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HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells
Published on: July 20, 2016
[Inhibition of 8-chloroadenosine on proteasome activity of HL-60 cells]
1National Research Laboratories of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, PR China.
Background & Objective:
The proteasome is a multicatalytic proteinase complex in eukaryotic cells, and it can be used as a new target for screening anti-tumor drugs according to certain previous researches. The inhibition activity of 8-CA (8-chloroadenosine, a new anti-tumor drug synthesized in our lab) in sarcoma 180, hepatocarcinoma 22, and human gastric carcinoma xenograft tumor has been confirmed. However,the relationship between the mechanism of the inhibition and proteasome is still unclear. The present study was designed to explore the effect of 8-CA on 3 kinds of enzyme activities (chymotrypsin-like activity, trypsin-like activity, and peptidyl-glutamyl- peptide hydrolyzing activity) of 20S proteasome in human progranulocyte leukemia HL-60 cells.
Methods:
HL-60 cells were treated with 8-CA at different concentrations for 24, 48, and 72 hours. Then total protein of HL-60 cells was extracted, and the three enzyme activities of 20S proteasomes in the total protein was examined. And the chymotrypsin-like activity of the purified proteasome from the total protein was also examined by immunoprecipitation. The enzyme activity was indicated by fluorescence absorbance (A) produced by degradation of specific substrates by proteasomes.
Results:
In HL-60 cells, three enzyme activities of the proteasomes were significantly inhibited after exposure to 0.1 micromol/L 8-CA for 48 hours; and the inhibition effect was concentration dependent. When the concentration of 8-CA reached 5 micromol/L, the inhibition rates of three proteasome activities (chymotrypsin-like activity,trypsin-like activity,and peptidyl- glutamyl-peptide hydrolyzing activity) were 44.96%, 54.52%, 48.36% for 48 hours and 67.53%, 70.48%, 64.08% for 72 hours, respectively. The inhibition rate of 1 micromol/L and 5 micromol/L 8-CA on chymotrypsin-like proteasome activity of HL-60 cells for 48 hours were 68.14% (P< 0.01) and 92.75% (P< 0.001), respectively.
Conclusion:
8-CA inhibits proteasome activity of HL-60 cells with a time- and concentration-dependent manner.
Insights
8-chloroadenosine (8-CA) effectively inhibits proteasome activity in human leukemia cells. This anti-tumor drug demonstrates a clear time- and concentration-dependent effect on key proteasome enzyme activities.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The proteasome is a validated target for anti-tumor drug development.
- 8-chloroadenosine (8-CA) has shown anti-tumor activity in various xenograft models.
- The precise mechanism of 8-CA's anti-tumor action, specifically its effect on proteasome activity, remains to be elucidated.
Purpose of the Study:
- To investigate the impact of 8-chloroadenosine (8-CA) on the chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide hydrolyzing activities of the 20S proteasome.
- To determine the effect of 8-CA on proteasome activity in human progranulocyte leukemia HL-60 cells.
Main Methods:
- HL-60 cells were treated with varying concentrations of 8-CA for 24, 48, and 72 hours.
- Total protein was extracted, and the three key enzyme activities of 20S proteasomes were measured.
- Immunoprecipitation was used to assess chymotrypsin-like activity in purified proteasomes.
Main Results:
- 8-CA significantly inhibited all three proteasome enzyme activities in HL-60 cells.
- Inhibition was observed at 0.1 micromol/L 8-CA after 48 hours and demonstrated a concentration-dependent effect.
- At 5 micromol/L 8-CA, inhibition rates reached 67.53% (chymotrypsin-like), 70.48% (trypsin-like), and 64.08% (peptidyl-glutamyl peptide hydrolyzing) after 72 hours.
Conclusions:
- 8-chloroadenosine (8-CA) effectively inhibits proteasome activity in HL-60 cells.
- The observed inhibition is both time-dependent and concentration-dependent, supporting its potential as an anti-cancer therapeutic targeting the proteasome.
