Date on Master's Thesis/Doctoral Dissertation

8-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

States, J. Christopher

Author's Keywords

Curcumin; Cell cycle progression; P53; BPDE; DNA repair; GSH

Subject

Curcumin--Therapeutic use; Lungs--Cancer

Abstract

Long-term exposure to harmful carcinogens like benzo[a]pyrene (BaP) are linked to lung cancer. Benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogen produced by BaP bioactivation, is believed to play an important role in lung carcinogenesis. The tumor suppressor protein is p53 signals cell cycle arrest, DNA repair, and apoptosis in response to BPDE-induced damage. However, physiological exposure to BPDE fails to activate p53. Although the bioactive principle of turmeric spice, curcumin, shows promising effects against BPDE-induced carcinogenesis, its exact mode of action remains unclear. Therefore the aim of this dissertation was to investigate whether curcumin prevents BPDE-induced DNA damage by regulating p53-mediated cellular responses in A549/LXSN (p53+) and A549/E6 (p53-) cells. I hypothesize that curcumin may reduce BPDE-induced DNA damage by lowering the threshold of p53 activation, thereby inducing p53-mediated mechanisms. Curcumin pretreatment reduced BPDEDNA adducts in a p53-dependent manner. However, p53-regulated proteins XPC and DDB2 did not change with curcumin pretreatment and p53-expression. Curcumin pretreatment did not change the overall repair rate in p53+ cells. Curcumin pretreatment, p53-expression, and BPDE exposure did not significantly change glutathione-S-transferase (GST) levels or activity as well as glutathione (GSH) levels. Curcumin increased levels of p53, phosphorylated p53 at Ser15 (P-p53S15), and CDKN1A, but decreased phosphorylated retinoblastoma at Ser807/811 (P-pRbS807/811) in p53+ cells with lower BPDE exposure. Curcumin and BPDE increased and decreased PpRbS807/ 811, respectively, in p53- cells. BPDE induced S phase arrest in both cell lines. P53+ curcumin pretreated cells escaped S phase arrest earlier with 300 nM BPDE in contrast to BPDE treatment alone. Yet, curcumin did not change S phase arrest in p53+ cells treated with 50 or 100 nM BPDE. P53- cells did not escape S phase arrest 24 h after low (50 and 100 nM) BPDE exposures; p53- curcumin pretreated cells entered S phase arrest earlier. Phosphorylated CHK1 Ser345 (pCHKS345) levels, an indicator of S phase arrest, was higher in p53- cells than p53+ cells; curcumin increased pCHK1 Ser345. Curcumin also induced apoptosis earlier in p53- cells exposed to 300nM BPDE. Therefore, this dissertation shows that curcumin prevents BPDE-induced DNA damage by activating a host of mechanisms independent of p53 expression.

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