Combination of coumarin and doxorubicin induces drug-resistant acute myeloid leukemia cell death

Abstract

Background: Chemotherapy remains to be the method of choice used by clinicians to treat acute myeloid leukemia (AML) patients. However, the most common problem usually faced in the course of treatment is multidrug resistance (MDR). Nowadays, combination therapy involving natural products as adjuvant therapy to chemotherapy and radiotherapy has been used for many of health problems. Coumarin is a natural compound with known chemotherapeutic activity, as well as other pharmacological properties. We focused on the combination of coumarin and doxorubicin in overcoming of drug-resistance in acute myeloid leukemia.

Methods: Cell viability, Apoptotic and necrotic cell death with FACS, oxidative stress detection, and protein expression analysis were used in this study.

Results: Coumarin as a single drug exerts a significant cell death on Human acute myeloid leukemia (HL60); however, it does not show the same effect on drug-resistant acute myeloid leukemia (HL60/ADR). Comparing the effects of doxorubicin and coumarin as single drugs versus a combination of coumarin and doxorubicin showed a significant apoptotic cell death.

Conclusion: In AML patients, the development of multiple drug resistance (MDR) is the biggest challenge in treating AML patients. Combination therapy with coumarin may be a good choice to overcome the drug resistance in AML patients.

Keywords: Anticancer drugs; Apoptosis; Cell viability; FACS assay; Natural medicine.

Figure 1

Western bloting of Cas-3, P53, PARP and Actin in HL60/ADR after co-treatment of 0.1 μg/ml of doxirubcin with (100, 250, 500, and 1000 μg/ml) coumarin) and (B-E) a quantification of western blot data using Image J softrware.

Figure 2

(A) cytotoxic effect of coumarin and doxrubcin treatment on HL60 cells using trypane blue exclusion dye. Apoptotic and necrotic cell death were assessed in HL60 using Annexin V and PI staining and analyzed using flow cytometer in ( B) negative control, (C) treated with 0.1 μg/ml of doxirubcin as a positive control, and (D -G) treated with (100, 250, 500, and 1000 μg/ml) coumarin.

Figure 3

Cell viability was assessed in HL60/ADR cells using trypane blue exclusion dye; (A) treated with different coumarin concentrations and (B) co-treated with 100 ng/ml doxrubcin with different coumarin concentrations.

Figure 4

Apoptotic and necrotic cell death were assessed in HL60/ADR cells using Annexin V and PI staining and analyzed using flow cytometer in ( B) (negative control), (C-F) HL60/ADR cells treated with (100, 250, 500, and 1000 μg/ml) coumarin , and (G-J) combination of 0.1 μg/ml of doxirubcin with (100, 250, 500, and 1000 μg/ml) coumarin and (K) quantification of Flowcytometry results.

Figure 5

Confocal imaging of ROS in HL60/ADR after treatment with; (A) negative control, (B) 0.1 μg/ml of doxirubcin, (C) 500 μM Pyocyanin, (D-G) treated with (100, 250, 500, and 1000 μg/ml) coumarin, and (H-K) combination of 0.1 μg/ml of doxirubcin with (100, 250, 500, and 1000 μg/ml) coumarin and (L) quantification of the intensity of fluorescence using imge J software.