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
Several flavonoids have been reported to be proteasome inhibitors, but whether prenylated flavonoids are able to inhibit proteasome function remains unknown. We report for the first time that Sanggenon C, a natural prenylated flavonoid, inhibits tumor cellular proteasomal activity and cell viability. We found that (1) Sanggenon C inhibited tumor cell viability and induced cell cycle arrest at G0/G1 phase; (2) Sanggenon C inhibited the chymotrypsin-like activity of purified human 20S proteasome and 26S proteasome in H22 cell lysate, and Sanggenon C was able to dose-dependently accumulate ubiquitinated proteins and proteasome substrate protein p27; (3) Sanggenon C-induced proteasome inhibition occurred prior to cell death in murine H22 and P388 cell lines; (4) Sanggenon C induced death of human K562 cancer cells and primary cells isolated from leukemic patients. We conclude that Sanggenon C inhibits tumor cell viability via induction of cell cycle arrest and cell death, which is associated with its ability to inhibit the proteasome function and that proteasome inhibition by Sanggenon C at least partially contributes to the observed tumor cell growth-inhibitory activity.
Sanggenon C; proteasome inhibitor; cell death; cell cycle; flavonoid
Shikonin, extracted from medicinal Chinese herb (Lithospermum erythrorhizo), was reported to exert anti-inflammatory and anti-cancer effects both in vitro and in vivo. We have found that proteasome was a molecular target of shikonin in tumor cells, but whether shikonin targets macrophage proteasome needs to be investigated. In the current study, we report that shikonin inhibited inflammation in mouse models as efficiently as dexamethasone. Shikonin at 4 μM reduced the Lipopolysaccharides (LPS)-mediated TNFα release in rat primary macrophage cultures, and blocked the translocation of p65-NF-κB from the cytoplasm to the nucleus, associated with decreased proteasomal activity. Consistently, shikonin accumulated IκB-α, an inhibitor of NF-κB, and ubiquitinated proteins in rat primary macrophage cultures, demonstrating that the proteasome is a target of shikonin under inflammatory conditions. Shikonin also induced macrophage cell apoptosis and cell death. These results demonstrate for the first time that proteasome inhibition by shikonin contributes to its anti-inflammatory effect. The novel finding about macrophage proteasome as a target of shikonin suggests that this medicinal compound has great potential to be developed into an anti-inflammatory agent.
Shikonin; Proteasome inhibitor; Macrophage; Inflammation
Ubiquitin-proteasome system (UPS) dysfunction has been implicated in cardiac pathogenesis. Understanding how cardiac UPS function is regulated will facilitate delineating the pathophysiological significance of UPS dysfunction and developing new therapeutic strategies. The COP9 signalosome (CSN) may regulate the UPS but this has not been tested in a critical vertebrate organ. Moreover, the role of CSN in a post-mitotic organ and the impact of cardiomyocyte-restricted UPS dysfunction on the heart have not been reported.
We sought to determine the role of CSN-mediated deneddylation in UPS function and postnatal cardiac development and function.
Methods and Results
Cardiomyocyte-restricted Csn8 gene knockout (CR-Csn8KO) in mice was achieved using a Cre-LoxP system. CR-Csn8KO impaired CSN holocomplex formation and cullin deneddylation and resulted in decreases in F-box proteins. Probing with a surrogate misfolded protein revealed severe impairment of UPS function in CR-Csn8KO hearts. Consequently, CR-Csn8KO mice developed cardiac hypertrophy, which rapidly progressed to heart failure and premature death. Massive cardiomyocyte necrosis rather than apoptosis appears to be the primary cause of the heart failure. This is because (1) massive necrotic cell death and increased infiltration of leukocytes were observed prior to increased apoptosis; (2) increased apoptosis was not detectable until overt heart failure was observed; and (3) cardiac overexpression of Bcl2 failed to ameliorate CR-Csn8KO mouse premature death.
Csn8/CSN plays an essential role in cullin deneddylation, UPS-mediated degradation of a subset of proteins, and the survival of cardiomyocytes; therefore is indispensible in postnatal development and function of the heart. Cardiomyocyte-restricted UPS malfunction can cause heart failure.
COP9 signalosome; ubiquitin E3 ligases; proteasome; cell death; heart failure
Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50–100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions.
ATP; proteasome; regulation; apoptosis
Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In the present study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2-16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB-α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin, and inhibition of the proteasome activity by shikonin contributes to its anti-tumor property.
Shikonin; medicinal compounds; proteasome inhibitor; hepatoma; prostate cancer