Dataset Information

Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

ABSTRACT: Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

SUBMITTER: Ko JK

PROVIDER: S-EPMC3059050 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

Ko Jae-Kyun JK Choi Kyoung-Han KH Peng Jun J He Feng F Zhang Zhi Z Weisleder Noah N Lin Jialing J Ma Jianjie J

The Journal of biological chemistry 20101228 11

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requ ...[more]

PMID: 21189256

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Project description:Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments. BH3 mimetics against members of the BCL-2 family have gained excitement with the recent success in hematological malignancies. However, single drug BH3 mimetic therapy in melanoma has limited effectiveness due to escape by the anti-apoptotic protein MCL-1 and/or survival of melanoma-initiating cells (MICs). We tested the efficacy of the BH3 mimetic combination of A-1210477 (an MCL-1 inhibitor) and ABT-263 (a BCL-2/BCL-XL/BCL-W inhibitor) in killing melanoma, especially MICs. We also sought to better define Dynamin-Related Protein 1 (DRP-1)'s role in melanoma; DRP-1 is known to interact with members of the BCL-2 family and is a possible therapeutic target for melanoma treatment. We used multiple assays (cell viability, apoptosis, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing techniques. For clinical relevance, we employed patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found the BH3 mimetic combination kill both the MICs and non-MICs (bulk of melanoma) in all cell lines and patient samples irrespective of the mutation status or relapsed state (p?<?0.05). Unexpectedly, the major pro-apoptotic proteins, NOXA and BIM, are not necessary for the combination-induced cell death. Furthermore, the combination impedes the activation of DRP-1, and inhibition of DRP-1 further enhances apoptosis (p?<?0.05). DRP-1 effects in melanoma differ from those seen in other cancer cells. These results provide new insights into BCL-2 family's regulation of the apoptotic pathway in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death even without involvement from major pro-apoptotic proteins. Importantly, our study also indicates that DRP-1 inhibition is a promising adjuvant for BH3 mimetics in melanoma treatment.

Project description:Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.

Dataset Information

Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

Publications

Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

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