Lung cancer, the leading cause of cancer-related deaths in America, is most commonly caused by long-term exposure to tobacco smoke carcinogens such as benzo[a]pyrene (BaP), which is metabolized into the ultimate metabolite benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE). Despite improvements in the treatment and management of lung cancer, these methods fail to reduce the overall mortality rate of this disease. While dietary components such as curcumin (turmic spice) show promising effects against BPDEinduced carinogenesis, its exact mode of action is not fully understood. The objective of this project is to elucidate whether pretreatment with curcumin inhibits BPDE-induced DMA damage by activating p53. We hypothesize that curcumin may reduce the mutagenic activity of low dose BPDE by lowering the threshold of p53 activation, thereby inducing DNA repair and cell cycle arrest at lower BPDE exposures.
In Specific aim 1 the expression of p53 and p53-regulated proteins as well as cell cycle progression will be monitored by Western blot and flow cytometric analysis, respectively, to determine whether curcumin influences cell cycle arrest in response to BPDE exposure. Because global genomic repair (GGR) removes most BPDE-DNA adducts in a p53-dependent manner, Specific aim 2 will use immunoslot blot, [32P]-postlabeling, and UvrABC mediated gene and strand-specific southern blot assays to analyze whether curcumin affects p53 activation and thus the relative extent of BPDE-DNA adducts formed and removed in genomic DNA.
In Specific aim 3 Northern and Western blot assays will be used to investigate the role of curcumin in the induction of damage recognition proteins of the GGR pathway (XPC, DDB2), which are p53-dependent. Furthermore it is believed that conjugation of glutathione (GSH) to BPDE by glutathione-S-transferase (GST) interacts with the expression of p53 to inactivate BPDE.
In Specific aim 4, Western blot and GST colorimetric assays will be used to monitor the expression of proteins involved in the GSH detoxification system as well as the overall activity of GST, respectively.
This aim will provide a better understanding of potential alternative routes that may inactivate BPDE and how curcumin may influence this activity. By investigating the effects of curcumin on p53-mediated responses to DNA damage, a better understanding of the potential role of curcumin in preventing tobacco carcinogen-induced mutagenesis and carcinogenesis will be gained. In addition, the results from this project will benefit the public by providing easy and inexpensive alternative methods in preventing initial and continuing signs of lung cancer.
