The antiangiogenic effect of digitoxin is dependent on a ROS-elicited RhoA/ROCK pathway activation

Area of Science:

• Molecular Biology
• Cell Biology
• Cancer Research

Background:

• Digitoxin previously demonstrated anti-angiogenic and anti-proliferative effects linked to protein tyrosine kinase 2 (FAK) inhibition.
• The Rho GTPases, RhoA and Rac1, are crucial regulators of cell cytoskeleton and motility.
• Understanding the specific Rho GTPase involvement in digitoxin's anti-angiogenic action is essential.

Purpose of the Study:

• To investigate the role of RhoA and Rac1 in the anti-angiogenic effects of digitoxin.
• To elucidate the signaling pathways mediating digitoxin's impact on endothelial cell behavior.
• To determine the relationship between digitoxin, reactive oxygen species (ROS), and RhoA activation.

Main Methods:

• Human umbilical vein endothelial cells (HUVECs) were treated with digitoxin.
• Phalloidin staining assessed stress fiber formation.
• Rhotekin and Pak1 pull-down assays detected GTP-bound RhoA and Rac1.
• RhoA silencing and Y-27632 treatment inhibited the RhoA-ROCK axis.
• Intracellular ROS levels were measured.
• NADPH oxidase (NOX) inhibitors (DPI, VAS-2870) were used.

Main Results:

• Digitoxin induced stress fiber formation and sustained RhoA activation in HUVECs, without affecting Rac1.
• Digitoxin's inhibition of HUVEC migration and tube formation was reversed by targeting the RhoA-ROCK pathway.
• Digitoxin treatment increased intracellular ROS levels in a dose-dependent manner.
• ROS production by endothelial NOX was identified as a key mediator of digitoxin-induced RhoA activation.
• RhoA activation by digitoxin was independent of FAK inhibition.

Conclusions:

• Digitoxin's anti-angiogenic effects are mediated by ROS production from endothelial NOX, leading to RhoA/ROCK pathway activation.
• The study reveals a novel mechanism involving ROS and RhoA in digitoxin's anti-angiogenic activity.
• These findings suggest broader implications for digitoxin's impact on cancer progression by affecting cytoskeletal organization and cell motility.