Pan-Bcl-2 Inhibitor Obatoclax Delays Cell Cycle Progression and Blocks Migration of Colorectal Cancer Cells

Abstract

Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC), prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

The BH3-mimetic obatoclax reduces HIF-1α levels and HIF-1 transcriptional activity and sensitizes hypoxic colon adenocarcinoma cells to 5-fluorouracil

“…1B), OBX was also found to cause cell cycle arrest (Fig. 1C), as described by Koehler et al in other colon cancer cell lines [45]; the fact that, at variance with the results of the present study, these Authors reported cell cycle arrest in G2/M rather than in G1 could depend on cell type-specific differences and/or on the Interestingly, while survival curves obtained for OBX under the three experimental conditions tested were totally superimposed in our preliminary experiments, the percentage of apoptotic cells induced by OBX increased significantly following hypoxic or chemical HIF-1α stabilization in HCT116 cells (Fig. 1B).…”

Prolyl Hydroxylase 3 Attenuates MCL-1–Mediated ATP Production to Suppress the Metastatic Potential of Colorectal Cancer Cells

“…This unique function, which goes beyond the antiapoptotic activity of full-length MCL-1 protein, makes MCL-1 indispensable for the survival of cancer cells (26,40). Furthermore, MCL-1 reportedly enhances the migratory and invasive potential of colorectal tumor cells, independently from its antiapoptotic effects (25,31). Consistently, we observed that improved ATP production, as well as enhanced migration and invasion of PHD3-deficient tumor cells, could be reverted by simultaneous knockdown of MCL-1.…”

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

“…The colorectal cancer cell lines HT29 and SW480 and the non-transformed colon cell line CCD 841 CoN were obtained from ATCC (Manassas, VA, USA) and cultured under standard conditions as described previously, supplemented with 10% fetal bovine serum (PAA laboratories, Cölbe, Germany) and 1% Penicillin/Streptomycin (Sigma-Aldrich, Munich, Germany) in a humidified atmosphere [45]. 5-Fluorouracil (5-FU) and Irinotecan were obtained from Sigma-Aldrich and dissolved in DMSO.…”

“…Flow cytometry analyses were performed using a FACS CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA) as described previously according to the protocol of Nicoletti et al [45]. Data were analyzed using the BD FACSDIVA ® software.…”

“…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].…”