Abstract
The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.
Highlights
The anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2, Bcl-xL, and Mcl-1 and XIAP, are over-expressed in chronic lymphocytic leukemia (CLL), whereas pro-apoptotic protein Bax is under-expressed.[3,4]
We previously demonstrated that the cell-penetrating VDAC1based Antp-LP4 peptide diminished the anti-apoptotic effects of HK-I, Bcl-2 or Bcl-xL,[14,15,17] and induced cell death in several cancer cell lines yet did not harm normal peripheral blood lymphocytes (PBMCs).[17]
Incubation of PBMCs for 90 min with Antp-LP4 resulted in marked cell death of the CLL-derived cells but only slightly affected cells isolated from healthy donors (Figure 1a)
Summary
The anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2, Bcl-xL, and Mcl-1 and XIAP, are over-expressed in CLL, whereas pro-apoptotic protein Bax is under-expressed.[3,4]. HK acts as an anti-apoptotic protein via direct association with the voltage-dependent anion channel[1] (VDAC1).[5,6,7,9] VDAC1, localized in the outer mitochondrial membrane, plays a central role in cell metabolism by mediating the transfer of metabolites between the mitochondrion and the cytosol.[10] VDAC1 is a key player in mitochondria-mediated apoptosis, participating in the release of mitochondrial pro-apoptotic proteins to the cytosol (e.g. cytochrome c (Cyto c), AIF and Smac/DIABLO),[5,11,12] and interacting with apoptosis regulatory proteins, such as. We demonstrate that the VDAC1-based peptides, Antp-LP4 and N-Terminal-Antp (N-Ter-Antp) peptides and improved versions thereof selectively induced cell death of PBMCs from CLL patients yet exhibited only minor effects on PBMCs from healthy donors. The mode of action of the peptides involves dysfunction of mitochondria energy production and apoptosis induction. These results demonstrate the potential of VDAC1-based peptides for treating CLL
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