Using Small Molecules to Dissect Non‐apoptotic Programmed Cell Death: Necroptosis,Ferroptosis, and Pyroptosis

Genetically programmed cell death is a universal and fundamental cellular process in multicellular organisms. Apoptosis and necroptosis, two common forms of programmed cell death, play vital roles in maintenance of homeostasis in metazoans. Dysfunction of the regulatory machinery of these processes can lead to carcinogenesis or autoimmune diseases. Inappropriate death of essential cells can lead to organ dysfunction or even death; ischemia–reperfusion injury and neurodegenerative disorders are examples of this. Recently, novel forms of non‐apoptotic programmed cell death have been identified. Although these forms of cell death play significant roles in both physiological and pathological conditions, the detailed molecular mechanisms underlying them are still poorly understood. Here, we discuss progress in using small molecules to dissect three forms of non‐apoptotic programmed cell death: necroptosis, ferroptosis, and pyroptosis.     

Role of Induced Programmed Cell Death in the Chemopreventive Potential of Apigenin

The flavonoid apigenin (4′,5,7-trihydroxyflavone), which is one of the most widely distributed phytochemicals in the plant kingdom, is one of the most thoroughly investigated phenolic components. Previous studies have attributed the physiological effects of apigenin to its anti-allergic, antibacterial, antidiabetic, anti-inflammatory, antioxidant, antiviral, and blood-pressure-lowering properties, and its documented anticancer properties have been attributed to the induction of apoptosis and autophagy, the inhibition of inflammation, angiogenesis, and cell proliferation, and the regulation of cellular responses to oxidative stress and DNA damage. The most well-known mechanism for the compound’s anticancer effects in human cancer cell lines is apoptosis, followed by autophagy, and studies have also reported that apigenin induces novel cell death mechanisms, such as necroptosis and ferroptosis. Therefore, the aim of this paper is to review the therapeutic potential of apigenin as a chemopreventive agent, as well as the roles of programmed cell death mechanisms in the compound’s chemopreventive properties.     

Mechanism of Bile Acid-Induced Programmed Cell Death and Drug Discovery against Cancer: A Review

Bile acids are major signaling molecules that play a significant role as emulsifiers in the digestion and absorption of dietary lipids. Bile acids are amphiphilic molecules produced by the reaction of enzymes with cholesterol as a substrate, and they are the primary metabolites of cholesterol in the body. Bile acids were initially considered as tumor promoters, but many studies have deemed them to be tumor suppressors. The tumor-suppressive effect of bile acids is associated with programmed cell death. Moreover, based on this fact, several synthetic bile acid derivatives have also been used to induce programmed cell death in several types of human cancers. This review comprehensively summarizes the literature related to bile acid-induced programmed cell death, such as apoptosis, autophagy, and necroptosis, and the status of drug development using synthetic bile acid derivatives against human cancers. We hope that this review will provide a reference for the future research and development of drugs against cancer.     

Small Molecules Targeting Programmed Cell Death in Breast Cancer Cells

Targeted chemotherapy has become the forefront for cancer treatment in recent years. The selective and specific features allow more effective treatment with reduced side effects. Most targeted therapies, which include small molecules, act on specific molecular targets that are altered in tumour cells, mainly in cancers such as breast, lung, colorectal, lymphoma and leukaemia. With the recent exponential progress in drug development, programmed cell death, which includes apoptosis and autophagy, has become a promising therapeutic target. The research in identifying effective small molecules that target compensatory mechanisms in tumour cells alleviates the emergence of drug resistance. Due to the heterogenous nature of breast cancer, various attempts were made to overcome chemoresistance. Amongst breast cancers, triple negative breast cancer (TNBC) is of particular interest due to its heterogeneous nature in response to chemotherapy. TNBC represents approximately 15% of all breast tumours, however, and still has a poor prognosis. Unlike other breast tumours, signature targets lack for TNBCs, causing high morbidity and mortality. This review highlights several small molecules with promising preclinical data that target autophagy and apoptosis to induce cell death in TNBC cells.     

An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP). This process generated nonfunctional autolysosomes, thereby inhibiting autophagy. In addition, Atz was found to promote LMP-mediated apoptosis. Specifically, LMP induced by Atz caused release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which subsequently activated Bax to induce mitochondrial outer membrane permeabilization (MOMP). This event led to cancer cell death via caspase activation. Overall, the findings suggest that Atz will serve as a new chemical probe in efforts aimed at gaining a better understanding of the autophagic process.     

Inhibiting Erastin-Induced Ferroptotic Cell Death by Purine-Based Chelators

Ferroptosis is a cell death event caused by increased lipid peroxidation leading to iron-dependent oxidative stress and is associated with a wide variety of diseases. In recent years, ferroptosis inhibition has emerged as a novel strategy to target different pathologies. Here, we report the synthesis of two purine derivatives, 1 and 2 , for iron chelation strategy and evaluate their potency to inhibit erastin-induced ferroptosis. Both compounds showed efficient iron chelation in solution as well as in cellular environment. The crystal structure of the purine derivatives with iron demonstrated a 2 : 1 (ligand to metal center) stoichiometry for iron and purine derivative complexation. The synthesized compounds also decrease the reactive oxygen species concentration in cell cultures. Compound 2 showed better potency towards the prevention of ferroptotic cell death as compared to commercially available iron chelator in the erastin-induced ferroptosis cell culture model. Such purine analogues are potential functional scaffolds for the development of target molecules for ferroptosis inhibition.     

Autophagic Cell Death and Cancer

Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Although apoptosis is the most common form of PCD, recent studies have provided evidence that there are other forms of cell death. One of such cell death is autophagic cell death, which occurs via the activation of autophagy. The present review summarizes recent knowledge about autophagic cell death and discusses the relationship with tumorigenesis.     

Primary and Secondary Cone Cell Death Mechanisms in Inherited Retinal Diseases and Potential Treatment Options

Inherited retinal diseases (IRDs) are a leading cause of blindness. To date, 260 disease-causing genes have been identified, but there is currently a lack of available and effective treatment options. Cone photoreceptors are responsible for daylight vision but are highly susceptible to disease progression, the loss of cone-mediated vision having the highest impact on the quality of life of IRD patients. Cone degeneration can occur either directly via mutations in cone-specific genes (primary cone death), or indirectly via the primary degeneration of rods followed by subsequent degeneration of cones (secondary cone death). How cones degenerate as a result of pathological mutations remains unclear, hindering the development of effective therapies for IRDs. This review aims to highlight similarities and differences between primary and secondary cone cell death in inherited retinal diseases in order to better define cone death mechanisms and further identify potential treatment options.     

C2-Ceramide Induces Cell Death and Protective Autophagy in Head and Neck Squamous Cell Carcinoma Cells

Ceramides are second messengers involved in several intracellular processes in cancer cells, amongst others. The aim of this study was to evaluate the anti-tumor efficacy of C2-ceramide (C2-Cer; N-acetyl-d-sphingosine) by investigating cell death and autophagy in head and neck squamous cell carcinoma (HNSCC) cells. C2-Cer showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. It simultaneously induced caspase-3-independent apoptosis and programmed necrosis. C2-Cer markedly increased the expression level of microtubule-associated protein 1 light chain 3B (LC3B) type II associated with protective autophagy. An autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity, while a programmed-necrosis inhibitor produced the opposite effect. Furthermore, C2-Cer up-regulated the phosphorylation of extracellular signal-regulated kinase 1/2, but down-regulated its downstream substrate phospho-mammalian target of rapamycin (p-mTOR) during the autophagy process. These results suggested that C2-Cer exerts anti-tumor effects by inducing programmed apoptosis and necrosis in HNSCC, and these cytotoxic effects are enhanced by an autophagy inhibitor.     

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on Cell Death

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated as a hypothalamic peptide based on its efficacy to increase adenylate cyclase (AC) activity. It has a widespread distribution throughout the body including the nervous system and peripheral organs, where PACAP exerts protective effects both in vivo and in vitro through its anti-apoptotic, anti-inflammatory, and antioxidant functions. The aim of the present paper was to review the currently available literature regarding the effects of PACAP on cell death in vitro in neural and non-neural cells. Among others, its effect on apoptosis can be detected in cerebellar granule cells against different toxic stimuli. Different neural cell types from the cerebral cortex are also prevented from cell death. PACAP also shows effects on cell death in cells belonging to the peripheral nervous system and protects both neural and non-neural cells of sensory organs. In addition, cell survival-promoting effect can be observed in different peripheral organ systems including cardiovascular, immune, respiratory, gastrointestinal, urinary, and reproductive systems. The studies summarized here indicate its noteworthy effect on cell death in different in vitro models, suggesting PACAP’s potential therapeutic usage in several pathological conditions.     

Cell Death Conversion under Hypoxic Condition in Tumor Development and Therapy

Hypoxia, which is common during tumor progression, plays important roles in tumor biology. Failure in cell death in response to hypoxia contributes to progression and metastasis of tumors. On the one hand, the metabolic and oxidative stress following hypoxia could lead to cell death by triggering signal cascades, like LKB1/AMPK, PI3K/AKT/mTOR, and altering the levels of effective components, such as the Bcl-2 family, Atg and p62. On the other hand, hypoxia-induced autophagy can serve as a mechanism to turn over nutrients, so as to mitigate the adverse condition and then avoid cell death potentially. Due to the effective role of hypoxia, this review focuses on the crosstalk in cell death under hypoxia in tumor progression. Additionally, the illumination of cell death in hypoxia could shed light on the clinical applications of cell death targeted therapy.     

Photodynamic Efficiency: From Molecular Photochemistry to Cell Death

Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research.     

Ursolic Acid Accelerates Paclitaxel-Induced Cell Death in Esophageal Cancer Cells by Suppressing Akt/FOXM1 Signaling Cascade

Ursolic acid (UA), a pentacyclic triterpenoid extracted from various plants, inhibits cell growth, metastasis, and tumorigenesis in various cancers. Chemotherapy resistance and the side effects of paclitaxel (PTX), a traditional chemotherapy reagent, have limited the curative effect of PTX in esophageal cancer. In this study, we investigate whether UA promotes the anti-tumor effect of PTX and explore the underlying mechanism of their combined effect in esophageal squamous cell carcinoma (ESCC). Combination treatment with UA and PTX inhibited cell proliferation and cell growth more effectively than either treatment alone by inducing more significant apoptosis, as indicated by increased sub-G1 phase distribution and protein levels of cleaved-PARP and cleaved caspase-9. Similar to the cell growth suppressive effect, the combination of UA and PTX significantly inhibited cell migration by targeting uPA, MMP-9, and E-cadherin in ESCC cells. In addition, combination treatment with UA and PTX significantly activated p-GSK-3β and suppressed the activation of Akt and FOXM1 in ESCC cells. Those effects were enhanced by the Akt inhibitor LY2940002 and inverted by the Akt agonist SC79. In an in vivo evaluation of a murine xenograft model of esophageal cancer, combination treatment with UA and PTX suppressed tumor growth significantly better than UA or PTX treatment alone. Thus, UA effectively potentiates the anti-tumor efficacy of PTX by targeting the Akt/FOXM1 cascade since combination treatment shows significantly more anti-tumor potential than PTX alone both in vitro and in vivo. Combination treatment with UA and PTX could be a new strategy for curing esophageal cancer patients.     

Current Trends in Neurodegeneration: Cross Talks between Oxidative Stress,Cell Death,and Inflammation

The human body is highly complex and comprises a variety of living cells and extracellular material, which forms tissues, organs, and organ systems. Human cells tend to turn over readily to maintain homeostasis in tissues. However, postmitotic nerve cells exceptionally have an ability to regenerate and be sustained for the entire life of an individual, to safeguard the physiological functioning of the central nervous system. For efficient functioning of the CNS, neuronal death is essential, but extreme loss of neurons diminishes the functioning of the nervous system and leads to the onset of neurodegenerative diseases. Neurodegenerative diseases range from acute to chronic severe life-altering conditions like Parkinson’s disease and Alzheimer’s disease. Millions of individuals worldwide are suffering from neurodegenerative disorders with little or negligible treatment available, thereby leading to a decline in their quality of life. Neuropathological studies have identified a series of factors that explain the etiology of neuronal degradation and its progression in neurodegenerative disease. The onset of neurological diseases depends on a combination of factors that causes a disruption of neurons, such as environmental, biological, physiological, and genetic factors. The current review highlights some of the major pathological factors responsible for neuronal degradation, such as oxidative stress, cell death, and neuroinflammation. All these factors have been described in detail to enhance the understanding of their mechanisms and target them for disease management.     

Mechanisms of Myofibre Death in Muscular Dystrophies: The Emergence of the Regulated Forms of Necrosis in Myology

Myofibre necrosis is a central pathogenic process in muscular dystrophies (MD). As post-lesional regeneration cannot fully compensate for chronic myofibre loss, interstitial tissue accumulates and impairs muscle function. Muscle regeneration has been extensively studied over the last decades, however, the pathway(s) controlling muscle necrosis remains largely unknown. The recent discovery of several regulated cell death (RCD) pathways with necrotic morphology challenged the dogma of necrosis as an uncontrolled process, opening interesting perspectives for many degenerative disorders. In this review, we focus on how cell death affects myofibres in MDs, integrating the latest research in the cell death field, with specific emphasis on Duchenne muscular dystrophy, the best-known and most common hereditary MD. The role of regulated forms of necrosis in myology is still in its infancy but there is increasing evidence that necroptosis, a genetically programmed form of necrosis, is involved in muscle degenerating disorders. The existence of apoptosis in myofibre demise will be questioned, while other forms of non-apoptotic RCDs may also have a role in myonecrosis, illustrating the complexity and possibly the heterogeneity of the cell death pathways in muscle degenerating conditions.     

Time to Classify Tumours of the Stomach and the Kidneys According to Cell of Origin

Malignant tumours are traditionally classified according to their organ of origin and whether they are of epithelial (carcinomas) or mesenchymal (sarcomas) origin. By histological appearance the site of origin may often be confirmed. Using same treatment for tumours from the same organ is rational only when there is no principal heterogeneity between the tumours of that organ. Organ tumour heterogeneity is typical for the lungs with small cell and non-small cell tumours, for the kidneys where clear cell renal carcinoma (CCRCC) is the dominating type among other subgroups, and in the stomach with adenocarcinomas of intestinal and diffuse types. In addition, a separate type of neuroendocrine tumours (NETs) is found in most organs. Every cell type able to divide may develop into a tumour, and the different subtypes most often reflect different cell origin. In this article the focus is on the cells of origin in tumours arising in the stomach and kidneys and the close relationship between normal neuroendocrine cells and NETs. Furthermore, that the erythropoietin producing cell may be the cell of origin of CCRCC (a cancer with many similarities to NETs), and that gastric carcinomas of diffuse type may originate from the ECL cell, whereas the endodermal stem cell most probably gives rise to cancers of intestinal type.     

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.     

Necrostatin-1 Attenuates Ischemia Injury Induced Cell Death in Rat Tubular Cell Line NRK-52E through Decreased Drp1 Expression

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In addition, we also investigated the mechanism of Nec-1 attenuates cell death in this renal ischemia model. The NRK-52E cells were incubated with TNF-α + antimycinA (TA) for 24 h with or without Nec-1. Cell death was observed under fluorescent microscope and quantified by flow cytometry. Cell viabilities were detected by MTT assay. The protein expression of dynamin-related protein 1 (Drp1) was detected by Western blotting and immunofluorescence assay. Increased cell death in simulated ischemia injury of NRK-52E cells were markedly attenuated in the Nec-1 pretreated ischemia injury group. Meanwhile, cell viability was significantly improved after using Nec-1. In addition, we also observed that the protein expression of Drp1, a mediator of mitochondrial fission, was significantly increased in simulated ischemia injury group. Increased Drp1 expression in the ischemia injury group can be abolished by Nec-1 or Drp1-knock down, accompanied with decreased cell death and improved cell viabilities. These results suggest that Nec-1 may inhibit cell death induced by simulated ischemia injury in the rat tubular cell line NRK-52E through decreased Drp1 expression.     

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1β converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.