Apoptosis induction resulting from proteasome inhibition.
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ABSTRACT:
SUBMITTER: Shinohara K
PROVIDER: S-EPMC1217499 | biostudies-other | 1996 Jul
REPOSITORIES: biostudies-other
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Project description:PURPOSE:Previously, compounds containing a piperidone structure have been shown to be highly cytotoxic to cancer cells. Recently, we found that the piperidone compound P2 exhibits a potent anti-neoplastic activity against human breast cancer-derived cells. Here, we aimed to evaluate two piperidone compounds, P1 and P2, for their potential anti-neoplastic activity against human leukemia/lymphoma-derived cells. METHODS:Cytotoxicity and apoptosis induction were evaluated using MTS, annexin V-FITC/PI and mitochondrial membrane potential polychromatic assays to confirm the mode of action of the piperidone compounds. The effects of compound P1 and P2 treatment on gene expression were assessed using AmpliSeq analysis and, subsequently, confirmed by RT-qPCR and Western blotting. RESULTS:We found that the two related piperidone compounds P1 and P2 selectively killed the leukemia/lymphoma cells tested at nanomolar concentrations through induction of the intrinsic apoptotic pathway, as demonstrated by mitochondrial depolarization and caspase-3 activation. AmpliSeq-based transcriptome analyses of the effects of compounds P1 and P2 on HL-60 acute leukemia cells revealed a differential expression of hundreds of genes, 358 of which were found to be affected by both. Additional pathway analyses revealed that a significant number of the common genes were related to the unfolded protein response, implying a possible role of the two compounds in the induction of proteotoxic stress. Subsequent analyses of the transcriptome data revealed that P1 and P2 induced similar gene expression alterations as other well-known proteasome inhibitors. Finally, we found that Noxa, an important mediator of the activity of proteasome inhibitors, was significantly upregulated at both the mRNA and protein levels, indicating a possible role in the cytotoxic mechanism induced by P1 and P2. CONCLUSIONS:Our data indicate that the cytotoxic activity of P1 and P2 on leukemia/lymphoma cells is mediated by proteasome inhibition, leading to activation of pro-apoptotic pathways.
| S-EPMC6241245 | biostudies-literature
Project description:Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin-proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base-copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and I?B-?, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi-pi, pi-cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents.
| S-EPMC3676669 | biostudies-literature
Project description:Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.
| S-EPMC3279386 | biostudies-literature
Project description:Aimsb-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition.ResultsWe report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation.InnovationThe findings establish that different modes of proteasome inhibition result in distinct cellular responses, a finding of potential therapeutic importance.ConclusionOur data show that enhanced oxidative stress and ER stress are major determinants of the strong apoptotic response elicited by the 19S DUB inhibitor b-AP15.
| S-EPMC4241954 | biostudies-literature
Project description:Proteasome inhibition represents an important anticancer strategy. Here, we studied the mechanisms at the basis of the pro-apoptotic activity of the standardized decoction of Hemidesmus indicus, a plant evoking a complex anticancer activity, and explored its inhibition of proteasome activity in human leukemia cells. Additionally, we preliminary tested the cytotoxicity of some H. indicus's phytochemicals on leukemia cells and their intestinal absorption on a human intestinal epithelium model consisting of a monolayer of differentiated Caco2 cells. We observed a potent antileukemic effect for H. indicus, imputable to the modulation of different critical targets at protein and mRNA levels and the reduction of the 26S proteasome expression. We found that some phytomarkers of H. indicus decoction passed through the enterocyte monolayer. Overall, our study supports the pharmacological potential of H. indicus, which can represent an interesting botanical drug in the oncological area.
| S-EPMC6510901 | biostudies-literature
Project description:Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.
| S-EPMC5940303 | biostudies-literature
Project description:Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.
| S-EPMC1219525 | biostudies-other
Project description:BACKGROUND AND PURPOSE: Proteasome inhibitors exhibit cytotoxic against tumours of different histology. However, the mechanism of apoptosis induction by these compounds remains unclear and is likely to be a complex cascade of events. Bcl-2-associated athanogene (BAG) family proteins are characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance, including heat shock proteins (HSP), Bcl-2, Raf-1. The role of BAG family proteins in proteasome inhibition has not been elucidated. EXPERIMENTAL APPROACH: Effects of proteasome inhibitors on BAG2 expression were evaluated using real-time reverse transcription-polymerase chain reaction (RT-PCR). BAG2 expression was knocked down by small interfering RNAs (siRNA). Cell death was evaluated using Annexin V/propidium iodide staining and subsequent FACS. KEY RESULTS: The proteasome inhibitors, MG132, PSI, lactacystin and epoxomicin, induced BAG2 at the transcriptional level. MG132-induced apoptosis was significantly suppressed by BAG2 knockdown using RNA interference. CONCLUSIONS AND IMPLICATIONS: Our results suggest that BAG2 is a novel molecule induced by proteasome inhibition, which exhibits a pro-apoptotic property in death of thyroid cancer cells induced by proteasome inhibition.
| S-EPMC2584920 | biostudies-other
Project description:BACKGROUND:The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction. METHODOLOGY/PRINCIPAL FINDINGS:Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells. CONCLUSIONS/SIGNIFICANCE:These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.
| S-EPMC2719015 | biostudies-literature
Project description:Heart is a multi-cellular organ made up of various cell types interacting with each other. Cardiomyocytes may benefit or suffer from crosstalk with noncardiomyocytes in response to diverse kinds of cardiac stresses. Proteasome dysfunction is a common cardiac stress which causes cardiac proteotoxicity and contributes to cardiac diseases such as heart failure and myocardial infarction. The role of crosstalk between cardiomyocytes and noncardiomyocytes in defense of cardiac proteotoxicity remains unknown. Here, we report a cardiomyocyte-specific survival upon proteasome inhibition in a heterogeneous culture consisting of cardiomyocytes and other three major cardiac cell types. Conversely, cardiomyocyte apoptosis is remarkably induced by proteasome inhibition in a homogeneous culture consisting of a majority of cardiomyocytes, demonstrating an indispensable role of noncardiomyocytes in the prevention of cardiomyocyte apoptosis resulting from proteasome inhibition. We further show that cardiomyocytes express brain natriuretic peptide (BNP) as an extracellular molecule in response to proteasome inhibition. Blockade of BNP receptor on noncardiomyocytes significantly exacerbated the cardiomyocyte apoptosis, indicating a paracrine function of cardiomyocyte-released extracellular BNP in activation of a protective feedback from noncardiomyocytes. Finally, we demonstrate that proteasome inhibition-activated transcriptional up-regulation of BNP in cardiomyocytes was associated with the dissociation of repressor element 1 silencing transcription factor (REST)/ histone deacetylase 1 (HDAC1) repressor complex from BNP gene promoter. Consistently, the induction of BNP could be further augmented by the treatment of HDAC inhibitors. We conclude that the crosstalk between cardiomyocytes and noncardiomyocytes plays a crucial role in the protection of cardiomyocytes from proteotoxicity stress, and identify cardiomyocyte-released BNP as a novel paracrine signaling molecule mediating this crosstalk. These findings provide new insights into the key regulators and cardioprotective mechanism in proteasome dysfunction-related cardiac diseases.
| S-EPMC7502079 | biostudies-literature