Pan-class I PI3-kinase inhibitor BKM120 induces MEK1/2-dependent mitotic catastrophe in non-Hodgkin lymphoma leading to apoptosis or polyploidy determined by Bax/Bak and p53.

Constitutive signaling of PI3K/Akt/mTOR plays a prominent role in malignant transformation and progression of B-cell non-Hodgkin lymphomas (B-NHL) underscoring the need for PI3K targeted therapies. The pan-class I PI3-kinase inhibitor BKM120 has shown preclinical activity in distinct malignancies and is currently tested in clinical trials. Intratumor heterogeneity is an intrinsic property of cancers that contributes to drug resistance and tumor recurrence. Here, we demonstrate that inhibition of PI3-kinases by BKM120 attenuates growth and survival of B-NHL cell lines by inducing mitotic arrest with subsequent induction of intrinsic apoptosis. BKM120-mediated downregulation of Cyclin A and activation of the CDK1/Cyclin B1 complex facilitates mitotic entry. In addition, concomitant BKM120-mediated upregulation of Cyclin B1 expression attenuates completion of mitosis, which results in mitotic catastrophe and apoptotic cell death. In Bax and Bak deficient B-NHL, which are resistant to BKM120-induced apoptosis, BKM120-induced mitotic catastrophe results in polyploidy. Upon re-expression of wt p53 in these p53 mutated cells, BKM120-induced polyploidy is strongly reduced demonstrating that the genetic status of the cells determines the outcome of a BKM120-mediated pathway inhibition. Mitotic catastrophe and unfavorable induction of polyploidy can be prevented in this setting by additional inhibition of MEK1/2 signaling. Combining MEK1/2 inhibitors with BKM120 enhances the anti-tumor effects of BKM120, prevents prognostic unfavorable polyploidy and might be a potential strategy for the treatment of B-NHL.

MACC1 regulates Fas mediated apoptosis through STAT1/3 - Mcl-1 signaling in solid cancers.

MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis.

Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC) is polyresistant to chemo- and radiotherapy and biologicals, including TNF-related apoptosis-inducing ligand (TRAIL). Sorafenib, a multikinase inhibitor approved for the treatment of RCC, has been shown to sensitize cancer cells to TRAIL-induced apoptosis, in particular by down-regulation of the Bak-inhibitory Bcl-2 family protein Mcl-1. Here we demonstrate that sorafenib overcomes TRAIL resistance in RCC by a mechanism that does not rely on Mcl-1 down-regulation. Instead, sorafenib induces rapid dissipation of the mitochondrial membrane potential (ΔΨm) that is accompanied by the accumulation of reactive oxygen species (ROS). Loss of ΔΨm and ROS production induced by sorafenib are independent of caspase activities and do not depend on the presence of the proapoptotic Bcl-2 family proteins Bax or Bak, indicating that both events are functionally upstream of the mitochondrial apoptosis signaling cascade. More intriguingly, we find that it is sorafenib-induced ROS accumulation that enables TRAIL to activate caspase-8 in RCC. This leads to apoptosis that involves activation of an amplification loop via the mitochondrial apoptosis pathway. Thus, our mechanistic data indicate that sorafenib bypasses central resistance mechanisms through a direct induction of ΔΨm breakdown and ROS production. Activation of this pathway might represent a useful strategy to overcome the cell-inherent resistance to cancer therapeutics, including TRAIL, in multiresistant cancers such as RCC.

Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak.

The pro-apoptotic multidomain Bcl-2 proteins Bax and Bak (also known as BAK1) are considered the gatekeepers of the intrinsic pathway of apoptosis by triggering the mitochondrial release of cytochrome c The role of the third Bax- and Bak-homologous multidomain protein Bok, however, is still unresolved. As cells doubly deficient for Bax and Bak are largely resistant to various apoptotic stimuli, it has been proposed that Bok is either dispensable for apoptosis or that its role is dependent on Bax and Bak. Here, we demonstrate, in several cell systems, that Bok efficiently induces cytochrome c release and apoptosis even in the complete absence of both Bak and Bax. Moreover, modulation of endogenous Bok levels affects the apoptosis response. By RNA interference and targeted deletion of the Bok gene, we demonstrate that Bok can significantly influence the apoptotic response to chemotherapeutic drugs in ovarian carcinoma cells. Hence, our results not only establish Bok as a Bak- and Bax-independent apoptosis inducer, but also suggest a potential impact of Bok expression in ovarian cancer therapy.

2D and 3D similarity landscape analysis identifies PARP as a novel off-target for the drug Vatalanib.

BACKGROUND: Searching for two-dimensional (2D) structural similarities is a useful tool to identify new active compounds in drug-discovery programs. However, as 2D similarity measures neglect important structural and functional features, similarity by 2D might be underestimated. In the present study, we used combined 2D and three-dimensional (3D) similarity comparisons to reveal possible new functions and/or side-effects of known bioactive compounds. RESULTS: We utilised more than 10,000 compounds from the SuperTarget database with known inhibition values for twelve different anti-cancer targets. We performed all-against-all comparisons resulting in 2D similarity landscapes. Among the regions with low 2D similarity scores are inhibitors of vascular endothelial growth factor receptor (VEGFR) and inhibitors of poly ADP-ribose polymerase (PARP). To demonstrate that 3D landscape comparison can identify similarities, which are untraceable in 2D similarity comparisons, we analysed this region in more detail. This 3D analysis showed the unexpected structural similarity between inhibitors of VEGFR and inhibitors of PARP. Among the VEGFR inhibitors that show similarities to PARP inhibitors was Vatalanib, an oral "multi-targeted" small molecule protein kinase inhibitor being studied in phase-III clinical trials in cancer therapy. An in silico docking simulation and an in vitro HT universal colorimetric PARP assay confirmed that the VEGFR inhibitor Vatalanib exhibits off-target activity as a PARP inhibitor, broadening its mode of action. CONCLUSION: In contrast to the 2D-similarity search, the 3D-similarity landscape comparison identifies new functions and side effects of the known VEGFR inhibitor Vatalanib.

Apoptosis resistance, mitotic catastrophe, and loss of ploidy control in Burkitt lymphoma.

UNLABELLED: Resistance to cell death is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphoma (BL). When analyzing therapy resistance in Burkitt lymphoma (BL), we discovered a link between apoptosis resistance and ploidy control. We therefore studied systematically a panel of 15 BL lines for apoptosis induction upon treatment with microtubule inhibitors and compared three types of microtubule toxins, i.e., paclitaxel, nocodazole and vincristine. We found an inverse relationship between apoptosis sensitivity and ploidy control. Thus, cells resistant to paclitaxel- or nocodazole-induced apoptosis underwent mitotic catastrophe and developed polyploidy (>4N). Mechanistically, apoptosis resistance was linked to failure of caspase activation, which was most pronounced in cells lacking the pro-apoptotic multidomain Bcl-2 homologs Bax and Bak. Pharmacological caspase inhibition promoted polyploidy upon exposure to paclitaxel and nocodazole supporting the relationship between resistance to apoptosis and polyploidization. Of note, vincristine induced persistent mitotic arrest but no loss of ploidy control. Considering targets to facilitate Bax/Bak-independent cell death and to avoid drug-induced mitotic catastrophe and consecutive mitotic catastrophe should be of great importance to overcome therapy resistance and therapy-related events that result in ploidy changes and tumor progression. KEY MESSAGE: Inverse relation of apoptosis and polyploidy induction by paclitaxel or nocodazole in BL. Resistant cells undergo mitotic catastrophe and develop polyploidy. Lack of Bax/Bak confers resistance and leads to induction of polyploidy in BL. Intact apoptosis response protects from polyploidy as a result of mitotic catastrophe.

p14ARF induces apoptosis via an entirely caspase-3-dependent mitochondrial amplification loop.

The p14(ARF) tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF-7 breast carcinoma cells, expression of the tumor suppressor gene p14(ARF) fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase-3 proficient MCF-7 cells upon expression of p14(ARF) . This occurred in the absence of S-phase progression or mitotic entry. In contrast, syngeneic, caspase-3-deficient MCF-7 cells remained entirely resistant to p14(ARF) -induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14(ARF) -induced cell death and promotes cell death via a caspase-3-dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan-caspase inhibitors and the caspase-3/7 inhibitor zDEVD-fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase-3 proficiency indicating that caspase-3 either acts "up-stream" of the mitochondria in a "non-canonical" pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14(ARF) -induced stress signaling.

Concurrent inhibition of PI3K and mTORC1/mTORC2 overcomes resistance to rapamycin induced apoptosis by down-regulation of Mcl-1 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive form of Non-Hodgkin-lymphoma (NHL) with an ongoing need for novel treatments. Apart from the translocation t(11:14), which facilitates constitutive transcription of cyclin D1, additional aberrations are frequently observed in MCL, including a recurrent dysregulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. mTOR, a key component of this pathway, is pivotal for the assembly of mTOR complex (mTORC) 1 and 2. Temsirolimus, an analog of the mTOR inhibitor rapamycin, is approved for the treatment of relapsed MCL. Response rates, however, are low and response durations are short. We demonstrate that inhibition of mTORC1 by rapamycin or blocking of mTORC1 and mTORC2 in conjunction with PI3K by NVP-BEZ235 reduces proliferation of MCL cell lines to a similar extent. However, only NVP-BEZ235 is able to sufficiently inhibit the downstream pathway of mTOR and to mediate cell death through activation of the intrinsic apoptosis pathway. Further analysis demonstrated that the anti-apoptotic Bcl-2 family member Mcl-1 plays a central role in regulation of MCL survival. While Mcl-1 protein levels remained unchanged after coculture with rapamycin, they were down-regulated in NVP-BEZ235 treated cells. Furthermore, inhibition of Mcl-1 by the BH3-only mimetic obatoclax or down-regulation of constitutive Mcl-1, but not of Bcl-2 or Bcl-xL, by siRNA facilitated cell death of MCL cells and enhanced NVP-BEZ235's capacity to induce cell death. Our findings may help to lay the foundation for further improvements in the treatment of MCL.

The BH3-only protein Bim(L) overrides Bcl-2-mediated apoptosis resistance in melanoma cells.

Melanoma cells are characterized by apoptosis deficiency coinciding with reduced expression of the proapoptotic Bcl-2 protein Bim. An adenoviral vector was constructed with the BimL cDNA controlled by an inducible promoter. Highly efficient apoptosis induction and abrogated cell proliferation was seen in melanoma cells upon BimL overexpression. Loss of mitochondrial membrane potential, release of mitochondrial apoptogenic factors and caspase-9 processing indicated the activation of mitochondrial apoptosis pathways. BimL activated both Bax and Bak, as shown by siRNA knockdown and activation-specific antibodies. Of note, BimL overrode the apoptosis blockade by Bcl-2 overexpression or by Bax/Bak single knockdown. The high efficacy correlated to BimL interaction with all antiapoptotic Bcl-2 family members in melanoma cells, shown by co-immunoprecipitation analyses for Bcl-2, Bcl-xL, Mcl-1 and Bcl-w. Thus, BimL reveals an outstanding proapoptotic potential in melanoma cells, and strategies for its re-expression appear of interest. These have been reported for B-Raf inhibitors, and their efficacy may be partly attributed to BimL.

Dysferlin-peptides reallocate mutated dysferlin thereby restoring function.

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca(2+)dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients' myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.

Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK).

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK), a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK) may trigger apoptosis.For efficient overexpression, Bcl-x(AK) was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK) appeared as an essential and initial step. Bcl-x(AK) was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L). A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins.

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.

Synthetic glycosidated phospholipids induce apoptosis through activation of FADD, caspase-8 and the mitochondrial death pathway.

Apoptosis is modulated by extrinsic and intrinsic signaling pathways through the formation of the death receptor-mediated death-inducing signaling complex (DISC) and the mitochondrial-derived apoptosome, respectively. Ino-C2-PAF, a novel synthetic phospholipid shows impressive antiproliferative and apoptosis-inducing activity. Little is known about the signaling pathway through which it stimulates apoptosis. Here, we show that this drug induces apoptosis through proteins of the death receptor pathway, which leads to an activation of the intrinsic apoptotic pathway. Apoptosis induced by Ino-C2-PAF and its glucosidated derivate, Glc-PAF, was dependent on the DISC components FADD and caspase-8. This can be inhibited in FADD--/-- and caspase-8--/-- cells, in which the breakdown of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-9, -8 and -3 do not occur. In addition, the overexpression of crmA, c-Flip or dominant negative FADD as well as treatment with the caspase-8 inhibitor z-IETD-fmk protected against Ino-C2-PAF-induced apoptosis. Apoptosis proceeds in the absence of CD95/Fas-ligand expression and is independent of blockade of a putative death-ligand/receptor interaction. Furthermore, apoptosis cannot be inhibited in CD95/Fas--/-- Jurkat cells. Expression of Bcl-2 in either the mitochondria or the endoplasmic reticulum (ER) strongly inhibited Ino-C2-PAF- and Glc-PAF-induced apoptosis. In conclusion, Ino-C2-PAF and Glc-PAF trigger a CD95/Fas ligand- and receptor-independent atypical DISC that relies on the intrinsic apoptotic pathway via the ER and the mitochondria.

MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria.

The BH3-only BID protein (BH3-interacting domain death agonist) has a critical function in the death-receptor pathway in the liver by triggering mitochondrial outer membrane permeabilization (MOMP). Here we show that MTCH2/MIMP (mitochondrial carrier homologue 2/Met-induced mitochondrial protein), a novel truncated BID (tBID)-interacting protein, is a surface-exposed outer mitochondrial membrane protein that facilitates the recruitment of tBID to mitochondria. Knockout of MTCH2/MIMP in embryonic stem cells and in mouse embryonic fibroblasts hinders the recruitment of tBID to mitochondria, the activation of Bax/Bak, MOMP, and apoptosis. Moreover, conditional knockout of MTCH2/MIMP in the liver decreases the sensitivity of mice to Fas-induced hepatocellular apoptosis and prevents the recruitment of tBID to liver mitochondria both in vivo and in vitro. In contrast, MTCH2/MIMP deletion had no effect on apoptosis induced by other pro-apoptotic Bcl-2 family members and no detectable effect on the outer membrane lipid composition. These loss-of-function models indicate that MTCH2/MIMP has a critical function in liver apoptosis by regulating the recruitment of tBID to mitochondria.

Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3.

Induction of cell death by p14(ARF) is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21(CDKN1). Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21(CDKN1) or p53-reconstituted DU145 prostate cancer cells, we show that p14(ARF)-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14(ARF) in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14(ARF)-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14(ARF)-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21(CDKN1) strongly enhances p14(ARF)-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14(ARF)-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14(ARF)-triggered apoptosis is enhanced by loss of p21(CDKN1)-mediated cell cycle checkpoint control.

Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma.

Tumor necrosis factor (alpha)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-xL. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-xL overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (alpha) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-xL is a major target for sensitization of Bax-deficient tumors for death receptor-induced apoptosis via the Bak pathway.

Aurora kinase inhibitor ZM447439 induces apoptosis via mitochondrial pathways.

ZM447439 (ZM) is a potent and selective inhibitor of aurora-A and -B kinase with putative anti-tumoral activity. Inhibitors of aurora kinases were shown to induce apoptosis in vitro and in vivo. To investigate the underlying mechanisms, cell death pathways triggered by ZM was analysed in HCT-116 colorectal cancer cells. Through correlation of polyploidization and apoptosis in different knockout cells, the interrelation of these cellular responses to ZM was investigated. ZM induced apoptosis in a concentration- and time-dependent manner. ZM-induced apoptosis was associated with an upregulation of p53, breakdown of the mitochondrial membrane potential (DeltaPsim) and activation of caspase-3. To precisely define key components for ZM-induced apoptosis, knockout cells lacking p53, Bak, Bax or both Bak and Bax were used. Lack of p53 reduced ZM-induced apoptosis and breakdown of DeltaPsim, while lack of Bak, Bax or both almost completely inhibited apoptosis and breakdown of DeltaPsim. Since no difference in apoptosis induction was detectable between HCT-116 cells lacking Bak, Bax or both, apoptosis induction depended non-redundantly on both Bak and Bax. Phenomenally, ZM induced notable polyploidization in all examined cells, especially in p53-/- cells. A correlation between polyploidization and apoptosis was observed in wild-type, and also in p53-/- cells, albeit with a modest extent of apoptosis. Moreover, in Bak-/-, Bax-/- and Bak/Bax-/- cells apoptosis was totally inhibited in spite of the strongest polyploidization, suggesting apoptosis may be a secondary event following polyploidization in HCT-116 cells. Thus ZM-induced apoptosis depends not only on polyploidization, but also on the intracellular apoptotic signaling.