Showing papers on "MG132 published in 2023"

Proteasome inhibitor MG132 enhances the sensitivity of human OSCC cells to cisplatin via a ROS/DNA damage/p53 axis

Abstract: Cis-diamine-dichloroplatinum II (cisplatin, CDDP) is a key chemotherapeutic regimen in the treatment of oral squamous cell carcinoma (OSCC). However, the therapeutic efficacy of cisplatin in OSCC may be hampered by chemoresistance. Therefore, the development of novel combination therapy strategies to overcome the limitations of CDDP is of great importance. The proteasome inhibitor MG132 exhibits anti-cancer properties against various types of cancer. However, our knowledge of its anti-cancer effects in combination with CDDP in OSCC cells remains limited. In the current study, the synergetic effects of MG132 and CDDP were evaluated in the human CAL27 OSCC cell line. CAL27 cells were treated with CDDP alone or in combination with MG132. The results showed that MG132 significantly reduced cell viability in a dose-dependent manner. Additionally, cell viability was significantly reduced in CAL27 cells treated with 0.2 µM MG132 and 2 µM CDDP compared with cells treated with MG132 or CDDP alone. In addition, MG132 significantly enhanced the CDDP-induced generation of intracellular reactive oxygen species and DNA damage in OSCC cells. Furthermore, treatment with CDDP or MG132 alone notably inhibited colony formation and proliferation of OSCC cells. However, co-treatment of OSCC cells with MG132 and CDDP further hampered colony formation and proliferation compared with cells treated with either MG132 or CDDP alone. Finally, in cells co-treated with MG132 and CDDP, the expression of p53 was markedly elevated and the p53-mediated apoptotic pathway was further activated compared with cells treated with MG132 or CDDP alone, as shown by the enhanced cell apoptosis, Bax upregulation, and Bcl-2 downregulation. Overall, the results of the current study support the synergistic anti-cancer effects of a combination of MG132 and CDDP against OSCC, thus suggesting that the combination of MG132 and CDDP may be a promising therapeutic strategy for the management of OSCC.

Cadmium Disrupted ER Ca2+ Homeostasis by Inhibiting SERCA2 Expression and Activity to Induce Apoptosis in Renal Proximal Tubular Cells

Abstract: Cadmium (Cd2+) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd2+ induced cytotoxicity by disrupting the intracellular Ca2+ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca2+ store. However, the molecular mechanism of ER Ca2+ homeostasis in Cd2+-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd2+ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca2+ homeostasis through the ER Ca2+ reuptake channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Cd2+-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd2+ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd2+-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd2+ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd2+-induced ER Ca2+ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd2+-induced cytotoxicity and renal injury.

Stapled peptide PROTAC induced significantly greater anti-PD-L1 effects than inhibitor in human cervical cancer cells

Abstract: Introduction Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that target immune checkpoints that suppress immune cell activity. Low efficiency and high resistance are currently the main barriers to their clinical application. As a representative technology of targeted protein degradation, proteolysis-targeting chimeras (PROTACs) are considered to have potential for addressing these limitations. Methods We synthesized a stapled peptide-based PROTAC (SP-PROTAC) that specifically targeted palmitoyltransferase ZDHHC3 and resulted in the decrease of PD-L1 in human cervical cancer cell lines. Flow cytometry, confocal microscopy, protein immunoblotting, Cellular Thermal Shift Assay (CETSA), and MTT assay analyses were conducted to evaluate the effects of the designed peptide and verify its safety in human cells. Results In cervical cancer celllines C33A and HeLa, the stapled peptide strongly downregulated PD-L1 to < 50% of baseline level at 0.1 μM. DHHC3 expression decreased in both dosedependentand time-dependent manners. MG132, the proteasome inhibitor, can alleviate the SP-PROTAC mediated degradation of PD-L1 in human cancer cells. In a co-culture model of C33A and T cells, treatment with the peptide induced IFN-γ and TNF-α release in a dose-dependent manner by degrading PD-L1. These effects were more significant than that of the PD-L1 inhibitor, BMS-8. Conclusions Cells treated with 0.1 μM of SP-PROTAC or BMS-8 for 4 h revealed that the stapled peptide decreased PD-L1 more effectively than BMS-8. DHHC3-targeting SP-PROTAC decreased PD-L1 in human cervical cancer more effectively than the inhibitor BMS-8.

Data from Proteasome Inhibition Potentiates Antitumor Effects of Photodynamic Therapy in Mice through Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response

Abstract: <div>Abstract<p>Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed <i>in vivo</i> in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors. [Cancer Res 2009;69(10):4235–43]</p></div>

Supplementary Figures S1 - S10 from Tyrosine Kinase Inhibitors Increase MCL1 Degradation and in Combination with BCLXL/BCL2 Inhibitors Drive Prostate Cancer Apoptosis

Abstract: <p>Fig. S1. Doxycycline-induced depletion of MCL1; Fig. S2. MCL1, BCLXL, and BCL2 expression in PCa databases, and comparison of the protein levels among benign prostate cell, PCa cells, and patient-derived xenograft (PDX) cells; Fig. S3. BH3 mimetics diminished the tumor growth of prostate cancer xenograft with MCL1 depletion; Fig. S4. Effects of lapatinib and ABT-737 in LNCaP and additional cell lines; Fig. S5. Effects of various kinase inhibitors on MCL1; Fig. S6. Effects of EGFR/ERBB2 inhibition on MCL1 mRNA and proteasome-dependent degradation of MCL1; Fig. S7. EGFR inhibition enhances MCL1 degradation by GSK3β-independent mechanism; Fig. S8. Erlotinib mediated MCL1 degradation is not through GSK3β or previously characterized ubiquitin ligases or DUBs; Fig. S9. Cabozantinib and sorafenib mediate MCL1 degradation that is independent of GSK3β; Fig. S10. Apoptosis mediated by single and combination therapies delivered locally into xenografts by implantable microdevice.</p>

Supplementary Figure 5 from Germline Mutations in BAP1 Impair Its Function in DNA Double-Strand Break Repair

Abstract: <p>PDF file - 39KB, Figure S5. Effect of MG132 on BAP1 recruitment to sites of DNA damage. (A) U2OS cells expressing GFP-BAP1 were either treated with DMSO or MG132 (1 uM) for 1 hr. Cells were monitored after micro-irradiation using time-lapse microscopy. The integrated intensity in the micro-irradiated areas was determined (n = 15) and the percentage of maximum value was plotted versus time. SEM of at least two independent experiments. (B) Flow cytometer profiles of the experiment done in figure 6A and B.</p>

Supplementary Figure 1 from Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations

Abstract: <p>PDF file, 71KB, The data are scaled by the positive control (1�M MG132) and the negative control (DMSO). The percentage of maximum activity (Amax) is represented in function of the crossing point (concentration in �M at 50% of MG132 activity). Each data point represents a cell line; the vertical and horizontal lines represent, respectively, the median of the crossing point values (1.33�M) and the median of the Amax values (-90.06%), of BKM120 across all cell lines. The populations of cell lines least responding to GDC-0941, among which some are sensitive to BKM120, are highlighted in green.</p>

Supplemental Figure S2 from A Bis-Indole–Derived NR4A1 Antagonist Induces PD-L1 Degradation and Enhances Antitumor Immunity

Abstract: <p>Supplemental Figure S2. Quantitation of results in Figure 1F and 1G. and mRNA results A. Breast cancer cells were treated with Cl-OCH3 (Fig. 1F) and effects on PD-L1, NR4A1 and Sp1 were determined by western blots and quantitation of the results are summarized. B. MDA-MB-231 and 4T1 cells were treated with NR4A1 antagonists, MG132 and their combinations, whole cell lysates were analyzed by western blots (Fig. 1G) and quantitation of the results are summarized. Results of both studies (A, B) are means {plus minus} SD for at least 3 replicate determinations for each treatment groups and changes in expression were determined relative to the control group (set ratio) significantly (p<0.05) decreased effects are indicated (*) and inhibition (reversal of degradation) by MG132 is also indicated (**). C. Basal PD-L1 and NR4A1 mRNA levels in cancer cell lines were determined as described in the Methods.</p>

Polypeptide antibiotic actinomycin D induces Mcl-1 uncanonical downregulation in lung cancer cell apoptosis.

Abstract: Actinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation. Human adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles. We found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis. All of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.

Data Supplement from Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

Abstract: <p>Supplementary Figure S1. Proteasome activity is involved in spindle pole localization of MYCN in human neuroblastoma cells. A, Representative images of MYCN localization to spindle poles during mitosis in SK-N-BE and CHP-212 cells with or without exposure of 10μM MG132 for 5h. 0.1% DMSO treated cells served as a control. NuMA is green, MYCN is red, and DAPI (DNA) is blue. Arrows show spindle poles. Scale bars indicate 10 μm. B, Proteasome activity in human neuroblastoma and control (Jurkat) cells.</p>

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation

Abstract: Abstract Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca 2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca 2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca 2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

Data from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Abstract: <div>Abstract<p>The human copper transporter 1 (hCTR1), the major transporter responsible for copper influx, mediates one component of the cellular accumulation of cisplatin (DDP). Both copper and DDP cause rapid down-regulation of hCTR1 expression in human ovarian carcinoma cells. In this study, we investigated the mechanism of this effect using digital deconvolution microscopy and Western blot analysis of cells stained with antibodies directed at both ends of the protein. Treatment of 2008 cells with DDP in combination with inhibitors of various endosomal pathways (amiloride, cytochalasin D, nystatin, and methyl-β-cyclodextrin) showed that hCTR1 degradation was blocked by amiloride and cytochalasin D, indicating that hCTR1 was internalized primarily by macropinocytosis. Expression of transdominant-negative forms of dynamin I and Rac showed that loss of hCTR1 was not dependent on pathways regulated by either of these proteins. DDP-induced loss of hCTR1 was blocked by the proteasome inhibitors lactacystin, proteasome inhibitor 1, and MG132. This study confirms that DDP triggers the rapid loss of hCTR1 from ovarian carcinoma cells at clinically relevant concentrations. The results indicate that DDP-induced loss of hCTR1 involves internalization from the plasma membrane by macropinocytosis followed by proteasomal degradation. Because hCTR1 is a major determinant of early DDP uptake, prevention of its degradation offers a potential approach to enhancing tumor sensitivity. (Cancer Res 2006; 66(22): 10944-52)</p></div>

CoCl2 triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299

Abstract: SIRT4 comprises together with SIRT3 and SIRT5 the mitochondrially localized subgroup of sirtuins. SIRT4 regulates via its NAD+-dependent enzymatic activities mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy). Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2-induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, within the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132 prevented CoCl2-induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms, (i) via mitochondrial autophagy/mitophagy, and (ii) as a separate process via proteasomal degradation within the cytoplasm.

HOS15 represses flowering by promoting GIGANTEA degradation in response to low temperature in Arabidopsis.

Abstract: Flowering is the primary stage of the plant developmental transition and is tightly regulated by environmental factors such as light and temperature. However, the mechanisms by which temperature signals are integrated into the photoperiodic flowering pathway are still poorly understood. Here, we demonstrate that HOS15, which is known as a GI transcriptional repressor in the photoperiodic flowering pathway, controls flowering time in response to low ambient temperature. At 16°C, the hos15 mutant exhibits an early flowering phenotype, and HOS15 acts upstream of photoperiodic flowering genes (GI, CO, and FT). GI protein abundance is increased in the hos15 mutant and is insensitive to the proteasome inhibitor MG132. Furthermore, the hos15 mutant has a defect in low ambient temperature-mediated GI degradation, and HOS15 interacts with COP1, an E3 ubiquitin ligase for GI degradation. Phenotypic analyses of the hos15 cop1 double mutant revealed that repression of flowering by HOS15 is dependent on COP1 at 16°C. However, the HOS15-COP1 interaction was attenuated at 16°C, and GI protein abundance was additively increased in the hos15 cop1 double mutant, indicating that HOS15 acts independently of COP1 in GI turnover at low ambient temperature. This study proposes that HOS15 controls GI abundance through multiple modes as an E3 ubiquitin ligase and transcriptional repressor to coordinate appropriate flowering time in response to ambient environmental conditions such as temperature and day length.

Protective role of FBXL19 in Streptococcus pneumoniae-induced lung injury in pneumonia immature mice

Abstract: Streptococcus pneumoniae (Spn) is a common pathogen for pediatric pneumonia and leads to severe lung injury. This study is conducted to analyze the role of F-box and leucine rich repeat protein 19 (FBXL19) in Spn-induced lung injury in immature mice.Immature mice were infected with Spn to record the survival rates and bacterial loads in bronchoalveolar lavage fluid. Levels of FBXL19 and FOXM1 in lung tissues were determined via real-time quantitative polymerase chain reaction or Western blotting. After the interference of FBXL19, its impacts on lung inflammatory injury were appraised by the lung wet/dry weight ratio, myeloperoxidase activity, hematoxylin and eosin staining, and enzyme-linked immunosorbent assay. The binding of FBXL19 to forkhead box M1 (FOXM1) in mouse lung epithelial cells was determined. After MG132 treatment, the protein and ubiquitination levels of FOXM1 were measured. The functional rescue experiments were performed to analyze the role of FOXM1 in FBXL19-regulated lung injury.FBXL19 was downregulated while FOXM1 was upregulated in lung tissues of Spn-infected immature mice. Overexpression of FBXL19 reduced the degree of lung injury and inflammation. FBXL19 can bind to FOXM1 to reduce its protein level via ubiquitination degradation. MG132 reduced the ubiquitination and increased the protein level of FOXM1. Overexpression of FOXM1 reversed the protective role of FBXL19 overexpression in lung injury of Spn immature mice.FBXL19 was downregulated by Spn and FBXL19 overexpression alleviated lung injury by inducing ubiquitination and degradation of FOXM1 in Spn immature mice.

Supplementary Figure 5 from Mutual Regulation between Raf/MEK/ERK Signaling and Y-Box–Binding Protein-1 Promotes Prostate Cancer Progression

Abstract: <p>Supplementary Figure 5 - Supplementary Figure 5. Densitometric analyses of Figure 4C - PDF file 83K, Protein expression level of Raf-1 in LNCaP and PC-3 cells transfected with control siRNA and not treated with cycloheximide (CHX) (A) or MG132 (B) was defined as 1. Bars, plus-minus s.d.; *P < 0.05 versus cells with transfected with control siRNA</p>

Data from Cell Type–Specific, Topoisomerase II–Dependent Inhibition of Hypoxia-Inducible Factor-1α Protein Accumulation by NSC 644221

Abstract: <div>Abstract<p><b>Purpose:</b> The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1α, NSC 644221.</p><p><b>Experimental Design:</b> We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1α.</p><p><b>Results:</b> NSC 644221 inhibited HIF-1–dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1α, but not HIF-1β, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1α protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1α as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [³⁵S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1α translation relative to untreated controls. Silencing of topoisomerase (topo) IIα, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1α. The data presented show that topo II is required for the inhibition of HIF-1α by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, γH2A.X, and G₂-M arrest in the majority of cell lines tested, it only inhibited HIF-1α in a distinct subset of cells, raising the possibility of pathway-specific “resistance” to HIF-1 inhibition in cancer cells.</p><p><b>Conclusions:</b> NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.</p></div>

Data from Nuclear factor κB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos

Abstract: <div>Abstract<p>Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor κB (NF-κB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-κB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-κB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-κB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-κB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IκB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-κB activation may be useful in alleviating radiation toxicity in patients. [Mol Cancer Ther 2009;8(9):2625–34]</p></div>

Black carp RNF5 inhibits STING/IFN signaling through promoting K48-linked ubiquitination and degradation of STING.

Abstract: Ubiquitination is one of the important post-translational modifications (PTMs) of proteins that plays a vital role in regulating substrate degradation to ensure cellular homeostasis. Ring finger protein 5 (RNF5) is an essential E3 ubiquitin ligase for inhibiting STING-mediated interferon (IFN) signaling in mammals. Nevertheless, the function of RNF5 in STING/IFN pathway remains obscure in teleost. Here, we reported that over-expression of black carp RNF5 (bcRNF5) inhibited STING-mediated transcription activity of bcIFNa, DrIFNφ1, NF-κB and ISRE promoters and antiviral activity against SVCV. Moreover, knockdown of bcRNF5 increased the expression of host genes, including bcIFNa, bcIFNb, bcILβ, bcMX1 and bcViperin, and also enhanced the antiviral capability of host cells. Immunofluorescence (IF) and Co-immunoprecipitation (Co-IP) assay confirmed that bcRNF5 was mainly localized in the cytoplasm and interacted with bcSTING. The expression level of bcSTING protein was attenuated by co-expressed bcRNF5 and MG132 treatment rescued this attenuating effect, suggesting that bcRNF5-mediated bcSTING degradation was dependent on the proteasome pathway. Subsequent, Co-IP and immunoblot (IB) experiments identified that bcRNF5 triggered the K48-linked but not K63-linked ubiquitination of bcSTING. Altogether, above results conclude that RNF5 suppresses STING/IFN signaling by enhancing K48-linked ubiquitination and protease degradation of STING in black carp.

Supplementary Methods, Figure Legends, Tables 1 - 2, Figures 1 - 7 from VR23: A Quinoline–Sulfonyl Hybrid Proteasome Inhibitor That Selectively Kills Cancer via Cyclin E–Mediated Centrosome Amplification

Abstract: <p>Supplementary Materials and Methods for cloning and sulforhodamine B (SRB) assay, analysis of cell cycle distribution, knockdown of cyclin E with siRNA, EdU labelling, molecular docking modeling, and reagents. Supplementary Table S1. VR23 sensitizes paclitaxel-mediated cell killing, as determined by a clonogenic assay of MCF7 cancer cells. Supplementary Table S2. Examination of VR23-induced toxicity, as determined by the levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in ATH490 athymic mice. Supplementary Figure S1. VR23 induces apoptosis in a cancer cell-specific manner. Figure S2. VR23 induces cell cycle arrest in prometaphase and, eventually, apoptosis through the p38 MAPK pathway. Supplementary Figure S3. Cells exposed to VR23 show a variety of different abnormal phenotypes. Supplementary Figure S4. In MCF10A, cyclin E is heavily ubiquitinated in response to bortezomib (Borz), but not to VR23 or MG132. Supplementary Figure S5. VR23 shows effective antitumor activity in mice engrafted with RPMI 8226. Supplementary Figure S6. VR23 does not show any notable adverse effects to animals. Supplementary Figure S7. VR23 does not cause any notable ill-effects on vital organs as determined by their weights.</p>

Supplemental Figure 2: from β-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation

Abstract: <p>A: Prostate cancer cells were seeded in 6 well plates. Twenty-four hours later, cells were treated with Geldanamycin (3 μM) for 1-24 hr, or were left untreated (NT). Protein extracts were subjected to western blotting. Actin served as a loading control. B: PC-3 cells were seeded in 6 well plates. Twenty-four hours later, cells were treated with 17-AAG (2 μM, 2 hr), proteasome inhibitor MG132 (10 µM, 2 hr) or were left untreated (NT). Protein extracts were collected and subjected to western blotting. Actin served as a loading control. C, D: PC-3 or LNCaP cells were treated with Geldanamycin as described above and nuclear or total protein extracts we collected and subjected to western blot analysis for detection of β-AR2 (C) and EGFR (D). AU: Arbitrary Units</p>

Supplementary Figure 6 from GSK-3α Promotes Oncogenic KRAS Function in Pancreatic Cancer via TAK1–TAB Stabilization and Regulation of Noncanonical NF-κB

Abstract: <p>PDF file - 118K, GSK-3 inhibition leads to a decrease in TAK1 protein Panc-1 and MiaPaCa-2 cells were treated with GSK-3 inhibitor, AR-A014418 or vehicle control, DMSO in the presence or absence of MG132 for 24 hours. Whole cell extracts were immunoblotted for indicated antibodies.</p>

Data Supplement from Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

Abstract: <p>Supplementary Figure S4. A GSK-3 inhibitor IX (BIO) suppresses spindle pole localization of MYCN under MG132 treatment. Percent of TGW (left) or SK-N-DZ (left) cells with MYCN accumulation to spindle poles. Error bars represent SEM from three experiments, P<0.0001 for TGW and P=0.006 for SK-N-DZ.</p>

Data from Polyphenon E Inhibits the Growth of Human Barrett's and Aerodigestive Adenocarcinoma Cells by Suppressing Cyclin D1 Expression

Abstract: <div>Abstract<p><b>Purpose:</b> Green tea consumption has been shown to exhibit cancer-preventive activities in preclinical studies. Polyphenon E (Poly E) is a well-defined green tea–derived catechin mixture. This study was designed to determine the effects of Poly E on the growth of human Barrett's and aerodigestive adenocarcinoma cells and the mechanisms involved in growth regulation by this agent.</p><p><b>Experimental Design:</b> Human adenocarcinoma cells and immortalized Barrett's epithelial cells were used as model systems.</p><p><b>Results:</b> Poly E inhibited the proliferation of immortalized Barrett's cells as well as various adenocarcinoma cells, and this was associated with the down-regulation of cyclin D1 protein expression. Inhibition of cyclin D1 led to dephosphorylation of the retinoblastoma protein in a dose-dependent manner; these changes were associated with G₁ cell cycle arrest. Poly E down-regulated cyclin D1 promoter activity and mRNA expression, suggesting transcriptional repression, and this correlated with decreased nuclear β-catenin and β-catenin/TCF4 transcriptional activity. MG132, an inhibitor of 26S proteosome, blocked the Poly E–induced down-regulation of cyclin D1, and Poly E promoted cyclin D1 polyubiquitination, suggesting that Poly E also inhibits cyclin D1 expression by promoting its degradation.</p><p><b>Conclusion:</b> Poly E inhibits growth of transformed aerodigestive epithelial cells by suppressing cyclin D1 expression through both transcriptional and posttranslational mechanisms. These results provide insight into the mechanisms by which Poly E inhibits growth of Barrett's and adenocarcinoma cells, and provides a rationale for using this agent as a potential chemopreventive and therapeutic strategy for esophageal adenocarcinoma and its precursor, Barrett's esophagus.</p></div>

Data from A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells

Abstract: <div>Abstract<p>Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either <i>p53</i> or <i>PUMA</i> led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome <i>c</i> release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor–induced apoptosis in colon cancer cells. [Mol Cancer Ther 2007;6(3):1062–9]</p></div>