Cannabidiol (CBD), a major nonpsychoactive constituent of cannabis, is considered an antineoplastic agent on the basis of its in vitro and in vivo activity against tumor cells. However, the exact molecular mechanism through which CBD mediates this activity is yet to be elucidated. Here, we have shown CBD-induced cell death of breast cancer cells, independent of cannabinoid and vallinoid receptor activation. Electron microscopy revealed morphologies consistent with the coexistence of autophagy and apoptosis. Western blot analysis confirmed these findings. We showed that CBD induces endoplasmic reticulum stress and, subsequently, inhibits AKT and mTOR signaling as shown by decreased levels of phosphorylated mTOR and 4EBP1, and cyclin D1. Analyzing further the cross-talk between the autophagic and apoptotic signaling pathways, we found that beclin1 plays a central role in the induction of CBD-mediated apoptosis in MDA-MB-231 breast cancer cells. Although CBD enhances the interaction between beclin1 and Vps34, it inhibits the association between beclin1 and Bcl-2. In addition, we showed that CBD reduces mitochondrial membrane potential, triggers the translocation of BID to the mitochondria, the release of cytochrome c to the cytosol, and, ultimately, the activation of the intrinsic apoptotic pathway in breast cancer cells. CBD increased the generation of reactive oxygen species (ROS), and ROS inhibition blocked the induction of apoptosis and autophagy. Our study revealed an intricate interplay between apoptosis and autophagy in CBD-treated breast cancer cells and highlighted the value of continued investigation into the potential use of CBD as an antineoplastic agent.

Transforming growth factor (TGF)-β family members exert strong effects on restoration of liver mass after injury. Bone morphogenetic proteins (BMPs) are members of the TGF-β family and are found in the liver, suggesting that these proteins may play a role in liver regeneration. We examined BMP signaling in the liver during hepatectomy. We found that BMP4 is constitutively expressed in the peribiliary stroma and endothelial cells of the liver and that expression is decreased after hepatectomy. Mice driven to maintain BMP4 expression in the liver display inhibited hepatocyte proliferation and restoration of liver mass after hepatectomy, suggesting that reduced BMP4 is necessary for normal regeneration. Consistent with this finding, hepatocyte-specific deletion of the BMP receptor activin receptor-like kinase 3 (Alk3) enhances regeneration and reduces phosphorylation of SMAD1/5/8, a transducer of BMP signaling. In contrast to experiments in wild-type mice, maintaining BMP4 levels has no effect on liver regeneration in hepatocyte-specific Alk3 null mice, providing evidence that BMP4 signals through Alk3 to inhibit liver regeneration. Consistent with these findings, the BMP4 antagonist Noggin enhances regeneration. Furthermore, high-dose BMP4 inhibits proliferation of primary hepatocytes and HepG2 cells in culture. These findings elucidate a new, potentially clinically relevant paradigm in which a constitutively expressed paracrine inhibitory factor plays a critical role in liver regeneration.

Dissemination of HIV in the host involves transit of the virus and virus-infected cells across the lymphatic endothelium. HIV may alter lymphatic endothelial permeability to foster dissemination, but the mechanism is largely unexplored. Using a primary human lymphatic endothelial cell model, we found that HIV-1 envelope protein gp120 induced lymphatic hyperpermeability by disturbing the normal function of Robo4, a novel regulator of endothelial permeability. HIV-1 gp120 induced fibronectin expression and integrin α5β1 phosphorylation, which led to the complexing of these three proteins, and their subsequent interaction with Robo4 through its fibronectin type III repeats. Moreover, pretreatment with an active N-terminus fragment of Slit2, a Robo4 agonist, protected lymphatic endothelial cells from HIV-1 gp120-induced hyperpermeability by inhibiting c-Src kinase activation. Our results indicate that targeting Slit2/Robo4 signaling may protect the integrity of the lymphatic barrier and limit the dissemination of HIV in the host.