Necroptosis In Cancer Cells Research Articles

Mts1 (S100A4) and Its Peptide Demonstrate Cytotoxic Activity in Complex with Tag7 (PGLYRP1) Peptide.

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10-8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.

Necroptosis induced by ruthenium (II) complexes as mitochondrial disruptors

Inducing necroptosis in cancer cells has emerged as an effective strategy to overcome drug resistance. However, while organic small molecules have been extensively studied for this purpose, metal-based compounds have received relatively little attention as triggers of necroptosis. The development of ruthenium (II) hybrid compounds, particularly those containing triazene (Ru-TRZ), highlights a novel avenue for modulating necroptotic cell death. Here we show that incorporating a methyltriazene moiety, a known alkylating warhead, confers superior mitochondrial-targeting properties and enhances cell death compared to amide-containing counterparts. Ru-hybrid TRZ2 exhibits also antitumor efficacy against in vivo drug-resistant cancer cells. Mechanistically, we demonstrate that Ru-TRZ hybrids induce apoptosis. In addition, by activating downstream RIPK3-driven cell death, TRZ2 proficiently restrains normal mitochondrial function and activity, leading to cancer cell necroptosis. Finally, TRZ2 synergizes anti-proliferative activity and cell death effects induced by conventional drugs. In conclusion, Ru-TRZ2 stands as a promising ruthenium-based chemotherapeutic agent inducing necroptosis in drug resistant cancer cells.

Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells

Evading the cellular apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in cancer cells, leading to the induction of ZBP1-mediated necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of necroptosis.

Abstract 3008: Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition

Abstract Cell proliferation and cell death are essential for maintaining homeostatic balance in eukaryotic organisms. Many diseases develop when cellular homeostasis is disturbed. Currently, several anti-cancer drugs are known to induce apoptosis through activation of intrinsic or extrinsic pathways. However, many types of cancer cells have acquired resistance due to quick adaptation to resist pro-apoptotic mechanisms. Therefore, simultaneous induction of other forms of cell deaths such as necroptosis, autophagy, etc., in addition to apoptosis, is becoming an attractive strategy for controlling tumor growth and metastasis. For this purpose, we have explored the role of XIAP in caspase-independent mechanisms of cell death during HDAC (Histone Deacetylase) inhibition. Our experiments with HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid) have revealed that the anti-apoptotic biomarkers, including survivin and XIAP, exhibit important regulatory functions for inducing necroptosis in cancer cells. Interestingly, many of the signal adaptors of apoptosis and necroptosis are known to show dual functions during drug-induced cancer cell death. For example, the formation of the survivin-XIAP complex prevents XIAP from undergoing proteasomal degradation and polyubiquitination, thereby stabilizing XIAP in cancer cells. When the survivin level is increased, it is also known to stop the inhibition of XIAP by Smac. In addition, since XIAP can inhibit caspase -9 and the executioner caspases -3 and -7, the apoptosis mechanisms will be blocked, and cell proliferation will continue to promote cell survival and tumorigenesis. Our western blot data revealed that the caspase -9, -3, -7, and -8 levels were significantly downregulated in MCF-7 breast cancer cells following SAHA treatment. Furthermore, SAHA treatment also increased the levels of necroptosis inducers such as phospho-RIP3 and MLKL in MCF-7 cells. However, treatment of the same MCF-7 cells with other drugs such as RG-7388, an MDM2 inhibitor, was able to induce apoptosis through activation of caspases. Our current results show that MCF-7 cells may switch to the necroptosis pathway due to the inhibition of caspases and increased levels of survivin and XIAP. (This research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Umamaheswari Natarajan, Grace Waldron, Thiagarajan Venkatesan, Appu Rathinavelu. Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3008.

Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

BackgroundImmunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term...

Necroptosis in Immuno-Oncology and Cancer Immunotherapy.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To “break” cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

ApoL6 Induces Dichotomous Cell Death Phenotype Involving Both Apoptosis and Necroptosis in Cancer Cells

Apolipoprotein L6 (ApoL6) is a pro-death, BH3-only and phospholipid-binding member of the Bcl-2 family. Previously, we showed that ectopic expression of ApoL6 induces mitochondria-mediated apoptosis. In this study, we hypothesized that ApoL6 plays a dichotomous role in regulated cell death (RCD) pathways that involves both apoptosis and necroptosis. Necroptosis, or programmed necrosis, can occur simultaneously or alternatively to apoptosis as a caspase-independent pathway, and is characterized by the formation of intracellular necrosomes and plasma membrane pores. The pseudokinase mixed lineage kinase domain-like protein (MLKL) interacts with the necrosome to become phosphorylated and activated. Phosphorylated (p-) MLKL molecules migrate to the plasma membrane to form pores, disrupt membrane integrity, and selectively release intracellular components. The proinflammatory cytokine IL-1β is amongst a number of molecules released in this process. To investigate the role of ApoL6 in RCD, ApoL6 was overexpressed via a Tet-Off gene inducible system in a p53 null colorectal cancer cell line DLD-1. Necroptotic cell death was marked by an increased level of intracellular and plasma membrane p-MLKL and extracellular release of IL-1β along with the disruption of the plasma membrane. We found that in the presence of apoptotic inhibitors, levels of necroptotic cell death increased, indicating an enhanced push away from apoptosis. We also showed that ApoL6 preferentially binds with phosphatidylinositol phosphate species (PIPs), which include PI (4,5) P2, PI4P and PI5P. Thus, we expand on the role of ApoL6 in RCD to induce a mixed cell-death phenotype that includes both apoptosis and necroptosis.

Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II.

Inducing necroptosis in cancer cells is an effective approach to circumvent drug-resistance. Metal-based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine were developed with a series of different ancillary ligands (Ru1-7). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus-targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug-resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru-based necroptosis-inducing chemotherapeutic agent.

Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II

AbstractInducing necroptosis in cancer cells is an effective approach to circumvent drug‐resistance. Metal‐based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1‐(pyrazin‐2‐yl)pyreno[4,5‐e][1,2,4]triazine were developed with a series of different ancillary ligands (Ru1‐7). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus‐targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug‐resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru‐based necroptosis‐inducing chemotherapeutic agent.

Exploring the prime site in caspases as a novel chemical strategy for understanding the mechanisms of cell death: a proof of concept study on necroptosis in cancer cells.

Caspases participate in regulated cell death mechanisms and are divided into apoptotic and proinflammatory caspases. The main problem in identifying the unique role of a particular caspase in the mechanisms of regulated cell death is their overlapping substrate specificity; caspases recognize and hydrolyze similar peptide substrates. Most studies focus on examining the non-prime sites of the caspases, yet there is a need for novel and more precise chemical tools to identify the molecular participants and mechanisms of programmed cell death pathways. Therefore, we developed an innovative chemical approach that examines the prime area of the caspase active sites. This method permits the agile parallel solid-phase synthesis of caspase inhibitors with a high yield and purity. Using synthesized compounds we have shown the similarities and differences in the prime area of the caspase active site and, as a proof of concept, we demonstrated the exclusive role of caspase-8 in necroptosis.

Complex roles of necroptosis in cancer.

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.

O42 MAM, a natural naphthoquinone, induces necroptosis in cancer cells

Necroptosis is a recently identified programmed cell death. Here, we reported that MAM, a natural naphthoquinone isolated from Huzhang, caused hydrogen peroxide dependent activation of JNK and induced the expression of iNOS, thereby leading to nitric oxide generation in A549 lung cancer cells. In addition, a decrease in GSH/GSSG levels accompanied by increased ROS production was also observed. Reversal of ROS generation and cell death in GSH pretreated cells indicated the involvement of GSH depletion in MAM mediated cytotoxicity. Furthermore, MAM treated HCT116 and HT29 cells, were characterised by mitochondrial ROS elevation, mitochondrial depolarization and ATP depletion. This process was identified as necroptosis, based on the experiments showing that MAM-induced cell death was attenuated by RIP1 inhibitor necrostatin-1s, siRNA-mediated gene silencing of RIP1 and RIP3. RIP1/RIP3 complex triggered necroptosis via the accumulation of cytosolic calcium, followed by sustained JNK activation. Finally, we determined that both the calcium chelator BAPTA-AM and JNK inhibitor SP600125 could ameliorate mitochondrial ROS elevation, mitochondrial depolarization and ATP depletion. In addition, MAM induced necroptosis was independent of TNF α , p53, MLKL, and LMP. By demonstrating that RIP1/RIP3 complex triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis by sustained activation of JNK, our study provides new insights into the molecular regulation of necroptosis in human colon cancer cells. In summary, MAM induced necroptosis in cancer cells. Acknowledgments This study was supported by the Science and Technology Development Fund of Macau (FDCT) (078/2016/A2) and the Research Fund of University of Macau (MYRG2016-00043-ICMS-QRCM).

Killing colon cancer cells through PCD pathways by a novel hyaluronic acid-modified shell-core nanoparticle loaded with RIP3 in combination with chloroquine.

Due to extensive apoptosis defects and multidrug resistance, there is great interest regarding non-apoptotic programmed cell death (PCD) pathways, such as lysosomal-mediated programmed cell death (LM-PCD), necroptosis and autophagy. Because there is an intricate effector network among these PCD pathways, it is expected that they may act synergistically in cancer therapy. In this study, chloroquine (CQ) was found to significantly upregulate receptor-interacting protein kinase 3 (RIP3) expression, and RIP3 were involved in CQ-related autophagy. Overexpressed-eGFP-RIP3 co-localized with the selective autophagy receptor p62. mRIP3 overexpression in combination with CQ markedly increased the inhibition rate relative to that observed in the CQ-treatment group. Several experiments, including Hoechst staining, transmission electron microscopy (TEM) observation, the high-mobility group box 1 (HMGB1) release assay, Annexin V/PI staining and immunoblotting of proteins included in PCD pathways, verified that mRIP3 overexpression in combination with CQ induced lysosomal membrane permeabilization (LMP) and necroptosis of cancer cells, leading to cancer cell death. For tumor-targeted delivery, hyaluronic acid (HA)-modified, lipid-coated PLGA nanoparticles loaded with mRIP3-pDNA were prepared and characterized using a particle sizer, differential scanning calorimetry (DSC) and TEM. The nanoparticles exhibited ideal biocompatibility and good tumor-targeting efficiency, and the tumor inhibition rate of HA-Lip-PEI-mRIP3-PLGA-NPs+CQ was 80.2% in the CT26 mouse model. In this study, we attempted to treat tumors by inducing several alternative PCD pathways to shed light on the combination therapy of alternative PCD inducers.

TRIP-Br1 oncoprotein inhibits autophagy, apoptosis, and necroptosis under nutrient/serum-deprived condition

TRIP-Br1 oncogenic protein has been shown to have multiple biological functions in cells. In this study, we demonstrate that TRIP-Br1 functions as an oncoprotein by inhibiting autophagy, apoptosis, and necroptosis of cancer cells and eventually helping them to survive under the nutrient/serum starved condition. TRIP-Br1 expression level was significantly increased in conditions with low levels of nutrients. Nutrient depleted conditions were induced by culturing cancer cells until they were overcrowded with high cell density or in media deprived of glucose, amino acids, or serum. Among them, serum starvation significantly enhanced the expression of TRIP-Br1 only in all tested breast cancer cell lines (MCF7, MDA-MB-231, T47D, MDA-MB-435, Hs578D, BT549, and MDA-MB-435) but not in the three normal cell lines (MCF10A, HfCH8, and NIH3T3). As compared with the control cells, the introduction of TRIP-Br1 silencing siRNA into MCF7 and MDA-MB-231 cells accelerated cell death by inducing apoptosis and necroptosis. In this process, TRIP-Br1 confers resistance to serum starvation-induced cell deaths by stabilizing the XIAP protein and inhibiting cellular ROS production. Moreover, our data also show that the intracellular increase of TRIP-Br1 protein resulting from serum starvation seems to occur in part through the blockage of PI3K/AKT signaling pathway.

Interferon-β-armed oncolytic adenovirus induces both apoptosis and necroptosis in cancer cells

Interferon-β (IFN-β) has been widely used in cancer therapy, but the clinical trial results are generally disappointing. Our previous studies have shown that an oncolytic adenovirus carrying IFN-β (ZD55-IFN-β) exhibits significant anti-tumor activities. However, the underlying mechanisms are not clear. Here we showed that ZD55-IFN-β infection-induced S-phase cell cycle arrest in a p53-dependent manner by activating the ataxia telangiectasia mutated-dependent DNA damage pathway. In addition, ZD55-IFN-β infection could initiate both caspase-dependent apoptosis and necroptosis in cancer cells. More importantly, ZD55-IFN-β showed a synergistic effect on cancer cells when combined with doxorubicin. These results suggest that the combination of ZD55-IFN-β with doxorubicin may represent a promising clinical strategy in cancer therapy.