Semester

Spring

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Yon Rojanasakul.

Abstract

Cr(VI) compounds are redox cycling carcinogens that induce apoptosis as a primary mode of cell death. Since defects in apoptosis regulatory mechanisms are considered to be indispensable foundation of carcinogenesis it is critical to understand the molecular effectors involved. Cellular energy powerhouses, the mitochondria, house the effectors involved in signaling cascades comprising commitment to apoptosis and are reported to be dysregulated in variety of tumors. The objective of this study was to characterize the key signaling effectors in Cr(VI)-induced apoptosis and determine the relationship between those effectors and mode of cellular energy production in cancerous vs. non-cancerous cells. Our data show that Cr(VI) induced mitochondrial ROS dependent apoptosis in human lung cancer (H460) and non-cancerous (BEAS-2B) cells. The intensity of mitochondrial ROS production and consequently apoptosis was highly dependent on the energy substrate available to the cells. Forcing of oxidative phosphorylation-only energy metabolism abrogated apoptosis resistance in cancerous H460 cells vs. non-cancerous BEAS-2B cells. Collectively, these findings present a mechanistic advancement in the understanding of molecular effectors underlying the Warburg's "mitochondrial dysregulation" hypothesis and put mitochondrial ROS as key mediators causal agents of the commitment phase of programmed cell death in a model of Cr(VI)-induced toxicity. In addition, long-term exposure to Cr(VI) leads to malignant transformation of human lung epithelial BEAS-2B cells, as indicated by their increased cell migration, invasion, proliferation, and colony formation activities. Cr(VI)-transformed cells induced tumorigenesis in nude mice comparable to that of well established human lung cancer H460 cells, whereas passage-matched control BEAS-2B cells showed no tumor development. To provide a mechanistic insight to the tumorigenic process, stable knockdown mutants of Cr(VI)-transformed cells and H460 cells exhibiting downregulated Bcl-2 phenotype were generated and evaluated for tumor associated properties in vitro and in vivo. The Bcl-2 knockdown mutants showed a substantial decrease in the rates of colony formation, invasion, migration and proliferation, as well as tumor formation in mice, in comparison to vector controls. These results indicate a novel role of Bcl-2, beyond apoptosis regulation, to be a key regulator of malignant transformation and tumorigenesis in vivo. The model described here may provide utility to other xeno-carcinogenesis studies, including those of heavy metals which currently lack effective in vivo tumor development experimental means.

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