Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion

Abstract

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo, but is non toxic to untransformed cells. In our previous study we observed that BetA consistently induced cell death in a broad panel of tumor cell lines. Apoptosis induced by BetA involves activation of caspases, PARP cleavage and DNA fragmentation and was suggested to depend on the mitochondrial pathway. However, conflicting results have been reported with respect to the role of the pro-and anti-apoptotic members of the Bcl-2 family, which are often aberrantly regulated in tumors and thereby confer growth and survival advantages. Here we show that BetA-induced apoptosis critically depends on the release of cytochrome c from the mitochondria and formation of the apoptosome. Nevertheless, over-expression of Bcl-2 or Bcl-XL only provides limited protection against BetAinduced apoptosis. More importantly, Bax/Bak deficient cells are as sensitive to BetA as their wild-type counterparts, suggesting that cytochrome c is released in a nonclassical fashion. In agreement, pre-incubation with cyclosporin A indicated a crucial role for the mitochondrial permeability transition pore (PT) in the induction of apoptosis. Our observations therefore indicate that BetA affects mitochondria and induces cytochrome c release directly via PT Pore. This is only temporarily prevented by anti-apoptotic members of the Bcl-2 family, but independent of Bax and Bak. These findings help to explain the remarkable broad efficacy of BetA against tumor cells of different origin and its effect in tumor cells that are resistant to other chemotherapeutic agents.

Betulinic Acid Induces Bax/Bak-Independent Cytochrome c Release in Human Nasopharyngeal Carcinoma Cells

“…Although insignifi- cant, the inhibition clearly confirms that mPTPs partially contribute to BetA-induced Bax/Bak-independent cyt c release. Mullauer et al recently published findings that show that BetA induces caspase-dependent, Bax/Bak-independent apoptosis and cyt c release (Mullauer et al, 2009); the results of our study partially agreed with their findings and confirmed the BetA pathway in the CNE2 cell line. However, Mullauer et al found a significant reduction of cyt c release after CsA treatment, in contrast with our system where a difference was found but it was not significant.…”

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“…A similar increase of ROS was also observed for oleanolic acid (25 µM) in astrocytoma cell lines [20]. While recently published data reported Bax/Bak-independent apoptosis induction by betulinic acid in various cancer cell lines [21], a number of publications show a modulation of anti-and pro-apoptotic proteins of the Bcl-2 family [13,[22][23][24][25]. The modulation of pro-and antiapoptotic factors is complex and probably cell-type-dependent.…”

“…These relatively low serum levels can be explained by the low solubility in water (BA, OA: 0.02 µg/mL and BE < 0.1 µg/mL) [62,122]. It is known that OA is able to bind to plasmin and albumin [17,21], so binding phenomena could support the bioavailability. Thus the prediction of in vivo effects and the concentrations necessary for them may be unreliable if based on in vitro results.…”

Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

“…20,21,25 Our findings on the Cytochrome c release and apoptosis can take place independent of Bax/Bak, but dependent on mPT. 39,40 Indeed, we observed that ISG12b2-mediated cytochrome c release in DKO MEFs was inhibited by CsA, a mPT inhibitor. These results suggest that ISG12b2 is likely involved in mPTdependent apoptosis.…”