Regulation of Anti-Proliferation in Melanoma and Breast Cancer Cells by Small Molecule Phytochemicals and Their Derivatives

Abstract/nRegulation of Anti-Proliferation in Melanoma and Breast Cancer Cells by Small Molecule Phytochemicals and Their Derivatives/nBy/nKevin Michael Poindexter/nDoctor of Philosophy in Endocrinology/nUniversity of California, Berkeley/nProfessor Gary L. Firestone, Chair/nMelanoma and breast cancer represent a significant portion of overall cancer burden, and many strategies have been developed to control their growth. Many of these current options have serious adverse effects and sometimes lead to therapy resistance which can complicate future treatment. Because of this, it is critical to investigate new classes and combinations of therapeutics that can overcome these drawbacks and retain effectiveness in multiple phenotypes. This thesis details the mechanism in which the combination of Indol-3-Carbinol (I3C) and Acetylsalicylic Acid (aspirin) act synergistically to slow the growth of human melanoma cells. In addition, this thesis demonstrates that the phytochemical Artemisinin and its derivative, Artesunate are able to inhibit the growth of luminal A estrogen receptor positive breast cancer cells. Finally, we utilize I3C derivatives to analyze the growth effects of different chemical functional groups in both melanoma and breast cancer cells. The combination of I3C and aspirin was able to synergistically control melanoma cell proliferation through an induction of G1 cell cycle arrest. Cell cycle arrest was due likely to a similar downregulation of Cyclin Dependent Kinase 2 (CDK2) levels which in turn is under the transcriptional control of the Microphthalmia-Associated Transcription Factor M (MITF-M). The combination of I3C and aspirin was able to significantly downregulate MITF-M protein and transcript expression. Luciferase analysis of the proximal region of the MITF-M promoter revealed that both compounds regulated promoter activation synergistically. The reduction in activation was mediated by two distinct pathways that met on the MITF-M promoter, I3C inhibited the activation of BRAF which caused a decrease in binding to the promoter of the transcriptional activator BRN-2 while aspirin downregulated β-catenin/LEF1 binding to the promoter, again decreasing activity. Site directed mutagenesis of these sites proved that they were sufficient for the compounds’ regulation of MITF-M transcriptional activity. These results uncover one mechanism in which aspirin controls melanoma growth, something that has not been explored, and the combination of aspirin and I3C could be utilized as a potential anti-cancer treatment in human melanoma. We also demonstrate that Artemisinin, a phytochemical derived from Artemisia annua and its derivative Artesunate are able to regulate the growth of human breast cancer cells through regulation of Cyclin A2, and cyclin dependent kinases 1 and 2 (CDK) critical regulators of the G1/M phase transition as well as G2 phase progression. Artesunate was able to achieve significant downregulation and induce growth effects at much smaller doses than its parent molecule indicating increased potency. Chromatin-immunoprecipitation indicated that Artemisinin was able to alter Sp1 binding to the Cyclin A promoter, potentially explaining its ability to regulate transcript levels. In the last chapter, we explore the effects on melanoma and breast cancer cell growth caused by 1-Benzyl-I3C derivatives. We find that small changes in structure alter growth inhibition by the compounds likely through changes in their ability to interact and alter function of target enzymes Human Neutrophil Elastase, and NEDD4-1. No compound was stronger than the parent molecule, 1-Benzyl-I3C, but all were able to induce significant changes in growth at concentrations lower than I3C. While this study is preliminary it provides information as to what potential derivatives might slow transformed cell growth in the future.

Advisor: Firestone, Gary L.