Shujue Li

Physiological levels of ATP Negatively Regulate Proteasome Function

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 and 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 upregulate proteasome activity on ATP reduction under stress conditions.

Shikonin extracted from medicinal Chinese herbs exerts anti-inflammatory effect via proteasome inhibition

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.

Gambogic Acid Is a Tissue-Specific Proteasome Inhibitor In Vitro and In Vivo

Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

Gambogic acid enhances proteasome inhibitor-induced anticancer activity

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.

The combination of proteasome inhibitors bortezomib and gambogic acid triggers synergistic cytotoxicity in vitro but not in vivo.

The proteasome inhibitor-based combinational therapy has been reported to be an efficient cancer treatment. Our recent studies demonstrated that the natural compound gambogic acid (GA) is a tissue-specific proteasome inhibitor, comparable to bortezomib (Bor), and sensitizes malignant cells to the proteasome inhibitor MG132/MG262 both in vitro and in vivo. The aim of this study was to further extend our investigation by combining GA with the clinically used proteasome inhibitor Bor to test their combined efficacy against human hepatoma HepG2 and mouse hepatoma H22 cells. GA and Bor synergistically induced cytotoxicity and cell death in human HepG2 and mouse H22 cells, and accelerated proteasome inhibition, endoplasmic reticulum (ER) stress and caspase activation in HepG2 cancer cells. However, unexpectedly, GA did not enhance or even antagonized Bor-induced tumor growth inhibition in H22 allograft and HepG2 xenograft tumor models. These findings demonstrated that GA increased Bor activity in vitro but limited the efficacy of Bor in vivo. We suggest that the combination of GA and Bor be avoided when administering these drugs to patients.

Intracellular ATP Concentration Contributes to the Cytotoxic and Cytoprotective Effects of Adenosine

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.

Autophagy inhibition attenuates hyperoxaluria-induced renal tubular oxidative injury and calcium oxalate crystal depositions in the rat kidney

Hyperoxaluria-induced oxidative injury of renal tubular epithelial cell is a casual and essential factor in kidney calcium oxalate (CaOx) stone formation. Autophagy has been shown to be critical for the regulation of oxidative stress-induced renal tubular injury; however, little is known about its role in kidney CaOx stone formation. In the present study, we found that the autophagy antagonist chloroquine could significantly attenuate oxalate-induced autophagy activation, oxidative injury and mitochondrial damage of renal tubular cells in vitro and in vivo, as well as hyperoxaluria-induced CaOx crystals depositions in rat kidney, whereas the autophagy agonist rapamycin exerted contrasting effects. In addition, oxalate-induced p38 phosphorylation was significantly attenuated by chloroquine pretreatment but was markedly enhanced by rapamycin pretreatment, whereas the protective effect of chloroquine on rat renal tubular cell oxidative injury was partly reversed by a p38 protein kinase activator anisomycin. Furthermore, the knockdown of Beclin1 represented similar effects to chloroquine on oxalate-induced cell oxidative injury and p38 phosphorylation in vitro. Taken together, our results revealed that autophagy inhibition could attenuate oxalate-induced oxidative injury of renal tubular cell and CaOx crystal depositions in the rat kidney via, at least in part, inhibiting the activation of p38 signaling pathway, thus representing a novel role of autophagy in the regulation of oxalate-induced renal oxidative injury and CaOx crystal depositions for the first time.

Prevalence of metabolic syndrome in China: An up-dated cross-sectional study

Metabolic syndrome (MS) is an increasing public health concern because of rapid lifestyle changes. Although there have been previous studies on the prevalence of MS in China, the prevalence may have changed with lifestyle changes over the last decade. To update this prevalence, we performed a cross-sectional survey among adults over 18 years old across China from May 2013 to July 2014. Participants underwent questionnaires and provided blood and urine samples for analysis. MS was defined according to the criteria of the China Diabetes Society. A total of 12570 individuals (45.2% men) with an average age of 48.8±15.3 (18–96) years were selected and invited to participate in the study. In total, 9310 (40.7% men) individuals completed the investigation, with a response rate of 74.1%. The prevalence of MS in China was 14.39% [95% confidence interval (CI): -3.75–32.53%], and the age-adjusted prevalence was 9.82% (95% CI: 9.03–10.61%; 7.78% in men and 6.76% in women; 7.39% in rural residents and 6.98% in urban residents). The highest prevalence occurred among adults aged 50–59 years (1.95%, 95% CI: 1.40–2.50%), and the lowest prevalence occurred among adults aged 40–49 years (0.74%, 95% CI: 0.38–1.10%); the prevalence was the highest in the south region and lowest in the east region (4.46% and 1.23%, respectively). The results of logistic regression analyses showed that age, urolithiasis, hyperuricemia, coronary artery disease, thiazide drugs intake, family history of diabetes and hypertension were all significantly associated with an increased risk of metabolic syndrome (OR>1). In addition, education, vitamin D intake and family history of urolithiasis are all protective factors (OR<1). Our results indicate that there was a high prevalence of MS in Chinese adults. Compared to the previous study 10 years ago, some preventive strategies have worked; however, further work on the prevention and treatment of MS remains necessary.

Integrative Analysis of miRNA and mRNA Expression Profiles in Calcium Oxalate Nephrolithiasis Rat Model

The microRNA (miRNA) expression profiles and their biological functions in calcium oxalate nephrolithiasis remain unclear. In this study, we investigate the miRNA and mRNA expression profiles of kidney tissues in calcium oxalate stone rats. 16 Sprague Dawley rats were divided into control group and stone-forming group. 24-hour urine samples and kidney tissues were collected for biochemical and histological determination after 4 weeks. MiRNA and mRNA microarray were applied to evaluate the miRNA and mRNA expression profiles. To validate the microarray results, the quantitative real-time PCR (qRT-PCR) was performed. A total of 38 miRNAs and 2728 mRNAs were significantly and differentially expressed in kidney tissues of stone-forming group versus control group. Gene Ontology (GO) analysis revealed that most of the target genes were enriched in terms of oxidation reduction, ion transport, inflammatory response, and response to wounding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these targets highlights their critical role in cytokine-cytokine receptor interaction, gap junction, and chemokine signaling pathway. Furthermore, the reliability of the microarray-based results was confirmed by using qRT-PCR determination. The miRNA and mRNA expressions in calcium oxalate stone rat kidneys might provide a basis for further research on urolithiasis mechanism.