“(Not) All (Dead) Things Share the Same Breath”: Identification of Cell Death Mechanisms in Anticancer Therapy

The main purpose of current anticancer therapies is to induce the specific removal of tumor cells without compromising the overall patient health. It soon became clear that the specific modality of cell death triggered could be of key importance to interpret treatment-related side effects, to understand therapeutic failure, and to help in generating patient cohorts based on their predicted sensibility to a specific agent (1–3). Our knowledge about cell death mechanisms (particularly at a molecular level) has evolved into a sophisticated body of knowledge with its own jargon, sometimes difficult to understand for nonspecialists. Since the beginning of the 21st century, a group of major research leaders in the field have been proposing and upgrading comprehensive nomenclature guidelines based on reliable markers and new developments in the field of cell death (4, 5).

However, those efforts have not stopped the increasing number of new cell death mechanisms described, some of them circumscribed to a very particular stimulus or cell type. We lack a proper characterization of which molecular modules are basal and which are derivates, meaning that nomenclature does not represent an attempt to provide a biologic classification of cell death modes based on phylogenetic relationships. Nevertheless, as an organized combination of morphologic and molecular descriptors, cell death nomenclature rules are the best frame we have to study cell demise (6).

In the development of the nomenclature consensus, there is a clear trend to embrace new molecular descriptors as the unique determinants for cell death mechanism attribution (4, 6). Comprehensive efforts have been carried out to organize the growing number of cell death players into hierarchically distributed signaling pathways (5, 7, 8). Despite recognizing the importance of the newly available biochemical tests, we advocate here for a cell death characterization without denying the old morphologic criteria. This defense of morphologic criteria has not to be viewed as a reductionist approach. Taxonomists use genetics or molecular characters to identify the varieties within a clearly recognizable genus and species. We consider that the important biochemical differences reported do not invalidate a higher division of cell death mechanisms, in which apoptosis and necrosis are considered to be major types, without denying the existence of several other minor or particular mechanisms.

We concisely present the current knowledge about cell death mechanisms involved in cancer therapy. However, the aim of the present review is to provide a guide about how to face accurately the description of cell death mechanisms when challenging the tumor cell physiology with different stimuli. For reasons of brevity, we focus on in vitro assays, as an accurate in vitro characterization could help to better design in vivo preclinical models.

The major form of cell death is called apoptosis because the dismantling of a cell in tiny detached pieces reminded Kerr, Willie, and Currie the leaves falling from the autumn trees (9). Apoptosis is a highly organized physiologic process by which a cell demolishes itself (10, 11). Within the body, apoptosis occurs continuously under physiologic conditions. Almost every step during apoptosis is finely orchestrated; it is a “clean” kind of death as the remnants (apoptotic bodies) are engulfed and recycled by neighboring cells, and generally it does not trigger inflammation (12, 13). In contrast, in cell cultures, apoptotic bodies are frequently identifiable for days after the challenge.

A complex molecular organization ensures the apoptotic demise. It is highly organized in different steps; any level amplifies the previous one, but internally undesired activations are prevented by sophisticated balances with inhibitor elements (10, 11). Multiple external signals and inner stimuli could serve as triggers. Many mediators have been identified, but the Bcl-2 family of proteins still keeps its central role, determining the initialization of the final apoptotic phase, executed by the caspase family of proteases (12).

The so-called mitochondrial apoptosis pathway is involved in reacting to stress or environmental signals (10, 14). This intrinsic pathway is characterized by the role of the mitochondria as encounter point of most of its initiators and mediators. The mitochondrial outer membrane is the place where the members of the three branches of the Bcl-2 family play their role. The multidomain proapoptotic members of the family (Bax and Bak) control this pathway by generating a pore at the inner mitochondrial membrane. Through this pore, signaling proteins (such as cytochrome c) are released into the cytoplasm, leading to the activation of executioner caspases. For this pore to be formed, antiapoptotic Bcl-2 proteins must be inactivated by BH3-only proteins, which also activate Bax and Bak. BH3-only proteins act as real sentinels of key cellular processes (ranging from correct gene expression to adequate cytoskeleton organization) and are activated in different ways (production, release, modification, etc.; ref. 15).

A second major apoptotic pathway is induced by the death receptors. Death ligands, which are cytokines of the TNF superfamily, interact with transmembrane receptors whose inner domains serve as sites for the recruitment of different adaptor proteins. The signaling cascade leads to the activation of caspase-8, the main mediator of this pathway (16). Caspase-8 cleaves and directly activates caspase-3 or it engages the mitochondrial pathway via the BH3-only protein Bid.

Some authors distinguish other apoptotic pathways, but those are rather trigger-specific variants than actual new mechanisms. An example is detachment-induced apoptosis, which has been traditionally termed anoikis (6). However, as with other types of stress, the onset of cell death is produced through BH3-only proteins and the mitochondrial pathway (17).

In clear contrast with the attention paid to apoptosis during the last decades, necrosis has suffered continuous ins and outs from the primary scientific focus. Long regarded as a mere accidental cellular explosion, it is now widely accepted that some forms of necrosis are regulated and executed by specific molecular machineries (8, 18). Furthermore, our knowledge about regulated necrosis is undergoing a boom in the recent years with a series of newly described cell death mechanisms (such as necroptosis and pyroptosis) that share the necrotic phenotype and basic biochemical descriptors (loss of plasma membrane barrier, water influx, swelling, and cell rupture with cytoplasmic content release). Despite these general similarities, the individualization of each regulated necrosis type is rooted in important signaling particularities (19–21).

Among the phenomena described that do not fit into morphologic apoptosis or necrosis, we want to comment briefly the particular case represented by “autophagic cell death” (ACD). ACD describes circumstances in which autophagy exert such an important role in cell death that its inhibition impedes its onset. The exact nature and relevance of ACD have been disputed for long, but the most recent findings point to some relevance in therapeutically induced cell death (5, 22).

The correct evaluation of cell death in every experimental setting related to cancer research is a must. A large number of valuable techniques could provide insights into cell death mechanisms, from microscopic observations to high-throughput quantitative assays (7, 18, 23). Our aim is to provide a series of guidelines of “good practices” for a reliable, quick assessment of cell death.

The first test comes with the simple observation of the cells under the cell culture room's bright-field microscope (Fig. 1). Observations will indicate whether the treated cells are actively dying (accumulation of cell remnants or formation of a population of detached and shrunk cells), blocked in their cell cycle (changes in the mitotic fraction or the rising of a giant cell population), or simply not growing as much as the controls. The isolation and staining of the detached cell population with DNA-binding dyes (such as DAPI, PI, or Hoechst-33342) could reveal the presence of typically apoptotic condensed and fragmented nuclei (karyorrhexis). Afterward, cationic dye exclusion assays (such as Trypan blue) are useful to quantify cell death scores.

An accurate observation could provide better information than cell-impedance sensors or metabolic proliferation tests (7). Impedance sensors can be fooled when cell morphology or plasma membrane potential changes drastically. MTT-like assays are indirect tests based on metabolic processes that could be affected by other cellular processes and be confused with growth arrest (and they are also quite susceptible to mycoplasma contaminations; ref. 23). However, once the exact nature of the induced process (cell death triggering vs. proliferation arrest) has been identified, both methods can provide reliable quantifications.

Despite powerful, it is important to understand that morphologic criteria are no longer sufficient as descriptors of cell death modalities. Researchers should provide valid biochemical data (Fig. 1) before attributing the observed phenotype to a mechanism of cell death (5, 6). To distinguish apoptosis from necrosis, the addition of caspase inhibitors (for instance Z-VAD-fmk or Q-VD-OPh) could provide useful information, as they should inhibit apoptotic cell death features and at least postpone membrane permeabilization, although not indefinitely (24, 25). Another way for a clear mitochondrial apoptosis identification is to test our experimental conditions in a model that overexpresses one of the multidomain antiapoptotic Bcl-2 proteins: cell death could not be possible or the phenotype should clearly change into a necrotic one (7). The best way to assess the precise involvement of the cell death receptors is, in our opinion, to repress its expression or signaling in the cell cultures when the treatment is performed. This might be done by using chemical inhibitors, neutralizing antibodies or gene-targeted means.

The next logical step is trying to quantify more finely the extent of cell death. Annexin V–propidium iodide (AnnV-PI) measurements by flow cytometry are probably the most common assay. This test is performed in living cells, and it is based on the flip of phosphatidyl-serine (PS) residues to the outer layer of the plasma membrane (recognized by AnnV) and the influx of PI to the cells that have lost the plasma membrane barrier. Thus, the test is able to discriminate among early apoptotic cells (AnnV⁺ PI⁻), necrotic cells (AnnV⁻ PI⁺), and late apoptotic cells (AnnV⁺ PI⁺). The major setback of this method is the possibility that the double positive staining (both AnnV and PI) could really represent primary necrotic cells (AnnV recognizing inner PS residues) rather than apoptotic cells that underwent secondary necrosis (26). Mitochondrial potential sensitive vital dyes (such as TMRM, DiOC₆(3), and JC-1) can be used in a similar manner. Cells with low mitochondrial staining and impermeable to PI could be considered early apoptotic events (26, 27). However, caution is advised as some forms of necrosis can present with mitochondrial depolarization (28). Quantification of the sub-G₁ fraction in cell-cycle profiles (with PI or other DNA-binding dyes) is an easy way to score apoptotic events but should be discouraged when the treatment courses with transient or long-standing episodes of G₂–M blockages or even sustained tetraploidization. Those cells start to degrade their genomic material when they possess a greater amount of DNA, so it takes them more time to reach the sub-G₁ levels. In any case, it would be wise to make different endpoint quantifications to understand better the dynamics of cell death. When accessible, new flow-cytometry platforms equipped for simultaneous imaging of events could provide really useful data (29).

Biochemical characterization of apoptosis can be enhanced by the use of common techniques, such as Western blotting for the detection of typically apoptotic cleaved proteins (PARP and caspase-3) or fluorescent immunolabeling and imaging of proteins like cytochrome c (contained in the mitochondria in healthy cells and released from them upon apoptotic commitment) or Bax (the other way around). As the major final executioner of the apoptotic demise, different caspase activation assays are available for microscopy, Western blotting, colorimetry, or flow cytometry (23). Morphologic analysis can be completed with more powerful imaging by electron microscopy, showing in detail the marked differences between the solid apoptotic cells and the ghostly appearance of the necrotic remnants (7, 18).

Much of the literature still focuses on apoptotic detection and description, but there is a recent increase in the interest of assessing necrosis, particularly regulated necrosis (8). Morphologically, necrosis is clearly identified under microscopic observations, especially in transmission and scanning electron microscopy (Fig. 1). Swollen and disorganized cytoplasm (oncosis) and the perforated plasma membrane are easily visualized as major hallmarks of late necrosis (7, 18). When the involvement of necrosis is supposed, early endpoint observations should be performed, as necrosis is a rather quick mechanism of cell demise. Early necrosis can be recognized under the inverted microscope by the formation of bubbles in the plasma membrane by the water influx resulted from the injury. Those bubbles are distinct from the apoptotic blebs, as the necrotic ones are mainly aqueous in nature whereas the apoptotic blebs are full of cellular content. Bubbles tend to converge and form one single structure as water continues to enter the cell, before it detaches, breaking the plasma membrane apart (18). Biochemically, necrosis detection has been performed by the measurement of cytosolic enzymes released to the culture media when the plasma membrane is broken. The recently described processes of regulated necrosis open the possibility of using specific inhibitors, such as necrostatin-1, to identify necroptosis (the regulated necrosis pathway that requires RIPKs and MLKL; refs. 7, 23).

There is also an increasing attention in the community to find better ways to clearly characterize the events of ACD, although not many inducers have been described (30). To clearly identify cell demise as ACD, there should be an increase in autophagic flux accompanied with a necrotic phenotype (as autophagy can lead to apoptosis) and vacuolated cytoplasm. Inhibition or silencing of autophagy-related proteins should prevent this form of cell death (6, 30).

Finally, we encourage the community to include long-term experiments in their in vitro settings. Even when minimal, the surviving fraction of cells could give rise to a resistant subpopulation by simple selection of preexisting characters (i.e., expression of membrane pumps able to reject the uptake of the challenging drug) or by enhanced evolutionary change (i.e., induction of mitotic catastrophe leading to major genomic rearrangements) stimulated by the treatment itself (31, 32). A simple washout of the cells after a determined endpoint, followed by observation of cell culture regrowth and another repetition of the experiment, could provide clues about the ability of tumor cells to escape cell death. The measurement of DNA content, karyotyping, or gene expression assays will generate valuable data about how the surviving cells manage to evolve and resist.

Cell death induction is the main aim of almost every anticancer therapeutic approach either approved, under evaluation, or still in research. However, not all cell deaths share the same characters, and the differences could be crucial. Necrosis induction is generally very effective, but the release of cytoplasmic content leads to undesired inflammation processes. Apoptosis is comparatively clean, but its highly regulated onset leaves a wide space for therapeutic resistance (1, 3). In a more general point of view, the knowledge of the processes involved in the induction of tumor cell clearance will help to provide susceptibility markers and to stratify patients. One example of the possibilities of the detailed characterization of cell death mechanisms is the identification of certain apoptotic processes that could combine their noninflammatory nature with the precise exhibition of signals that lead to lymphocyte recruitment and immune response. This process, so-called immunogenic cell death, is thought to provide tremendous progress in future anticancer research and treatment (3).

It is crucial to unveil the precise molecular signaling networks underlying cell death phenotypes, but that effort has to be rooted in a complete assessment that includes a detailed description of the sequence of events involved both in the short and in the long term. Following that way, researchers will have more reliable data, planning much better the animal experiments and moving toward preclinical experimentation based on a more solid ground.

No potential conflicts of interest were disclosed.

The authors apologize to those colleagues whose work could not be cited.

S. Rello-Varona is a MarieCurieCOFUND-BeatriuDePinòs Researcher (The European Union 7th Framework Program for R+D and the Generalitat de Catalunya's Department for Economy and Knowledge: Secretary for Universities and Research). D Herrero-Martín is funded by Asociación Española contra el Cáncer-AECC. Studies related to the topic of this review are supported by Fondo de Investigaciones Sanitarias (FIS) grants PI13/00139 (C. Muñoz-Pinedo) and CP06/00151, CES12/021, and PI11/00038 (O.M. Tirado).