Mesenchymal stem cell-induced doxorubicin resistance in triple negative breast cancer

Abstract

Triple negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and a worse clinical outcome compared with other breast cancer subtypes. Doxorubicin is considered to be one of the most effective agents in the treatment of TNBC. Unfortunately, resistance to this agent is common. In some drug-resistant cells, drug efflux is mediated by adenosine triphosphate-dependent membrane transporter termed adenosine triphosphate-binding cassette (ABC) transporter, which can drive the substrates across membranes against concentration gradient. In the tumor microenvironment, upon interaction with mesenchymal stem cells (MSCs), tumor cells exhibit altered biological functions of certain gene clusters, hence increasing stemness of tumor cells, migration ability, angiogenesis, and drug resistance. In our present study, we investigated the mechanism of TNBC drug resistance induced by adipose-derived MSCs. Upon exposure of TNBC to MSC-secreted conditioned medium (CM), noticeable drug resistance against doxorubicin with markedly increased BCRP protein expression was observed. Intracellular doxorubicin accumulation of TNBC was also decreased by MSC-secreted CM. Furthermore, we found that doxorubicin resistance of TNBC was mediated by IL-8 presented in the MSC-secreted CM. These findings may enrich the list of potential targets for overcoming drug resistance induced by MSCs in TNBC patients.

Figure 1

Adipose-derived mesenchymal stem cells-secreted conditioned medium induced doxorubicin resistance in MDA-MB-231 cells. Cells were treated by 200 nM doxorubicin with L15 (a) or MSCM medium (b) in a time-dependent manner and cell viability was examined by performing crystal violet staining. (c) Cells were treated by 200 nM doxorubicin with MSC-ad conditioned medium for 24 hours and cell viability was also examined by performing crystal violet staining. Graphs showed mean ± SEM of three independent experiments. *P < 0.05 to doxorubicin-untreated group; ^# P < 0.05 to doxorubicin in MSCM group. CM, conditioned medium from adipose-derived mesenchymal stem cells (MSC-ad); MSCM, mesenchymal stem cell medium (fresh MSC culture medium).

Figure 2

Adipose-derived mesenchymal stem cells-secreted conditioned medium increased BCRP protein expression in MDA-MB-231 cells. Cells were treated with L15 (control medium), MSCM, or MSC-ad conditioned medium for 24 hours and protein expression of (a) P-gp, (b) MRP, and (c) BCRP was examined by Western blotting. Graphs showed mean ± SEM of three independent experiments. *P < 0.05 to MSCM group. CM, conditioned medium from adipose-derived mesenchymal stem cells (MSC-ad); MSCM, mesenchymal stem cell medium (fresh MSC culture medium).

Figure 3

Adipose-derived mesenchymal stem cells-secreted conditioned medium decreased intracellular doxorubicin accumulation in MDA-MB-231 cells. Intracellular doxorubicin accumulation was measured by intensity of doxorubicin fluorescence using flow cytometry. (a) Cells were treated with different concentrations of doxorubicin for 1 hour without wash period. (b) Cells were treated with 2 μM doxorubicin for 1 hour and then washed for 1, 2, or 4 hour(s). (c) Cells were treated with 2 μM doxorubicin for 1 hour with or without conditioned medium and then washed for 4 hours. When using Ko143 as BCRP specific inhibitor, Ko143 was present during both doxorubicin-treated period and wash period. Each histogram image was a representative from three independent experiments (n = 3). Doxo, doxorubicin; CM, conditioned medium from adipose-derived mesenchymal stem cells (MSC-ad).

Figure 4

Cytokine expression of adipose-derived mesenchymal stem cells-secreted conditioned medium. Analysis of human cytokine expression of (a) MSCM (fully supplemented MSC culture medium) only and (b) MSC-ad conditioned medium by human cytokine array. R, reference spot; 1, IL-6; 2, IL-8; 3, Serpin E1.

Figure 5

IL-8 induced BCRP protein expression in MDA-MB-231 cells. (a) Human recombinant IL-8 dose-dependently elevated BCRP protein expression after 24 hours of examination by Western blotting. (b) IL-8 neutralizing antibody (5 μg/mL) antagonized MSC-ad conditioned medium-induced BCRP protein expression. IgG isotype control antibody was used as negative control. Graphs showed mean ± SEM of three independent experiments. *P < 0.05 to control group; ^# P < 0.05 to CM-treated group. CM, conditioned medium from adipose-derived mesenchymal stem cells (MSC-ad); NeuAb, IL-8 neutralizing antibody.

Figure 6

IL-8 induced drug resistance against doxorubicin in MDA-MB-231 cells. (a) Cells were treated by 200 nM doxorubicin for 24 hours with or without pretreatment of human recombinant IL-8 (50 or 100 ng/mL) for 24 hours and cell viability was examined by performing crystal violet staining. *P < 0.05 to control group; ^# P < 0.05 to doxorubicin-treated group. (b) IL-8 neutralizing antibody (5 μg/mL) antagonized MSC-ad conditioned medium-induced doxorubicin (200 nM) resistance. IgG isotype control antibody was used as negative control. *P < 0.05 to MSCM with doxorubicin-treated group; ^# P < 0.05 to CM with doxorubicin-treated group. Graphs showed mean ± SEM of three independent experiments. Doxo, doxorubicin; MSCM, mesenchymal stem cell medium (fresh MSC culture medium); CM, conditioned medium from adipose-derived mesenchymal stem cells (MSC-ad); NeuAb, IL-8 neutralizing antibody.