Borneol and Luteolin from Chrysanthemum morifolium Regulate Ubiquitin Signal Degradation.

Targeting the two degradation systems, ubiquitin proteasome pathway and ubiquitin signal autophagy lysosome system, plays an important function in cancer prevention. Borneol is called an "upper guiding drug". Luteolin has demonstrated anticancer activity. The fact that borneol regulates luteolin can be sufficient to serve as an alternative strategy. Borneol activates luteolin to inhibit E1 and 20S activity (IC50 = 118.8 ± 15.7 µM) and perturb the 26S proteasome structure in vitro. Borneol regulates luteolin to inhibit 26S activity (IC50 = 157 ± 19 µM), induces apoptosis (LC50 = 134 ± 4 µM), and causes pre-G1 and G0/G1 arrest in HepG2 cells. Borneol regulates luteolin to induce ubiquitin signal autophagic degradation, resulting in induction of E1, reduction of USP47, and accumulation of p62 in HepG2 reporter cells. Interestingly, luteolin decreased Ub conjugates, while borneol increased the accumulation of Ub conjugates in HepG2 reporter cells. E1, p62, and ubiquitin levels were downregulated in borneol-treated HepG2 reporter cells at 24 h. These observations suggest a potential autophagic inhibitor of borneol that may guide luteolin in the ubiquitin proteasome pathway and the ubiquitin signal autophagic degradation.

Regulation of ubiquitin and 26S proteasome mediated by phenolic compounds during oxidative stress.

Little attention has been devoted to studying the roles of natural antioxidants in the ubiquitin-proteasome pathway during oxidative stress. We demonstrated that a time course revealed that the reassociation of the 19S regulators with the 20S proteasomes occurred automatically and rapidly to reconstitute the 26S proteasomes, with up to 80% completion, within 5 min after H2O2 treatment. Ubiquitin, methyl gallate and tannic acid are able to prevent H2O2 from inhibiting the 26S activity. We further show that the level of the ubiquitin, S5a and 20S core subunits decreased within 30 min and increased after 24 h of H2O2 treatment in Hep-2 cells. Phenolic compounds not only inhibited the 26S activity but also decreased the USP47 levels, which reduce the DNA damage repair rate during oxidative stress; in addition, the presence of DNA fragments, procaspase-3 and a decreased poly (ADP-ribose) polymerase also appeared as a result of the above conditions. Ubiquitin could serve as a protective substrate in H2O2 and phenolic compound-treated Hep-2 cells. Methyl gallate and tannic acid, as prooxidants, can attenuate the apoptotic response resulting from long-term oxidative stress. Collectively, these data demonstrate an important role for phenolic compounds in regulating the 26S proteasome and ubiquitin during oxidative stress.

Combination of quercetin and tannic acid in inhibiting 26S proteasome affects S5a and 20S expression, and accumulation of ubiquitin resulted in apoptosis in cancer chemoprevention.

To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or ß-casein, and (5) inhibit ß-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination.

5,6,3',4'-Tetrahydroxy-7-methoxyflavone as a novel potential proteasome inhibitor.

Proteasome inhibition is one of the new pharmaceutical approaches to study antitumor activity. Although the active components are not yet identified for either treating or preventing cancer, the low toxicity plant Anisomeles ovata R. Br. has been used in traditional herbal medicine for more than 1000 years. In this study, the methanol extract from Anisomeles ovata showed potent inhibition of proteasome activity. Twenty compounds, two macrocycylic diterpenoids, six aromatics, seven flavonoids, and five phenylethanoids were isolated from Fang Feng Cao the dried aerial parts of A. ovata. Their structures have been established on the basis of spectral evidence. Using a proteolysis assay for inhibition of 26S proteasome from pig red blood cells, we found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 90.5 %, 85.4 % and 73.1 % the chymotrypsin-like, caspase-like and trypsin-like activities of 26S proteasome with IC (50) values of 14.0, 5.4 and 24.1 microM, respectively, when Suc-LLVY-AMC, Z-LLE-AMC and Ac-RLR-AMC were used as substrates. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone had a higher inhibitory at 15 minutes. A combination of 5,6,3',4'-tetrahydroxy-7-methoxyflavone and 5,6,4'-trihydroxy-7,3'-dimethoxyflavone increased the inhibition ability on 26S enzymatic activity. This combination appears to be a potentially attractive chemotherapy approach. We have found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone has the highest inhibitory effects on 26S proteasome activities when compared to the other 11 flavonoids. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S proteasome activity.

Inhibitory effect of flavonoids on 26S proteasome activity.

Inhibiting proteasomal degradation has been shown to induce apoptosis in tumor cells. Utilization of proteasome inhibition is therefore one approach to anticancer therapy. Some of the flavonoids can induce cell apoptosis via inhibiting proteasome 26S activity. In this study, the inhibition of 26S proteasome from pig red blood cells was analyzed on 12 flavones, 5 flavanones, and 9 isoflavones by using a proteolysis assay. Several flavonoids such as apigenin-6-hydroxy-7-O-beta-D-glucoside, quercetin, rutin, 6-hydroxyapigenin, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, 5,6,3',4'-tetrahydroxy-7-methoxyflavone, glycitecin, and 6,7,4'-trihydroxyisoflavone inhibited the chymotrypsin-like, caspase-like, or trypsin-like activity of 26S proteasome when Suc-LLVY-AMC, Z-LLE-AMC, and Ac-RLR-AMC were used as substrates. Three peptidase activities of flavonoids were found to be significantly correlated with one another. Flavones had significantly stronger inhibitory effects on chymotrypsin-like and caspase-like activities than flavanones and isoflavones. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, and quercetin displayed a mixed type inhibition of 26S by Lineweaver-Burk plots analysis. Furthermore, 5,6,3',4'-tetrahydroxy-7-methoxyflavone is found to have a higher inhibitory effect on 26S proteasome activities and is the only flavonoid to inhibit all three peptidase activities, whereas the inhibition of flavonoids was not affected by ubiquitin-induced stimulation of the three peptidase activities of 26S proteasome; 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 75% casein degradation. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S activity.

The roles of ubiquitin and 26S proteasome in human obesity.

The ubiquitin-proteasome pathway is responsible for the degradation of most intracellular proteins in eukaryotes. It may also play a role in the modulation of inflammatory process and pathogenesis of cancer. Immunoglobulin levels are higher in cancer. Obesity is a risk factor for several common diseases, particularity type 2 diabetes mellitus, cardiovascular diseases, and tumors. The aim of this study was to study a possible correlation between plasma ubiquitin, 26S proteasome levels, and obesity. The body mass index (BMI), plasma ubiquitin levels, and 26S proteasome activity levels were determined and statistically analyzed in 31 volunteers, aged 19 to 58 years and including 9 men and 22 women, from the general population of Southern Taiwan. We also compared the immunoglobulin among the underweight, normal-weight, and overweight groups. We demonstrated that plasma ubiquitin is significantly decreased in obese individuals vs normal controls (65.2 +/- 23.4 vs 159.5 +/- 73.1 ng/mL). Plasma ubiquitin levels were found to be inversely correlated with the BMI of individuals (r = -0.39, P < .001). In addition, there was an inverse relationship between 20S proteasome levels in red blood cells and BMI (r = -0.33, P < .001), whereas 26S proteasome activity was found to be dependent quantitatively to S5a in erythrocytes (r = 0.88, P < .001). Immunoglobulin is significantly decreased in overweight individuals vs normal controls. Plasma ubiquitin and 20S proteasome levels are potential biomarkers for the risk assessment and possibly serve as one of the targeted studies for the development of human obesity.

Role of ginsenoside Rd in inhibiting 26S proteasome activity.

Drugs targeting 26S proteasome as antitumor agents are considered to be important for cancer therapy. Although the active components are yet to be identified, for more than 1000 years, the low-toxicity Panax ginseng has been used in traditional herbal medicine for either treating or preventing cancer. Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 are distinct components that can be isolated from P. ginseng C.A. Meyer. In this study 26S proteasome was purified from pig red blood cells, and the activity of the seven isolated ginsenosides was analyzed by proteolysis assay. It was found that ginsenoside Rd inhibited 52.9% the chymotrypsin-like activity of 26S proteasome with an IC(50) value of 107.5 microM when Suc-LLVY-AMC was used as a substrate. Ginsenoside Rd displayed a mixed type inhibition of 26S proteasome when analyzed by Lineweaver-Burk plots of the inhibition kinetics. Unlike ginsenoside Rd, the other ginsenosides showed low inhibitory effect of the chymotrypsin-like activity of 26S proteasome. Seven ginsenosides did not inhibit the trypsin-like and caspase-like activities of 26S when Ac-RLR-AMC or Z-LLE-AMC was used as substrate. These results suggest that ginsenoside Rd is a potential drug for cancer prevention due to its specific 26S proteasome inhibitory effect and known low toxicity. Furthermore, both 3-O-Glc(2)-Glc and 20-O-beta-Glc positions of the ginsenoside may play a role in the inhibitory property of the chymotrypsin-like activity in 26S proteasome.

Alterations of metastasis-related genes identified using an oligonucleotide microarray of genistein-treated HCC1395 breast cancer cells.

Genistein, one of the major isoflavones, potently inhibits the growth and metastasis of breast cancer. However, the precise molecular mechanism in metastasis inhibition is not clear. We investigated the effect of genistein in HCC1395 cells, a cell line derived from an early-stage primary breast cancer. Genistein dose dependently both decreased cell viability and inhibited the invasion potential. We used human oligonucleotide microarrays to determine the gene expression profile altered by genistein treatment. TFPI-2, ATF3, DNMT1, and MTCBP-1, which inhibit invasion and metastasis, were upregulated, and MMP-2, MMP-7, and CXCL12, which promote invasion and metastasis, were downregulated. We used quantitative real-time polymerase chain reaction to verify the microarray data at the mRNA level. We conclude that genistein-induced alternations of gene expression involving metastasis may be exploited for devising chemopreventive and therapeutic strategies, particularly for early-stage breast cancer.

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome.

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells.

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its alpha subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (alpha' spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the alpha'/alpha spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the alpha'/alpha spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/E3 activity is an important determinant of sickle cell severity.