Abstract
Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.
Highlights
As unresectable liver metastases from colorectal cancer are difficult to treat by single modality, we have spent several years developing a multimodality approach for hyperthermic isolated hepatic perfusion (IHP) therapy
This study revealed that the combinatorial treatment altered cellular metabolism and induced energy sensor AMP-activated protein kinase (AMPK) activation at two stages in the process, resulting in Beclin-1 phosphorylation and autophagy starting with the early stage and Beclin-1 cleavage by caspase-8 and apoptosis concurrent with the late stage
We have presented a novel combination treatment of melphalan in combination with the proteasome inhibitor bortezomib and the mTOR inhibitor rapamycin for colon cancer cells, as well as colon cancer stem cells, and demonstrated that this multidrug treatment-induced synergistic apoptosis mediated by AMPK activation through facilitation of Beclin-1 cleavage
Summary
As unresectable liver metastases from colorectal cancer are difficult to treat by single modality, we have spent several years developing a multimodality approach for hyperthermic IHP therapy. Bortezomib, the first clinically available proteasome inhibitor, possesses antitumor activity in a variety of human cancers and is often used in the treatment of hematological malignancies. It can induce both proapoptotic effects, including the induction of Bik, Bim and Noxa proteins, and antiapoptotic effects, including the accumulation of Mcl-1 and HSP70, as well as autophagic formation.[15,16,17]. This study revealed that the combinatorial treatment altered cellular metabolism and induced energy sensor AMP-activated protein kinase (AMPK) activation at two stages in the process, resulting in Beclin-1 phosphorylation and autophagy starting with the early stage and Beclin-1 cleavage by caspase-8 and apoptosis concurrent with the late stage. Our observations provided evidence that AMPK has an important role in cross-talk between autophagy and apoptosis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
