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[The effect of arsenic trioxide (As2O3) combined with BSO on K562/ADM cell and its mechanisms]
Tao Wang1, Liang-Ming Ma, Hua-Ping Zhang
1Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, China.
Objective:
To investigate the apoptosis-induction, P-glycoprotein (P-gp) and mdr1 mRNA inhibition effects of arsenic trioxide (As2O3) and buthionine sulfoximine (BSO) on multidrug-resistant cell line K562/ADM cells, and to determine the relationship between intracellular GSH content and arsenic effect.
Methods:
K562/ADM cells were treated with arsenic (0.5, 2.0, 5.0 micromol/L) alone or combined with BSO (100 micromol/L). The cell proliferating capacity was assessed with MTT assay, and cell apoptosis by Annexin V and propidium iodide (PI) staining. Intracellular GSH contents were measured using a glutathione assay kit by spectrophotometry. P-gp expression was determined by flow cytometry, and mdr1 mRNA expression by semi-quantitative RT-PCR.
Results:
The GSH contents in K562/ADM cell was (81.13 +/- 3.91) mg/g protein. After the GSH contents were degraded by BSO, the K562/ADM cell proliferating capacity was obviously inhibited and the cells were induced apoptosis in 24 hours by the combination of clinic dose arsenic group (0.5, 2.0 micromol/L) and BSO (100 micromol/L). The cell apoptosis rates at 48 hours in arsenic alone group and combination group were (59.29 +/- 6.01)% and (65.06 +/- 8.29)%, and at 72 hours were (82.15 +/- 9.28)% and (92.72 +/- 9.41)% retrospectively. At 48 hours, the mdr1 mRNA inhibition effect of the combination group was obviously stronger than that of high dose arsenic alone group. At 72 hours, the P-gp inhibition effect of the combination group (clinic dose arsenic group, 0.5, 2.0 micromol/L) was obviously stronger than that of high dose arsenic alone group (5.0 micromol/L).
Conclusion:
The intracellular GSH contents are closely correlated with the arsenic effect. The combination of conventional dose arsenic and BSO significantly induces K562/ADM cell apoptosis and inhibits P-gp and mdr1 mRNA expression in the cells.
Insights
Combining arsenic trioxide (As2O3) with buthionine sulfoximine (BSO) effectively induces apoptosis in multidrug-resistant K562/ADM cells. This combination also inhibits P-glycoprotein (P-gp) and mdr1 mRNA, overcoming drug resistance by targeting intracellular glutathione levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer is a significant clinical challenge, often mediated by efflux pumps like P-glycoprotein (P-gp).
- Intracellular glutathione (GSH) plays a crucial role in cellular defense mechanisms and can contribute to drug resistance by detoxifying chemotherapeutic agents.
- Arsenic trioxide (As2O3) has shown potential in cancer therapy, but its efficacy can be limited by MDR.
Purpose of the Study:
- To investigate the combined effects of As2O3 and buthionine sulfoximine (BSO) on apoptosis induction in K562/ADM cells.
- To assess the impact of this combination on P-glycoprotein (P-gp) and mdr1 mRNA expression.
- To determine the relationship between intracellular GSH levels and the therapeutic effect of As2O3.
Main Methods:
- K562/ADM cells were treated with varying concentrations of As2O3 alone or in combination with BSO.
- Cell proliferation was assessed using MTT assay.
- Apoptosis was quantified by Annexin V and propidium iodide (PI) staining.
- Intracellular GSH content was measured spectrophotometrically.
- P-gp expression was analyzed by flow cytometry, and mdr1 mRNA levels by RT-PCR.
Main Results:
- BSO treatment significantly reduced intracellular GSH content in K562/ADM cells.
- The combination of As2O3 and BSO markedly enhanced apoptosis induction compared to As2O3 alone.
- Combined treatment showed superior inhibition of mdr1 mRNA and P-gp expression at 48 and 72 hours, respectively, compared to high-dose As2O3 monotherapy.
- Apoptosis rates increased significantly with combination therapy, reaching over 92% at 72 hours.
Conclusions:
- Intracellular GSH levels are closely correlated with the efficacy of As2O3 in K562/ADM cells.
- The combination of conventional doses of As2O3 and BSO effectively induces apoptosis and overcomes MDR.
- This synergistic approach inhibits key mediators of drug resistance, including P-gp and mdr1 mRNA.
