Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage.

Macroautophagy (hereafter autophagy) is a multistep intracellular catabolic process with pleiotropic implications in cell fate. Attending to its activation, autophagy can be classified into inducible or constitutive. Constitutive, or basal autophagy, unfolds under nutrient-replete conditions to maintain the cellular homeostasis. Autophagy inhibitory drugs are powerful tools to interrogate the role of autophagy and its consequences on cell fate. However, 3-methyladenine and various of these compounds present an intrinsic capacity to trigger cell death, for instance the broadly-employed 3-methyladenine. To elucidate whether the inhibition of basal autophagy is causative of cell demise, we have employed several representative compounds acting at different phases of the autophagic process: initiation (SBI0206965 and MHY1485), nucleation (3-methyladenine, SAR405, Spautin-1 and Cpd18), and completion (Bafilomycin A1 and Chloroquine). These compounds inhibited the basal autophagy of MEF cultures in growing conditions. Among them, 3-methyladenine, SBI-0206965, Chloroquine, and Bafilomycin A1 triggered BAX- and/or BAK-dependent cytotoxicity and caspase activation. 3-methyladenine was the only compound to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. 3-methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis. Autophagy-deficient Fip200-/- MEFs displayed an increased sensitivity to activate caspases and to undergo cell death in response to 3-methyladenine. The cytotoxicity induced by 3-methyladenine correlated with a massive DNA damage, as shown by γ-H2A.X. This genotoxicity was observed at 10 mM 3-methyladenine, the usual concentration to inhibit autophagy and was maximized in Fip200-/- MEFs. In sum, our results suggest that, in growing conditions, autophagy acts as a protective mechanism to diminish the intrinsic cytotoxicity of 3-methyladenine. However, when the cellular stress exerted by 3-methyladenine surpasses the protective effect of basal autophagy, caspase activation and DNA damage compromise the cell viability.

Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation.

Autophagy is generally regarded as a mechanism to promote cell survival. However, autophagy can occasionally be the mechanism responsible of cell demise. We have found that a concomitant depletion of glucose, nutrients and growth factors provoked cell death in a variety of cell lines. This death process was contingent upon caspase activation and was mediated by BAX/BAK proteins, thus indicating its apoptotic nature and the engagement of an intrinsic pathway. In order to abrogate autophagy, 3-methyladenine (3-MA), BECLIN-1 siRNA and Atg5 knock-out (Tet-Off type) approaches were alternatively employed. Irrespective of the procedure, at short times of starvation, we found that the ongoing autophagy was sensitizing cells to the permeabilization of the mitochondrial outer membrane (MOMP), caspase activation and, therefore, apoptosis. On the contrary, at longer times of starvation, autophagy displayed its characteristic pro-survival effect on cells. As far as we know, we provide the first experimental paradigm where time is the only variable determining the final outcome of autophagy. In other words, we have circumscribed in time the shift transforming autophagy from a cell death to a protection mechanism. Moreover, at short times, starvation-driven autophagy exacerbated the apoptotic cell death caused by several antitumor agents. In agreement with this fact, their apoptotic effects were greatly diminished by autophagy inhibition. The implications of these facts in tumor biology will be discussed.

Pharmacological modulation of reactive oxygen species in cancer treatment.

Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective antioxidant systems like glutathione. Current anticancer therapies focus on the cancer dependence on oncogenes and non-oncogenes. Tumors trigger mechanisms to circumvent the oncogenic stress and to escape cell death. In this context we have studied 2-phenylethinesulfoxamine (PES), which disables the cell protective mechanisms to confront the proteotoxicity of damaged and unfolded proteins. Proteotoxic stress is increased in tumor cells, thus providing an explanation for the anticancer selectivity of PES. In addition, we have found that PES induces a severe oxidative stress and the activation of p53. The reduction of the cell content in glutathione by means of L-buthionine-sulfoximine (BSO) synergizes with PES. In conclusion, we have found that ROS constitutes a central element in a series of positive feed-back loops in the cell. ROS, p53, proteotoxicity, autophagy and mitochondrial dynamics are interconnected with the mechanisms leading to cell death, either apoptotic or necrotic. This network of interactions provides multiple targets for drug discovery and development in cancer.

2-Phenylethynesulfonamide (PES) uncovers a necrotic process regulated by oxidative stress and p53.

2-Phenylethynesulfonamide (PES) or pifithrin-µ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with l-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis.

Cell death induced by 2-phenylethynesulfonamide uncovers a pro-survival function of BAX.

PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX.

Transcriptional modulation of apoptosis regulators by roscovitine and related compounds.

Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis (PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.

BAX and BAK proteins are required for cyclin-dependent kinase inhibitory drugs to cause apoptosis.

In previous reports, we have shown in SH-SY5 cells that olomoucine and roscovitine, two inhibitory drugs of cyclin-dependent kinases, caused apoptosis independent of the extrinsic pathway. In this experimental paradigm, apoptosis was refractory to the protective effects of either Bcl-2 or Bcl-XL overexpression. We are now reporting that the failure of Bcl-XL to prevent dell death was consistent with no effect on the kinetics of caspase activation and cytochrome c release. To further characterize this issue, we have discarded a direct effect of either olomoucine or roscovitine on mitochondrial permeability transition. Moreover, we have evidence that an intrinsic pathway took place in SH-SY5Y cells by showing the mitochondrial translocation of a GFP-Bax construct on transfection and treatment with cyclin-dependent kinase inhibitory drugs. Finally, we tested the effect of olomoucine and roscovitine on wild-type, bax-/-, bak-/-, and double bax-/-bak-/- mouse embryonic fibroblasts (MEF). In wild-type MEFs, both drugs induced cell death by apoptosis in a dose-dependent manner. In bax-/-, bak-/-, and, particularly, double bax-/-bak-/- MEFs, we observed the inhibition of apoptosis. In conclusion, olomoucine and roscovitine caused apoptosis through an intrinsic pathway, with Bax and Bak proteins being involved.

7-Bromoindirubin-3'-oxime uncovers a serine protease-mediated paradigm of necrotic cell death.

The new 7-bromoindirubin-3'-oxime (7BIO) compound induces caspase-independent cell death in all cell lines tested to date. Irrespective of the cell line, a 25 microM treatment for 24 h is lethal for the entire cell population. In SH-SY5Y and Jurkat cells, 7BIO (25 microM) was found to collapse the mitochondrial transmembrane potential (DeltaPsi m) at only 2-3 h of treatment. Concomitantly mitochondria swelled, cristae disrupted and, after 9 h, external cell membranes ruptured. In addition, endoplasmic reticulum dilated and, unexpectedly, the acute cytoplasmic destruction yielded isolated nuclei with preserved morphology and DNA integrity. Furthermore, the process was independent of both Bax and Bak, since cell viability and DeltaPsi m decayed indistinguishably in double Bax-/-Bak-/- mouse embryonic fibroblasts (MEFs) and their wild type counterparts. Pharmacological inhibition of the mitochondrial permeability transition pore (MPTP) did not prevent 7BIO-induced DeltaPsi m loss in none of the aforementioned cell lines. Caspase-independent inducers of cell death like AIF (Apoptosis Inducing Factor), cathepsins and calpains were not involved. Only the chemical inhibitors of serine proteases and, particularly, AEBSF afforded a significant protection thus suggesting a process regulated by this type of enzymes. As far as we know, these features are quite unique once taken together. Therefore, we propose 7BIO is triggering a specific type of necrotic cell death. Finally, the cytotoxicity of 7BIO on apoptosis-resistant cells like double Bax-/-Bak-/- MEFs seems of great interest envisaging cancer therapy.

(R)-roscovitine (CYC202, Seliciclib) sensitizes SH-SY5Y neuroblastoma cells to nutlin-3-induced apoptosis.

In this study, we have analyzed the consequences, on several neuroblastoma cell lines, of combined treatments with (R)-roscovitine (CYC202, Seliciclib), a CDK inhibitory drug, and nutlin-3, a p53 activating drug. Both compounds were found to synergize, causing significant levels of apoptosis in cultured cells when combined at sublethal concentrations. In SH-SY5Y cells, Bcl-XL protein overexpression protected from apoptosis induced by either nutlin-3 alone or the (R)-roscovitine plus nutlin-3 association but failed to prevent apoptosis triggered by (R)-roscovitine alone. Moreover, Western blot studies showed that (R)-roscovitine increased nutlin-3-mediated p53 stabilization. Therefore, we conclude the contribution of (R)-roscovitine to the synergism is basically the sensitization of SH-SY5Y cells to the action of nutlin-3 on p53. The relevance of this pharmacological synergism with respect to the treatment of neuroblastoma is discussed.

Caspase 8/10 are not mediating apoptosis in neuroblastoma cells treated with CDK inhibitory drugs.

Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK inhibitory drugs. Exp. Cell Res. 295 9-24.], which depends on either Caspase 8 or Caspase 10 activation. However, neither Caspase 8 nor Caspase 10 is expressed in SH-SY5Y cells because of gene silencing. Upon Olomoucine or Roscovitine treatment, no re-expression of Caspase 8 or Caspase 10 was found. Therefore, in SH-SY5Y cells, this type of drugs is not triggering a canonical, Caspase 8/10-mediated, extrinsic apoptotic pathway.

Cell differentiation, caspase inhibition, and macromolecular synthesis blockage, but not BCL-2 or BCL-XL proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK inhibitory drugs.

Olomoucine and Roscovitine are two ATP-competing compounds described as specific inhibitors of cyclin-dependent kinases (CDK). Both drugs showed to induce apoptosis in SH-SY5Y, a neuroblastoma-derived cell line. In these cells, neither Bcl-2 nor Bcl-XL overexpression conferred any resistance to both drugs. However, a partial protective effect was detected when cells were treated with a general inhibitor of caspases (zVADfmk), cycloheximide (CHX), or actinomycin D (DAct). Interestingly, a synergism in cell protection was observed between zVADfmk and macromolecular synthesis inhibitors, thus suggesting different apoptotic pathways in distinct subpopulations of the cell culture. On the other hand, no lethality was found when cells were treated with either PD98059 or UO126. This discarded Erk1/Erk2 inhibition as the cause of apoptosis. Furthermore, SH-SY5Y cells became resistant to either Olomoucine or Roscovitine upon the induction of differentiation. This resistance correlated with the extent of differentiation and, therefore, the number of cells entering a quiescent state. In conclusion, our results seem to support a role for CDK inhibition as the cause of the apoptotic process triggered by Olomoucine and Roscovitine. In addition, we contribute to define a promising profile as anticancer drugs for both compounds, at least in the treatment of neuroblastoma.

The prevention of the staurosporine-induced apoptosis by Bcl-X(L), but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells.

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.