FTY720 Increases Doxorubicin Sensitivity in Resistant Acute Myeloid Leukemia Cells

Summary

This protocol evaluates how FTY720 increases doxorubicin sensitivity in resistant acute myeloid leukemia cells by assessing cell viability, apoptosis, intracellular doxorubicin accumulation, drug-efflux transporter expression, and protein kinase B/mechanistic target of rapamycin signaling.

Abstract

Chemoresistance remains a major barrier to effective treatment of acute myeloid leukemia, partly because resistant leukemic cells reduce intracellular drug exposure and evade apoptosis. FTY720, a sphingosine-1-phosphate receptor modulator, has reported antiproliferative and chemosensitizing activity in several tumor models. However, whether FTY720 increases doxorubicin sensitivity in doxorubicin-resistant acute myeloid leukemia cells, and whether this effect is associated with changes in drug-efflux transporters and protein kinase B/mechanistic target of rapamycin signaling, remains insufficiently defined. This study evaluated the effects of FTY720 alone and in combination with doxorubicin in parental acute myeloid leukemia cell lines (HL60 and K562) and their doxorubicin-resistant derivatives (HL60/A and K562/A). Cell viability was assessed using Cell Counting Kit-8 assays, apoptosis was measured by Annexin V/propidium iodide flow cytometry, intracellular doxorubicin accumulation was quantified by flow cytometry, and P-glycoprotein, multidrug resistance-associated protein 1, protein kinase B/mechanistic target of rapamycin pathway proteins, and apoptosis-related proteins were examined by flow cytometry and Western blotting. FTY720 showed dose-dependent cytotoxicity and increased doxorubicin sensitivity in resistant leukemia cells. Combined treatment reduced cell viability, increased apoptotic cell fractions, enhanced intracellular doxorubicin accumulation, and decreased P-glycoprotein and multidrug resistance-associated protein 1 expression. FTY720 treatment was also associated with reduced phosphorylation of protein kinase B and mechanistic target of rapamycin, whereas the protein kinase B activator SC79 partially reversed the observed changes in signaling, transporter expression, and apoptosis. These findings indicate that FTY720 increases doxorubicin sensitivity in HL60/A and K562/A cells and that this effect is associated with reduced protein kinase B/mechanistic target of rapamycin signaling, decreased drug-efflux transporter expression, enhanced intracellular doxorubicin accumulation, and increased apoptosis. Because this study was limited to in vitro cell-line models, further validation in primary leukemia samples and in vivo systems is required before therapeutic conclusions can be drawn.

Introduction

Acute myeloid leukemia is a heterogeneous hematological malignancy arising from the malignant transformation of hematopoietic stem and progenitor cells. It is the most common form of acute leukemia in adults and remains associated with poor long-term survival. Although treatment strategies have improved, induction chemotherapy based on cytarabine combined with an anthracycline remains a cornerstone of treatment. However, many patients develop primary or acquired resistance, leading to relapse, refractory disease, and poor clinical outcomes^1,2,3. Understanding the mechanisms t....

Protocol

This study was performed using established human acute myeloid leukemia cell lines and their doxorubicin-resistant derivatives. No human participants, animals, or primary patient-derived samples were involved. Therefore, institutional review board approval for human-subject research and animal ethics approval were not required. The cell lines were obtained from the Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China, and maintained at the Fujian Institute of Hematology, Fujian Medical University Union Hospital. Cell identity was confirmed by karyotype and morphological assessment prior to experimentation, and all cell cultures were tested for ....

Results

FTY720 reduced the viability of parental and doxorubicin-resistant acute myeloid leukemia cells

HL60, K562, HL60/A, and K562/A cells were exposed to increasing concentrations of FTY720 (0, 3.125, 6.25, 12.5, 25, 50, and 100 µM) for 48 h, and cell viability was measured using the Cell Counting Kit-8 assay. FTY720 reduced cell viability in all four cell lines in a concentration-dependent manner (Figure 1A). The calculated half-maximal inhibitory con.......

Discussion

Chemoresistance remains one of the major barriers to effective acute myeloid leukemia treatment. In patients who fail to respond to induction therapy or develop relapse after initial response, resistant leukemia cells can persist despite exposure to cytarabine- and anthracycline-based regimens, making the identification of approaches that increase drug sensitivity an important experimental and clinical question²³. Doxorubicin is an anthracycline with broad antitumor activity, but its clinical use .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well tissue culture plates	Corning / Costar	3516 or laboratory-verified equivalent	Used for treatment, apoptosis, doxorubicin accumulation, and protein-expression experiments.
96-well tissue culture plates	Corning / Costar	3599 or laboratory-verified equivalent	Used for Cell Counting Kit-8 assays.
Alexa Fluor 647 mouse immunoglobulin G1 isotype control	BioLegend	400130 or laboratory-verified equivalent	Isotype control for Alexa Fluor 647-conjugated antibody staining. Use the exact catalog number from the reagent vial.
Alexa Fluor 647-conjugated anti-human multidrug resistance-associated protein 1 / ABCC1 antibody	BioLegend	370103	Used for flow cytometric detection of multidrug resistance-associated protein 1. Product availability should be confirmed because some BioLegend MRP1 formats have been discontinued or replaced.
Annexin V apoptosis detection kit with propidium iodide	BioLegend	640914 or laboratory-verified equivalent	Used for Annexin V and propidium iodide apoptosis analysis by flow cytometry. If Annexin V-allophycocyanin was used instead of Annexin V-fluorescein isothiocyanate, replace this with the exact kit catalog number.
Bicinchoninic acid protein assay kit	Thermo Fisher Scientific / Beyotime / laboratory-verified supplier	Use the actual catalog number	Used for protein concentration measurement.
Carbon dioxide cell culture incubator	Thermo Fisher Scientific / Eppendorf / laboratory-verified instrument	Model to be inserted from laboratory record	Maintained cells at 37 °C and 5% carbon dioxide.
Caspase-3 antibody	Cell Signaling Technology	9662; RRID:AB_331439	Detects full-length and cleaved caspase-3 by Western blot. If a cleaved caspase-3-specific antibody was used, replace with the exact catalog number.
Cell Counting Kit-8	Dojindo Molecular Technologies	CK04	Used for cell viability and half-maximal inhibitory concentration assays.
Cell staining buffer	BioLegend	420201	Used for antibody dilution and washing during flow cytometric staining.
Cleaved poly(adenosine diphosphate-ribose) polymerase antibody	Cell Signaling Technology	94885; RRID:AB_2800237	Western blot antibody for cleaved poly(adenosine diphosphate-ribose) polymerase.
CompuSyn	ComboSyn, Inc.	Version 1.0 or laboratory-verified version	Used for Chou-Talalay combination-index analysis if complete combination-dose data are available.
Dimethyl sulfoxide	Sigma-Aldrich / Merck	D2650 or laboratory-verified equivalent	Vehicle for FTY720 and SC79 stock solutions; final concentration did not exceed 0.1%.
Doxorubicin hydrochloride	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Anthracycline used for resistance maintenance and treatment assays. This entry must match the reagent actually used in the experiments.
Enhanced chemiluminescence substrate	Thermo Fisher Scientific / MilliporeSigma / laboratory-verified supplier	Use the actual catalog number	Used for Western blot signal detection.
Fetal bovine serum	Gibco, Thermo Fisher Scientific	10099-141 or laboratory-verified equivalent	Added to culture medium at 10%. Use heat-inactivated or non-heat-inactivated serum consistently across all experiments.
Flow cytometer	BD Biosciences	BD FACScan or actual instrument model	Used for apoptosis, intracellular doxorubicin accumulation, and transporter-expression assays. Record laser/filter configuration in the supplementary methods if available.
Flow cytometry analysis software	BD Biosciences / FlowJo / laboratory-verified software	Version to be inserted from laboratory record	Used for compensation, gating, and median fluorescence intensity analysis.
FTY720 / fingolimod hydrochloride	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Drug-treatment reagent. This entry must match the reagent actually used in the experiments. Do not replace with a different supplier unless the experiment was performed with that product.
Glyceraldehyde-3-phosphate dehydrogenase antibody	Abcam	ab181602	Loading control for Western blot. Confirm RRID from the product insert before submission.
GraphPad Prism	GraphPad Software	Version 9.5	Used for dose-response curve fitting, half-maximal inhibitory concentration calculation, and statistical analysis.
HL60 human acute myeloid leukemia cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued in this study	Human acute myeloid leukemia parental cell line; authenticated by karyotype and morphology before use. If a public reference is required, HL60 corresponds to RRID:CVCL_0002.
HL60/A doxorubicin-resistant cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued	Doxorubicin-resistant derivative of HL60 cells; maintained at the Fujian Institute of Hematology. Resistance was maintained with 1 µg/mL doxorubicin before drug-free culture.
Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody	Cell Signaling Technology / Abcam / laboratory-verified supplier	Use the actual catalog number	Secondary antibody for Western blot. Insert exact supplier and catalog number from laboratory records.
IBM SPSS Statistics	IBM	Version 23.0	Used for statistical analysis.
ImageJ	National Institutes of Health	Version 1.53t	Used for Western blot densitometric quantification.
K562 human leukemia cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued in this study	Human leukemia parental cell line; authenticated by karyotype and morphology before use. If a public reference is required, K562 corresponds to RRID:CVCL_0004.
K562/A doxorubicin-resistant cells	Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China	Not commercially catalogued	Doxorubicin-resistant derivative of K562 cells; maintained at the Fujian Institute of Hematology. Resistance was maintained with 5 µg/mL doxorubicin before drug-free culture.
Microplate reader	BioTek / Thermo Fisher Scientific / Molecular Devices / laboratory-verified instrument	Model to be inserted from laboratory record	Used to measure absorbance at 450 nm for Cell Counting Kit-8 assays.
Multidrug resistance-associated protein 1 antibody for Western blot	Cell Signaling Technology	72202	Used for Western blot detection of multidrug resistance-associated protein 1. Confirm RRID from the product insert or Antibody Registry before submission.
Penicillin-streptomycin solution	Gibco, Thermo Fisher Scientific	15140122 or laboratory-verified equivalent	Added to complete medium at 1%.
P-glycoprotein / MDR1 / ABCB1 antibody for Western blot	Cell Signaling Technology	13978; RRID:AB_2798357	Used for Western blot detection of P-glycoprotein.
Phosphate-buffered saline	Gibco, Thermo Fisher Scientific	10010023 or laboratory-verified equivalent	Used for cell washing and sample preparation.
Phosphorylated mechanistic target of rapamycin antibody	Cell Signaling Technology	2971; RRID:AB_330970	Western blot antibody for phosphorylated mechanistic target of rapamycin at Ser2448.
Phosphorylated protein kinase B antibody	Cell Signaling Technology	4060; RRID:AB_2315049	Western blot antibody for phosphorylated protein kinase B at Ser473.
Phycoerythrin mouse immunoglobulin G2a isotype control	BioLegend	400213	Isotype control for phycoerythrin-conjugated antibody staining. Confirm the isotype matches the anti-P-glycoprotein antibody used.
Phycoerythrin-conjugated anti-human P-glycoprotein / CD243 / ABCB1 antibody	BioLegend	919405	Used for flow cytometric detection of P-glycoprotein. Include clone and RRID if confirmed from the product insert.
Polyvinylidene difluoride membrane	MilliporeSigma	IPVH00010 or laboratory-verified equivalent	Used for Western blot transfer.
Protease and phosphatase inhibitor cocktail	Thermo Fisher Scientific / Roche / laboratory-verified supplier	Use the actual catalog number	Added to lysis buffer before protein extraction.
Radioimmunoprecipitation assay lysis buffer	Beyotime / Thermo Fisher Scientific / laboratory-verified supplier	Use the actual catalog number	Used for total protein extraction.
Roswell Park Memorial Institute 1640 medium	HyClone / Cytiva	SH30809.01 or laboratory-verified equivalent	Basal culture medium for all leukemia cell lines. Use the exact catalog number from the laboratory purchase record if different.
SC79, protein kinase B activator	Use the actual supplier from the laboratory purchase record	Use the actual catalog number and lot number	Used in rescue experiments. The manuscript should consistently report the same final SC79 concentration across Protocol, Results, and Figure 6 legend.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reagents	Bio-Rad / Beyotime / laboratory-verified supplier	Use the actual catalog number	Used for Western blot protein separation.
Total mechanistic target of rapamycin antibody	Cell Signaling Technology	2972; RRID:AB_330978	Western blot antibody for total mechanistic target of rapamycin.
Total protein kinase B antibody	Cell Signaling Technology	4691; RRID:AB_915783	Western blot antibody for total protein kinase B.
Western blot imaging system	Bio-Rad / Cytiva / laboratory-verified instrument	Model to be inserted from laboratory record	Used for enhanced chemiluminescence image acquisition.