Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with the ability to resist apoptosis by the activation of survival promoting and anti-apoptotic proteins. METHODS: Efficacy of the apoptosis-inducing agent betulinic acid (BA) was determined in RMS cell cultures and in vivo by measuring cell viability, survival, apoptosis, hedgehog signalling activity, and neovascularisation. RESULTS: Betulinic acid had a strong cytotoxic effect on RMS cells in a dose-dependent manner. The BA treatment caused a massive induction of apoptosis mediated by the intrinsic mitochondrial pathway, which could be inhibited by the broad-range caspase inhibitor zVAD.fmk. Exposure of hedgehog-activated RMS-13 cells to BA resulted in a strong decrease in GLI1, GLI2, PTCH1, and IGF2 expression as well as hedgehog-responsive luciferase activity. Intraperitoneal injection of 20 mg BA per kg per day significantly retarded growth of RMS-13 xenografts in association with markedly higher counts of apoptotic cells and down-regulation of GLI1 expression compared with control tumours, while leaving microvascular density, cell proliferation, and myogenic differentiation unaffected. CONCLUSION: Our data show that induction of apoptosis and inhibition of hedgehog signalling are important features of the anti-tumourigenic effect of BA in RMS and advices this compound for the use in a multimodal therapy of this highly aggressive paediatric tumour.