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Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting
Published on: March 8, 2018
Proteasomes are a target of the anti-tumour drug vinblastine
M Piccinini1, O Tazartes, C Mezzatesta
1Department of Experimental Medicine and Oncology, Section of Biochemistry, University of Turin, Via Michelangelo 27/B, 10126 Turin, Italy.
Abstract:
Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.
Insights
The anti-cancer drug vinblastine inhibits proteasome activity, affecting protein degradation pathways. This study reveals proteasomes as a novel target for vinblastine, impacting cell cycle progression and apoptosis.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Proteasomes are crucial for cellular protein degradation via the ubiquitin/ATP-dependent pathway.
- Proteasome inhibitors block cell cycle progression and induce apoptosis, making them valuable anti-cancer agents.
- Vinblastine, an anti-tumor drug, primarily acts by disrupting mitotic spindle dynamics.
Purpose of the Study:
- To investigate the impact of vinblastine on the peptidase and proteolytic activities of human 20 S and 26 S proteasomes.
- To determine if proteasomal function is affected by vinblastine in vitro and in vivo.
- To explore proteasomes as a potential new target for vinblastine's anti-cancer effects.
Main Methods:
- Assessed proteasome peptidase activities using fluorogenic peptides and ubiquitin-protein conjugates.
- Measured 26 S proteasome proteolytic activity with (125)I-lysozyme-ubiquitin conjugates.
- Utilized Western blotting to analyze intracellular proteasome substrates like ubiquitin-protein conjugates and IκBα in vinblastine-treated HL60 cells.
- Evaluated the signal-induced degradation of IκBα.
Main Results:
- Vinblastine (3–110 μM) reversibly inhibited chymotrypsin-like activity of the 20 S proteasome and trypsin-like and chymotrypsin-like activities of both 20 S and 26 S proteasomes.
- At 110 μM, vinblastine inhibited the chymotrypsin-like activity of the 26 S proteasome.
- Vinblastine (25–200 μM) inhibited the degradation of ubiquitinated lysozyme.
- In HL60 cells, vinblastine (0.5–10 μM) caused a dose-related accumulation of polyubiquitinated proteins and a high-molecular-mass form of IκBα.
- Vinblastine impaired the signal-induced degradation of IκBα, with minimal impact on cell viability (~95%).
Conclusions:
- Proteasomal function is significantly affected by vinblastine in a dose-dependent manner.
- Vinblastine inhibits multiple proteasome activities, including those involved in protein degradation.
- These findings identify proteasomes as a novel and additional target for the anti-cancer drug vinblastine.
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