Blockade of Chemotherapy-Induced Thrombocytopenia by Bax Inhibiting Peptides (BIPs) in Mouse Model.

Thrombocytopenia is a major side effect of cancer chemotherapy producing bleeding problems as well as cancer treatment delays. To preclude it, the platelets progenitors megakaryocytes need to be protected from drug-induced apoptosis. According to previous reports, anti-apoptotic molecules such as Bcl-2, Bcl-xL, and caspase inhibitors were found to interfere platelet formation by megakaryocytes, thus these apoptosis inhibitors are not appropriate therapeutic candidates for the prevention of drug-induced thrombocytopenia. Recently we developed a new type of cell permeable apoptosis inhibitor, Bax Inhibiting Peptides (BIPs), which are derived from the Bax binding domain of Ku70. Bax is a key mediator of apoptosis, and Ku70 is a multifunctional protein playing roles in cell survival and DNA repair. In the present study, we determined whether BIPs prevent chemotherapy-induced thrombocytopenia both in vitro and in vivo. Importantly, there are several cancer cell types that acquired mutations in genes of Bax and/or Bax activating factors. Therefore, we hypothesize that protection of non-tumorigenic essential cells (such as megakaryocytes) by BIPs is beneficial for patients suffering from Bax-negative cancer. For in vitro study, Dami cells (human pro-megakaryocyte cell line) and mouse primary cultured megakaryocytes were used. BIPs significantly inhibited apoptosis induction by anti-cancer drugs such as etoposide, cisplatin, doxorubicin, or paclitaxel. Importantly, BIPs did not interfere the activity of megakaryocytes to produce platelet-like particle in cell culture, suggesting that BIPs protect megakaryocytes from drug-induced apoptosis without suppressing their platelets production. For in vivo study, C57BL/6J mice and Bax-/- mice (backcrossed with C57BL/6J) were used. Etoposide (150 mg/kg, i.p.) induced a significant decrease of platelet count in whole blood within 2 days after the drug administration in wild type C57BL/6J mice. Pretreatment of mice with BIPs (166 mg/kg, i.p., 30 min before etoposide injection) blocked etoposide-induced platelet count decrease in these wild type mice. The same dose of etoposide did not induce significant platelet count decrease in Bax-/- mice. These results suggest that chemotherapy-induced thrombocytopenia, at least in part, is due to the stimulation of Bax-mediated cell death probably both in megakaryocytes and platelets. In our preliminary experiments, three times injection of caspase inhibitor (z-VAD-fmk, 166 mg/kg, i.p. every 24 hrs) caused prolongation of bleeding time from the wound (tail cut). Thus, as previously reported by other groups using in vitro system, caspase inhibitor may have its own side effect to inhibit platelet formation or/and activation in vivo. On the other hand, BIPs (166 mg/kg, 3 times every 24 hrs, i.p.) did not extend bleeding time from the tail. Taken together, BIP may become a useful supplemental therapeutic agent to block chemotherapy-induced thrombocytopenia.