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.     

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.     

Stress and IGF-I differentially control cell fate through mammalian target of rapamycin (mTOR) and retinoblastoma protein (pRB)

Significant discoveries have recently contributed to our knowledge of intracellular growth factor and nutrient signaling via mTOR (mammalian target of rapamycin). This signaling pathway is essential in cellular metabolism and cell survival by enhancing protein translation through phosphorylation of 4EBP-1 and p70S6K. Growth factors like insulin-like growth factor-I induce mTOR to prevent cell death during cellular stress. Agents targeting mTOR are of major interest as anticancer agents. We show here, using human breast cancer cells, that certain types of stress activate mTOR leading to 4E-BP1 and p70S6K phosphorylation. UV treatment increased phosphorylation of the translation inhibitor eIF2alpha, suggesting a potential mechanism for UV activation of Akt and mTOR. c-Myc, a survival protein regulated by cap-dependent protein translation, increased with IGF-I treatment, but this response was not inhibited by rapamycin. Additionally, UV treatment potently increased c-Myc degradation, which was reduced by co-treatment with the proteasomal inhibitor, MG-132. Together, these data suggest that protein translation does not strongly mediate cell survival in these models. In contrast, the phosphorylation status of retinoblastoma protein (pRB) was mediated by mTOR through its inhibitory effects on phosphatase activity. This effect was most notable during DNA damage and rapamycin treatment. Hypophosphorylated pRB was susceptible to inactivation by caspase-mediated cleavage, resulting in cell death. Reduction of pRB expression inhibited IGF-I survival effects. Our data support an important role of phosphatases and pRB in IGF-I/mTOR-mediated cell survival. These studies provide new directions in optimizing anticancer efficacy of mTOR inhibitors when used in combination with DNA-damaging agents.     

Investigation of the eIF2alpha phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2alpha) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2alpha in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2alpha. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2alpha phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2alpha phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2alpha phosphorylation; futhermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eLF2alpha. Zinc (II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2alpha phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.     

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.     

Investigation of the eIF2α phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2α in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2α. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2α phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2α phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2α phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eIF2α. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2α phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.     

Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation

S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.     

Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.     

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.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

Oxaliplatin down-regulates survivin by p38 MAP kinase and proteasome in human colon cancer cells

Oxaliplatin, a platinum derivative cancer drug, has been used for treating human colorectal cancers. Survivin has been proposed as a cancer target, which highly expressed in most cancer cells but not normal adult cells. In this study, we investigated the regulation of survivin expression by exposure to oxaliplatin in human colon cancer cells. Oxaliplatin (3–9 μM for 24 h) markedly induced cytotoxicity, proliferation inhibition and apoptosis in the human RKO colon cancer cells. The survivin protein expression of RKO cells is dramatically reduced by oxaliplatin; however, the survivin gene expression is slightly altered. The survivin blockage of oxaliplatin elevated caspase-3 activation and apoptosis in RKO cells. Over-expression of survivin proteins by transfection with a survivin-expressed vector resisted the oxaliplatin-induced cancer cell death. Meantime, oxaliplatin elicited the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated oxaliplatin-induced cancer cell death. In addition, oxaliplatin increased the levels of phospho-p53 (Ser-15) and total p53 proteins. Inhibition of p53 expression by a specific p53 inhibitor pifithrin-α reduced the phosphorylated p38 MAP kinase and active caspase-3 proteins in the oxaliplatin-exposed RKO cells. In contrast, SB202190 did not alter the oxaliplatin-induced p53 protein level. Furthermore, treatment with a specific proteasome inhibitor MG132 restored survivin protein level in the oxaliplatin-treated colon cancer cells. Taken together, our results demonstrate for the first time that survivin is down-regulated by p38 MAP kinase and proteasome degradation pathway after treatment with oxaliplatin in the human colon cancer cells.     

D-glucosamine inhibits proliferation of human cancer cells through inhibition of p70S6K

Although D-glucosamine has been reported as an inhibitor of tumor growth both in vivo and in vitro, the mechanism for the anticancer effect of D-glucosamine is still unclear. Since there are several reports suggesting D-glucosamine inhibits protein synthesis, we examined whether D-glucosamine affects p70S6K activity, an important signaling molecule involved in protein translation. In the present study, we found D-glucosamine inhibited the activity of p70S6K and the proliferation of DU145 prostate cancer cells and MDA-MB-231 breast cancer cells. D-glucosamine decreased phosphorylation of p70S6K, and its downstream substrates RPS6, and eIF-4B, but not mTOR and 4EBP1 in DU145 cells, suggesting that D-glucosamine induced inhibition of p70S6K is not through the inhibition of mTOR. In addition, D-glucosamine enhanced the growth inhibitory effects of rapamycin, a specific inhibitor of mTOR. These findings suggest that D-glucosamine can inhibit growth of cancer cells through dephosphorylation of p70S6K.     

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.     

GSK3 beta-dependent phosphorylation of the alpha NAC coactivator regulates its nuclear translocation and proteasome-mediated degradation

c-Jun is an immediate-early gene whose degradation by the proteasome pathway is required for an efficient transactivation. In this report, we demonstrated that the c-Jun coactivator, nascent polypeptide associated complex and coactivator alpha (alphaNAC) was also a target for degradation by the 26S proteasome. The proteasome inhibitor lactacystin increased the metabolic stability of alphaNAC in vivo, and lactacystin, MG-132, or epoxomicin treatment of cells induced nuclear translocation of alphaNAC. We have shown that the ubiquitous kinase glycogen synthase kinase 3beta (GSK3beta) directly phosphorylated alphaNAC in vitro and in vivo. Inhibition of the endogenous GSKappa3beta activity resulted in the stabilization of this coactivator in vivo. We identified the phosphoacceptor site in the C-terminal end of the coactivator, on position threonine 159. We demonstrated that the inhibition of GSK3beta activity by treatment of cells with the inhibitor 5-iodo-indirubin-3'-monoxime, as well as with a dominant-negative GSK3beta mutant, induced the accumulation of alphaNAC in the nuclei of cells. Mutation of the GSK3beta phosphoacceptor site on alphaNAC induced a significant increase of its coactivation potency. We conclude that GSK3beta-dependent phosphorylation of alphaNAC was the signal that directed the protein to the proteasome. The accumulation of alphaNAC caused by the inhibition of the proteasome pathway or the activity of GSK3beta contributes to its nuclear translocation and impacts on its coactivating function.     

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 enhances whole-body protein turnover in rat

Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.