leupeptins and Osteosarcoma

Osteosarcoma (OS) is the most malignant primary bone tumor in children and adolescents with limited treatment options and poor prognosis. Recently, aberrant expression of Runx2 has been found in OS, thereby contributing to the development, and progression of OS. However, the upstream signaling molecules that regulate its expression in OS remain largely unknown. In the present study, we first confirmed that the inhibition of HSP90 with 17-AAG caused significant apoptosis of OS cells via a caspase-3-dependent mechanism, and that inhibition or knockdown of HSP90 by 17-AAG or siRNAs significantly suppressed mRNA and protein expression of Runx2. Furthermore, we provided evidence that Runx2 was transcriptionally regulated by HSP90 when using MG132 and CHX chase assay. We also demonstrated that β-catenin was overexpressed in OS tissue, and that knockdown of β-catenin induced pronounced apoptosis of OS cells in the presence or absence of 17-AAG. Interestingly, this phenomenon was accompanied with a significant reduction of Runx2 and Cyclin D1 expression, indicating an essential role of Runx2/Cyclin D1 in 17-AAG-induced cells apoptosis. Moreover, we demonstrated that the apoptosis of OS cells induced by 17-AAG did require the involvement of the AKT/GSK-3β/β-catenin signaling pathway by using pharmacological inhibitor GSK-3β (LiCl) or siGSK-3β. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3β/β-catenin signaling pathway, and by which leads to apoptosis of OS cells.

Topics: Benzoquinones; beta Catenin; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Core Binding Factor Alpha 1 Subunit; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Osteosarcoma; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription, Genetic

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; Humans; Leupeptins; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Proteasome Inhibitors; Xenograft Model Antitumor Assays

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Leupeptins; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA Interference; RNA, Small Interfering; Signal Transduction; Sp7 Transcription Factor; Transcription Factors

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27(kip1) and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27(kip1), down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10 μM MG132 and 100 ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p<0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2-M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.

Topics: Antineoplastic Agents; Bone and Bones; Bone Neoplasms; Caspase 3; Caspase 8; Cell Cycle; Cell Line, Tumor; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Humans; Leupeptins; M Phase Cell Cycle Checkpoints; Neoplasm Invasiveness; Osteosarcoma; Proteasome Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand

Clusterin/apolipoprotein J (CLU) is a secreted glycoprotein associated with many severe physiological disturbances that represent states of increased oxidative stress, such as aging, cancer, atherosclerosis, diabetes, and renal and neurodegenerative diseases. The aim of our work was to examine the effect of proteasome and lysosome inhibition on CLU expression and to determine whether those proteolytic pathways are implicated in CLU gene regulation and protein degradation. To this end we used two different model systems, namely the U-2 OS osteosarcoma cell line and the WI38 primary human embryonic lung fibroblasts. We report that proteasome inhibition promotes both heat-shock factor 1 (HSF-1)-dependent CLU gene expression induction and protein accumulation due to reduced degradation. In contrast, lysosome inhibition results in elevated levels of CLU protein but does not affect the CLU mRNA levels. We also provide direct evidence that both the intracellular precursor, psCLU, and the mature secreted, sCLU, isoforms constitute proteolytic substrates of the proteasome and the lysosome. Overall our findings indicate that CLU overexpression after proteasome inhibition relates to both positive gene transcriptional regulation by HSF-1 and posttranslational protein accumulation due to reduced proteasomal and lysosomal degradation.

Topics: Antibodies, Blocking; Cell Line, Tumor; Clusterin; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation; Heat Shock Transcription Factors; Humans; Hydrolysis; Leupeptins; Lysosomes; Male; Oligopeptides; Osteosarcoma; Proteasome Inhibitors; Protein Processing, Post-Translational; Transcription Factors; Transcriptional Activation; Transfection; Ubiquitin

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.

Topics: Animals; Cell Cycle; Cell Line; Cell Line, Tumor; Core Binding Factor Alpha 1 Subunit; Core Binding Factor beta Subunit; Cysteine Proteinase Inhibitors; G1 Phase; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Mice; Mitosis; Osteoblasts; Osteosarcoma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Ubiquitination

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Drug Evaluation, Preclinical; Ellipticines; Green Fluorescent Proteins; Humans; Leupeptins; Osteosarcoma; Recombinant Fusion Proteins; Thapsigargin; Tumor Suppressor Protein p53

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG-63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis-inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132-treated cells and MG132 mostly caused MG-63 cell arrest at G(2)-M-phase by cell cycle analysis. Increased activation of caspase-8, accumulation of p27(Kip1), and an increased ratio of Bax:Bcl-2 were detected by RT-PCR and Western blot analysis. Activation of caspase-3 and caspase-9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase-8 dependent, p27 and bcl-2 family related.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Bone Neoplasms; Caspase 8; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; Fibroblasts; Humans; Leupeptins; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The p53 oncosuppressor protein is subject to negative regulation by MDM2, which efficiently inhibits its activity through an autoregulatory loop. In response to stress, however, p53 undergoes post-translational modifications that allow the protein to escape MDM2 control, accumulate, and become active. Recent studies have shown that, following DNA damage, the HIPK2 serine/threonine kinase binds and phosphorylates p53, inducing p53 transcriptional activity and apoptotic function. Here, we investigated the role of HIPK2 in the activation of p53 in the presence of MDM2. We found that HIPK2 rescues p53 transcriptional activity overcoming MDM2 inhibition, and that restoration of this p53 function induces apoptosis. Recovery of p53-dependent apoptosis is achieved by preventing p53 nuclear export and ubiquitination mediated by MDM2 in vitro and in vivo following genotoxic stress. These results shed new light on the mechanisms by which the HIPK2/p53 pathway promotes apoptosis and suppression of tumorigenesis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Cell Nucleus; Cisplatin; Colonic Neoplasms; Cysteine Proteinase Inhibitors; DNA Damage; Humans; Leupeptins; Luciferases; Lung Neoplasms; Nuclear Proteins; Osteosarcoma; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transcriptional Activation; Tumor Suppressor Protein p53; Ubiquitin

E2F family of transcription factors regulates the transcription of genes required for DNA synthesis. E2F is itself controlled by a series of transcriptional and post-transcriptional pathways. Here we provide evidence that proteasome inhibitor-mediated E2F1 gene down-regulation is regulated by transcriptional events. Using the proteasome-specific inhibitors, MG132 and lactacystin, we show that the p53, the cdk inhibitors p21 and p27, and cyclin A are degraded by the ubiquitin-proteasome pathway in human osteosarcoma cells. Interestingly, the expression levels of E2F1 and E2F2 are down-regulated by proteasome inhibitors. E2F promoter and RT-PCR assay clearly demonstrated that proteasome inhibitors could reduce E2F transcriptional activation. However, MG132-induced repression of E2F1 and E2F2 is not associated with ROS generation.

Topics: Acetylcysteine; Antioxidants; Cell Cycle Proteins; Cell Line, Tumor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Down-Regulation; E2F Transcription Factors; E2F1 Transcription Factor; E2F2 Transcription Factor; Glutathione; Humans; Leupeptins; Multienzyme Complexes; Osteosarcoma; Transcription Factors; Transcription, Genetic; Transfection

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Topics: 3T3 Cells; Acetylcysteine; Animals; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Deletion; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Hemagglutinins; Humans; Leupeptins; Mice; Models, Genetic; Multienzyme Complexes; Mutagenesis; Osteosarcoma; Proliferating Cell Nuclear Antigen; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Time Factors; Transfection; Tumor Cells, Cultured; Ubiquitins

The AF-2 helix of nuclear receptors is essential for ligand-activated transcription, and it may function to couple the receptor to transcriptional coactivator proteins. This domain also contacts components of the proteasome machinery, suggesting that nuclear receptors may be targets for proteasome-mediated proteolysis. In the present study, we demonstrate that mSUG1 (P45), a component of the 26S proteasome, interacts in a 1,25-(OH)2D3-dependent manner with the AF-2 domain of the vitamin D receptor (VDR). Furthermore, treatment of ROS 17/2.8 osteosarcoma cells with the proteasome inhibitors MG132 or beta-lactone increased steady-state levels of the VDR protein. In the presence cycloheximide (10 microg/ml), the liganded VDR protein was degraded with a half-life of approximately 8 h, and this rate of degradation was completely blocked by 0.05 mM MG132. The role of SUG1 -VDR interaction in this process was investigated in transient expression studies. Overexpression of wild-type mSUG1 in ROS17/2.8 cells generated a novel proteolytic VDR fragment of approximately 50 kDa, and its production was blocked by proteasome inhibitors or by a nonhydrolyzable ATP analog. Parallel studies with SUG1 (K196H), a mutant that does not interact with the VDR, did not produce the 50 kDa VDR fragment. Functionally, expression of SUG1 in a VDR-responsive reporter gene assay resulted in a profound inhibition of 1,25-(OH)2D3-activated transcription, while expression of SUG1 (K196H) had no significant effect in this system. These data show that the AF-2 domain of VDR interacts with SUG1 in a 1,25-(OH)2D3-dependent fashion and that this interaction may target VDR to proteasome-mediated degradation as a means to downregulate the 1,25-(OH)2D3-activated transcriptional response.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Animals; ATPases Associated with Diverse Cellular Activities; Calcitriol; Carrier Proteins; Cloning, Molecular; Cycloheximide; Cysteine Endopeptidases; Gene Expression Regulation; Half-Life; Intracellular Signaling Peptides and Proteins; Lactones; Leupeptins; Ligands; LIM Domain Proteins; Mice; Multienzyme Complexes; Osteosarcoma; Peptide Fragments; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Structure, Secondary; Receptors, Calcitriol; Transcription Factors; Transfection; Tumor Cells, Cultured

Topics: Cyclic AMP; Dinoprost; Dinoprostone; DNA; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Leupeptins; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Prostaglandins F; Receptors, Cell Surface; Time Factors