We demonstrated for the first time that the COP9 signalosome (COPS) controls the degradation of a surrogate and a bona fide misfolded protein in the cytosol of cardiomyocytes likely via supporting ubiquitination by CUL/cullin-RING ligases, and that Cops8 hypomorphism exacerbates cardiac proteinopathy in mice, in which autophagic impairment appears to be in play. It will be extremely imprtant to investigate cardiac ablation of another Cops gene to decipher whether COPS8 deficiency phenotypes are attributable to the COPS or unique to COPS8.
autophagy; cullin-RING ligase; desmin-related cardiomyopathy; protein quality control; COP9 signalosome
Acquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
Cell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients’ bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses.
NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death.
These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.
Nickel pyrithione; Apoptosis; Chronic myelogenous leukemia; Imatinib resistance; Bcr-Abl
Impaired degradation of misfolded proteins is associated with a large subset of heart diseases. Misfolded proteins are degraded primarily by the ubiquitin-proteasome system (UPS) but the ubiquitin ligases responsible for the degradation remain largely unidentified. The cullin deneddylation activity of the COP9 signalosome (CSN) requires all 8 CSN subunits (CSN1 through CSN8) and regulates cullin-RING ligases (CRLs), thereby controlling ubiquitination of a large number of proteins; however, neither CSN nor CRLs are known to regulate the degradation of cytosolic misfolded proteins.
We sought to investigate the role of CSN8/CSN in misfolded protein degradation and cardiac proteinopathy.
Methods and Results
Cardiac CSN8 knockout causes mouse premature death; hence, CSN8 haploinsufficiency (CSN8hypo) mice were used. Myocardial neddylated forms of cullins were markedly increased and myocardial capacity of degrading a surrogate misfolded protein was significantly reduced by CSN8hypo. When introduced into proteinopathic mice in which a bona fide misfolded protein CryABR120G is overexpressed in the heart, CSN8hypo aggravated CryABR120G-induced restrictive cardiomyopathy and shortened the lifespan of CryABR120G mice, which was associated with augmented accumulation of protein aggregates, increased neddylated proteins, and reduced levels of total ubiquitinated proteins and LC3-II in the heart. In cultured cardiomyocytes, both CSN8 knockdown and CRL inactivation suppressed the ubiquitination and degradation of CryABR120G but not native CryAB, resulting in accumulation of protein aggregates and exacerbation of CryABR120G cytotoxicity.
(1) CSN8/CSN promotes the ubiquitination and degradation of misfolded proteins and protects against cardiac proteotoxicity and (2) CRLs participate in degradation of cytosolic misfolded proteins.
Cardiomyopathy; Heart Failure; Myocardial Biology; Genetically Altered and Transgenic Models; COP9 signalosome; ubiquitin; autophagy; misfolded proteins; Cops8; desmin-related cardiomyopathy; ubiquitin-proteosome system genetics; proteasome; crystalin; proteotoxicity
The in vivo function status of the ubiquitin-proteasome system (UPS) in pressure overloaded hearts remains undefined. Cardiotoxicity was observed during proteasome inhibitor chemotherapy, especially in those with preexisting cardiovascular conditions; however, proteasome inhibition (PsmI) was also suggested by some experimental studies as a potential therapeutic strategy to curtail cardiac hypertrophy. Here we used genetic approaches to probe cardiac UPS performance and determine the impact of cardiomyocyte-restricted PsmI (CR-PsmI) on cardiac responses to systolic overload. Transgenic mice expressing an inverse reporter of the UPS (GFPdgn) were subject to transverse aortic constriction (TAC) to probe myocardial UPS performance during systolic overload. Mice with or without moderate CR-PsmI were subject to TAC and temporally characterized for cardiac responses to moderate and severe systolic overload. After moderate TAC (pressure gradient: ~40mmHg), cardiac UPS function was upregulated during the first two weeks but turned to functional insufficiency between 6 and 12 weeks as evidenced by the dynamic changes in GFPdgn protein levels, proteasome peptidase activities, and total ubiquitin conjugates. Severe TAC (pressure gradients >60mmHg) led to UPS functional insufficiency within a week. Moderate TAC elicited comparable hypertrophic responses between mice with and without genetic CR-PsmI but caused cardiac malfunction in CR-PsmI mice significantly earlier than those without CR-PsmI. In mice subject to severe TAC, CR-PsmI inhibited cardiac hypertrophy but led to rapidly progressed heart failure and premature death, associated with a pronounced increase in cardiomyocyte death. It is concluded that cardiac UPS function is dynamically altered, with the initial brief upregulation of proteasome function being adaptive; and CR-PsmI facilitates cardiac malfunction during systolic overload.
ubiquitin-proteasome system; proteasome inhibition; pressure overload; cardiac hypertrophy; heart failure; transgenic mice
Deubiquitinating-enzymes (DUBs) are key components of the ubiquitin-proteasome-system (UPS). The fundamental role of DUBs is specific removal of ubiquitin from substrates. DUBs contribute to activation/deactivation, recycling and localization of numerous regulatory-proteins, thus playing major roles in diverse cellular-processes. Altered DUB activity is associated with multitudes of pathologies including cancer. Therefore, DUBs represent novel candidates for target-directed drug development.
The article is a thorough review/accounting of patented compounds targeting DUBs stratifying/classifying the patented compounds based on: chemical-structures, nucleic-acid compositions, modes-of-action and targeting-sites. The review provides a brief background on the UPS and DUBs involvement. Furthermore, methods for assessing efficacy and potential pharmacological utility of DUB inhibitor (DUBi) are discussed.
The FDA’s approval of the 20S proteasome inhibitors: bortezomib and carfilzomib for treatment of hematological malignancies established the UPS as an anti-cancer target. Unfortunately, many patients are inherently resistant or develop resistance to proteasome inhibitors (PIs). One potential strategy to combat PI resistance is targeting upstream components of the UPS such as DUBs. DUBs represent a promising potential therapeutic target due to their critical roles in various cellular processes including protein-turnover, localization and cellular homeostasis. While considerable efforts have been undertaken to develop DUB modulators, significant advancement is necessary move DUB inhibitors into the clinic.
ubiquitin–proteasome system; deubiquitinating enzymes; DUB inhibitors; cancer; drug discovery; molecular targeting
MicroRNAs (miRs) play pivotal roles in carcinogenesis and endoplasmic reticulum (ER) that performs the folding, modification and trafficking of proteins targeted to the secretory pathway. Cancer cells often endure ER stress during tumor progression but use the adaptive ER stress response to gain survival advantage. Here we report: (i) A group of miRs, including miR-30b-5p and miR-30c-5p, are upregulated by proteasome inhibitor PS-341 treatment, in HepG2 and MDA-MB-453 cells. (ii) Two representative PS-341-induced miRs: miR-30b-5p and miR-30c-5p are found to promote cell proliferation and anti-apoptosis in both tumor cells. (iii) eIF2α is confirmed as the congenerous target of miR-30b-5p and miR-30c-5p, essential to the anti-apoptotic function of these miRs. (iv) Upregulation of miR-30b-5p or miR-30c-5p, which occurs latter than the increase of phosphorylated eIF2α (p-eIF2α) in the cell under ER stress, suppresses the p-eIF2α/ATF4/CHOP pro-apoptotic pathway. (v) Inhibition of the miR-30b-5p or miR-30c-5p sensitizes the cancer cells to the cytotoxicity of proteasome inhibition. In conclusion, we unravels a new miRs-based mechanism that helps maintain intracellular proteostasis and promote cell survival during ER stress through upregulation of miR-30b-5p and miR-30c-5p which target eIF2α and thereby inhibit the p-eIF2α/ATF4/CHOP pro-apoptotic pathway, identifying miR-30b-5p and miR-30c-5p as potentially new targets for anti-cancer therapies.
Inhibition of proteasome-associated deubiquitinases (DUBs) is emerging as a novel strategy for cancer therapy. It was recently reported that auranofin (Aur), a gold (I)-containing compound used clinically to treat rheumatoid arthritis, is a proteasome-associated DUB inhibitor. Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, is currently in clinical use for treating alcoholism. Recent studies have indicated that DSF can also act as an antitumor agent. We investigated the effect of combining DSF and Aur on apoptosis induction and tumor growth in hepatoma cancer cells. Here we report that (i) the combined treatment of Aur and DSF results in synergistic cytotoxicity to hepatoma cells in vitro and in vivo; (ii) Aur and DSF in combination induces caspase activation, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production; (iii) pan-caspase inhibitor z-VAD-FMK could efficiently block apoptosis but not proteasome inhibition induced by Aur and DSF combined treatment, and ROS is not required for Aur+DSF to induce apoptosis. Collectively, we demonstrate a model of synergism between DSF and proteasome-associated DUB inhibitor Aur in the induction of apoptosis in hepatoma cancer cells, identifying a potential novel anticancer strategy for clinical use in the future.
auranofin; deubiquitinase inhibitor; disulfiram; anticancer strategy
Cystic fibrosis (CF) patients suffer from chronic airway inflammation with excessive neutrophil infiltration. Migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), which plays an important role in mediating adhesive interactions between effector and target cells in the immune system. In this study, we investigated the relationship between ICAM-1 and epithelium-specific ETS-like transcription factor 1 (ESE-1) and found that ICAM-1 expression is upregulated in cell lines of CF (IB3-1) as well as non-CF (BEAS-2B and A549) epithelial origin in response to inflammatory cytokine stimulation. Since ESE-1 is highly expressed in A549 cells without stimulation, we examined the effect of ESE-1 knockdown on ICAM-1 expression in these cells. We found that ICAM-1 expression was downregulated when ESE-1 was knocked down in A549 cells. We also tested the effect of ESE-1 knockdown on cell-cell interactions and demonstrate that the knocking down ESE-1 in A549 cells reduce their interactions with HL-60 cells (human promyelocytic leukemia cell line). These results suggest that ESE-1 may play a role in regulating airway inflammation by regulating ICAM-1 expression.
Copper and gold complexes have clinical activity in several diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is associated with targeting the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. Here we discuss metal DUB inhibitors in treating cancer and other diseases. (from Editor). Several copper and gold complexes have clinical activity in treating some human diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is tightly associated with their ability to target and inhibit the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. In this article we review small molecule inhibitors of DUBs and 19S proteasome-associated DUBs. We then describe and discuss the ubique nature of CuPT and auranofin, which is inhibition of 19S proteasome-associated UCHL5 and USP14. We finally suggest the potential to develop novel, specific metal-based DUB inhibitors for treating cancer and other diseases
19S-associated deubiquitinases; copper complexes; gold complexes
Patients with chronic myeloid leukemia (CML) are commonly treated with a specific inhibitor of BCR-ABL tyrosine kinase, imatinib mesylate (IM). Unfortunately, CML patients develop IM-resistance, which has emerged as a significant clinical problem. Somatic mutations, especially T315I mutation, in BCR-ABL kinase domain represent the most common mechanism underlying drug resistance to tyrosine kinase inhibitors (TKI), including imatinib. Thus, it is urgent to develop novel therapeutic strategies to overcome TKI-resistance. The anti-rheumatic gold (I) compound Auranofin (AF), was recently approved by US Food and Drug Administration for Phase II clinical trials to treat leukemia. In a recent study, we discovered that AF can selectively inhibit 19S proteasome-associated deubiquitinases (UCHL5 and USP14), which mediates its anticancer effects. More recently studies we have shown that AF inhibits the growth of both Bcr-Abl wild-type cells and IM-resistant Bcr-Abl-T315I mutation cells in vitro and in vivo. AF-induced Bcr-Abl down regulation is associated with diminished mRNA expression and caspase-dependent Bcr-Abl cleavage. More importantly, we unraveled that AF cytotoxicity is mediated by proteasome inhibition rather than previously suspected reactive oxygen species (ROS) generation. These findings support that AF overcomes IM-resistance through Bcr/Abl-dependent and -independent mechanisms, identifying a potentially new strategy for cancer treatment.
auranofin; CML; Bcr-Abl; imatinib resistance; proteasomal deubiquitinases; reactive oxygen species
Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment.
Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts.
Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid–induced Bcr-Abl downregulation and apoptotic cell death.
These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.
Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.
Auranofin; proteasome; chronic myelogenous leukemia; imatinib resistance; Bcr-Abl
The COP9 signalosome (CSN) is an evolutionarily conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8). We have recently discovered in perinatal mouse hearts that CSN regulates not only proteasome-mediated proteolysis but also macroautophagy. However, the physiological significance of CSN in a pots-mitotic organ of adult vertebrates has not been determined. We sought to study the physiological role of CSN8/CSN in adult mouse hearts.
Methods and Results
Csn8 was conditionally ablated in the cardiomyocytes of adult mice (CSN8CKO) using a temporally controlled Cre-LoxP system. Loss of CSN8 accumulated the neddylated forms of cullins and non-cullin proteins, increased ubiquitinated proteins, and stabilized a surrogate substrate of the proteasome in the heart. Autophagic flux was significantly decreased while autophagosomes were markedly increased in CSN8CKO hearts, indicative of impaired autophagosome removal. Furthermore, we observed increased oxidized proteins, massive necrotic cardiomyocytes, and morphological and functional changes characteristic of dilated cardiomyopathy in CSN8CKO mice.
CSN deneddylates substrates more than cullins and is indispensable to cardiomyocyte survival in not only perinatal hearts but also adult hearts. CSN8/CSN regulates both proteasome-mediated proteolysis and the autophagic-lysosomal pathway, critical to the removal of oxidized proteins in the heart.
COP9 signalosome; autophagy; proteasome; NEDD8; heart
Proteasomes are attractive emerging targets for anti-cancer therapies. Auranofin
(Aur), a gold-containing compound clinically used to treat rheumatic arthritis, was
recently approved by US Food and Drug Administration for Phase II clinical trial to
treat cancer but its anti-cancer mechanism is poorly understood. Here we report that
(i) Aur shows proteasome-inhibitory effect that is comparable to that of
bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits
proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S
proteasome; (iii) inhibition of the proteasome-associated DUBs is required for
Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in
vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia
patients. This study provides important novel insight into understanding the
proteasome-inhibiting property of metal-containing compounds. Although several DUB
inhibitors were reported, this study uncovers the first drug already used in clinic
that can inhibit proteasome-associated DUBs with promising anti-tumor effects.
cancer; deubiquitinase; proteasome; auranofin
Intracellular protein degradation is primarily performed by the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). The interplay between these two pathways has been rarely examined in intact animals and the mechanism underlying the interplay remains unclear. Hence, we sought to test in vivo and in vitro the impact of inhibition of the ALP on UPS proteolytic performance in cardiomyocytes and to explore the underlying mechanism. Transgenic mice ubiquitously expressing a surrogate UPS substrate (GFPdgn) were treated with bafilomycin-A1 (BFA) to inhibit the ALP. Myocardial and renal GFPdgn protein levels but not mRNA levels were increased at 24 hours but not 3 hours after the first injection of BFA. Myocardial protein abundance of key proteasome subunits and the activities of proteasomal peptidases were not discernibly altered by the treatment. In cultured neonatal rat ventricular myocytes (NRVMs), the surrogate UPS substrate GFPu and a control red fluorescence protein (RFP) were co-expressed to probe UPS performance. At 12 hours or 24 hours after ALP inhibition by 3-methyladenine (3-MA) or BFA, GFPu/RFP protein ratios and the protein half-life of GFPu were significantly increased, which is accompanied by increases in p62 proteins. Similar findings were obtained when ALP was inhibited genetically via silencing Atg7 or Rab7. ALP inhibition-induced increases in GFPu and p62 are co-localized in NRVMs. siRNA-mediated p62 knockdown prevented ALP inhibition from inducing GFPu accumulation in NRVMs. We conclude that in a p62-dependent fashion, ALP inhibition impairs cardiac UPS proteolytic performance in cardiomyocytes in vitro and in vivo.
The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy.
Cardiac hypertrophy is a common response of the heart to a variety of cardiovascular stimuli. Pathological cardiac hypertrophy eventually leads to heart failure. Gambogic acid (GA) is a main active ingredient isolated from the gamboge resin of Garcinia hanburyi trees and has potent anti-tumor and anti-inflammatory effects that are associated with inhibition of the NF-κB pathway. We and others recently reported that GA can significantly inhibit the function of the proteasome with much less toxicity than conventional proteasome inhibitors. The increasing lines of evidence indicate that the inhibition of the proteasome can promote the regression of cardiac hypertrophy induced by pressure overload through the blockade of the NF-κB pathway. In the present study, we examined the effect of GA on pressure overload or isoproterenol infusion induced cardiac hypertrophy and fibrosis, and changes in myocardial NF-κB signaling. We observed that the heart weight/body weight ratio, the size of cardiomyocytes, interstitial fibrosis, and the reactivation of fetal genes (α-SK-actin and BNP mRNA) were markedly increased by abdominal aorta constriction (AAC) or isoproterenol infusion (ISO), all of which were effectively inhibited by GA treatment. Furthermore, GA treatment abolished proteasome chymotrypsin-like activity increases induced by AAC or ISO, led to increased myocardial IκB protein, decreased NF-κB p65 subunit levels in the nuclear fraction, decreased NF-κB DNA-binding activity, and reduced IL2 levels in the myocardium of rats subject to AAC or ISO. In conclusion, GA treatment can suppress cardiac hypertrophy and fibrosis induced by pressure overload or isoproterenol possibly through the inhibition of the proteasome and the NF-κB pathway, suggesting that GA treatment may provide a new strategy to treat cardiac hypertrophy.
Gambogic acid; cardiac hypertrophy; pressure overload; isoproterenol; proteasome; NF-κB
Extracellular adenosine (Ade) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.
Gambogic acid (GA) is the principal active ingredient of gamboges. GA was reported to exert anti-tumor and anti-inflammatory effects both in vitro and in vivo. Previously, we have shown that GA is a more tissue-specific proteasome inhibitor than bortezomib and it is less toxic to peripheral white blood cells compared to bortezomib. Ubiquitous proteasome inhibition was shown by some reports, but not by others, to prevent cardiac remodeling in response to pressure overload by blocking the NF-κB pathway; however, whether GA modulates the development of chronic hypoxia-induced right ventricular hypertrophy has not been investigated yet. Here we report that GA can significantly attenuate right ventricular hypertrophy induced by chronic hypoxia, reduce cardiac fibrosis, and remarkably block the reactivation of bona fide fetal genes in the cardiac tissue. Furthermore, we also investigated the potential molecular targets of GA on right ventricular hypertrophy. The results showed that GA could accumulate the IκB levels associated with decreased proteasomal activity, block the translocation of NF-κB from the cytoplasm to the nucleus, decrease NF-κB DNA-binding activity, and reduce IL-2 levels. In conclusion, GA is capable of preventing the development of chronic hypoxia-induced right ventricular hypertrophy. GA has great potential to be developed into an effective anti-hypertrophy agent.
Gambogic acid; chronic hypoxia; right ventricular hypertrophy; NF-κB
The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.
Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy.
Gambogic acid; Proteasome inhibitors; Antitumor activity; Synergistic effect
Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.
L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21cip1 gene, mRNA and protein in cancer cells but not p27kip1; (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21cip1 gene but not p27kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.
The proteasome is a multicatalytic protease complex whose activity is required for the growth of normal or tumor cells. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that the proteasome could be a target of chemotherapy. Studies suggest that traditional Chinese medicine (TCM) is an effective approach for cancer treatment. Here we reviewed several TCMs for their potential in treatment of cancer. This short review focuses mainly on the TCMs that potentially target the tumor cellular proteasome and NF-κB pathway whose activation is dependent on the proteasome activity.
Proteasome inhibitors; natural products; medicinal compounds; cancer; prevention; treatment