Synergistic Apoptosis Induction by Betulinic Acid and Established Cytotoxic Drugs in Leukemia Cells.

Betulinic acid (BA) represents an effective inducer of apoptosis in a broad spectrum of solid tumor cells in vitro and in small animal models in vivo. Due to its low toxicity in animal trials, it represents a putative future anti-cancer drug. We first described that in addition to solid tumor cells, BA potently induces apoptosis in leukemia cells expanding the therapeutic use of BA to hematological malignancies (Ehrhardt et al., Leukemia 2004).

Here we asked how BA might best be incorporated into polychemotherapy protocols used to treat acute leukemia and therefore searched for conventional cytotoxic drugs which enhance the anti-tumor effect of BA. Of suitable drugs discovered, we characterized the molecular mechanisms determining the synergistic interaction with BA.

We used both leukemia cell lines and primary tumor cells obtained from children with acute lymphoblastic or myeloid leukemia at diagnosis of disease or relapse. Primary, patient-derived tumor cells were further amplified in NOD/SCID mice. Most importantly, we transfected primary, patient-derived tumor cells to knock down endogenous apoptosis signaling proteins.

When conventional cytotoxic drugs in routine use to treat acute leukemia were screened on leukemic cell lines, three drugs were identified which induce synergistic induction when given together with BA: doxorubicin, asparaginase and vincristine. Accordingly, clonogenic survival was reduced in a super-additive way, when one of these drugs was combined with BA. Importantly, synergistic apoptosis induction by these drugs with BA was also found in primary, patient-derived leukemic tumor samples. Both in primary samples and cell lines, BA-induced apoptosis was enhanced by addition of the second drug, even if doxorubicin, asparaginase or vincristine alone were unable to induce apoptosis due to apoptosis resistance.

Synergistic apoptosis induction was accompanied by increased caspase activation and was inhibited by the addition of zVAD, by overexpression of XIAP or knockdown of Caspase-9.

p53 was activated nearly exclusively, when doxorubicin, asparaginase or vincristine was combined with BA and knockdown of p53 inhibited synergistic apoptosis induction of the drug combinations.

While expression of Bak, Bim, Bid, Bcl-2, Bcl-xL and PUMA remained unchanged by stimulation with BA and doxorubicin, asparaginase or vincristine, the p53 target gene NOXA was strongly upregulated exclusively when drugs were combined. When doxorubicin, asparaginase or vincristine were given together with BA, more apoptogenic factors like Smac and Cytochrome c were released from mitochondria and synergistic apoptosis induction by BA with either doxorubicin, asparaginase or vincristine depended on increased mitochondrial signaling. Knockdown of Bim, Bid or PUMA did not alter synergistic apoptosis induction by BA and doxorubicin, asparaginase or vincristine. In contrast, overexpression of Bcl-2 or Bcl-xL or knockdown of either Bak or NOXA inhibited synergistic apoptosis induction. Knockdown of p53 and NOXA in primary, patient-derived leukemia cells completely inhibited synergistic apoptosis induction by BA and doxorubicin, asparaginase or vincristine.

Our data show that BA induces synergistic apoptosis induction when given in combination with doxorubicin, asparaginase and vincristine based on increased activation of p53 which enables expression of NOXA and a NOXA – Bak dependent activation of mitochondria. BA should best be incorporated into future anti-leukemia polychemotherapy protocols in close proximity to doxorubicin, asparaginase or vincristine.

No relevant conflicts of interest to declare.