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

DataSheet_1_The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.pdf

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.</p

DataSheet_2_The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.pdf

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.</p