Showing papers on "MG132 published in 2015"
TL;DR: The studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α, and it is shown that the BBR reducing effects on H NF1α protein andPCSK9 gene transcription can be eradicated by proteasome inhibitors.
106 citations
TL;DR: Investigation of the molecular basis of insulin resistance elicited by FFAs using the human hepatocyte cell line HepG2 revealed that palmitate facilitates ubiquitination of the key insulin signaling molecules, and subsequently elicits their proteasomal degradation.
84 citations
TL;DR: It is shown that the C-terminal LRLRGG of ISG15 interacts with the binder of ubiquitin zinc finger (BUZ) domain of histone deacetylase 6 (HDAC6) that marks proteins for interaction with HDAC6 and p62 upon forced stressful conditions likely as a step toward autophagic clearance.
75 citations
TL;DR: Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival, and this means of rebalancing proteostasis is conserved from yeast to humans.
Abstract: Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.
69 citations
TL;DR: The results suggest that TLR-mediated Sesn2 induction is dependent on AP-1, Nrf2, and the inhibition of ubiquitin-mediated degradation of SesN2 and might protect cells against endotoxin toxicity.
63 citations
TL;DR: The data suggest that p27Kip1 down-regulation in cervical cancer cells is primarily regulated through PI3K/AKT-mediated proteasomal degradation and may form a basis for the design of novel cervical cancer therapies.
Abstract: The cyclin-dependent kinase inhibitor p27Kip1 is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27Kip1 may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27Kip1 down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27Kip1 and its effects on proliferation, cell cycle progression and (inhibition of) apoptosis in cervical cancer cells. Primary cervical cancer samples (n = 70), normal cervical tissue samples (n = 30) and cervical cancer-derived cell lines (n = 8) were used to assess the expression of p27Kip1 and AKT1 by RT-PCR, Western blotting and immunohistochemistry, respectively. The effects of the PI3K inhibitor LY294004 and the proteasome inhibitor MG132 on cervical cancer cell proliferation were investigated using a MTT assay. Apoptosis and cell cycle analyses were carried out using flow cytometry, and sub-cellular p27Kip1 localization analyses were carried out using immunofluorescence assays. We observed p27Kip1 down-regulation (p = 0.045) and AKT1 up-regulation (p = 0.046) in both the primary cervical cancer samples and the cervical cancer-derived cell lines, compared to the normal cervical tissue samples tested. Treatment of cervical cancer-derived cell lines with the PI3K inhibitor LY294002 resulted in a reduced AKT1 activity. We also observed a dose-dependent inhibition of cell viability after treatment of these cell lines with the proteasome inhibitor MG132. Treatment of the cells with LY294002 resulted in a G1 cell cycle arrest, a nuclear expression of p27Kip1, and a cytoplasmic p27Kip1 accumulation after subsequent treatment with MG132. Additionally, we found that the synergistic effect of MG132 and LY294002 resulted in a sub-G1 cell cycle arrest and apoptosis induction through poly (ADP-ribose) polymerase (PARP) cleavage. Our data suggest that p27Kip1 down-regulation in cervical cancer cells is primarily regulated through PI3K/AKT-mediated proteasomal degradation. The observed synergistic effect of the MG132 and LY294002 inhibitors may form a basis for the design of novel cervical cancer therapies.
60 citations
TL;DR: It is demonstrated that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.
Abstract: The proteasome inhibitors Bortezomib (BZM) and MG132 trigger cancer cell death via induction of endoplasmic reticulum (ER) stress and unfolded protein response. Epigallocatechin gallate (EGCG), the most bioactive green tea polyphenol, is known to display strong anticancer properties as it inhibits proteasome activity and induces ER stress. We investigated whether combined delivery of a proteasome inhibitor with EGCG enhances prostate cancer cell death through increased induction of ER stress. Paradoxically, EGCG antagonized BZM cytotoxicity even when used at low concentrations. Conversely, the MG132 dose-response curve was unaffected by co-administration of EGCG. Moreover, apoptosis, proteasome inhibition and ER stress were inhibited in PC3 cells simultaneously treated with BZM and EGCG but not with a combination of MG132 and EGCG; EGCG enhanced autophagy induction in BZM-treated cells only. Autophagy inhibition restored cytotoxicity concomitantly with CHOP and p-eIF2α up-regulation in cells treated with BZM and EGCG. Overall, these findings demonstrate that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.
55 citations
TL;DR: It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway.
52 citations
TL;DR: It is shown that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway in the CHO cell line, and it is found that APP interacts with USP25.
Abstract: Beta-amyloid (Aβ), a major pathological hallmark of Alzheimer's disease (AD), is derived from amyloid precursor protein (APP) through sequential cleavage by β-secretase and γ-secretase enzymes. APP is an integral membrane protein, and plays a key role in the pathogenesis of AD; however, the biological function of APP is still unclear. The present study shows that APP is rapidly degraded by the ubiquitin-proteasome system (UPS) in the CHO cell line in response to endoplasmic reticulum (ER) stress, such as calcium ionophore, A23187, induced calcium influx. Increased levels of intracellular calcium by A23187 induces polyubiquitination of APP, causing its degradation. A23187-induced reduction of APP is prevented by the proteasome inhibitor MG132. Furthermore, an increase in levels of the endoplasmic reticulum-associated degradation (ERAD) marker, E3 ubiquitin ligase HRD1, proteasome activity, and decreased levels of the deubiquitinating enzyme USP25 were observed during ER stress. In addition, we found that APP interacts with USP25. These findings suggest that acute ER stress induces degradation of full-length APP via the ubiquitin-proteasome proteolytic pathway.
49 citations
TL;DR: A potential clinical application of ATO is suggested in sarcoma therapy and that combined treatment with a proteasome inhibitor can increase the therapeutic efficacy.
43 citations
TL;DR: The results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.
Abstract: Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na(+)/K(+))-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.
TL;DR: It is demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated withHDAC4 sumoylation to induce these protective effects, and TSA treatment resulted in a decrease in HDAC 4 in cardiomyocytes infected with adenoviral HDac4, and HDAC3‐induced injury was attenuated by TSA.
Abstract: Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition-mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 h of hypoxia (H) (1% O2 ) and 2 h of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R-elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA-induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R-induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4-induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects.
TL;DR: In this article, the authors defined a role for survivin in cellular protection by insulin against doxorubicin-associated injury and showed that Sp1 is a critical factor in the transcriptional regulation of survivin.
Abstract: Insulin inhibits ischemia/reperfusion-induced myocardial apoptosis through the PI3K/Akt/mTOR pathway. Survivin is a key regulator of anti-apoptosis against doxorubicin-induced cardiotoxicity. Insulin increases survivin expression in cardiac myocytes to mediate cytoprotection. However, the mechanism by which survivin mediates the protective effect of insulin against doxorubicin-associated injury remains to be determined. In this study, we demonstrated that pretreatment of H9c2 cardiac myocytes with insulin resulted in a significant decrease in doxorubicin-induced apoptotic cell death by reducing cytochrome c release and caspase-3 activation. Doxorubicin-induced reduction of survivin mRNA and protein levels was also significantly perturbed by insulin pretreatment. Reducing survivin expression with survivin siRNA abrogated insulin-mediated inhibition of caspase-3 activation, suggesting that insulin signals to survivin inhibited caspase-3 activation. Interestingly, pretreatment of H9c2 cells with insulin or MG132, a proteasome inhibitor, inhibited doxorubicin-induced degradation of the transcription factor Sp1. ChIP assay showed that pretreatment with insulin inhibited doxorubicin-stimulated Sp1 dissociation from the survivin promoter. Finally using pharmacological inhibitors of the PI3K pathway, we showed that insulin-mediated activation of the PI3K/Akt/mTORC1 pathway prevented doxorubicin-induced proteasome-mediated degradation of Sp1. Taken together, insulin pretreatment confers a protective effect against doxorubicin-induced cardiotoxicity by promoting Sp1-mediated transactivation of survivin to inhibit apoptosis. Our study is the first to define a role for survivin in cellular protection by insulin against doxorubicin-associated injury and show that Sp1 is a critical factor in the transcriptional regulation of survivin.
TL;DR: An inhibitor of ubiquitin activating E1 enzyme inhibits Ubiquitin conjugation and aggresome formation.
Abstract: Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy–lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies.
TL;DR: The results indicate that accumulation of ubiquitinated proteins alter cellular bioenergetics and redox status, leading to AMPK activation.
TL;DR: The findings in this study highlight a novel biological function of JQ1 in modulating apoptosis and warrant further study of the potential treatment of cancer with the J Q1 and TRAIL combination.
Abstract: Inhibition of BET bromodomains (BRDs) has emerged as a promising cancer therapeutic strategy. Accordingly, inhibitors of BRDs such as JQ1 have been actively developed and some have reached clinical testing. However, the mechanisms by which this group of inhibitors exerts their anticancer activity, including induction of apoptosis, have not been fully elucidated. This report reveals a previously uncovered activity of JQ1 in inducing c-FLIP degradation and enhancing TRAIL-induced apoptosis. JQ1 potently decreased c-FLIP (both long and short forms) levels in multiple cancer cell lines without apparently increasing the expression of DR5 and DR4. Consequently, JQ1, when combined with TRAIL, synergistically induced apoptosis; this enhanced apoptosis-inducing activity could be abolished by enforced expression of ectopic FLIPL or FLIPS. Hence it appears that JQ1 decreases c-FLIP levels, resulting in enhancement of TRAIL-induced apoptosis. Inhibition of proteasome with MG132 prevented JQ1-induced c-FLIP reduction. Moreover, JQ1 decreased c-FLIP stability. Therefore, JQ1 apparently decreases c-FLIP levels through facilitating its proteasomal degradation. Genetic inhibition of either BRD4 or c-Myc by knocking down their expression failed to mimic JQ1 in decreasing c-FLIP and enhancing TRAIL-induced apoptosis, suggesting that JQ1 induces c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 or c-Myc inhibition. In summary, our findings in this study highlights a novel biological function of JQ1 in modulating apoptosis and warrant further study of the potential treatment of cancer with the JQ1 and TRAIL combination.
TL;DR: The results suggest that PI-induced autophagy may act as a compensatory degradation system for degradation of A53T α-syn when the ubiquitin-proteasome system is impaired and may directly contribute to the survival of PC12 cells treated with proteasome inhibitors.
Abstract: The aim of the present study was to examine the effects of proteasome inhibitor (PI)‑induced autophagy on PC12 cells overexpressing A53T mutant α‑synuclein (α‑syn) by detecting alterations in the levels of microtubule‑associated protein 1A/1B light chain (LC3)+ autophagosomes and the lysotracker‑positive autolysosomes using immunofluorescence, the expression of LC3‑II using western blot analysis and the morphology of PC12 cells using transmission electron microscopy It was found that the addition of MG132 (500 nmol/l) significantly increased the number of autophagosomes and autolysosomes and upregulated the expression of LC3‑II The autophagy inhibitor 3‑methyladenine (3‑MA) completely inhibited the autophagy induced by MG132 (500 nmol/l) The autophagy enhancer trehalose significantly increased the number of autophagosomes and autolysosomes and improved the protein level of LC3‑II induced by MG132 To examine the effect of PI‑induced autophagy on the degradation of A53T mutant α‑syn, the expression of α‑syn was detected by western blot analysis It was revealed that MG132 increased the expression of A53T α‑syn and trehalose counteracted the increase of A53T α‑syn induced by MG132 Combined inhibition of 3‑MA and PI significantly increased the accumulation of A53T α‑syn as compared with treatment using either single agent In addition, combination of MG132 (500 nmol/l) with trehalose (50 mmol/l) or 3‑MA (2 mmol/l) markedly decreased the cell viability as compared with treatment using either single agent individually as demonstrated using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay These results suggest that the PI, MG132, could induce autophagy in PC12 cells overexpressing A53T mutant α‑syn and this autophagy could be completely inhibited by 3‑MA, indicating that PI‑induced autophagy is mediated by the upregulation of the macroautophagy class III PI3K pathway PI‑induced autophagy may act as a compensatory degradation system for degradation of A53T α‑syn when the ubiquitin‑proteasome system is impaired Autophagy activation may directly contribute to the survival of PC12 cells treated with proteasome inhibitors The present study may assist in illuminating the association between PI and autophagy in the pathogenesis of Parkinson's disease
TL;DR: Results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astroCytic dysfunction and the maintenance of the neuroinflammatory state.
TL;DR: IR combined with a proteasome inhibitor or TRAF6 inhibition could represent a new therapeutic strategy for human pancreatic cancer and display synergistic cell killing effects and induced endoplasmic reticulum stress.
TL;DR: The data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression and opens avenues of research to determine whether the prote asome, or K48-linked ubiquitinated substrates, are potential therapeutic targets in OA.
Abstract: Objectives To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). Methods Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin–proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). Results Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. Conclusions Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are potential therapeutic targets in OA.
TL;DR: It is demonstrated that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins, suggesting that targeting the UPS and BCl-2 protein expression may be a valuable strategy to prevent β-cell demise in type 2 diabetes.
Abstract: Pancreatic β-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of β-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in β-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected β-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent β-cell demise in type 2 diabetes.
TL;DR: It is demonstrated that HT upregulates HO‐1 expression by stimulating the nuclear accumulation and stabilization of Nrf2, leading to the wound repair of VECs crucial in the prevention of atherosclerosis.
Abstract: Hydroxytyrosol (HT), an olive plant (Olea europaea L.) polyphenol, has proven atheroprotective effects. We previously demonstrated that heme oxygenase-1 (HO-1) is involved in the HT dependent prevention of dysfunction induced by oxidative stress in vascular endothelial cells (VECs). Here, we further investigated the signaling pathway of HT-dependent HO-1 expression in VECs. HT dose- and time-dependently increased HO-1 mRNA and protein levels through the PI3K/Akt and ERK1/2 pathways. Cycloheximide and actinomycin D inhibited both increases, suggesting that HT-triggered HO-1 induction is transcriptionally regulated and that de novo protein synthesis is necessary for this HT effect. HT stimulated nuclear accumulation of nuclear factor E2-related factor 2 (Nrf2). This Nrf2 accumulation was blocked by actinomycin D and cycloheximide whereas HT in combination with the 26S proteasome inhibitor MG132 enhanced the accumulation. HT also extended the half-life of Nrf2 proteins by decelerating its turnover. Moreover, HO-1 inhibitor, ZnppIX and CO scavenger, hemoglobin impaired HT-dependent wound healing while CORM-2, a CO generator, accelerated wound closure. Together, these data demonstrate that HT upregulates HO-1 expression by stimulating the nuclear accumulation and stabilization of Nrf2, leading to the wound repair of VECs crucial in the prevention of atherosclerosis.
TL;DR: The role of c‐Myc in mediating anti‐proliferative effects of γ‐tocotrienol in mammary cancer cells was determined and it was found that c‐ myc activity plays a central role in cancer transformation.
Abstract: Objectives
Aberrant c-Myc activity plays a central role in cancer transformation. γ-tocotrienol is a member of the vitamin E family that displays potent anti-cancer activity. Here, studies were conducted to determine the role of c-Myc in mediating anti-proliferative effects of γ-tocotrienol in mammary cancer cells.
Materials and methods
Treatment effects on mouse +SA and human MCF-7 mammary cancer cell proliferation were determined by MTT assay and Ki-67 staining. Protein expression was determined by western blot analysis. Immunofluorescence staining and qRT-PCR were used to characterize cellular c-Myc and MYC levels respectively.
Results
Anti-proliferative effects of γ-tocotrienol were associated with reduction in total c-Myc and phosphorylated-c-Myc-serine 62, and increase in phosphorylated-c-Myc-threonine 58 levels. γ-tocotrienol also reduced PI3K/Akt/mTOR and Ras/MEK/Erk mitogenic signalling, cyclin D1 and cyclin-dependent kinase 4 levels, and increased p27 levels. However, γ-tocotrienol had no effect on MYC mRNA levels. γ-tocotrienol also increased levels of FBW7 (E3 ligase that initiates ubiquitination of c-Myc), but had no effect on serine/threonine phosphatase PP2A or isomerase Pin 1 levels. Combined treatment with GSK3α/β inhibitor LiCl or proteasome inhibitor MG132 blocked γ-tocotrienol-induced reductions in c-Myc.
Conclusions
These findings indicate that anti-proliferative effects of γ-tocotrienol are associated with reduction in c-Myc that results from increase in GSK-3α/β-dependent ubiquitination and degradation, rather than from reduction in c-Myc synthesis in +SA and MCF-7 mammary cancer cells.
TL;DR: The results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.
TL;DR: Results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.
Abstract: It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.
TL;DR: The results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.
Abstract: Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.
TL;DR: It is observed that synergistic cytotoxicity of GOS and VPA could be extended to human A375, HeLa, and PC-3 cancer cells and induced robust apoptosis by suppressing the cyclin-A2/Akt/FOXO3a pathway.
Abstract: // Gao-Xiang Zhao 1, * , Li-Hui Xu 2, * , Hao Pan 1 , Qiu-Ru Lin 1 , Mei-Yun Huang 1 , Ji-Ye Cai 3 , Dong-Yun Ouyang 1 , Xian-Hui He 1 1 Department of Immunobiology, College of Life Science and Technology, Jinan University, Guangzhou, China 2 Department of Cell Biology, College of Life Science and Technology, Jinan University, Guangzhou, China 3 Department of Chemistry, College of Life Science and Technology, Jinan University, Guangzhou, China * These authors have contributed equally to this work Correspondence to: Xian-Hui He, e-mail: thexh@jnu.edu.cn Dong-Yun Ouyang, e-mail: dongyun1967@aliyun.com Keywords: gossypol, valproic acid, apoptosis, akt, FOXO3a Received: April 29, 2015 Accepted: October 06, 2015 Published: October 16, 2015 ABSTRACT Previously we reported that valproic acid (VPA) acts in synergy with GOS to enhance cell death in human DU145 cells. However, the underlying mechanism remains elusive. In this study, we observed that such synergistic cytotoxicity of GOS and VPA could be extended to human A375, HeLa, and PC-3 cancer cells. GOS and VPA co-treatment induced robust apoptosis as evidenced by caspase-8/-9/-3 activation, PARP cleavage, and nuclear fragmentation. GOS and VPA also markedly decreased cyclin A2 protein expression. Owing to the reduction of cyclin A2, Akt signaling was suppressed, leading to dephosphorylation of FOXO3a. Consequently, FOXO3a was activated and the expression of its target genes, including pro-apoptotic FasL and Bim , was upregulated. Supporting this, FOXO3a knockdown attenuated FasL and Bim upregulation and apoptosis induction in GOS+VPA-treated cells. Furthermore, blocking proteasome activity by MG132 prevented the downregulation of cyclin A2, dephosphorylation of Akt and FOXO3a, and induction of apoptosis in cells co-treated with GOS and VPA. In mouse model, GOS and VPA combination significantly inhibited the growth of A375 melanoma xenografts. Our findings indicate that GOS and VPA co-treatment induces apoptosis in human cancer cells by suppressing the cyclin-A2/Akt/FOXO3a pathway.
TL;DR: This study suggests that insulin upregulates SESN2 content via the PI3K/mTOR signaling pathway and this effect is attributed to decreased SESn2 degradation.
Abstract: Sestrin (SESN) is known as a cysteine sulfinic acid reductase. Recently, nonredox functions of SESN in metabolic regulation and antitumor property have been recognized. While mechanisms underlying the expression of SESN are not fully understood. Here we report that insulin markedly increased SESN2 level in HepG2 cells through mTOR activation. To determine whether insulin affects SESN2 degradation, we assessed SESN2 turnover by applying the protein synthesis inhibitor, cycloheximide (CHX), and found that following insulin treatment SESN2 protein levels were reduced significantly slower than non-insulin-treated cells. Furthermore, the proteasomal inhibitor, MG132, dramatically increased SESN2 protein and its ubiquitination level while in the presence of MG132 insulin did not further increase SESN2 content, suggesting that insulin increases SESN2 content mainly via inhibiting its proteasomal degradation. We then explored the potential feedback role of SESN2 in insulin signaling by SESN2 siRNA knockdown in HepG2 cells. Following SESN2 knockdown insulin-stimulated PKB phosphorylation was enhanced and accompanied by reduced PTEN content. Taken together, our study suggests that insulin upregulates SESN2 content via the PI3K/mTOR signaling pathway and this effect is attributed to decreased SESN2 degradation. Furthermore, SESN2 via modulating PTEN plays a negative feedback role in insulin signaling.
TL;DR: Together, this study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.
Abstract: Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.
TL;DR: It is found that reconstitution of HSF4b into Hsf4-/- lens epithelial cells can simultaneously downregulate Hsp70 expression and upregulate the expression of small heat shock proteins Hsp25 and αB-crystallin at both RNA and protein levels.