Chymotrypsin-like Activity Of 26S Proteasome Research Articles

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Chemical characterization and cytotoxic potential of an ellagitannin-enriched fraction from Fragaria vesca leaves

The hepatocellular carcinoma, a primary malignancy of the liver, has a very poor prognosis and a lower survival rate. Moreover, the inefficacy of conventional therapies emphasizes the importance of discovering new bioactive compounds. Several studies clearly state that plant-derived polyphenols, namely ellagitannins, have several health benefits. Fragaria vesca leaves contain high amounts of polyphenols, being especially rich in ellagitannins. Therefore, this study aimed to characterize an ellagitannin-enriched fraction (EEF) from F. vesca leaves and to unveil the anticancer potential of this fraction on human hepatocellular carcinoma cells.The analysis of EEF by HPLC-PDA-ESI/MSn allowed the detection of 12 ellagitannins. The cell viability of both EEF and crude extract was determined after 24h of cells treatment and the half-maximal inhibitory concentration (IC50) was evaluated. The IC50 of the EEF (113μg/mL) was about 6 times lower than the IC50 of the crude extract (690μg/mL). Furthermore, EEF induced cell cycle arrest at G2/M checkpoint and decreased cell proliferation in a dose-dependent way. This fraction also induced an accumulation of LC3-II protein through blockage of autophagic flux, and inhibited chymotrypsin-like activity of 26S proteasome. These results showed, for the first time, that EEF from F. vesca leaves inhibits both, autophagic and ubiquitin-proteasome system pathways, two main intracellular protein degradation systems that are targets for anticancer therapies. Additionally, a proteomic analysis allowed the identification of 914 proteins, among which 133 were modulated after cells treatment with EEF, most of them related to metabolic pathways. Overall, this study shows that the EEF from F. vesca leaves decreased cell proliferation, inhibited the proteolytic mechanisms and modulated the metabolic pathways of the cell. Additionally this study points out F. vesca as a source of valuable molecules with anticancer potential, suggesting that ellagitannins, the polyphenols identified in this fraction, could be useful in the development of new fine-tuned therapeutic strategies against carcinogenesis.

The role of ginsenosides in inhibiting ubiquitin activating enzyme (E1) activity

Functional foods targeting the ubiquitin-activating enzyme (E1) of the ubiquitin–proteasome pathway as anti-tumour agents are considered to be important for cancer chemoprevention. The low-toxicity Panax ginseng was used in traditional herbal medicine or food use for either treating or preventing cancer for more than 1000years. In this study, we constructed an ubiquitin-tagged protein as a substrate to analyze the six ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1 by ubiquitination assay. More ubiquitinated E1 levels were induced by increasing concentrations of ubiquitin-activating enzyme. Ginsenosides Re and Rg1 inhibited ubiquitin-activating enzyme. Unlike ginsenosides Re and Rg1, ginsenosides Rb1, Rb2, Rc, and Rd increased ubiquitination on E1 enzyme. Interestingly, ginsenoside Rg1 inhibits both the chymotrypsin-like activity of 26S proteasome and E1 activity. These results suggest that ginsenoside Rg1 is a potential functional food application for cancer prevention because of its specific E1 inhibitory effects. Furthermore, the 6-O-Glu position of the ginsenoside may play a role in its E1 inhibitory property. Ginsenosides could be further developed as a useful drug for cancer therapy.

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.

Blocking of NF-κB activation enhances the tumor necrosis factor α-induced apoptosis of a human gastric cancer cell line

The transcription factor NF-κB is constitutively activated in many human cancers and induces the expression of multiple genes, including those of anti-apoptotic proteins. This study investigated the mechanism by which human gastric cancer cells (MKN45) are resistant to apoptosis induced by tumor necrosis factor α (TNF-α). Confluent monolayers of MKN45 cells were either pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant human TNF-α, and cell viabilities were determined by the WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342, and DNA fragmentation was determined by a DNA fragmentation detection kit. A 24-h incubation with either TNF-α or PSI alone did not affect cell viabilities; however, pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-α. Therefore, this study suggests the possibility that blocking of NF-κB activity renders gastric cancer cells susceptible to the apoptosis induced by TNF-α.

Ubiquitin-proteasome inhibitor enhances tumour necrosis factor-alpha-induced apoptosis in rat gastric epithelial cells.

Tumour necrosis factor (TNF-alpha) is a candidate factor for involvement in inflammation-mediated gastric mucosal injury. However, the effect of this cytokine on gastric epithelial cells has been poorly investigated. In the present study, we examined whether gastric epithelial cells are resistant to TNF-alpha-induced apoptosis, and whether this resistance is related to ubiquitin-proteasome-associated nuclear factor-kappaB (NF-kappaB) activation. The rat gastric mucosal cell line RGM-1 was grown in DMEM/F12 medium supplemented with 10% FCS. Confluent monolayers of cells were pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant rat TNF-alpha and their viability was determined by WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342 and propidium iodide, and DNA fragmentation was determined by flow cytometry using an APO-BRDU kit. IkappaB-alpha and the p65 binding subunit of NF-kappaB were detected by Western blots. Twenty-four-hour incubation with TNF-alpha alone or PSI alone did not affect the cell viability of RGM-1 cells. Pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-alpha. In RGM-1 cells treated with TNF-alpha, cytoplasmic IkappaB-alpha decreased and p65 in nuclear extracts increased markedly 30 min after cytokine stimulation. Pretreatment with PSI at 12.5 micromol/L blocked these TNF-alpha-induced changes. PSI enhances TNF-alpha-induced apoptosis through inhibition of NF-kappaB activation in RGM-1 cells.

Temperature-Dependent Enhancement of Proteolysis in C2C12 Myotubes in Association with the Activation of 26S Proteasome

The effect of temperature on protein metabolism of C2C12 myotubes was investigated in order to estimate the potential effect of fever on muscle catabolism. The half-life of long-lived proteins in C2C12 myotubes was significantly (13%) shorter when incubated at 40°C than at 37°C. The activities of cathepsins B and L were not significantly different at 37 and 40°C, nor were the levels of the protein and mRNA of the two cathepsins. In contrast, the chymotrypsin-like activity of 26S proteasome was elevated by 53% at 40°C, compared to that at 37°C, although it was not associated with an increase in the levels of the protein and mRNA of proteasome subunits. mRNA levels of calpain and ubiquitin were not affected by temperature. It is concluded that temperature-dependent enhancement of proteolysis in C2C12 myotubes is associated with an increase in 26S proteasome activity.