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Background and Purpose Experimental Approach Key Results Conclusion Emerging evidence has highlighted that paraptosis may be an effective strategy for treating liver cancer. In our previous studies, Compound 4a induced paraptosis in cancer cells. Here, the characteristics of Compound 4a–induced paraptosis were further revealed and, for the first time, the target and related molecular mechanisms of Compound 4a–induced paraptosis in liver cancer were defined.The effects and mechanism of Compound 4a in liver cancer cells were studied in in vitro and in vivo (BALB/c‐nude xenograft model) experiments, and the targets of Compound 4a that trigger paraptosis were identified and confirmed via mass spectrometry–based drug affinity responsive target stability (DARTS) analyses, siRNA experiments and a cellular thermal shift assay (CETSA). The function and distribution of calreticulin (CRT) protein were detected via Cal‐520 AM and immunofluorescence staining, respectively.Compound 4a effectively induced paraptosis‐like cell death in liver cancer, both in vitro and in vivo, and its effect was comparable with the first‐line anti–liver cancer drug oxaliplatin but with a higher safety profile. We identified the CRT protein as a target of Compound 4a, which caused cellular endoplasmic reticulum stress (ERS) and calcium overload. CRT knockdown weakened the anti–liver cancer activity of Compound 4a, which may be related to the inhibition of paraptosis.Compound 4a represents a potentially safe and effective agent for the treatment of liver cancer. The characteristics of Compound 4a–triggered paraptosis was clarified and a unique function of CRT in paraptosis was revealed. [ABSTRACT FROM AUTHOR]

Cell death is a critical biological process necessary for development, tissue maintenance, and defense against diseases. To date, more than 20 forms of cell death have been identified, each defined by unique molecular pathways. Understanding these different forms of cell death is essential for investigating the pathogenesis of diseases such as cancer, neurodegenerative disorders, and autoimmune conditions and developing appropriate therapies. Paraptosis is a distinct form of regulated cell death characterized by cytoplasmic vacuolation and dilatation of cellular organelles like the mitochondria and endoplasmic reticulum (ER). It is regulated by several signaling pathways, for instance, those associated with ER stress, calcium overload, oxidative stress, and specific cascades such as insulin-like growth factor I receptor (IGF-IR) and its downstream signaling pathways comprising mitogen-activated protein kinases (MAPKs) and Jun N-terminal kinase (JNK). Paraptosis has been observed in diverse biological contexts, including development and cellular stress responses in neuronal, retinal, endothelial, and muscle cells. The induction of paraptosis is increasingly important in anticancer therapy, as it targets non-apoptotic stress responses in tumor cells, which can be utilized to induce cell death. This approach enhances treatment efficacy and addresses drug resistance, particularly in cases where cancer cells are resistant to apoptosis. Combining paraptosis-inducing agents with traditional therapies holds promise for enhancing treatment efficacy and overcoming drug resistance, suggesting a valuable strategy in anticancer therapy. [ABSTRACT FROM AUTHOR]

Paraptosis is a non‐apoptotic and caspase‐independent programmed cell death that can trigger immunogenic cell death (ICD) in tumor cells, representing a potent tactic to overcome immune tolerance to apoptosis. Here, this study demonstrates the construction of a dual‐responsive nanoreactor (MCGDH) to achieve paraptosis‐mediated ICD for chemo‐immunotherapy. Specifically, by doping Cu2+ into glutathione (GSH)‐responsive dendritic mesoporous silica nanoparticles, the platform (CGDMSN) is endowed with partial acid‐sensitivity. After loaded with 8‐hydroxyquinoline (8‐HQ), cell membrane fragments are coated onto the ammoniated CGDMSN surface to construct MCGDH. Upon internalization by tumor cells, the release of Cu2+ and 8‐HQ from MCGDH in response to the acidic pH and high concentration of GSH in the tumor microenvironment stimulates in situ generation of Cu(8‐HQ)2, inducing tumor cells paraptosis at a low copper dose. Moreover, MCGDH‐mediated paraptosis amplifies the immunogenicity of tumor cells, facilitating antigen presentation to dendritic cells and activating CD8+/CD4+ T cells immune responses. Furthermore, the combination of MCGDH and anti‐PD‐1 antibodies (αPD‐1) promotes the systemic anti‐tumor immune responses and long‐term immunological effect to vastly inhibit the primary/distant tumor growth and prevent tumor metastasis. This GSH/pH dual‐responsive nanoreactor serves as a selective platform for accelerating the development of chemo‐immunotherapy. [ABSTRACT FROM AUTHOR]

This article explores the concept of paraptosis, a type of programmed cell death that is different from apoptosis and necrosis. It discusses the mechanisms of paraptosis and highlights the potential of natural products to induce paraptosis in cancer cells, offering an alternative to traditional cancer therapies. The article also mentions the potential of paraptosis in enhancing tumor immunogenicity and triggering immune responses against cancer cells. Clinical trials are being conducted to test the efficacy and safety of paraptosis in cancer treatment, although challenges such as the lack of rigorous clinical trials and difficulties in large-scale isolation and standardization of natural products exist. Overall, the article provides valuable insights into the field of paraptosis and its potential applications in cancer therapy. [Extracted from the article]

We report on the synthesis of optically pure cyclometalated iridium(III) complex‐peptide hybrids as amphiphilic peptide conjugates (IPH‐ACs) and the effect of the stereochemistry with respect to their Ir(tpy)3 (tpy: 2‐(4′‐tolyl)pyridine) core on their cytotoxicity against cancer cells. Stereochemically pure IPH‐ACs were synthesized from Δ‐ and Λ‐Ir(tpyCO2H)3 by optical resolution via the diastereomeric intermediates that are conjugated with the chiral alcohol, (1R,2R)‐2‐aminocyclohexanol ((R,R)‐11), followed by the hydrolysis of the ester moieties, as we reported very recently, and their spectroscopic spectra are reported. It was found that both optically pure Δ‐ and Λ‐forms of IPH‐ACs induce paraptotic cell death in Jurkat cells and the EC50 values were evaluated by MTT assays. We also performed TEM (transmission electron microscope) analyses of Jurkat cells treated with Λ‐13 to observe morphological changes in paraptosis processes. The intracellular uptake of Λ‐forms of IPH‐ACs in the cells measured by ICP‐MS (inductively coupled plasma‐mass spectrometry) was higher than those of Δ‐forms and the EC50 values of the Λ‐forms were smaller than those of the Δ‐IPH‐ACs. The analysis of these results suggests that the intrinsic cytotoxicity is almost equal for the Δ‐ and Λ‐forms and that the difference in cytotoxicity against Jurkat cells is due to the selectivity in the intracellular uptake of each stereoisomer. [ABSTRACT FROM AUTHOR]

Background: Paraptosis is a programmed cell death characterized by cytoplasmic vacuolation, which has been explored as an alternative method for cancer treatment and is associated with cancer resistance. However, the mechanisms underlying the progression of paraptosis in cancer cells remain largely unknown. Methods: Paraptosis-inducing agents, CPYPP, cyclosporin A, and curcumin, were utilized to investigate the underlying mechanism of paraptosis. Next-generation sequencing and liquid chromatography-mass spectrometry analysis revealed significant changes in gene and protein expressions. Pharmacological and genetic approaches were employed to elucidate the transcriptional events related to paraptosis. Xenograft mouse models were employed to evaluate the potential of paraptosis as an anti-cancer strategy. Results: CPYPP, cyclosporin A, and curcumin induced cytoplasmic vacuolization and triggered paraptosis in cancer cells. The paraptotic program involved reactive oxygen species (ROS) provocation and the activation of proteostatic dynamics, leading to transcriptional activation associated with redox homeostasis and proteostasis. Both pharmacological and genetic approaches suggested that cyclin-dependent kinase (CDK) 7/9 drive paraptotic progression in a mutually-dependent manner with heat shock proteins (HSPs). Proteostatic stress, such as accumulated cysteine-thiols, HSPs, ubiquitin-proteasome system, endoplasmic reticulum stress, and unfolded protein response, as well as ROS provocation primarily within the nucleus, enforced CDK7/CDK9–Rpb1 (RNAPII subunit B1) activation by potentiating its interaction with HSPs and protein kinase R in a forward loop, amplifying transcriptional regulation and thereby exacerbating proteotoxicity leading to initiate paraptosis. The xenograft mouse models of MDA-MB-231 breast cancer and docetaxel-resistant OECM-1 head and neck cancer cells further confirmed the induction of paraptosis against tumor growth. Conclusions: We propose a novel regulatory paradigm in which the activation of CDK7/CDK9–Rpb1 by nuclear proteostatic stress mediates transcriptional regulation to prime cancer cell paraptosis. [ABSTRACT FROM AUTHOR]

Background: Non‐apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α‐hederin (α‐hed) induces caspase‐independent paraptotic modes of cell death. Purpose: The present study is aimed to investigate the role of α‐hed induces paraptosis and the associated mechanism of it. Methods: The cell proliferation was detected by CCK‐8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α‐hed. Molecular docking methods were used to evaluate binding abilities of α‐hed with targets. The expressions of genes and proteins were analyzed by RT‐qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo. Results: α‐hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg‐2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α‐hed. Besides, both Swiss prediction and molecular docking showed that the structure of α‐hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)‐β3/ inositol 1,4,5‐trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α‐hed. After blocking the GPCR signaling, α‐hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α‐hed activated MAPK cascade by elevating Ca2+ flux. Since non‐apoptotic cell death is presently emerging as a potential direction to overcome chemo‐drug resistance, we then found α‐hed also induced paraptosis in 5‐fluorouracil‐resistant (5‐FU‐R) CRC cells, and it reduced the growth of 5‐FU‐R CRC xenografts. Conclusions: Collectively, our findings proved α‐hed as a promising candidate for inducing non‐apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic‐based chemo‐resistance in CRC. [ABSTRACT FROM AUTHOR]

The mechanisms of neuronal cell death in neurodegenerative disease remain incompletely understood, although recent studies have made significant advances. Apoptosis was previously considered to be the only mechanism of neuronal cell death in neurodegenerative diseases. However, recent findings have challenged this dogma, identifying new subtypes of necrotic neuronal cell death. The present review provides an updated summary of necrosis subtypes and discusses their potential roles in neurodegenerative cell death. Among numerous necrosis subtypes, including necroptosis, paraptosis, ferroptosis, and pyroptosis, transcriptional repression-induced atypical cell death (TRIAD) has been identified as a potential mechanism of neuronal cell death. TRIAD is induced by functional deficiency of TEAD-YAP and self-amplifies via the release of HMGB1. TRIAD is a feasible potential mechanism of neuronal cell death in Alzheimer's disease and other neurodegenerative diseases. In addition to induction of cell death, HMGB1 released during TRIAD activates brain inflammatory responses, which is a potential link between neurodegeneration and neuroinflammation. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEA) is present worldwide as a serious contaminant of food and feed and causes male reproductive toxicity. The implication of paraptosis, which is a nonclassical paradigm of cell death, is unclear in ZEA-induced male reproductive disorders. In this study, the toxic effects of ZEA on the blood-testis barrier (BTB) and the related mechanisms of paraptosis were detected in goats. ZEA exposure, in vivo, caused a significant decrease in spermatozoon quality, the destruction of seminiferous tubules, and damage to the BTB integrity. Furthermore, ZEA exposure to Sertoli cells (SCs) in vitro showed similar dysfunction in structure and barrier function. Importantly, the formation of massive cytoplasmic vacuoles in ZEA-treated SCs corresponded to the highly swollen and dilative endoplasmic reticulum (ER), and paraptosis inhibition significantly alleviated ZEA-induced SC death and vacuolization, which indicated the important contribution of paraptosis in ZEA-induced BTB damage. Meanwhile, the expression of ER stress marker proteins was increased after ZEA treatment but decreased under the inhibition of paraptosis. The vacuole formation and SC death, induced by ZEA, were remarkably blocked by ER stress inhibition. In conclusion, these results facilitate the exploration of the mechanisms of the SC paraptosis involved in ZEA-induced BTB damage in goats. [ABSTRACT FROM AUTHOR]

Currently, hepatocellular carcinoma (HCC) is characterized by its unfavorable prognosis and resistance to conventional chemotherapy and radiotherapy. Drug repositioning, an approach aimed at identifying novel therapeutic applications for existing drugs, presents a cost-effective strategy for developing new anticancer agents. We explored the anticancer properties of Ezetimibe, a widely used oral lipid-lowering drug, in the context of HCC. Our findings demonstrate that Ezetimibe effectively suppresses HCC cell proliferation through paraptosis, an apoptotic-independent cell death pathway. The examination of HCC cells lines treated with Ezetimibe using light microscopy and transmission electron microscopy (TEM) showed cytoplasmic vacuolation in the perinuclear region. Notably, the nuclear membrane remained intact in both Ezetimibe-treated and untreated HCC cell lines. Probe staining assays confirmed that the cytoplasmic vacuoles originated from dilated endoplasmic reticulum (ER) compartments rather than mitochondria. Furthermore, a dose-dependent accumulation of reactive oxygen species (ROS) was observed in Ezetimibe-treated HCC cell lines. Co-treatment with the general antioxidant NAC attenuated vacuolation and improved cell viability in Ezetimibe-treated HCC cells. Moreover, Ezetimibe induced paraptosis through proteasome activity inhibition and initiation of the unfolded protein response (UPR) in HCC cell lines. In our in vivo experiment, Ezetimibe significantly impeded the growth of HCC tumors. Furthermore, when combined with Sorafenib, Ezetimibe exhibited a synergistic antitumor effect on HCC cell lines. Mechanistically, Ezetimibe induced paraptosis by targeting NPC1L1 to inhibit the PI3K/AKT/mTOR signaling pathway. In conclusion, our study highlights the potential of Ezetimibe as an anticancer agent by triggering paraptosis in HCC cells. [ABSTRACT FROM AUTHOR]

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF‐7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal‐related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy‐treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK‐dependent paraptosis. [ABSTRACT FROM AUTHOR]

Simple Summary: Cell death was once thought of as two distinct processes involving mainly apoptosis or necrosis. In recent years, several different forms of cell death have gained importance, such as autophagy, paraptosis, and ferroptosis. Brassinin (BSN) is a phytoalexin identified from Cruciferase vegetables (Brassica) and various pharmacological properties have been reported. However, the efficacy of BSN for chronic myelogenous leukemia has not been elucidated. In our study, we confirmed whether BSN modulates the different cell death processes including apoptosis, autophagy, and paraptosis in chronic myelogenous leukemia cells. In addition, we confirmed that BSN promotes the activation of the MAPK signaling pathway. In this study, BSN could stimulate the activation of apoptosis, autophagy, and paraptosis by modulating the MAPK signaling cascade. Brassinin (BSN), a potent phytoalexin found in cruciferous vegetables, has been found to exhibit diverse anti-neoplastic effects on different cancers. However, the impact of BSN on chronic myelogenous leukemia (CML) cells and the possible mode of its actions have not been described earlier. We investigated the anti-cytotoxic effects of BSN on the KBM5, KCL22, K562, and LAMA84 CML cells and its underlying mechanisms of action in inducing programmed cell death. We noted that BSN could induce apoptosis, autophagy, and paraptosis in CML cells. BSN induced PARP cleavage, subG1 peak increase, and early apoptosis. The potential action of BSN on autophagy activation was confirmed by an LC3 expression and acridine orange assay. In addition, BSN induced paraptosis through increasing the reactive oxygen species (ROS) production, mitochondria damage, and endoplasmic reticulum (ER) stress. Moreover, BSN promoted the activation of the MAPK signaling pathway, and pharmacological inhibitors of this signaling pathway could alleviate all three forms of cell death induced by BSN. Our data indicated that BSN could initiate the activation of apoptosis, autophagy, and paraptosis through modulating the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

The mulberry tree (Morus alba) has been cultivated in China for thousands of years. Mulberry Diels-Alder-type adducts (MDAAs) are characteristic constituents of the genus Morus. The unique structure and diverse bioactivities of MDAAs have attracted the attention of researchers. Kuwanon M (KWM) is an MDAA isolated from the root bark of Morus alba. This research reports the growth inhibitory effects of KWM on human lung cancer cells and its possible mechanism. In A549 and NCI-H292 cells, KWM treatment induced suppression of cell proliferation and migration. The appearance of chromatin condensation, phosphatidyl serine exposure and caspase cleavage indicated the arising of apoptosis. The loss of mitochondrial membrane potential (MMP), release of cytochrome c and dysregulation of Bax/Bcl-2 demonstrated that the KWM-induced apoptosis was through the mitochondrial pathway. Paraptosis was simultaneously detected under KWM treatment, as evidenced by the exhibition of cytoplasmic vacuolation, down-regulation of Alix and up-regulation of endoplasmic reticulum (ER) stress-related proteins. Mechanistically, ER stress induced activation of unfolded protein response (UPR) pathways and activation of the MAPK (JNK and ERK) pathway, all of which were critical for KWM-induced apoptosis and paraptosis. These findings suggested the possibility that KWM might be considered as a potential lung cancer therapeutic agent. [ABSTRACT FROM AUTHOR]

Micheliolide (MCL), a naturally occurring sesquiterpene lactone, has demonstrated significant anticancer properties through the induction of various programmed cell death mechanisms. This study aimed to explore MCL's effects on autophagy, paraptosis, and ferroptosis in pancreatic and colon cancer cells, along with its modulation of the MAPK signaling pathway. MCL was found to substantially suppress cell viability in these cancer cells, particularly in MIA PaCa-2 and HT-29 cell lines. The study identified that MCL induced autophagy by enhancing the levels of autophagy markers such as Atg7, p-Beclin-1, and Beclin-1, which was attenuated by the autophagy inhibitor 3-MA. Furthermore, MCL was found to facilitate paraptosis, indicated by decreased Alix and in-creased ATF4 and CHOP levels. It also promoted ferroptosis, as demonstrated by the reduced expression of SLC7A11, elevated TFRC levels, and increased intracellular iron. Additionally, MCL activated the MAPK signaling pathway, marked by the phosphorylation of JNK, p38, and ERK, linked with an increase in ROS production that is vital in regulating these cell death mechanisms. These findings propose that MCL is a versatile anticancer agent, capable of activating various cell death pathways by modulating MAPK signaling and ROS levels. These results emphasize the therapeutic promise of MCL in treating cancer, pointing to the necessity of further in vivo investigations to confirm these effects and determine its potential clinical uses. [ABSTRACT FROM AUTHOR]

[Display omitted] Although photodynamic therapy (PDT) has been well-known as a promising anti-tumor treatment, its limited therapeutic efficiency remains to be a large challenge. In this study, a carrier free nanomedicine (designated as PyroMor) is developed to greatly initiate cell apoptosis and paraptosis for synergistic cancer therapy. Pyropheophorbide-a (Pyro) and morusin (Mor) are capable of self-assembling into PyroMor, which has been testified to have superiority of improved stability, cellular internalization, and biocompatibility. Because of efficient cellular uptake behavior, PyroMor could induce cellular paraptosis by Mor-caused vacuolation in mitochondria, ER and cytoplasm, contributing to improving the PDT efficacy of Pyro. Therefore, self-delivery PyroMor is able to accomplish synergistic anti-tumor effect via stimulation of cell apoptosis as well as paraptosis. In addition, in vivo studies also clarify that PyroMor presents passive tumor targeting delivery, leading to robust repressive effect on tumor proliferation with negligible systemic toxicity. This strategy of combined cancer therapy by initiating both cell apoptosis and paraptosis extremely benefits to the development of precise and effective cancer therapy in clinic. [ABSTRACT FROM AUTHOR]

Deregulated activation of the Wnt/β-catenin pathway is observed in many types of human malignancies including colon cancer. Abrogation of the Wnt/β-catenin pathway has been demonstrated as an effective way of inducing cancer cell death. Herein, a new isoxazolyl-urea (QR-5) was synthesized and examined its efficacy on the viability of colon cancer cell lines. QR-5 displayed selective cytotoxicity towards colon cancer cells over normal counterparts. QR-5 induced apoptosis as evidenced by elevation in sub-G1 cells, decrease in Bcl-2, MMP-9, COX-2, VEGF and cleavage of PARP and caspase-3. QR-5 reduced the mitochondrial membrane potential, decreased the expression of Alix and elevated the expression of ATF4 and CHOP indicating the induction of paraptosis. The inhibitor of apoptosis (Z-DEVD-FMK) and paraptosis (CHX) could not restore Alix expression and PARP cleavage in QR-5 treated cells, respectively suggesting the complementation between the two cell death pathways. QR-5 suppressed the expression of Wnt/β-catenin pathway proteins which was also evidenced by the downregulation of nuclear and cytoplasmic β-catenin. The dependency of QR-5 on β-catenin for inducing apoptosis and paraptosis was demonstrated by knockdown experiments using β-catenin specific siRNA. Overall, QR-5 induces apoptosis as well as paraptosis by mitigating the Wnt/β-catenin axis in colon cancer cells. • A new isoxazolyl-urea (QR-5) was synthesized and examined for cytotoxicity. • QR-5 displayed selective cytotoxicity on colon cancer cells over normal cells. • QR-5 induced apoptosis and paraptosis in colon cancer cells. • QR-5 abrogated Wnt/β-catenin pathway to induce cytotoxicity in colon cancer cells. [ABSTRACT FROM AUTHOR]

The process of apoptosis is one of the essential processes involved in maintenance of homeostasis in the human body. It can aid to remove misfolded proteins or cellular organelles. This sequence is especially necessary in cancer cells. However, specifically targeting already apoptotic pathways can induce drug resistance in cancer cells and hence drugs can induce cell death by alternative mechanism. We investigated whether fangchinoline (FCN) can target renal carcinoma cells by inducing multiple cell death mechanisms. Both paraptosis, autophagy, and apoptosis were induced by FCN through stimulation of diverse molecular signaling pathways. FCN induced ROS production with GSH/GSSG imbalance, and ER stress. In addition, formation of autophagosome and autophagy related markers were stimulated by FCN. Moreover, FCN induced cell cycle arrest and PARP cleavage. Except for blocking protein synthesis, these three cell death pathways were found to be complementarily working together with each other. FCN also exhibited synergistic effects with paclitaxel in inducing programmed cell death in RCC cells. Our data indicates that FCN could induce apoptotic cell death and non-apoptotic cell death pathways and can be con-tribute to development of novel cancer prevention or therapy. [ABSTRACT FROM AUTHOR]

Although Erythrina senegalensis is a plant widely used in traditional medicine in sub-Saharan Africa, its biological properties have been poorly investigated to date. We first characterized by conventional reactions the composition of several stem bark extracts and evaluated in acellular and cellular assays their pro- or antioxidant properties supported by their high phenolic and flavonoid content, particularly with the methanolic extract. The pro- or antioxidant effects observed did not correlate with their IC50 concentrations against five cancer cell lines determined by MTT assay. Indeed, the CH2Cl2 extract and its ethyl acetate (EtOAc) subfraction appeared more potent although they harbored lower pro- or antioxidant effects. Nevertheless, at equipotent concentration, both extracts induced ER- and mitochondria-derived vacuoles observed by fluorescent microscopy that further led to non-apoptotic cell death. LC coupled to high resolution MS investigations have been performed to identify chemical compounds of the extracts. These investigations highlighted the presence of compounds formerly isolated from E. senegalensis including senegalensein that could be retrieved only in the EtOAc subfraction but also thirteen other compounds, such as 16:3-Glc-stigmasterol and hexadecanoic acid, whose anticancer properties have been previously reported. Nineteen other compounds remain to be identified. In conclusion, E. senegalensis appeared rich in compounds with antioxidant and anticancer properties, supporting its use in traditional practice and its status as a species of interest for further investigations in anticancer drug research. [ABSTRACT FROM AUTHOR]

Despite the success of proteasome inhibitors (PIs) in treating hematopoietic malignancies, including multiple myeloma (MM), their clinical efficacy is limited in solid tumors. In this study, we investigated the involvement of the integrated stress response (ISR), a central cellular adaptive program that responds to proteostatic defects by tuning protein synthesis rates, in determining the fates of cells treated with PI, bortezomib (Bz). We found that Bz induces ISR, and this can be reversed by ISRIB, a small molecule that restores eIF2B-mediated translation during ISR, in both Bz-sensitive MM cells and Bz-insensitive breast cancer cells. Interestingly, while ISRIB protected MM cells from Bz-induced apoptosis, it enhanced Bz sensitivity in breast cancer cells by inducing paraptosis, the cell death mode that is accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Combined treatment with ISRIB and Bz may shift the fate of Bz-insensitive cancer cells toward paraptosis by inducing translational rescue, leading to irresolvable proteotoxic stress. • Bz-induced ISR can have a pro-survival or pro-death role depending on the cell types. • Inhibition of ISR by ISRIB switches fates of Bz-sensitive/insensitive cancer cells. • ISRIB plus Bz triggers paraptosis in breast cancer cells via enhanced proteotoxicity. [ABSTRACT FROM AUTHOR]

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB + mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer. [ABSTRACT FROM AUTHOR]

In our previous study, 20 nm-sized amorphous silica nanoparticles (20-SiNPs), but not 50 nm-sized amorphous silica nanoparticles (50-SiNPs), induced pulmonary inflammatory response in rats exposed repeatedly for 14 days (12.5, 25, and 50 μg/time, total six times). In this study, we tried to clarify the causes of different responses induced by both SiNPs using mice (12.5, 25, and 50 μg/lung) and mouse alveolar macrophage cells. When exposed to alveolar macrophage cells for 24 h, both SiNPs decreased cell viability and enhanced ROS generation compared to controls. The 20- and 50-SiNPs also formed giant and autophagosome-like vacuoles in the cytoplasm, respectively. Structural damage of organelles was more pronounced in 20-SiNPs-treated cells than in 50-SiNPs-treated cells, and an increased mitochondrial membrane potential and mitochondrial calcium accumulation were observed only in the 20-SiNPs-treated cells. Additionally, a single intratracheal instillation of both sizes of SiNPs to mice clearly elevated the relative proportion of neutrophils and inhibited differentiation of macrophages and expression of an adhesion molecule. Meanwhile, interestingly, the total number of pulmonary cells and the levels of pro-inflammatory mediators more notably increased in the lungs of mice exposed to 20-SiNPs compared to 50-SiNPs. Given that accumulation of giant vacuoles and dilation of the ER and mitochondria are key indicators of paraptosis, we suggest that 20-SiNPs-induced pulmonary inflammation may be associated with paraptosis of alveolar macrophages. [ABSTRACT FROM AUTHOR]

In today's world, cancer is still the leading cause of human death. Among them, the incidence and mortality of lung cancer remain high, and have become the focus of research in the world. BEAP, a protein with anti-lung cancer activity, was isolated and purified from the edible mushroom Boletus edulis. Previous studies have shown that BEAP can inhibit the proliferation of non-small cell lung cancer A549 cells by inducing apoptosis and cell cycle arrest in vitro and in vivo. However, there are many ways in which antitumor proteins from edible and medicinal mushroom play their roles. It is worth exploring whether there are other antitumor mechanisms of BEAP, which can provide reference value for the development of new drugs targeting non-small cell lung cancer and the repurposing of existing drugs. [ABSTRACT FROM AUTHOR]

Apoptosis resistance of tumor cells often results in chemoresistance and treatment failure in clinic. In this work, a Cu2+-coordinated morusin/doxorubicin biological organizer (designated as COMBO) is designed to combat cellular resistance to apoptosis for combined tumor therapy. By virtue of the coordination and π-π stacking effects, the self-assembled COMBO possesses nanometer particle size, narrow and homogenous graininess distribution as well as a good dispersion stability. Moreover, COMBO could be disassembled by glutathione (GSH) with an effective drug release and fluorescence recovery. Morusin-mediated paraptosis could induce extensive vacuolization through the dilation of endoplasmic reticulum (ER) and mitochondria, leading to non-apoptotic programmed cell death (PCD) regardless of the cellular resistance to apoptosis. Furthermore, the released doxorubicin prefers to locate in cell nucleus to cause cell apoptosis for combined chemotherapy. By the joint action of paraptosis and apoptosis, COMBO exhibits a great superiority over monotherapy in tumor inhibition with a low system toxicity. This study may open a window in the development of self-delivery nanomedicine for overcoming apoptosis resistance in tumor therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Non‐small cell lung cancer (NSCLC) with wild‐type epidermal growth factor receptor (EGFR) is intrinsic resistance to EGFR‐tyrosine kinase inhibitors (TKIs), such as afatinib. Celastrol, a natural compound with antitumor activity, was reported to induce paraptosis in cancer cells. In this study, intrinsic EGFR‐TKI‐resistant NSCLC cell lines H23 (EGFR wild‐type and KRAS mutation) and H292 (EGFR wild‐type and overexpression) were used to test whether celastrol could overcome primary afatinib resistance through paraptosis induction. The synergistic effect of celastrol and afatinib on survival inhibition of the NSCLC cells was evaluated by CCK‐8 assay and isobologram analysis. The paraptosis and its modulation were assessed by light and electron microscopy, Western blot analysis, and immunofluorescence. Xenografts models were established to investigate the inhibitory effect of celastrol plus afatinib on the growth of the NSCLC tumors in vivo. Results showed that celastrol acted synergistically with afatinib to suppress the survival of H23 and H292 cells by inducing paraptosis characterized by extensive cytoplasmic vacuolation. This process was independent of apoptosis and not associated with autophagy induction. Afatinib plus celastrol‐induced cytoplasmic vacuolation was preceded by endoplasmic reticulum stress and unfolded protein response. Accumulation of intracellular reactive oxygen species and mitochondrial Ca2+ overload may be initiating factors of celastrol/afatinib‐induced paraptosis and subsequent cell death. Furthermore, Celastrol and afatinib synergistically suppressed the growth of H23 cell xenograft tumors in vivo. The data indicate that a combination of afatinib and celastrol may be a promising therapeutic strategy to surmount intrinsic afatinib resistance in NSCLC cells. [ABSTRACT FROM AUTHOR]

Nowadays, non-small cell lung cancer (NSCLC) is still one of the most life-threatening diseases in the world. In previous studies, a fungal protein PFAP with anti-NSCLC properties was isolated and identified from Pleurotus ferulae lanzi. In this study, the amino acid sequence of PFAP was analyzed and found to be highly homologous to the aegerolysin family. PFAP, like other members of the aegerolysin family, specifically recognizes lipid raft domains rich in cholesterol and sphingomyelin, which is probably its specific anti-tumor mechanism. Previous studies have shown that PFAP can induce AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells. This study further revealed that PFAP can also induce paraptosis and endoplasmic reticulum stress (ERS) in A549 cells in vitro by targeting AMPK. PFAP induces multi-pathway death of A549 cells, and thus demonstrates its potential value for developing new drugs for NSCLC. [ABSTRACT FROM AUTHOR]

Cyclometalated iridium (Ir) complexes having Ir(tpy)3 (tpy=2- (4'-tolyl)pyridine) scaffold exhibit promising potential for the diagnosis of cancer, as phosphorescent probes for cellular imaging and in the treatment of cancer, due to their high stability, tunable photophysical properties, and biological activities. We previously reported on the synthesis of some cationic amphiphilic Ir(III) complex-peptide hybrids (IPHs) such as 2 and 3 that contain KK(K)GG peptide units (K: lysine, G: glycine) that are attached via alkyl chain (C2~C16) linkers and induce paraptosis-like cell death in Jurkat (T-lymphocyte leukemia) cancer cells through multiple cell death pathways. Herein, we report on the design and synthesis of two IPHs, 4 and 5, which possess two and one KKKGG sequences, respectively, and an examination of the effect of the number of peptide units on their anticancer activity. Our evaluations of the anticancer properties of 3-5 suggested that 3 and 4 are more cytotoxic than 5 against Jurkat cells, indicating a positive relationship between the number of H2N-KKKGG units in 3-5 and their anticancer activity. In addition, a parallel relationship was observed between the cytotoxicity of 3-5 and Ca2+ overload in mitochondria, suggesting the direct transfer of Ca2+ from the endoplasmic reticulum to mitochondria. [ABSTRACT FROM AUTHOR]

Objective: There were rarely investigations on the effects and molecular mechanisms of oral squamous cell carcinoma (OSCC) cells when treated with isorhamnetin. This article assesses the anti‐cancer effect of isorhamnetin. Methods and materials: Oral squamous cell carcinoma cells were treated with or without isorhamnetin. Cell proliferation, cell cycle arrest, cell migration, cell death, and the related signaling pathways were evaluated. Results: The results revealed that cell proliferation was inhibited in a dose‐ and time‐dependent manner, which was confirmed by diminished cell viability and revealed by decreased in the number of cell colonies. In addition, the cell cycle arrested in the G2/M phase, and the protein levels of cyclin B1 and CDC2 were suppressed. Moreover, the cell migration was inhibited, and the protein levels of related proteins were modulated. Furthermore, it could be observed that abundant cytoplasmic vacuoles existed which that were derived from mitochondria and the endoplasmic reticulum. It was confirmed that cell death did not result from apoptosis and may have which may be apt to paraptosis. Isorhamnetin was observed to upregulate phosphorylated ERK cascades and increase intracellular reactive oxygen species levels. Conclusions: Our study suggested that the anti‐cancer effect of isorhamnetin might trigger paraptosis, which may indicate a new therapeutic approach to OSCC. [ABSTRACT FROM AUTHOR]

Purpose: Paclitaxel is an anticancer drug for the treatment of non-small cell lung cancer (NSCLC). However, drug-resistance remains a major problem. Honokiol is a natural component which has been found to exhibit anti-tumor activity. Paclitaxel and honokiol have been reported to be able to induce paraptosis. The aim of this study was to investigate whether honokiol can reverse paclitaxel resistance by inducing paraptosis in NSCLC cells. Methods: NSCLC cell lines H1650 (paclitaxel-sensitive), H1299 and H1650/PTX (intrinsic and acquired paclitaxel-resistant, respectively) were used to assess the cytotoxic effects of paclitaxel and honokiol. Light and transmission electron microscopy were performed to detect cytoplasmic vacuolation. In vitro cell viability and clonogenic survival assays, as well as in vivo xenograft assays were conducted to test synergistic killing effects of paclitaxel and honokiol on NSCLC cells. Western blotting, flow cytometry and immunofluorescence were performed to evaluate paraptosis-regulating mechanisms. Results: We found that combination treatment with paclitaxel and honokiol synergistically killed H1650, H1299 and H1650/PTX cells by inducing paraptosis, which is characterized by cytoplasmic vacuolation. Moreover, paclitaxel/honokiol treatment resulted in a significant growth delay in H1299 xenograft tumors that showed extensive cytoplasmic vacuolation. Mechanistically, proteasomal inhibition-mediated endoplasmic reticulum (ER) stress and unfolded protein responses leading to ER dilation, and the disruption of intracellular Ca2+ homeostasis and mitochondrial Ca2+ overload resulting in mitochondrial disfunction, were found to be involved in paclitaxel/honokiol-induced paraptosis. Cellular protein light chain 3 (LC3) may play an important role in paclitaxel/honokiol induced cytoplasmic vacuolation and NSCLC cell death. Conclusions: Combination of honokiol and paclitaxel may represent a novel strategy for the treatment of paclitaxel-resistant NSCLC. [ABSTRACT FROM AUTHOR]

Thrombospondin‐1 (TSP1) is involved in corneal wound healing caused by chemical injury. Herein, we examined the effects of TSP1 on hypoxia‐induced damages and wound‐healing activity in human corneal epithelial (HCE) cells. Exosomal protein expression was determined using liquid chromatography‐tandem mass spectrometry, and HCE cell migration and motility were examined through wound‐healing assay and time‐lapse microscopy. Reestablishment of cell junctions by TSP1 was assessed through confocal microscopy and 3D image reconstruction. Our results show that CoCl2‐induced hypoxia promoted HCE cell death by paraptosis. TSP1 protected these cells against paraptosis by attenuating mitochondrial membrane potential depletion, swelling and dilation of endoplasmic reticulum and mitochondria, and mitochondrial fission. Exosomes isolated from HCE cells treated with TSP1 contained wound healing‐associated proteins that were taken up by HCE cells to promote tissue remodeling and repair. TSP1 protected HCE cells against hypoxia‐induced damages and inhibited paraptosis progression by promoting cell migration, cell‐cell adhesion, and extracellular matrix remodeling. These findings indicate that TSP1 ameliorates hypoxia‐induced paraptosis in HCE cells and promotes wound healing and remodeling by regulating exosomal protein expression. TSP1 may, therefore, play important roles in the treatment of hypoxia‐associated corneal diseases. [ABSTRACT FROM AUTHOR]

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2′‐hydroxy‐retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin‐like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin‐like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy. [ABSTRACT FROM AUTHOR]

Aims: Due to their significant biological activity, thiosemicarbazones (TSCs) are promising candidates for anticancer therapy. In part, the efficacy of TSCs is linked to their ability to chelate essential metal ions such as copper and iron. Triapine, the best-studied anticancer TSC, has been tested clinically with promising results in hematological diseases. During the past few years, a novel subclass of TSCs with improved anticancer activity was found to induce paraptosis, a recently characterized form of cell death. The aim of this study was to identify structural and chemical properties associated with anticancer activity and paraptosis induction of TSCs. Results: When testing a panel of structurally related TSCs, compounds with nanomolar anticancer activity and paraptosis-inducing properties showed higher copper(II) complex solution stability and a slower reduction rate, which resulted in reduced redox activity. In contrast, TSCs with lower anticancer activity induced higher levels of superoxide that rapidly stimulated superoxide dismutase expression in treated cells, effectively protecting the cells from drug-induced redox stress. Innovation: Consequently, we hypothesize that in the case of close Triapine derivatives, intracellular reduction leads to rapid dissociation of intracellularly formed copper complexes. In contrast, TSCs characterized by highly stable, slowly reducible copper(II) complexes are able to reach new intracellular targets such as the endoplasmic reticulum-resident protein disulfide isomerase. Conclusion: The additional modes of actions observed with highly active TSC derivatives are based on intracellular formation of stable copper complexes, offering a new approach to combat (drug-resistant) cancer cells. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family. It is an arbovirus that can cause congenital abnormalities and is sexually transmissible. A series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital ZIKV syndrome and its underlying pathophysiological mechanisms. Endoplasmic reticulum (ER) and ER-related proteins are essential in ZIKV genome replication. This review highlights the subcellular localization of ZIKV to the ER and ZIKV modulation on the architecture of the ER. This review also discusses ZIKV interaction with ER proteins such as signal peptidase complex subunit 1 (SPCS1), ER membrane complex (EMC) subunits, and ER translocon for viral replication. Furthermore, the review covers several important resulting effects of ZIKV infection to the ER and cellular processes including ER stress, reticulophagy, and paraptosis-like death. Pharmacological targeting of ZIKV-affected ER-resident proteins and ER-associated components demonstrate promising signs of combating ZIKV infection and rescuing host organisms from severe neurologic sequelae. [ABSTRACT FROM AUTHOR]

The superior inhibitory effect of DpdtbA on ferroptosis-resistant melanoma cells stemmed from ferritinophagy mediating the ROS production which triggered G1 phase arrest, apoptosis, paraptosis and inactivation of PI3K/AKT. [Display omitted] Melanoma cells were more resistant to ferroptosis with still poor therapy outcomes. Sensitizing melanoma cell to the ferroptosis inducer was a crucial strategy for treatment of melanoma. In the present study, 2,2′-di-pyridylketone hydrazone dithiocarbamate s-butyric acid (DpdtbA) displayed superior inhibitory activity than ferroptosis inducer Erastin in melanoma cells, which prompt us to explore the underlying mechanism. The analyses from flow cytometry and Western blot showed that the growth inhibition of DpdtbA against SK-MEL-28 and A375 cells involved apoptosis induction and G1 phase arrest. Surprisingly, the cytoplasmic vacuoles were found upon the treatment; transmission electron microscopy and endoplasmic reticulum (ER) staining revealed that the cytoplasmic vacuoles were in ER; while down-regulation of alix and requirement of protein synthesis suggested there was an occurrence of paraptosis. However, both NAC and 3-MA could significantly attenuate the cytoplasmic vacuolization and growth inhibition, hinting that both ROS and autophagy involved the paraptosis induction. The additional evidence revealed that there was an occurrence of continuous ferritinophagy, which was responsible for the ROS production. Downregulation of NCOA4 clearly attenuated the apoptosis and paraptosis induction. In addition, activation of MAPK involved regulation of paraptosis, but only ERK and JNK had role in the formation of cytoplasmic vacuoles and growth inhibition. Furthermore, a ROS dependent regulation of PI3K/AKT pathway was also involved. Taken together, our result firstly demonstrated that a continuous ferritinophagy contributed to the apoptosis and paraptosis induction, highlighting that the lysosomal labile iron pool had a crucial role in control of melanoma cell fate. [ABSTRACT FROM AUTHOR]

Objectives: Prostate cancer, after the phase of androgen dependence, may progress to the castration‐resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E‐derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti‐cancer properties of δ‐TT in CRPC cells (PC3 and DU145) and the related mechanisms of action. Materials and methods: MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy. Results: We demonstrated that δ‐TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ‐TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ‐TT; in autophagy‐defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ‐TT; (c) in both CRPC cell lines, δ‐TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ‐TT. Conclusions: These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti‐cancer activity of δ‐TT in CRPC cells. [ABSTRACT FROM AUTHOR]

This study revealed that iturin A‐like lipopeptides produced by Bacillus subtillis induced both paraptosis and apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco‐2) cells. Autophagy was simultaneously induced in Caco‐2 cells treated with iturin A‐like lipopeptides at the early stage and inhibited at the later stage. A western blot analysis showed that the lipopeptides induced apoptosis in Caco‐2 cells via a mitochondrial‐dependent pathway, as indicated by upregulated expression of the apoptotic genes bax and bad and downregulated expression of the antiapoptotic gene bcl‐2. The induction of paraptosis in Caco‐2 cells was indicated by the occurrence of many cytoplasmic vacuoles accompanied by endoplasmic reticulum (ER) dilatation and mitochondrial swelling and dysfunction. ER stress also occurred with significant increases in reactive oxygen species and Ca2+ levels in cells. Autophagy was detected by a transmission electron microscopy analysis and by upregulated expression of LC3‐II and downregulated expression of LC3‐I. The inhibition of autophagy at the later stage was shown by upregulated expression of p62. This study revealed the capability of iturin A‐like B. subtilis lipopeptides to simultaneously execute antitumor potential via multiple pathways. Endoplasmic reticulum stress caused by Iturin A‐like lipopeptides human epithelial colorectal adenocarcinoma (Caco‐2) cells the appeared reaction, resulting in the increase of intracellular calcium and swelling and dysfunction of mitochondria. A large number of vacuoles appeared in the cell, and the parabed apoptosis occurred. Reactive oxygen species (ROS) was released in mitochondria, DNA was damaged and mitochondria permeability increased. Then the endogenous apoptosis pathway is triggered. A large number of damaged organelles and proteins in the cell lead to the activation of autophagy pathway. [ABSTRACT FROM AUTHOR]

Rifampicin-induced hepatotoxicity is a primary cause of drug-induced liver injury (DILI), posing a significant challenge to its continued clinical application. Moreover, the mechanism underlying rifampicin-induced hepatotoxicity remains unclear. Human hepatocyte line-17 (HHL-17) cells were treated with an increasing dose of rifampicin for 24 h, and male Wistar rats were given rifampicin [150 mg/kg body weight (bw)] orally for 28 days. Viability assay, protein expression, and cell death assays were analyzed in vitro. Moreover, liver serum markers, body/organ weight, H&E staining, protein expression, etc., were assayed in vivo. Rifampicin induced a dose-dependent hepatotoxicity in HHL-17 cells (IC 50 ; 600 μM), and increased the serum levels of liver injury markers, e.g., alanine transaminase (ALT) and aspartate transaminase (AST) in rats. Rifampicin-induced cell death was non-apoptotic and non-necroptotic both in vitro and in vivo. Further, excessive cellular vacuolization and reduced expression of Alix protein confirmed the induction of paraptosis both in vitro and in vivo. In addition, a significant increase in the endoplasmic reticulum (ER) stress markers (e.g., BiP, CHOP, and total polyubiquitinated proteins) was detected, demonstrating the induction of ER stress and altered protein homeostasis. Interestingly, rifampicin-induced hepatotoxicity was associated with the inhibition of autophagy and enhanced reactive oxygen species (ROS) generation in HHL-17 cells. Furthermore, inhibition of protein synthesis by cycloheximide (CHX) suppressed paraptosis by alleviating rifampicin-induced ER stress and ROS generation. Rifampicin-induced hepatotoxicity involves ER stress-driven paraptosis as a novel mechanism of its toxicity that may be targeted to protect liver cells from rifampicin toxicity. [Display omitted] • Rifampicin induces hepatotoxicity via alternate programmed cell death paraptosis. • Undue cellular vacuolization and reduction of Alix protein underlying paraptosis • Rifampicin-induced ER stress and ROS played a crucial role in hepatotoxicity. • Autophagy suppression by rifampicin promotes ER stress-mediated proteotoxicity. • Paraptosis inhibition by cycloheximide prevents rifampicin-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

[Display omitted] • Three new cyclometalated Ir(III) complexes were synthesized and characterized. • Complex Ir1 is mainly located lysosome in HepG2 cancer cells. • Ir1 is the less cytotoxic to normal cells than to tumor cells. • Ir1 is the first reported iridium(III) complex-based paraptosis inducer to successfully induce tumor cell ICD. Immunotherapy has been shown to provide superior antitumor efficacy by activating the innate immune system to recognize, attack and eliminate tumor cells without seriously harming normal cells. Herein, we designed and synthesized three new cyclometalated iridium(III) complexes (Ir1 , Ir2 , Ir3) then evaluated their antitumor activity. When co-incubated with HepG2 cells, the complex Ir1 localized in the lysosome, where it induced paraptosis and endoplasmic reticulum stress (ER stress). Notably, Ir1 also induced immunogenic cell death (ICD), promoted dendritic cell maturation that enhanced effector T cell chemotaxis to tumor tissues, down-regulated proportions of immunosuppressive regulatory T cells within tumor tissues and triggered activation of antitumor immunity throughout the body. To date, Ir1 is the first reported iridium(III) complex-based paraptosis inducer to successfully induce tumor cell ICD. Furthermore, Ir1 induced ICD of HepG2 cells without affecting cell cycle or reactive oxygen species levels. [ABSTRACT FROM AUTHOR]

The polyphenolic component of citrus fruits, hesperetin (Hst), is a metabolite of hesperidin. In this study, we examined the effect of varying doses and exposure times of hesperetin on MCF-7 and MDA-MB-231 cancer cells, as well as MCF-10A normal cells. By using MTT assay, real-time PCR, western blot, and flow cytometry, we determined the effects of Hst on cell viability, ROS levels, and markers of cell death. Furthermore, molecular docking was used to identify Hst targets that might be involved in ROS-dependent cell death. According to the results, different concentrations of Hst induced different modes of cell death at specific ROS levels. Paraptosis occurred in all cell lines at concentration ranges of IC 35 to IC 60 , and apoptosis occurred at concentrations greater than IC 65. In addition, MDA-MB-231 cells were subjected to senescence at sub-toxic doses when treated for a long period of time. When Hst levels were higher, N -acetylcysteine (NAC)'s effect on neutralizing ROS was more pronounced. According to the docking results, Hst may interact with several proteins involved in the regulation of ROS. As an example, the interaction of CCS (Copper chaperone for superoxide dismutase) with Hst might interfere with its chaperone function in folding SOD-1 (superoxide dismutase enzyme), contributing to an increase in cytoplasmic ROS levels. Finally, depending on the ROS level, Hst induces various modes of cell death. • Hesperetin induces paraptosis in breast cancer and normal cell lines. • At subtoxic concentrations, hesperetin induces senescence in the MDA-MB-231 cell line. • Hesperetin induces different models of cell death at different levels of ROS. • Hesperetin may increase Ras by interfering with SOD1's ROS regulation. [ABSTRACT FROM AUTHOR]

Introduction: As a commonly used chemotherapeutic agent, fluorouracil (5-FU) has serious dose-limiting side effects. In this study, we evaluated the synergy between red American ginseng (RAG) and 5-FU on human colorectal cancer cells, and explored the potential mechanisms. Methods: Ginsenoside contents of white American ginseng (WAG) and RAG were determined by HPLC. Cell proliferation was evaluated by MTS assay. Combination Index (CI) analysis was executed using CompuSyn software. Paraptotic events were observed after crystal violet staining. Cell cycle distribution, cyclin A expression and apoptotic induction were analyzed using flow cytometry. Results: We observed the heat treatment remarkably increased levels of ginsenoside Rg3, 20R-Rg3, Rk1 and Rg5. When the combinations of 5-FU and RAG were applied, cell proliferation inhibition rates were notably increased, indicating that RAG significantly enhanced 5-FU's effect. Additionally, CI analysis suggested that there was a synergistic action of 5-FU and RAG when combined. The cell cycle data indicated 5-FU induced S phase arrest, and the combination of 5-FU and RAG increased G1 phase. Further, the RAG's ability to enhance the anti-cancer effects of 5-FU was linked to both paraptosis and apoptosis inductions. Conclusion: RAG may have clinical utility to decrease the dosage of 5-FU in colorectal cancer therapeutics. [ABSTRACT FROM AUTHOR]

Curcumin is a polyphenol compound extracted from Curcuma longa plant, is a molecule with pleiotropic effects that suppresses transformation, proliferation and metastasis of malignant tumors. Curcumin can cause different kinds of cell death depending of its concentration on the exposed cell type. Here we show that exposure of the glioblastoma cell line A172 to curcumin at 50 μM, the IC50, causes morphological change characteristic of paraptosis cell-death. Vesicles derived from the endoplasmic reticulum (ER) and low membrane potential of the mitochondria were constantly found in the exposed cells. Furthermore, changes in expression of the ER Stress Response (ERSR) genes IRE1 and ATF6 , and the microRNAs (miRNAs) miR-27a , miR-222 , miR-449 was observed after exposure to curcumin. AKT-Insulin and p53-BCL2 networks were predicted being modulated by the affected miRNAs. Furthermore, AKT protein levels reduction was confirmed. Our data, strongly suggest that curcumin exerts its cell-death properties by affecting the integrity of the reticulum, leading to paraptosis in the glioblastoma cells. These data unveils the versatility of curcumin to control cancer progression. [ABSTRACT FROM AUTHOR]

An α, β-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of β5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells. [ABSTRACT FROM AUTHOR]

In the Mid-19th century, Rudolf Virchow considered necrosis to be a prominent form of cell death; since then, pathologists have recognized necrosis as both a cause and a consequence of disease. About a century later, the mechanism of apoptosis, another form of cell death, was discovered, and we now know that this process is regulated by several molecular mechanisms that "programme" the cell to die. However, discoveries on cell death mechanisms are not limited to these, and recent studies have allowed the identification of novel cell death pathways that can be molecularly distinguished from necrotic and apoptotic cell death mechanisms. Moreover, the main goal of current cancer therapy is to discover and develop drugs that target apoptosis. However, resistance to chemotherapeutic agents targeting apoptosis is mainly responsible for the failure of clinical therapy and adverse side effects of the chemotherapeutic agents currently in use pose a major threat to the well-being and lives of patients. Therefore, the development of natural-based anticancer drugs with low cellular and organismal side effects is of great interest. In this comprehensive review, we thoroughly examine and discuss natural anticancer compounds that specifically target non-canonical cell death mechanisms. [ABSTRACT FROM AUTHOR]

The effects of the exposure of proliferating MDCK cells to thallium [Tl(I) or Tl(III)] on cell viability and proliferation were investigated. Although Tl stopped cell proliferation, the viability was > 95%. After 3 h, two autophagy markers (SQSTM-1 expression and LC3β localization) were altered, and at 48 h increased expression of SQSTM-1 (60%) and beclin-1 (50–100%) were found. At 24 h, the expression of endoplasmic reticulum (ER) stress markers ATF-6 and IRE-1 were increased in 100% and 150%, respectively, accompanied by XBP-1 splicing and nuclear translocation. At 48 h, major ultrastructure abnormalities were found, including ER enlargement and cytoplasmic vacuolation which was not prevented by protein synthesis inhibition. Increased PHB (85% and 40% for Tl(I) and Tl(III), respectively) and decreased β-tubulin (45%) expression were found which may be related to the promotion of paraptosis. In summary, Tl(I) and Tl(III) promoted ER stress and probably paraptosis in MDCK cells, impairing their proliferation. • Tl(I) and Tl(III) stop MDCK cell proliferation without affecting their viability. • Tl(I) and Tl(III) do not induce MDCK cell necrosis or apoptosis. • Tl(I) and Tl(III) induce the formation of cytosolic vacuoles. • Tl(I) and Tl(III) cause early autophagy in MDCK cells. • Tl(I) and Tl(III) induce reticulum stress and paraptosis in MDCK cells. [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) plays a central role in the regulation of proteins that control cell growth and apoptosis and has therefore become an important target for anticancer therapy. Several constitutive subunits of the 19S proteasome display deubiquitinase (DUB) activity, suggesting that ubiquitin modification of proteins is dynamically regulated. Our study and others have shown that metal complexes, such as copper complexes, can induce cancer cell apoptosis through inhibiting 19S proteasome-associated DUBs and/or 20S proteasome activity. In this study, we found that (1) Hinokitiol copper complex (HK-Cu) induces striking accumulation of ubiquitinated proteins in A549 and K562 cells (2) HK-Cu potently inhibits the activity of the 19S proteasomal DUBs much more effectively than it does to the chymotrypsin-like activity of the 20S proteasome (3) HK-Cu effectively induces caspase-independent and paraptosis-like cell death in A549 and K562 cells, and (4) HK-Cu-induced cell death depends on ATF4-assosiated ER stress but is apparently not related to ROS generation. Altogether, these data indicate that HK-Cu can inhibit the activity of the 19S proteasomal DUBs and induce paraptosis-like cell death, representing a new drug candidate for cancer treatment. [ABSTRACT FROM AUTHOR]

Objective To detect the expression profile of transcription factor C/EBPβ in human immortalized normal hepatic cell lines and hepatocellular carcinoma cell lines so as to determine the correlation between C/EBPβ with cell death mediated by endoplasmic reticulum stress in hepatocellular cells. Methods We cultured the human immortalized normal hepatic cells lines HHL5 and HL7702 and hepatocellular carcinoma cell lines SMMC7721; Bel7402, HepG2 and Hep3B. Hep3B cells were used as the cell model in tunicamycin-induced endoplasmic reticulum stress. Cellular morphology was observed under an inverted optical microscope. MTT assay was used to assess the inhibition of cell growth. To detect cell apoptosis, the cells were dyed with Hoechst 33258 and observed using a fluorescence microscope. RT-PCR and Western blotting were used to detect the expression of at mRNA and protein levels, respectively. Results We found that normally the mRNA and protein isoform of C/EBPβ, C/EBPβ-1, were both expressed in all of the four hepatocellular cell lines and the two immortalized normal hepatic cell lines, while C/EBPβ protein isoform C/EBPβ-3 was only expressed in the two immortalized normal hepatic cell lines. Tunicamycin increased the expressions of both mRNA and protein of C/EBPβ in Hep3B cells and the increase of protein isoform C/EBPβ-3 was the most remarkable. In Hep3B cells, cell death was induced by tunicamycin through endoplasmic reticulum stress activity. Apoptosis as well as paraptosis was observed in tunicamycin-induced cell death. Conclusion C/EBPβ-3, one of the protein isoforms of C/EBPβ, is only expressed in normal hepatic cell lines, but not in hepatocellular cell lines. C/EBPβ is involved in cell death mediated by endoplasmic reticulum stress activity in hepatocellular carcinoma cells. [ABSTRACT FROM AUTHOR]

The cytopathic effects of Zika virus ( ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins ( IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by 'implosive' cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum ( ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death. [ABSTRACT FROM AUTHOR]

[Display omitted] Glioblastoma multiforme (GBM) is one of the most lethal malignant tumors in the human brain, with only a few chemotherapeutic drugs available after surgery. Nitrovin (difurazone) is widely used as an antibacterial growth promoter in livestock. Here, we reported that nitrovin might be a potential anticancer lead. Nitrovin showed significant cytotoxicity to a panel of cancer cell lines. Nitrovin induced cytoplasmic vacuolation, reactive oxygen species (ROS) generation, MAPK activation, and Alix inhibition but had no effect on caspase-3 cleavage and activity, suggesting paraptosis activation. Nitrovin-induced cell death of GBM cells was significantly reversed by cycloheximide (CHX), N-acetyl-l-cysteine (NAC), glutathione (GSH), and thioredoxin reductase 1 (TrxR1) overexpression. Vitamins C and E, inhibitors of pan-caspase, MAPKs, and endoplasmic reticulum (ER) stress failed to do so. Nitrovin-triggered cytoplasmic vacuolation was reversed by CHX, NAC, GSH, and TrxR1 overexpression but not by Alix overexpression. Furthermore, nitrovin interacted with TrxR1 and significantly inhibited its activity. In addition, nitrovin showed a significant anticancer effect in a zebrafish xenograft model, which was reversed by NAC. In conclusion, our results showed that nitrovin induced non-apoptotic and paraptosis-like cell death mediated by ROS through targeting TrxR1. Nitrovin might be a promising anticancer lead for further development. [ABSTRACT FROM AUTHOR]

Programmed cell death is a vital biological process for multicellular organisms to maintain cellular homeostasis, which is regulated in a complex manner. Over the past several years, apart from apoptosis, which is the principal mechanism of caspase-dependent cell death, research on non-apoptotic forms of programmed cell death has gained momentum. p53 is a well characterized tumor suppressor that controls cell proliferation and apoptosis and has also been linked to non-apoptotic, non-canonical cell death mechanisms. p53 impacts these non-canonical forms of cell death through transcriptional regulation of its downstream targets, as well as direct interactions with key players involved in these mechanisms, in a cell type- or tissue context-dependent manner. In this review article, we summarize and discuss the involvement of p53 in several non-canonical modes of cell death, including caspase-independent apoptosis (CIA), ferroptosis, necroptosis, autophagic cell death, mitotic catastrophe, paraptosis, and pyroptosis, as well as its role in efferocytosis which is the process of clearing dead or dying cells. [ABSTRACT FROM AUTHOR]

Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Manystudies have shown that prolonged paraptosis activation improves tumorimmunogenicity; this treatment reproduces the vaccinating effects of mM-CSFtransduced cells. In this short communication, we want to highlight the paraptosisprocess as a valuable strategy for clinical immunotherapy against cancer. [ABSTRACT FROM AUTHOR]