Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Inhibition of proteasome by MG-132 treatment causes extra phragmoplast formation and cortical microtubule disorganization during M/G1 transition in synchronized tobacco cells

The 26S proteasome plays essential roles in cell cycle progression in various types of cell. We previously reported that the inhibition of 26S proteasome activities by a proteasome inhibitor, MG-132, exclusively caused cell cycle arrest in synchronized tobacco BY-2 cells. Here we report a further observation of 26S proteasome involvement during M/G1 transition utilizing a transgenetic BY-2 cell line that stably expresses a GFP-alpha-tubulin fusion protein (BY-GT16). Interestingly, MG-132 treatment caused the arrest of cell cycle progression prior to entering the G1 phase. Indeed, phragmoplast-like structures were formed and cortical microtubules were not organized after the collapse of the original phragmoplasts. Additionally, actin microfilaments showed irregular rearrangements when further incubated with MG-132 and as the phragmoplast-like structures developed. Since these phragmoplast-like structures had a similar configuration and ability to form cell plates to that of the original phragmoplasts, we designated these phragmoplast-like structures as extra phragmoplasts. Furthermore, we showed that a tobacco kinesin-related polypeptide of 125 kDa (TKRP125) localized in the extra phragmoplasts and that its protein level remained unchanged during MG-132 treatment. We propose that TKRP125 might be one of the possible targets of the ubiquitin-proteasome degradation pathway during M/G1 transition.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21^Waf1/Cip1 mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21^Waf1/Cip1 induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.     

Proteasome activator enhances survival of Huntington's disease neuronal model cells

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28gamma, but not subunit S5a, recovered proteasome function in the HD cells. PA28gamma also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells.     

The Proteasome Inhibitor MG-132 Induces AIF Nuclear Translocation Through Down-Regulation of ERK and Akt/mTOR Pathway

Anticancer activity of proteasome inhibitors has been demonstrated in various cancer cell types. However, mechanisms by which they exert anticancer action were not fully understood. The present study was undertaken to examine the effect of the proteasome inhibitor MG-132 and the underlying mechanism in glioma cells. MG-132 caused alterations in mitochondrial membrane potential and apoptosis-inducing factor (AIF) nuclear translocation. MG-132 induced reduction in ERK and Akt activation. The transient transfection of constitutively active forms of MEK, an upstream of ERK, and Akt blocked the MG-132-induced cell death. Similarly to down-regulation of Akt, expression levels of mTOR were inhibited by MG-132. Addition of rapamycin, an inhibitor of mTOR, caused stimulation of the MG-132-induced cell death. There were no significant changes in levels of XIAP, survivin, and Bax. Overexpression of constitutively active forms of MEK and Akt blocked the MG-132-induced AIF nuclear translocation. These findings indicate that MG-132 induces AIF nuclear translocation through down-regulation of ERK and Akt/mTOR pathways. These data suggest that proteasome inhibitors may serve as potential therapeutic agents for malignant human gliomas.     

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.     

Green tea polyphenol (-) -epigallocatechin-3-gallate promotes the rapid protein kinase C- and proteasome-mediated degradation of Bad: implications for neuroprotection

The aim of the present study was to gain a deeper insight into the cell signaling pathways involved in the neuroprotection/neurorescue activity of the major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG). EGCG (1 micro m) caused an immediate (30 min) down-regulation (approximately 40%) of Bad protein levels, and a more pronounced reduction after 24 h (55%) in the human neuroblastoma cell line SH-SY5Y. Co-treatment with EGCG and the protein synthesis inhibitor cycloheximide prominently shortened Bad half-life, with as little as 30% of the Bad protein content remaining after 2 h, suggesting an effect of EGCG on Bad protein degradation. Accordingly, the proteasome inhibitors MG-132 and lactacystin damped Bad down-regulation by EGCG. The general protein kinase C (PKC) inhibitor GF109203X, or the down-regulation of conventional and novel PKC isoforms, abolished EGCG-induced Bad decline. However, no inhibition was seen with the cell-permeable myristoylated pseudosubstrate inhibitor of the atypical PKCzeta isoform. The enforced expression of Bad for up to 72 h rendered the cells more susceptible to serum deprivation-induced cell death, whereas EGCG treatment significantly improved cell viability (up to 1.6-fold). The present study reveals a novel pathway in the neuroprotective mechanism of the action of EGCG, which involves a rapid PKC-mediated degradation of Bad by the proteasome.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Rescue from a two hit, high-throughput model of neurodegeneration with N-acetyl cysteine

Postmortem tissue from patients with neurodegeneration exhibits protein-misfolding stress and reduced proteasome activity. This hallmark burden of proteotoxic stress has led to the term "proteinopathies" for neurodegenerative diseases. Proteinopathies may also be exacerbated by previous insults, according to the two hit hypothesis of accelerated neurodegeneration. In order to model the response to two successive insults in a high-throughput fashion, we exposed the neuronal cell line N2a to two hits of the proteasome inhibitor MG132 and performed three unbiased viability assays. MG132 toxicity was synergistically exacerbated following sequential hits provided the first hit was high enough to be toxic. This accelerated viability loss was apparent by (1) a nuclear and cytoplasmic stain (DRAQ5+Sapphire), (2) immunocytochemistry for a cytoskeletal marker (α-tubulin), and (3) ATP levels (Cell Titer Glo). Ubiquitin-conjugated proteins were raised by toxic, but not subtoxic MG132, and were thus correlated with toxicity exacerbation at higher doses. We hypothesized that levels of autophagic and antioxidant defenses would be reduced with toxic, but not subtoxic MG132, explaining their differential impact on a second hit. However, proteins involved in chaperone-mediated autophagy were raised by toxic MG132, not reduced. Furthermore, inhibiting autophagy enhanced the toxicity of both subtoxic and toxic MG132 as well as of dual hits, suggesting that autophagic removal of cellular debris protected against proteasome inhibition. Two toxic hits of MG132 synergistically decreased the antioxidant glutathione. The glutathione precursor N-acetyl cysteine reversed this glutathione loss and prevented the toxic response to dual hits by all three assays. Dietary supplementation with N-acetyl cysteine benefits Alzheimer's patients and is currently undergoing clinical trials in Parkinson's disease. The present report is the first demonstration that this versatile compound is protective against synergistic loss of viability as well as of glutathione following unrelenting, sequential hits of proteotoxic stress as may occur in the diseased brain.