Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling

Abstract

BackgroundColorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials.MethodsFlow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux.ResultsThis study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12.ConclusionsThe objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1929-y) contains supplementary material, which is available to authorized users.

Knockdown of Atg7 Induces Nuclear-LC3 Dependent Apoptosis and Augments Chemotherapy in Colorectal Cancer Cells

“…Cell lysis, SDS-page and Western blotting were performed according to standard procedures as described previously [45]. The following antibodies were used: anti-Beclin-1 (Santa Cruz Biotechnology, Heidelberg In order to quantify protein expression, we used ImageJ ® (by Wayne Rasband at NIH, Bethesda, MD, USA) for densitometric analysis as described previously [49]. In brief, band density was measured relative to the untreated control and then adjusted to tubulin as loading control.…”

Combination of an Autophagy Inducer and an Autophagy Inhibitor: A Smarter Strategy Emerging in Cancer Therapy

“…The Bcl-2 inhibitor ABT-737 has been identified as an autophagy inhibitor (Yang et al, 2016). Moreover, obatoclax which exhibits pan-Bcl-2 inhibition effect has been demonstrated to cause a striking inhibition of autophagy at late-stage in colorectal cancer and bladder cancer cells (Huang and Sinicrope, 2010;Koehler et al, 2015;Jiménez-Guerrero et al, 2018). Clarithromycin is a macrolide antibiotic frequently utilized in the treatment of upper and lower respiratory tract infections and Helicobacter pylori that has demonstrated inhibitory effects on autophagy (Altman and Platanias, 2012;Giulia Petroni et al, 2020).…”

Cyclin-dependent kinase 2 inhibitor SU9516 increases sensitivity of colorectal carcinoma cells Caco-2 but not HT29 to BH3 mimetic ABT-737

“…Interestingly, multikinase inhibitor BAY 43-9006 that downregulates Mcl-1 through inhibition of translation (Rahmani et al 2005) or proteasomal degradation (Yu et al 2005) was shown to downregulate Mcl-1 also in HT29 cells (Yu et al 2005). Unlike ABT-737, pan-Bcl-2 inhibitor Obatoclax that binds to Bcl-2, Bcl-X L , Bcl-w and Mcl-1 (Konopleva et al 2008) was shown to be a potent late stage autophagy inhibitor in HT29 cells, causing cell growth arrest and cell death (Koehler et al 2015).…”