Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (<b>LCA</b>), deoxycholic acid (<b>DCA</b>), and cholic acid (<b>CA</b>). These bile acid–tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The <i>in vitro</i> anticancer activities of these bile acid–tamoxifen conjugates show that the free amine headgroup based cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid–tamoxifen conjugates. The cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor −ve breast cancer cell lines. The enhanced anticancer activity of <b>CA-Tam</b>_<b>3</b><b>-Am</b> is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G₀ phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome <i>c</i>, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER −ve cells. <i>In vivo</i> studies in the 4T1 tumor model showed that <b>CA-Tam</b>_<b>3</b><b>-Am</b> is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid–drug conjugates

Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (<b>LCA</b>), deoxycholic acid (<b>DCA</b>), and cholic acid (<b>CA</b>). These bile acid–tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The <i>in vitro</i> anticancer activities of these bile acid–tamoxifen conjugates show that the free amine headgroup based cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid–tamoxifen conjugates. The cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor −ve breast cancer cell lines. The enhanced anticancer activity of <b>CA-Tam</b>_<b>3</b><b>-Am</b> is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G₀ phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome <i>c</i>, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER −ve cells. <i>In vivo</i> studies in the 4T1 tumor model showed that <b>CA-Tam</b>_<b>3</b><b>-Am</b> is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid–drug conjugates

Bisphosphonate-Generated ATP-Analogs Inhibit Cell Signaling Pathways

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget’s disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl₂p). We screened both ApppI as well as AppCCl₂p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl₂p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads