Bax Inhibiting Peptides (BIPs) Derived from Ku70 Suppress Chemotherapy Induced-Cell Death of Megakaryocytes.

Thrombocytopenia is one of the major side effects of cancer chemotherapy. To prevent the chemotherapy-induced thrombocytopenia, the protection of megakaryocytes from drug-induced apoptosis is necessary. Caspase inhibitor and anti-apoptotic protein Bcl-2 are often employed for the protection of cells from drug-induced apoptosis in other cell types. However, it has been reported that (1) caspase inhibitors markedly decrease platelet formation because caspase activity is required for platelet cleavage from megakaryocytes, and (2) Bcl-2 exhibited reduced platelet formation probably due to the attenuation of cell division. Therefore, new cell death inhibitors are needed to maintain platelet formation during cancer chemotherapy. Previously, we reported a new type of cell death inhibitors, named Bax Inhibiting Peptide (BIP) (Nature Cell Biol, p352-357, 5, 2003, BBRC, p961-966, 321, 2004). BIP is designed from the amino acid sequence of Ku70 protein that protect cells from apoptosis by inhibiting proapoptotic protein Bax. BIP directly binds Bax and inhibits cytotoxic activity of Bax. Bax is widely expressed in human cells including megakaryocytes and platelets, suggesting that BIP may be utilized as a new tool to protect megakaryocytes from chemotherapy. In this study, we examined the effects of BIP on chemotherapy-induced cell death in a megakaryocyte-like cell line (DAMI cells) and in primary mouse megakaryocytes. Three versions of BIPs were used; VPMLK (derived from human Ku70), VPTLK (from mouse Ku70) and VPALR (from rat Ku70). First, cell permeability of BIPs was examined by incubating cells with FITC-labeled BIPs and by detecting FITC using confocal microscope and FACS. All BIPs showed cell permeability in DAMI cells and mouse megakaryocytes. Interestingly, VPTLK showed the strongest cell permeability in these cell types. Next, we examined whether these BIPs bind Bax expressed in DAMI cells. Biotin-labeled BIPs were added to the cell lysates of DAMI cells and biotin-BIPs were precipitated by streptavidin beads. Specific interaction of Bax with BIPs was confirmed by the detection of Bax in the Western blot of the precipitated samples. Then, we examined the effects of BIPs against chemotherapy-induced apoptosis in DAMI cells. Apoptosis was induced by the treatment of etoposide, cisplatin, paclitaxel, and doxorubicin, that are commonly used anti-cancer drugs. BIPs suppressed apoptosis of DAMI cells in a dose dependent manner (50–400 μM). We also tested the effects of BIPs on etoposide-induced cell death in primary mouse megakaryocytes, and confirmed that BIPs significantly blocked apoptosis in megakaryocytes. Finally, we examined the effects of BIPs on platelet formation in mouse megakaryocytes and observed that platelet formation was normal even in the presence of BIPs. These results suggest that BIPs may become a useful therapeutic to prevent thrombocytopenia induced by chemotherapy, and other cancer therapies (e.g. radiation) that trigger Bax-mediated apoptosis. The present study was performed in cell culture. Further studies using in vivo system to examine the effectiveness of BIPs on thrombocytopenia are currently in progress.