Cell death mechanisms include accidental cell death (ACD) and regulated cell death (RCD)
[1]. External extreme temperature and pressure, chemical stress, and osmotic pressure exceeding the capability of the cell to restore a physiologic condition lead to ACD. This type of cell death is an uncontrolled process in which the hallmark is the cell membrane rupturing, causing the spillover of the cytoplasm into the extracellular environment. Alterations in the cell membrane and the release of cellular content are also typical features of RCD
[1][2]. RCD is controlled by molecules of specific signal cascades governing biochemical, morphological, and immunological consequences in a regulated and programmed manner. RCD guides pivotal steps of physiological and pathological processes
[1]. Besides the most studied apoptosis, RCD includes autophagy, pyroptosis,
ferroptosis, and necroptosis (NEC)
[3][4][5][6][7][8][9]. RCD is a concept in continuous evolution. Very recently, PANoptosis, which embraces pyroptosis,
apoptosis, and NEC, has emerged as a new RCD mode controlled by the aggregation of specific effector molecules leading to the formation of the PANoptosome
[10][11]. The role played by PANoptosis in tumors was reported in colorectal cancer
[10].
2. Apoptosis and Necroptosis: Overview on Molecular Mechanisms
In 2005, Degterev and coworkers described NEC as a peculiar type of necrosis controlled by a specific signaling cascade that is inhibited by necrostatin 1 (Nec1)
[22]. Taking advantage of a siRNA-mediated approach, receptor-interacting serine/threonine protein kinase 1 (RIP1) was identified as the target of Nec1. In 2018, such a type of programmed necrosis, not dependent on the activation of cysteine-aspartic proteases (caspases), and mainly governed by RIP1, receptor-interacting serine/threonine protein kinase 3 (RIP3) and substrate mixed lineage kinase domain like pseudokinase (MLKL) was called NEC
[23]. NEC controls cell homeostasis, neurodegeneration, infectious diseases, inflammation, cardiovascular and skin diseases, acute kidney injury, and cancer
[24].
Cells undergoing NEC and necrosis show similar behavior
[25][26]. Although trauma, toxic stress, and infection are responsible for both necrosis and NEC, the latter differs from necrosis as a finely controlled type of RCD. Specific signaling pathways, including TNRF1 and RIP, govern NEC induction via the formation of the necroptosome complex. No important differences in morphology characterize cells undergoing NEC and necrosis (e.g., organelle and cell swelling, loss of membrane integrity, and the release of intracellular content). Moreover, both cell death mechanisms induce mitochondrial dysfunction with mitochondrial membrane collapse. However, only during NEC the production of reactive oxygen species (ROS) and the release of apoptosis-inducing factor (AIF) occur. Proinflammatory response characterizes both NEC and necrosis. Nec1 counteracts only NEC, while it is ineffective on necrosis.
Tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) are death-inducing molecules acting on FasL/TNF receptor (TNFR)1, which can induce either apoptosis or NEC depending on caspase functionality (
Figure 1)
[27][28].
Figure 1. Schematic representation of cellular pathways involved in necroptosis. TNF-α, tumor necrosis factor α; FasL, Fas ligand; TRAIL, TNF-related apoptosis-inducing ligand; TNFR1, TNF receptor 1; RIP1/3, receptor-interacting serine/threonine-protein kinase 1/3; MLKL, mixed lineage linase domain like pseudokinase; TRADD, TNF receptor associated death domain protein; FADD, Fas-associated death domain; TRAF2, TNF receptor associated factor 2; cIAP1/2, cellular inhibitor of apoptosis proteins 1/2; TAK complex formed by TAK1, TAB2 and TAB3; IKK complex, formed by IKKα and IKKβ. Figure was prepared using tools from Servier Medical Art (
http://www.servier.fr/servier-medical-art, accessed on 15 March 2022).
Cells undergoing apoptosis arrest their growth and division, and activate plasma membrane modifications, chromatin condensation, and DNA fragmentation (DNA ladder), and cell shrinkage favoring the formation of apoptotic bodies engulfing the surrounding environmental phagocytes (Table 1). During NEC, a cell increases its volume, and shows organelle shrinkage and plasma membrane disintegration. Cellular contents, including damage-related pattern molecules (DAMPs), high-mobility group box 1 (HMGB1), and mitochondrial DNA, are released into the extracellular environment. Conversely to apoptosis, which is characterized by limited DMAP release, the massive cytoplasm spillover observed during NEC induces an important immune response.
Table 1. Comparison of necroptosis-, necrosis-, and apoptosis-regulated cell death properties.
Both membrane death receptors activation (via TNF-α, TRAIL, FasL) and intracellular stimulation (via genetic damage, hypoxia, osmotic stress, or starvation) trigger apoptosis
[29]. The latter, known as intrinsic (e.g., mitochondrial) apoptosis, depends on mitochondria-secreted factors including cytochrome c, SMAC/DIABLO, HrtA2/Omi, and AIF that stimulate apoptosis. Intracellular cytotoxic stimuli activate BH3-only proteins leading to BAX and BAK activation, inducing the formation of
mitochondrial permeability transition pores on the outer mitochondrial membrane. This feature stimulates the release of cytochrome c into the cytoplasm and its interaction with apoptotic protease activating factor 1 (APAF1) favoring the formation of the apoptosome. This molecular complex in turn stimulates procaspase 9, 3, and 7 cleavage leading to the dysfunction or disruption of cellular components and apoptosis.
The interaction of membrane death receptors and death ligands initiates extrinsic (e.g., extracellular) apoptosis. The formation of death-included signaling complex (DISC), which contains TNFR1, Fas-FasL, death receptor 4 (DR4), death receptor 3 (DR3), and tumor necrosis factor superfamily 10 (known as TRAIL/Apo2L), is the first step of the pathway
[30]. The interaction of Fas with FasL induces conformational changes of the complex that favors the exposure of Fas death effector domains, facilitating the recruitment of adaptor FADD, and the activation of procaspase 8 and 10. In turn, the cleavage or activation of procaspases 8 and 10 stimulates procaspase 3 and 7 cleavage leading to the enzymatic activation of target proteins and apoptosis
[30].
Similar to apoptosis, NEC is triggered by the interaction of death receptors (Fas, TNFR1, and TNFR2) with death ligands (TNF-α, FasL, and TRAIL). In apoptosis, NEC induction requires the inhibition of caspase signaling pathways (
Figure 1)
[22][31]. The interaction of death ligands with membrane receptors provokes conformational changes that induce the recruitment of TNF receptor-associated death domain protein (TRADD), RIP1, tumor necrosis factor-related factor 2 (TRAF2), the cellular inhibitors of apoptosis cIAP1/2 and ubiquitination complex, favoring the formation of a TNFR complex (complex I)
[32]. This protein complex is responsible for cell fate, i.e., death or survival, depending on the activation of downstream pathways via ubiquitination and phosphorylation activities. Through the activation of NF-kB, complex I stimulates cell survival and promotes inflammation
[33][34][35]. The ubiquitination status of RIP1 favors the recruitment of transforming growth factor β kinase 1 (TAK1), TAK1-binding protein 2 (TAB2), and TAB3 (TAK complex), and the IKK complex (IKKα and IKKβ). In this condition, RIP1 functions as a scaffold protein that favors the activation of NF-kB (e.g., cIAP2-mediated degradation of NF-kB inhibitory protein Ikβ of the IKK complex), which, following nuclear translocation, stimulates cell survival (e.g., activation of antiapoptotic genes including cFLIP) and inflammation. Apart from
enzymatic activity, cFLIP is highly homologous to caspase 8, and interacting with caspase 8 inhibits its activation, favoring cell survival
[36][37][38][39].
The ubiquitination status of complex I elements stabilizes its localization on the cell membrane, impeding the formation of complex II and favoring cell survival
[38]. Reduced RIP1 ubiquitination favors the formation and the cytoplasmic localization of complex II (IIa, IIb, and IIc/necrosome) leading to apoptosis or NEC
[37][38][39]. The formation of complex IIa, which contains procaspase 8, TRADD, and FADD, allows for caspase 8 and subsequently caspase 3 activation, leading to apoptosis. The inhibition of RIP1 polyubiquitination in complex I favors its cytoplasmic localization and the interaction with FADD and procaspase 8, leading to the formation of complex IIb (RIP1/FADD/procaspase 8). Regarding complex IIa, the activation of caspase 8 induces caspase 8-dependent apoptosis as well.
Phosphorylated RIP1 (p-RIP1) interacts with RIP3 and favors its phosphorylation (p-RIP3). Following the inhibition of caspase 8 activation (e.g., via cFLIP), the p-RIP1/p-RIP3 complex recruits MLKL favoring the formation of complex IIc/necrosome. Phosphorylation on threonine 357/serine 358 of MLKL provokes its translocation on the plasma membrane leading to the activation of sodium and calcium channels, and inducing membrane dysfunction, cell rupture, and NEC execution
[40][41][42].
The levels of cFLIP influence the switch apoptosis/NEC. Elevated cFLIP expression inhibits caspase 8 by forming heteromeric complex caspase 8–cFLIP, thereby blocking apoptosis dependent on complexes IIa and IIb.
Besides plasma membrane localization, the RIP1/RIP3/MLKL complex translocates to the mitochondrial membrane provoking mitochondrial dysfunction (e.g., mitochondrial permeability transition), and inducing the production of ROS and the activation of mitochondrial phosphoglycerate mutase 5 (PGAM5). Activated PGAM5 recruits mitochondrial dynamin-related protein (DRP1) leading to mitochondrial fragmentation
[43]. ROS are crucial for NEC, and some evidence links RIP3 to ROS levels. RIP3 stimulates mitochondrial enzymes, including pyruvate dehydrogenase complex, glutamine synthetase, and glutamate dehydrogenase, promoting ROS production and NEC
[44]. However, the involvement of mitochondria in NEC is controversial. Indeed, cells depleted of mitochondria through forced mitophagy undergo NEC, implying that mitochondria or mitochondrial metabolism are not essential for this type of RCD
[45]. This scenario reveals that the kinase-dependent NEC can be viewed as a rescue mechanism of cell death functioning when caspase-mediated apoptosis fails
[26][40][46].
3. Necroptosis and Necroptosis Inducers in Prostate Cancer
Though reported since 2005, the induction of NEC for fighting PCa is still scarcely considered. RIP3 is decreased in PCa specimens and in cell lines (e.g., PC3, DU145, and 22Rv1). Moreover, in advanced PCa samples, RIP3 is significantly downregulated compared to normal tissue
[47]. The reduced expression of RIP3 correlates with tumor size and prostate-specific antigen (PSA) levels
[48]. High or normal levels of RIP3 counteract disease progression by favoring MLKL phosphorylation, leading to NEC. The overexpression of RIP3 in PC3 and 22Rv1 cell lines induces G2 cell-cycle arrest, reduces cell survival, proliferation, invasion (increased MMP2 and MMP9, vimentin, fibronectin, and N-cadherin), and favors NEC (phosphorylation of MLKL)
[47]. In addition, compared to corresponding parental tumors, tumorigenesis and tumor volume are reduced in mice bearing RIP3-overexpressing PC3 and 22Rv1 tumors
[47].
The seven members’ sirtuin (SIRT) family of proteins shows enzymatic activity implicated in numerous cellular functions, including DNA damage repair, senescence, metabolism, and tumor development and progression. Two enzymes of this family, SIRT3 and SIRT6, are involved in NEC induction in PCa. The increased expression of SIRT3 and SIRT6 is significantly associated with nodal metastasis and Gleason score, and negatively impacts on overall patient survival
[49]. Therefore, the silencing of SIRT3 and SIRT6 in LNCaP, DU145, and PC3 cells significantly reduces cell growth, which is paralleled by RIP3-mediated NEC induction (e.g., increased phosphorylation of RIP3 and MLKL). These results were confirmed in vivo in SIRT3 and SIRT6 silenced LNCaP tumor xenografts that showed reduced tumor volume compared to the corresponding tumors expressing normal enzyme levels.
Compounds that induce NEC are receiving considerable attention, and several molecules, though not specific NEC inducers, showed antitumor activity on PCa cell lines (Figure 2 and Figure 3). Available compounds are: (i) of natural origin, (ii) synthetic and semisynthetic small molecules, and (iii) selenium and selenium-based nanoparticles (SeNPs).
Figure 2. Chemical structures of compounds of natural origin that induce necroptosis in prostate cancer cell lines. These compounds stimulate necroptosis by inducing (i) the dysfunction of mitochondrial membrane potential,
curcumin, and arctigenin; (ii) cell-cycle arrest, shikonin; (iii) FADD stabilization, ophiopogonin D’; (iv) modulation of multiple necroptosis pathways, deslanoside; (v) reactive oxygen species, resveratrol; (vi) RIP1/3 activation following combination with autophagy inhibitors, artepillin C.
Figure 3. Chemical structures of synthetic and semisynthetic small molecules that induce necroptosis in prostate cancer cell lines. These compounds stimulate necroptosis by (i) inducing mitochondrial membrane permeability, (17E)-5α-androst-3-en-17-one oxime and (17E)-androst-4-en-17-one oxime; (ii) inducing reactive oxygen species and cell-cycle arrest, 17-cyanopyridine pregnenolone; (iii) inducing cell growth inhibition following combination with necrostatin, HUHS1015; (iv) inhibiting Polo-like kinase 1, BI2536; (v) inhibiting kinases, sorafenib.
4. Ongoing Clinical Trials Containing Necroptosis Inducers in Prostate Cancer
Some compounds reported here are contained in clinical trials enrolling patients suffering from PCa (
Table 2,
www.clinicaltrials.gov, accessed on 23 February 2022).
Table 2. Clinical trials containing necroptosis inducers ongoing in prostate cancer a.
a Clinical data are from ClinicalTrials.gov, a service of the U.S. National Institutes of Health.
www.clinicaltrial.gov (accessed on 23 February 2022). nd, not defined; na, not available.
Curcumin alone (NCT03769766, phase III; NCT03211104; NCT02064673, phase III) or in combination with piperine (NCT04731844, phase II), ursolic acid (NCT04403568, phase I), vitamin D, omega 3 and turmeric (NCT03290417), taxotere (NCT02095717, phase II) and radiotherapy (NCT01917890; NCT02724618, phase II; NCT03493997, phase II) is studied in PCa patients. Despite studies implying the measure of different biomarkers, mostly PSA, no specific markers of NEC are considered. Only trials NCT04403568 and NCT01917890 contemplate the evaluation of TNF-α and NF-kB. These two molecules are involved in NEC, though they are not specific biomarkers.
Polyphenon E was included in numerous studies as supplementary diet in subjects at high risk of developing PCa (prostatic hyperplasia) or in postsurgery patients (NCT00596011, phase II; NCT00676780, phase II; NCT01340599, phase II; NCT00459407, phase I; NCT00253643; NCT04597359, phase II). Polyphenon E-based studies contemplated the measure of numerous biological markers (e.g., PSA, Ki67, Bcl2, Cyclin D, p27, VEGF, CD31, MMP2 and 9, IGF1, HGF, FASN) but not specifically related to NEC.
Sorafenib alone (NCT00090545, phase II; NCT00694291, phase II; NCT00466752, phase I; NCT00093457, phase II) or in combination with leuprolide acetate or bicalutamide (NCT00924807, phase I-II), docetaxel (NCT00589420, phase II; NCT00619996, phase II), taxotere (NCT00405210, phase I) mitoxantrone (NCT00452387, phase II) or gleevec (NCT00424385, phase I) is investigated in PCa patients. In all these studies, PSA is used as biological marker. The trial NCT00619996 contemplates the measure of p-ERK and VEGF-R2 as well. Interestingly, in trial NCT00466752, samples from PCa patients collected before and after sorafenib administration were analyzed for gene and protein expression by microarray, Western blot, and immunohistochemistry. In this context, the levels of p-ERK, p-AKT, p-S6- kinase and caspase-3 expression, and the Ki67 index were considered. Though the study is completed, results are not yet available. In sorafenib-containing clinical trials, specific biomarkers of NEC are also not measured.
Selenite in combination with docetaxel (NCT01155791, phase I) or with radiotherapy (NCT02184533, phase I) is studied in PCa-affected patients. In these studies, PSA was the only considered biomarker.