Semester

Fall

Date of Graduation

2004

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Jeannine S. Strobl.

Abstract

An impeding challenge to breast cancer drug therapies is the availability of more effective and less toxic chemotherapeutic agents that do not relay harm to neighboring normal breast cells and tissues. Preliminary studies showed that the quinoline antimalarials, chloroquine (CQ and hydroxychloroquine (HCQ), inhibit proliferation and induce differentiation in breast cancer cell lines without toxicity to normal MCF-10A breast cells. Hence, the goal of these studies was to explore: (1) whether a drug combination modulating epigenetic events would sensitize breast cancer cells to the antitumor activity of CQ or HCQ, (2) and if so, which would be the most promising combination of agents for the generation of safer and less toxic chemotherapeutic agents for the prevention and treatment of advanced breast cancer. We hypothesized that the use of a drug combination modulating epigenetic events would lower the concentration of CQ or HCQ needed to produce the differentiation response. Hence, the quinoline agents were used in combination with the demethylating agent, 5-Aza-2'-deoxycytidine (5-Aza-dC or Aza), or with the differentiating agent, all-trans-Retinoic acid (ATRA) in order to lower the threshold for chemotherapy-induced cell death in breast cancer cells. Cell survival, cellular differentiation, and histone H3/H4 acetylation status were measured to show that combination of 5-Aza-dC or ATRA with the quinolines sensitized breast cancer cells to enhanced growth inhibition, differentiation, and histone hyperacetylation compared to cells treated with either CQ or HCQ alone. Furthermore, ATRA was identified as a direct HDAC inhibitor and upon combination treatment with HCQ had the most significant inhibitory effect on tumor cell clonogenic survival in both estrogen receptor (ER+) and (ER-) breast cancers. Thus, ATRA combined with HCQ served as the most promising combination of chemotherapeutic agents and were subsequently assessed using a new and highly sensitive assay for histone acetylation by mass spectrometry to generate a profile on lysine modification (acetylation/deacetylation). The combination of ATRA with HCQ increased acetylation of N-terminal lysine residues on histones H3/H4 as well as caused direct HAT activation. The overall data suggest that HCQ might contribute to breast cancer cell differentiation by modulating HAT activity; whereas, ATRA sensitizes breast cancer cells to the antitumor activity of HCQ via regulation of both HAT and HDAC enzymatic activities.

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