Induced Cell Death Research Articles

Effect of impaired autophagic flux on breast carcinogenesis through the enhanced LC3-p62-NRF2 feedback loop.

6 Background: Breast cancer affects more women worldwide than any other type of cancer. Although some genes have been identified as potential causes, the exact molecular mechanism of the disease is still unknown. In our study, we aimed to investigate the role of NRF2 and the autophagy markers p62 and LC3 in breast cancer cell lines that have different Tp53 status using a new compound called 6,6'dihydroxythiobinupharidine (DTBN). In our previous research, we have shown that DTBN modulates key cellular signal transduction pathways that are relevant to disease biology, including cancer. Furthermore, we analyzed online databases to determine the correlation between NRF2, p62, LC3, and Tp53 in breast cancer patients. Methods: To evaluate the effect of DTBN on NRF2, p62 and LC3 in different types of breast cancer cell lines with varying TP53 status, we conducted molecular studies by inhibiting NRF2 activity. We synthesized the correlation between NRF2, p62, LC3 and TP53 expression and clinical parameters by analyzing TCGA and GEO datasets. Results: Our findings suggest that DTBN induces cell death and autophagy in all breast cancer cell lines, regardless of their Tp53 status. However, they also led us to hypothesize that NRF2 may hinder the sensitivity of cancer cells to DTBN-induced cell death and autophagy. To test this hypothesis, we inhibited NRF2 activity using an NRF2 inhibitor called brusatol. The results of the study indicate that the inhibition of NRF2 significantly increased cell death and autophagy in DTBN-treated cells. We observed a decrease in the expression of the autophagy marker p62, while an increase in the expression of another autophagy marker LC3. These findings suggest that the interaction between NRF2, LC3 and p62, which is activated in response to DTBN treatment, may serve as a potential anticancer drug target. We have validated the positive correlation between NRF2 and the autophagic gene p62 and LC3, in different Tp53 status breast cancer cell lines by analyzing TCGA and GEO data. Conclusions: Our study shows that high levels of Nrf2, LC3 and p62 expression are significantly associated with increased breast cancer cell proliferation and migration. This implies that patients with breast cancer have lower overall survival and a higher rate of recurrence. Our findings highlight the role of DTBN-induced NRF2 and LC3 expression and reduced p62 expression in breast cancer cells with varying Tp53 status. These results may represent a paradigm for better understanding the cancer cell response to therapies and designing more efficient combined anticancer therapies targeting NRF2, LC3 p62, and Tp53.

SLC7A11 protects luminal A breast cancer cells against ferroptosis induced by CDK4/6 inhibitors

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6 inhibitors) can significantly extend tumor response in patients with metastatic luminal A breast cancer, yet intrinsic and acquired resistance remains a prevalent issue. Understanding the molecular features of CDK4/6 inhibitor sensitivity and the potential efficacy of their combination with novel targeted cell death inducers may lead to improved patient outcomes. Herein, we demonstrate that ferroptosis, a form of regulated cell death driven by iron-dependent phospholipid peroxidation, partly underpins the efficacy of CDK4/6 inhibitors. Mechanistically, CDK4/6 inhibitors downregulate the cystine transporter SLC7A11 by inhibiting SP1 binding to the SLC7A11 promoter region. Furthermore, SLC7A11 is identified as critical for the intrinsic sensitivity of luminal A breast cancer to CDK4/6 inhibitors. Both genetic and pharmacological inhibition of SP1 or SLC7A11 enhances cell sensitivity to CDK4/6 inhibitors and synergistically inhibits luminal A breast cancer growth when combined with CDK4/6 inhibitors in vitro and in vivo. Our data highlight the potential of targeting SLC7A11 in combination with CDK4/6 inhibitors, supporting further investigation of combination therapy in luminal A breast cancer.

Cinobufagin treatments suppress tumor growth by enhancing the expression of cuproptosis-related genes in liver cancer.

Cuproptosis is a recently discovered form of regulated cell death triggered by excess copper (Cu) strongly influenced by the import, export, and intracellular utilization of Cu known as Cu homeostasis. Cinobufagin (CB) is a well-known Chinese medicine for its apoptosis-inducing role; however, its function on cuproptosis is poorly understood. To evaluate the effect of CB on inducing cell death through cuproptosis, we used RNA-seq data of HepG2-treated cells with CB to understand Cuproptosis genes. By using CCK-8 assay, Ross assay, GSH assay, and qRT-PCR, we found that CB could enhance cuproptosis in primary liver cancer cell lines, especially by increasing copper transporters CTR1, CTR2, and LIAS and downregulation of copper efflux transporters ATP7A and ATP7B resulted in increased reactive oxygen species (ROS) production, copper ionophores while reduced intracellular copper chelator glutathione (GSH) synthesis. In conclusion, our findings indicated that CB by increasing cuproptosis-related genes can mediate higher cell cytotoxicity against HepG2 and HUH7 and could provide a new insight into mechanisms of CB as an anti-tumor agent for targeting liver cancer.

Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

Arsenic trioxide (ATO) has exhibited remarkable efficacy in treating acute promyelocytic leukemia (APL), primarily through promoting the degradation of the PML-RARα fusion protein. However, ATO alone fails to confer any survival benefit to non-APL acute myeloid leukemia (AML) patients and exhibits limited efficacy when used in combination with other agents. Here, we explored the general toxicity mechanisms of ATO in APL and potential drugs that could be combined with ATO to exhibit synergistic lethal effects on other AML. We demonstrated that PML-RARα degradation and ROS upregulation were insufficient to cause APL cell death. Based on the protein synthesis of different AML cells and their sensitivity to ATO, we established a correlation between ATO-induced cell death and protein synthesis. Our findings indicated that ATO induced cell death by damaging nascent polypeptides and causing ribosome stalling, accompanied by the activation of the ZAKα-JNK pathway. Furthermore, ATO-induced stress activated the GCN2–ATF4 pathway, and ribosome-associated quality control cleared damaged proteins with the assistance of p97. Importantly, our data revealed that inhibiting p97 enhanced the effectiveness of ATO in killing AML cells. These explorations paved the way for identifying optimal synthetic lethal drugs to enhance ATO treatment on non-APL AML.

Pyrimidine Triones as Potential Activators of p53 Mutants.

p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy. Prior research indicates that reactivating p53 suppresses cancer cell proliferation and tumor growth in animal models. Early clinical evidence with a compound selectively targeting p53 mutants with substitutions of tyrosine 220 suggests potential therapeutic benefits of reactivating p53 in patients. This study identifies and examines the UCI-1001 compound series as a potential corrector for several p53 mutations. The findings indicate that UCI-1001 treatment in p53 mutant cancer cell lines inhibits growth and reinstates wild-type p53 activities, including DNA binding, target gene activation, and induction of cell death. Cellular thermal shift assays, conformation-specific immunofluorescence staining, and differential scanning fluorometry suggest that UCI-1001 interacts with and alters the conformation of mutant p53 in cancer cells. These initial results identify pyrimidine trione derivatives of the UCI-1001 series as candidates for p53 corrector drug development.

Mechanisms of Cell Death Induced by Erastin in Human Ovarian Tumor Cells.

Erastin (ER) induces cell death through the formation of reactive oxygen species (ROS), resulting in ferroptosis. Ferroptosis is characterized by an accumulation of ROS within the cell, leading to an iron-dependent oxidative damage-mediated cell death. ER-induced ferroptosis may have potential as an alternative for ovarian cancers that have become resistant due to the presence of Ras mutation or multi-drug resistance1 (MDR1) gene expression. We used K-Ras mutant human ovarian tumor OVCAR-8 and NCI/ADR-RES, P-glycoprotein-expressing cells, to study the mechanisms of ER-induced cell death. We used these cell lines as NCI/ADR-RES cells also overexpresses superoxide dismutase, catalase, glutathione peroxidase, and transferase compared to OVCAR-8 cells, leading to the detoxification of reactive oxygen species. We found that ER was similarly cytotoxic to both cells. Ferrostatin, an inhibitor of ferroptosis, reduced ER cytotoxicity. In contrast, RSL3 (RAS-Selective Ligand3), an inducer of ferroptosis, markedly enhanced ER cytotoxicity in both cells. More ROS was detected in OVCAR-8 cells than NCI/ADR-RES cells, causing more malondialdehyde (MDA) formation in OVCAR-8 cells than in NCI/ADR-RES cells. RSL3, which was more cytotoxic to NCI/ADR-RES cells, significantly enhanced MDA formation in both cells, suggesting that glutathione peroxidase 4 (GPX4) was involved in ER-mediated ferroptosis. ER treatment modulated several ferroptosis-related genes (e.g., CHAC1, GSR, and HMOX1/OX1) in both cells. Our study indicates that ER-induced ferroptotic cell death may be mediated similarly in both NCI/ADR-RES and OVCAR-8 cells. Additionally, our results indicate that ER is not a substrate of P-gp and that combinations of ER and RSL3 may hold promise as more effective treatment routes for ovarian cancers, including those that are resistant to other current therapeutic agents.

Exotoxins secreted by Clostridium septicum induce macrophage death: Implications for bacterial immune evasion mechanisms at infection sites

The induction of macrophage death is considered a potential mechanism by which components secreted by Clostridium septicum are used to evade the innate immune response and cause tissue damage. This study aimed to determine the effects of partially purified fractions of extracellular proteins secreted by C. septicum on the death of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages were incubated with partially purified fractions of proteins secreted by C. septicum into the culture medium. After incubation, the protein fraction with a molecular weight ≥100 kDa caused significant cell death in macrophages, altered cell morphology, increased the expression of markers of apoptosis and autophagy, and increased the expression (protein and mRNA) of IL-10 and TNFα. Our data suggest that the proteins secreted by C. septicum (MW, ≥100 kDa) induce cell death in macrophages by promoting autophagy-triggered apoptosis. This study may contribute to our understanding of the molecular mechanism of immune evasion by C. septicum at the infection site.

Morin overcomes doxorubicin resistance in human breast cancer by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 signaling pathway.

Breast cancer chemoresistance hampers chemotherapy efficacy; researchers investigate the pharmacological activities of natural products for potential solutions. This study aimed to determine the effect of morin, a bioflavonoid isolated from Maclura pomifera, on two Dox-resistant human breast cancer cell lines MDA-MB-231 (MDA-DR) and MCF-7 (MCF-DR). Sulforhodamine B and colony-forming assays demonstrated the cytotoxic effect of morin on both cell lines. Morin induced DNA damage and reduced the DNA repair mechanism, a feature of chemoresistance. In addition, morin reduced the protein expressions of cell cycle regulators, such as cyclin D1, CDK4, cyclin E1, cyclin B1, and p-Rb, thereby halting cell cycle progression. Moreover, morin slightly reduced PARP and Bcl-xL expressions but left LC3-II and RIPK3 expressions unchanged. Annexin-V/7-AAD analysis showed morin increased 7-AAD positive cells and annexin-V positive cells among MDA-DR and MCF-DR cells, respectively. In addition, morin increased p-AMPK and p-LKB1 levels; and, thus, inhibited phosphorylation of the mTOR pathway, but decreased t-AMPK levels by inducing lysosomal degradation, and AICAR, an AMPK activator, reduced Raptor, cyclin D1, CDK4, cyclin E1 and phosphorylated, and total mTOR levels, indicating AMPK is a key player in inducing cell death. Also, morin modulated MAPK phosphorylation and attenuated p-Akt and p-GSK3αβ levels; and thus, inhibited cell survival. In addition, morin suppressed tumor growth in our MDA-DR xenografted mouse model. These findings indicate that morin is a potential treatment for Dox-resistant breast cancer and that it does so by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 pathway, along with regulating the MAPK, and Akt/GSK3αβ signaling pathways.

Discovery of an 8-oxoguanine regulator PCBP1 inhibitor by virtual screening and its synergistic effects with ROS-modulating agents in pancreatic cancer.

Introduction: Drugs that target reactive oxygen species (ROS) metabolism have progressed the treatment of pancreatic cancer treatment, yet their efficacy remains poor because of the adaptation of cancer cells to high concentration of ROS. Cells cope with ROS by recognizing 8-oxoguanine residues and processing severely oxidized RNA, which make it feasible to improve the efficacy of ROS-modulating drugs in pancreatic cancer by targeting 8-oxoguanine regulators. Methods: Poly(rC)-binding protein 1 (PCBP1) was identified as a potential oncogene in pancreatic cancer through datasets of The Cancer Genome Atlas (TCGA) project and Gene Expression Omnibus (GEO). High-throughput virtual screening was used to screen out potential inhibitors for PCBP1. Computational molecular dynamics simulations was used to verify the stable interaction between the two compounds and PCBP1 and their structure-activity relationships. In vitro experiments were performed for functional validation of silychristin. Results: In this study, we identified PCBP1 as a potential oncogene in pancreatic cancer. By applying high-throughput virtual screening, we identified Compound 102 and Compound 934 (silychristin) as potential PCBP1 inhibitors. Computational molecular dynamics simulations and virtual alanine mutagenesis verified the structure-activity correlation between PCBP1 and the two identified compounds. These two compounds interfere with the PCBP1-RNA interaction and impair the ability of PCBP1 to process RNA, leading to intracellular R loop accumulation. Compound 934 synergized with ROS agent hydrogen peroxide to strongly improve induced cell death in pancreatic cancer cells. Discussion: Our results provide valuable insights into the development of drugs that target PCBP1 and identified promising synergistic agents for ROS-modulating drugs in pancreatic cancer.

The first-in-class pro-apoptotic peptide PEP-010 is effective in monotherapy and in combination with paclitaxel on resistant ovarian adenocarcinoma cell models.

Ovarian adenocarcinoma is the gynecological malignancy with the worst prognosis and the highest mortality rate. In the first stages of treatment, chemotherapy results effective, but its prolonged use and high doses lead to the appearance of resistance to treatments and relapse in most patients, representing a major challenge for clinicians. We developed PEP-010, a cell penetrating proapoptotic peptide disrupting the protein-protein interaction between caspase-9 and protein phosphatase 2A, thereby leading to the recovery of their activity in the apoptotic pathway. MTT assay or Annexin-V/Propidium Iodide staining and flow cytometry analysis were used to assess sensitivity to chemotherapies and apoptosis after treatment with PEP-010 in monotherapy or in combination with paclitaxel in ovarian carcinoma cell lines. DNA damage was assessed by immunofluorescence using γH2AX marker. We show here that PEP-010 effectively induces cell death in monotherapy on in up to 55% of cells from ovarian adenocarcinoma cell models resistant to different chemotherapies. Moreover, when used in combination with paclitaxel, one of the therapeutic options for recurrent ovarian carcinoma, PEP-010 showed a beneficial effect leading to the reduction of the IC50 of paclitaxel of 2.2 times and to apoptosis in 87% of cells. The described results suggest the potential therapeutic interest for PEP-010 and lead to the choice of ovarian adenocarcinoma as one of the major indications of the ongoing clinical trial.

Zinc transporter 1 functions in copper uptake and cuproptosis

Copper (Cu) is a co-factor for several essential metabolic enzymes. Disruption of Cu homeostasis results in genetic diseases such as Wilson's disease. Here, we show that the zinc transporter 1 (ZnT1), known to export zinc (Zn) out of the cell, also mediates Cu2+ entry into cells and is required for Cu2+-induced cell death, cuproptosis. Structural analysis and functional characterization indicate that Cu2+ and Zn2+ share the same primary binding site, allowing Zn2+ to compete for Cu2+ uptake. Among ZnT members, ZnT1 harbors a unique inter-subunit disulfide bond that stabilizes the outward-open conformations of both protomers to facilitate efficient Cu2+ transport. Specific knockout of the ZnT1 gene in the intestinal epithelium caused the loss of Lgr5+ stem cells due to Cu deficiency. ZnT1, therefore, functions as a dual Zn2+ and Cu2+ transporter and potentially serves as a target for using Zn2+ in the treatment of Wilson's disease caused by Cu overload.

Transferrin Receptor-Mediated Cellular Uptake of Fluorinated Chlorido[N,N'-bis(salicylidene)-1,2-phenylenediamine]iron(III) Complexes.

Fluorinated chlorido[salophene]iron(III) complexes (salophene = N,N'-bis(salicylidene)-1,2-phenylenediamine) are promising anticancer agents. Apoptosis and necrosis induction have already been described as part of their mode of action. However, the involvement of ferroptosis in cell death induction, as confirmed for other chlorido[salophene]iron(III) complexes, has not yet been investigated. Furthermore, the mechanism of cellular uptake of these compounds is unknown. Therefore, the biological activity of the fluorescent chlorido[salophene]iron(III) complexes with a fluorine substituent at positions 3, 4, 5, or 6 at the salicylidene moieties (C1-C4) was evaluated in malignant and nonmalignant cell lines with focus on the involvement of the transferrin receptor-1 (TfR-1) in cellular uptake, the influence of the complexes on mitochondrial function, and the analysis of the molecular mechanism of cell death. All complexes significantly decreased the metabolic activity in the tested ovarian cancer (A2780, A2780cis), breast cancer (MDA-MB 231), and leukemia (HL-60) cell lines, while the nonmalignant human stroma cell line HS-5 at a concentration of 0.5 μM, which represents the IC50 of the complexes in most of the used tumorigenic cell lines, was not affected. The mitochondrial function was impaired, as evidenced by a reduced mitochondrial membrane potential ΔΨm and decreased mitochondrial activity. Besides apoptosis and necroptosis, ferroptosis was identified as part of the mode of action. It was further demonstrated for the first time that fluorinated chlorido[salophene]iron(III) complexes downregulate TfR-1 expression, comparable to ferristatin II, an iron transport inhibitor that acts via TfR-1 degradation. FerroOrange staining further indicated that the complexes strongly increased the intracellular iron(II) level as a driving force to induce ferroptosis. In conclusion, these fluorinated chlorido[salophene]iron(III) complexes are potent, tumor cell-specific chemotherapeutic agents, with the potential to treat various types of cancers.

Commiphora myrrha n-hexane extract suppressed breast cancer progression through induction of G0/G1 phase arrest and apoptotic cell death by inhibiting the Cyclin D1/CDK4-Rb signaling pathway.

Breast cancer (BC) is one of the most frequently observed malignancies globally, yet drug development for BC has been encountering escalating challenges. Commiphora myrrha is derived from the dried resin of C. myrrha (T. Nees) Engl., and is widely adopted in China for treating BC. However, the anti-BC effect and underlying mechanism of C. myrrha remain largely unclear. MTT assay, EdU assay, and colony formation were used to determine the effect of C. myrrha n-hexane extract (CMHE) on the proliferation of human BC cells. Cell cycle distribution and apoptosis were assessed via flow cytometry analysis. Moreover, metastatic potential was evaluated using wound-scratch assay and matrigel invasion assay. The 4T1 breast cancer-bearing mouse model was established to evaluate the anti-BC efficacy of CMHE in vivo. RNA-sequencing analysis, quantitative real-time PCR, immunoblotting, immunohistochemical analysis, RNA interference assay, and database analysis were conducted to uncover the underlying mechanism of the anti-BC effect of CMHE. We demonstrated the significant inhibition in the proliferative capability of BC cell lines MDA-MB-231 and MCF-7 by CMHE. Moreover, CMHE-induced G0/G1 phase arrest and apoptosis of the above two BC cell lines were also observed. CMHE dramatically repressed the metastatic potential of these two cells in vitro. Additionally, the administration of CMHE remarkably suppressed tumor growth in 4T1 tumor-bearing mice. No obvious toxic or side effects of CMHE administration in mice were noted. Furthermore, immunohistochemical (IHC) analysis demonstrated that CMHE treatment inhibited the proliferative and metastatic abilities of cancer cells, while also promoting apoptosis in the tumor tissues of mice. Based on RNA sequencing analysis, quantitative real-time PCR, immunoblotting, and IHC assay, the administration of CMHE downregulated Cyclin D1/CDK4-Rb signaling pathway in BC. Furthermore, RNA interference assay and database analysis showed that downregulated Cyclin D1/CDK4 signaling cascade participated in the anti-BC activity of CMHE. CMHE treatment resulted in the suppression of BC cell growth through the stimulation of cell cycle arrest at the G0/G1 phase and the induction of apoptotic cell death via the inhibition of the Cyclin D1/CDK4-Rb pathway, thereby enhancing the anti-BC effect of CMHE. CMHE has potential anti-BC effects, particularly in those harboring aberrant activation of Cyclin D1/CDK4-Rb signaling.

The E3 ubiquitin ligase Herc1 modulates the response to nucleoside analogs in acute myeloid leukemia

AbstractFor several decades, induction therapy with nucleoside analogs, in particular cytarabine (Ara-C) and, to a lesser extent, fludarabine, has been the standard of care for patients diagnosed with acute myeloid leukemia (AML). However, the antitumor efficacy of nucleoside analogs is often limited by intrinsic and acquired drug resistance, thereby leading to poor therapeutic response and suboptimal clinical outcomes. In this study, we used genome-wide CRISPR-based pharmacogenomic screening to map the genetic factors that modulate the response to nucleoside analogs in AML and identified the E3 ubiquitin ligase, Herc1, as a key modulator of Ara-C response in mouse AML models driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1, both in vitro and in vivo. Loss of HERC1 enhanced nucleoside analog–induced cell death in both murine and human AML cell lines by compromising cell cycle progression. In-depth proteomic analysis and subsequent validation identified deoxycytidine kinase as a novel target of Herc1 in both mouse AML models. We observed that HERC1 is overexpressed in AML when compared with other cancer types and that higher HERC1 expression was associated with shorter overall survival in patients with AML in the The Cancer Gene Atlas program (TCGA) and BEAT-AML cohorts. Collectively, this study highlights the importance of HERC1 in the response of AML cells to nucleoside analogs, thereby establishing this E3 ubiquitin ligase as a novel predictive biomarker and potential therapeutic target for the treatment of AML.

Chrysophanol Induces Cell Death and Inhibits Invasiveness through Alteration of Calcium Levels in HepG2 Human Liver Cancer Cells.

To investigate the effect of chrysophanol, a phytochemical derived from Radix et Rhizoma Rhei on HepG2 liver cancer cells. HepG2 cell line was treated with different concentrations chrysophanol (0-100 μmol/L) for 24 h. The cell counting kit 8 assay was employed to assess cell viability. Intracellular calcium levels were examined using Fluo-4 AM and Mag-fluo-4 AM staining, followed by flow cytometry analysis. Mitochondrial membrane potential was measured with JC-1 assay kit. Additionally, the expressions of key proteins such as p-JNK, Bax, cytochrome c (Cyt C), cleaved caspase-3 (cCaspase-3), and caspase-8 were analyzed by Western blot. The inhibitory effects of chrysophanol on the invasion of cells were determined using a Transwell assay. Analysis of invasiveness was conducted by wound healing assay. Chrysophanol significantly reduced the proliferation of HepG2 liver cancer cells by affecting intracellular calcium distribution, diminishing mitochondrial membrane potential, and enhancing the expressions of p-JNK, Bax, Cyt C, cCaspase-3, and caspase-8 in the groups treated with 75 or 100 μmol/L chrysophanol compared to the control group (P<0.05). Additionally, 75 and 100 μmol/L chrysophanol exhibited inhibitory effects on cell migration and wound healing. Chrysophanol demonstrates potential against HepG2 liver cancer cells, suggesting its potential use as a therapeutic agent for liver cancer treatment.

Multiple mechanisms contribute to acquired TRAIL resistance in multiple myeloma

Multiple Myeloma (MM) prognosis has recently improved thanks to the incorporation of new therapies to the clinic. Nonetheless, it is still a non-curable malignancy. Targeting cancer cells with agents inducing cell death has been an appealing alternative investigated over the years, as is the case of TRAIL, an agonist of DR4 and DR5 death receptors. This pathway, involved in apoptosis triggering, has demonstrated efficacy on MM cells. In this research, we have investigated the sensitivity of a panel of MM cells to this agent and generated TRAIL-resistant models by continuous culture of sensitive cells with this peptide. Using genomic and biochemical approaches, the mechanisms underlying resistance were investigated. In TRAIL-resistant cells, a strong reduction in cell-surface receptor levels was detected and impaired the apoptotic machinery to respond to the treatment, enabling cells to efficiently form the Death Inducing Signalling Complex. In addition, an upregulation of the inhibitory protein c-FLIP was detected. Even though the manipulation of these proteins was able to modify cellular responses to TRAIL, it was not complete, pointing to other mechanisms involved in TRAIL resistance.

Synergistic metabolism intervention and immune activation for antitumor therapy by biomineralized lactate oxidase

Depleting tumoral lactate is a promising strategy to enhance the immune response and thereby suppressing tumorigenesis. However, the use of lactate oxidase (LOx), the most straightforward lactate-eliminating agent, faces several issues including low stability, no targeting capacity. To solve these problems, a “carrier-free” nanodrug LOx@manganese sulfide (LOx@MnS) was facilely prepared via biomineralization. This way, all the constituents integrated in the nanodrug, including LOx, Mn2+, and hydrogen sulfide (H2S), could be readily delivered into tumor cells and exert their effects. LOx combined with Mn2+ to convert lactate to cytotoxic reactive oxygen species (ROS) through the cascade reaction, meanwhile the crosstalk between H2S and ROS induced metabolism suppression to further augment the therapeutic efficacy. Consequently, the multi-mode modalities evoked by LOx@MnS led to effective induction of immunogenic cell death (ICD) and reversing high-lactate, immunosuppressive microenvironment, suggesting the great potential for anti-tumor immunotherapy. We believe this strategy opens a new avenue to construct functional materials from frangible biomolecules and expand their applications.

PINK1 insufficiency can be exploited as a specific target for drug combinations inducing mitochondrial pathology-mediated cell death in gastric adenocarcinoma

There exist very limited non-hazardous therapeutic strategies except for surgical resection and lymphadenectomy against gastric cancer (GC) despite being the third leading cause of cancer deaths worldwide. This study proposes an innovative treatment approach against GC using a drug combination strategy that manipulates mitochondrial dynamics in conjunction with the induction of mitochondrial pathology-mediated cell death. Comparative analysis was done with gastric adenocarcinoma and normal cells by qPCR, western blot, microscopic immunocytochemistry, and live cell imaging. In this study, impairment of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission by Mdivi-1 created an imbalance in mitochondrial structural dynamics in indomethacin-treated AGS cells in which mitophagy-regulator protein PINK1 is downregulated. These drug combinations with the individual sub-lethal doses ultimately led to the activation of cell death machinery upregulating pro-apoptotic proteins like Bax, Puma, and Noxa. Interestingly, this combinatorial therapy did not affect normal gastric epithelial cells significantly and also no significant upregulation of death markers was observed. Moreover, the drug combination strategy also retarded cell migration and reduced stemness in GC cells. In summary, this study offers a pioneering specific therapeutic strategy for GC treatment, sparing normal cells providing opportunities for minimal drug-mediated toxicity utilizing mitochondria as a viable and specific target for anti-cancer therapy in gastric cancer.

Dissecting transposable elements and endogenous retroviruses upregulation by HDAC inhibitors in leiomyosarcoma cells: Implications for the interferon response

Transposable elements (TEs) are of interest as immunomodulators for cancer therapies. TEs can fold into dsRNAs that trigger the interferon response. Here, we investigated the effect of different HDAC inhibitors (HDACIs) on the expression of TEs in leiomyosarcoma cells. Our data show that endogenous retroviruses (ERVs), especially ERV1 elements, are upregulated after treatment with HDAC1/2/3-specific inhibitors. Surprisingly, the interferon response was not activated. We observed an increase in A-to-I editing of upregulated ERV1. This could have an impact on the stability of dsRNAs and the activation of the interferon response. We also found that H3K27ac levels are increased in the LTR12 subfamilies, which could be regulatory elements controlling the expression of proapoptotic genes such as TNFRSF10B. In summary, we provide a detailed characterization of TEs modulation in response to HDACIs and suggest the use of HDACIs in combination with ADAR inhibitors to induce cell death and support immunotherapy in cancer.

Abstract Mo020: Loss of TRAF2 Signaling Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy

Rationale: Cytokines such as TNFα have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). While TNFα can elicit different cellular responses including survival or death, the mechanisms underlying these divergent outcomes in the heart remains cryptic. The E3 ubiquitin ligase TRAF2 provides a critical signaling platform for K63 - linked ubiquitination of RIPK1, crucial for NF-kB activation by TNFα. Whether alterations in TNFα-TRAF2 signaling underlie the cardiotoxic effects of DOX, remains poorly understood. Results: In contrast to vehicle treated mice, severe ultrastructural defects including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFa levels were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFa in DOX cardiotoxicity, we discovered that in the absence of DOX, NF-kB was readily activated by TNFa. However, TNFα -mediated NF-kB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked poly-ubiquitination of RIPK1, attributed to the proteasomal degradation of TRAF2. Further, TRAF2 protein expression was markedly reduced in hearts of cancer patients treated with DOX. Impaired TRAF2 signaling resulted in the activation of Bnip3 and mitochondrial perturbations, including disrupted bioenergetics, loss of membrane potential and permeability transition pore opening. We further established that the reciprocal actions of the ubiquitinating and de-ubiquitinating enzymes c-IAP1 and USP19 respectively regulated the proteasomal degradation of TRAF2 in DOX treated cardiac myocytes. Importantly, an E3 ligase mutant of c-IAP1(c-IAP1 H588A) or gain of function of USP19, prevented proteasomal degradation of TRAF2 and DOX -induced cell death. Further, wild type TRAF2 but not a RING finger mutant defective for K63 ubiquitination of RIPK1, restored NF-kB signaling and suppressed DOX-induced cardiac cell death. Finally, cardiomyocyte-restricted expression of TRAF2 (AAV9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Conclusions: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX, sensitized cardiac myocytes to TNFa mediated necrotic cell death.