, in Chapter 3 taking advantage of proteomic techniques and proteins identified in Chapter 2, we hypothesized that dynamic changes in protein expression profiles in the blood plasma of smokers characterized by a proteomics approach would assist in identifying early molecular changes related to tobacco-induced diseases such as lung cancer. Selenium-containing compounds are promising chemopreventive agents against lung cancer development. Developments of agents that can target molecular pathways that are critical in lung carcinogenesis, such as nitric oxide synthase (iNOS) and nitric oxide production, known to be involved in the promotion/progression phases of lung carcinogenesis, are needed and may be beneficial in chemoprevention of lung cancer in both smokers and former smokers. Therefore, in Chapter 4 we hypothesized that substitution of sulfur for selenium in an inducible nitric oxide synthase inhibitor, S,S¡¦-(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea (PBIT), would enhance its chemopreventive activity. Methods: (Chapter 2) The proteomic techniques two-dimensional differential in-gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantitation (iTRAQ) were employed to achieve a global protein expression profile of the lung during the progression of disease development in the NNK-induced lung tumorigenesis A/J mouse model. Furthermore, the synthetic organoselenium chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was administered to validate potential protein biomarkers. (Chapter 3) In order to gain an understanding of some of the dynamic protein changes in human plasma as a result of chronic exposure to mainstream tobacco smoke, we used iTRAQ and immunoblot analyses in blood plasma samples derived from healthy male smokers and non-smokers. (Chapter 4) To compare the efficacy of the novel selenium analog of PBIT (Se-PBIT) with PBIT and p-XSC, various in vitro molecular assays were performed in NSCLC cells including detection of nitric oxide production, cellular proliferation and apoptosis, cell cycle distribution, and protein expression related to important cellular activities. Results: (Chapter 2) We discovered proteins, including the 14-3-3 protein isoforms (ƒá, ƒÕ, £m, and ƒê), annexin A5, Clara cell 10 kDa protein (CC10), high mobility group box 1, and carbonyl reductase 2 that were differentially expressed in the lungs of mice treated with the tobacco carcinogen NNK versus vehicle (control)-treated mice during the progression of adenocarcinoma development; some of these changes were further modulated by p-XSC. These proteins are involved in a variety of biological functions that are critical in lung carcinogenesis, as well as in its prevention. (Chapter 3) We identified differentially expressed plasma proteins in healthy chronic cigarette smokers compared to healthy non-smokers. Several of the proteins identified, including ITI-HC3 and VDBP, associated with immunity and inflammatory responses, and 14-3-3 £m and £a, identified in Chapter 2, have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. (Chapter 4) We found Se-PBIT to be superior to both PBIT and p-XSC as an inducer of apoptosis and inhibitor of cell growth in NSCLC cells, as determined by analysis of its effects on important molecular targets involved in cell growth inhibition, induction of apoptosis, and cell cycle regulation. Conclusions: (Chapter 2) Based on our results, we were able to demonstrate the utility of a well-defined animal model in developing candidate protein biomarkers for lung cancer. Such biomarkers may be useful in early detection of the disease, as well as in the efficacy of chemopreventive agents. (Chapter 3) Our clinical pilot study results demonstrated for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome and these changes may be indicative of future health concerns. (Chapter 4) We demonstrated that selenium in the form of Se-PBIT is a promising candidate for chemoprevention of NSCLC and appears superior to PBIT and p-XSC.