用于监测蒽环类药物进入哺乳动物细胞的摄取分析

Nicolas Brosseau; Emil Andreev; Dindial Ramotar

doi:10.21769/BioProtoc.2555

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Peer-reviewed

Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells

用于监测蒽环类药物进入哺乳动物细胞的摄取分析

NB Nicolas Brosseau

EA Emil Andreev

Dindial Ramotar email

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2555 浏览次数: 7103

评审: Nicoletta CordaniAnonymous reviewer(s)

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Abstract

Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.

Keywords: Influx transporters (流入转运蛋白)

Anthracyclines (蒽环类药物)

Autofluorescent drugs (自体荧光药)

FACS analysis (FACS分析)

Epifluorescence microscopy (荧光显微镜检查)

Fluoroskan reader (Fluoroskan读数仪)

Background

The anthracyclines, such as doxorubicin and daunorubicin, act by damaging the DNA and are used for treating various types of cancers including acute myeloid leukemia. When cancer patients are given anthracyclines systemically, there are several factors limiting the amount of the drugs that reach the tumor sites (Chauncey, 2001; Deng et al., 2014; Riganti et al., 2015). In the tumor, the drug activity on the cancer cells is limited by poor drug uptake, excessive drug efflux as well as changes in the cellular targets. For several decades, it remains unclear how these DNA damaging agents enter cancer cells (Aouida et al., 2010; Cesar-Razquin et al., 2015; Zhang et al., 2015). To address this question, we developed three reliable in vitro assays to monitor daunorubicin accumulation into cells. Two of these assays are quantitative and required access to a Fluorescence-Activated Cell Sorting (FACS) caliber and a Fluoroskan instrumentation and the third is semi-quantitative using epifluorescence microscopy. Using these assays, we established that daunorubicin enter into cells in a time- and concentration-dependent manner and that each cell type showed a different rate of uptake, suggesting that an active process is involved in the uptake of anthracyclines (Andreev et al., 2016). We applied these assays and uncovered the organic cation transporter 1 (OCT1) as a key protein for the uptake of daunorubicin into the cells (Andreev et al., 2016). Modulating the level of OCT1 resulting in cells with altered uptake and sensitivity towards daunorubicin (Andreev et al., 2016). These assays provide hints that additional transporters exist to allow uptake of daunorubicin into the cells. We believe that these uptake assays can be exploited further to identify additional factors such as kinases (Tanaka et al., 2004; Ciarimboli and Schlatter, 2005; Zhou et al., 2005; Pelis et al., 2006; Filippo et al., 2011; Sprowl et al., 2016) that influence the rate of daunorubicin uptake. In this protocol, we describe three analyses to monitor the uptake of anthracyclines into cells.

Materials and Reagents

Mammalian cell culture containers for adherent cells (100 and 60 mm Petri dishes and T-75 flask)
100 mm Petri dishes (SARSTEDT, catalog number: 83.3902 )
60 mm Petri dishes (SARSTEDT, catalog number: 83.3901 )
T-75 flask (Corning, catalog number: 430641 )
Notes:
1. However, any other Petri dishes or flask suitable for your cell line may work.
2. Note that you can grow suspension cells in containers for adherent cells.
Eppendorf tubes
FACS tubes that will adapt to the flow cytometer:
Falcon 5 ml polystyrene round-bottom tube (Corning, Falcon^®, catalog number: 352058 )
Microscope slides (UltiDent Scientific, catalog number: 170-7107A )
Note: Any microscope slide that fit on your microscope can be used.
Cover glass (UltiDent Scientific, catalog number: 170-C1818 )
Note: Any cover glass can be used. The best cover glasses are the #1.5, however #1.0 or #0 may be used if high quality images are not required (e.g., confocal microscopy).
Black clear flat bottom 96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 165305 )
Mammalian adherent cells, for example, HeLa, HEK293 and TOV112D
Note: Suspension cells such as HL60 and K562 can be used, although the protocol would need to be adjusted.
Required complete culture media
1. DMEM (WISENT, catalog number: 319-005-EL )
2. 1x RPMI 1640 (WISENT, catalog number: 350-000-CL )
3. Ovarian Surface Epithelial (OSE) (WISENT, catalog number: 316-030-CL )
Note: We used the above growth media depending on the cell line. Verify with the cell line suppliers to determine the optimal growth medium for the cells and the necessary supplements.
1. Fetal bovine serum (FBS) (WISENT, catalog number: 095150 )
  Note: It is usually used at 10%, but some cell lines will require different concentrations. See with your cell line supplier.
2. Penicillin/streptomycin (WISENT, catalog number: 450-201-EL )
  Note: It is usually supplied as a 100x stock and therefore must be diluted 1:100 in DMEM and RPMI1640.
3. Gentamycin Sulfate 50 mg/ml Solution (WISENT, catalog number: 450-135-XL ) supplied at 1,000x and must be diluted 1:1,000 in the media
4. Amphotericin B (Sigma-Aldrich, catalog number: A4888 ) used at 0.5 μg/ml in OSE
  Note: We usually prepare a stock solution of 250 μg/ml and add 1 ml of to 500 ml of OSE.
Trypsin (0.05% or 0.25% in PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200072 )
Daunorubicin
Notes:
1. Provided by the pharmacy (Maisonneuve-Rosemont Hospital), but can be purchased from Sigma (Sigma-Aldrich, catalog number: 30450 ). Prepared at 5 mg/ml in sterile water to a final molar concentration of 8.87 mM.
2. Daunorubicin may be replaced by doxorubicin (Sigma-Aldrich, catalog number: D1515 ).
4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 ) solution in PBS
Mounting medium with DAPI (Vectashield, Vector Laboratories, catalog number: H-1200 )
Note: This component may be replaced by 50% glycerol (Bio Basic, catalog number: GB0232 ) containing 1 to 10 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: H1399 ).
Nail polish
Phosphate buffer saline (PBS) (see Recipes)
Sodium chloride (NaCl) (WISENT, catalog number: 600-082-IK )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
Sodium phosphate dibasic (Na₂HPO₄) (Bio Basic, catalog number: S0404 )
Potassium phosphate monobasic (KH₂PO₄) (Bio Basic, catalog number: PB0445 )
Uptake buffer (see Recipes)
Sodium chloride (NaCl) (WISENT, catalog number: 600-082-IK )
HEPES pH 7.4 (Bio Basic, catalog number: HB0265 )
Glucose (WISENT, catalog number: 600-350-IK )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
Potassium phosphate monobasic (KH₂PO₄) (Bio Basic, catalog number: PB0445 )
Calcium chloride (CaCl₂) (Fisher Scientific, catalog number: C79-500 )
Magnesium sulfate (MgSO₄) (Bio Basic, catalog number: MN1988 )

Equipment

Pipettes (any pipettes will do)
Incubator (any incubator is fine if it maintains 5% CO₂ and moisture)
pH meter (any pH meter will work)
Eppendorf centrifuge (any centrifuge that can adapt 1.5 ml microcentrifuge tube)
Hemocytometer (any hemocytometer will do)
Flow cytometer (FACS)
Note: This protocol is using a FACS Calibur from Becton-Dickson (BD, model: FACS Calibur^TM ).
Epifluorescence microscope (Olympus, model: BX53 )
Note: We use an Olympus BX53 , but any epifluorescence microscope with the required filter sets may be used (see in Procedure D for the required filters).
Fluoroskan Ascent (Thermo Fisher Scientific, Thermo Scientific^TM, model: Fluoroskan Ascent^TM )

Software

ImageJ
Note: We used the one at the National institute of Health website; https://imagej.nih.gov/ij/.

Procedure

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Brosseau, N., Andreev, E. and Ramotar, D. (2017). Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells. Bio-protocol 7(18): e2555. DOI: 10.21769/BioProtoc.2555.