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. 2009 Aug;30(8):1298-304.
doi: 10.1093/carcin/bgp120. Epub 2009 May 14.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase

Elizabeth W LaPensee  1 , Sandy J Schwemberger, Christopher R LaPensee, El Mustapha Bahassi, Scott E Afton, Nira Ben-Jonathan
Affiliations

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase

Elizabeth W LaPensee et al. Carcinogenesis. 2009 Aug.

Abstract

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

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Figures

Fig. 1.
Fig. 1.
PRL protects breast cancer cells from chemotherapeutic agents. In all panels, cells were treated with the drug alone for 4 days (filled triangles marker) or pretreated with PRL (25 ng/ml) for 24 h (filled squares marker) before exposure to the drug for 4 days. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl tetrazolium bromide assay. (A) MDA-MB-468 cells were treated with increasing concentrations of taxol, vinblastine or doxorubicin. (B–C) MDA-MB-468 or T47D cells were treated with cisplatin alone or pretreated with 25 ng/ml PRL followed by cisplatin. (D) MDA-MB-468 cells were treated for 24 h with increasing concentrations of PRL before treatment with 100 ng/ml cisplatin for 4 days. Each value is a mean ± SEM of six replicates. In panels A–C, * designates significant differences (P < 0.05) between PRL treatment and drug alone. In panel D, * designates significant difference compared with control, and ** designates significant difference compared with cisplatin.
Fig. 2.
Fig. 2.
PRL protects MDA-MB-468 cells from cisplatin cytotoxicity via Jak-Stat and MAPK-signaling pathways. (A) Cells were treated with PRL (100 ng/ml) for 0–240 min. Cell lysates were analyzed by western blotting, using antibodies against phospho-ERK1/2 (p-ERK1/2), ERK1/2, phospho-Stat5 (p-Stat5), Stat5 or phospho-Akt (p-Akt) and Akt. Shown are representative blots, repeated three times. (B) Cells were treated with AG490 (10 μM), UO126 (10 μM) or wortmannin (Wort; 250 nM) for 1 h before addition of 100 ng/ml PRL for 24 h. The next day, cisplatin (800 ng/ml) was added for 24 h. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl tetrazolium bromide assay. Each value is a mean ± SEM of six replicates. * designates significant difference (P < 0.05) compared with control and ** designates significant difference compared with cisplatin.
Fig. 3.
Fig. 3.
PRL overrides cisplatin-induced G2/M cell cycle arrest and apoptosis. (A) MDA-MB-468 cells were treated with 100 ng/ml PRL for 24 h followed by 200 ng/ml cisplatin (cis) for 24 h. After staining with PI, fluorescence was analyzed by flow cytometry. The percentages of cells in G0/G1, S and G2/M phases are displayed for each histogram. Shown are representative histograms, repeated three times. (B) Cells were treated as in (A) and incubated with anti-phospho-histone H3 antibody and 4′,6-diamidino-2-phenylindole and photographed. (C) The number of mitotic cells (histone H3 positive) in (C) was normalized to the total number of cells in the population. Con-control; Cis-cisplatin. (D) Cells were treated with 100 ng/ml PRL for 24 h, followed by cisplatin (200 ng/ml) for 24 h. Following staining with Annexin V and PI, cells were analyzed by flow cytometry. Shown are representative histograms, repeated three times. (E) Table depicting the percentage of cells in each treatment (Tx) group that are live (no staining), in early apoptosis (Annexin V positive), in late apoptosis/necrosis (Annexin V + PI positive) or dead (PI positive). Pretreatment with PRL antagonized cisplatin by suppressing the number of cells undergoing early and late apoptosis.
Fig. 4.
Fig. 4.
PRL reduces cisplatin binding to DNA. (A) MDA-MB-468 cells were treated with 100 ng/ml PRL for 24 h followed by 200 ng/ml cisplatin (cis) for 72 h. Following incubation with anti-phospho-H2AX antibody and 4′,6-diamidino-2-phenylindole, cells were photographed. Pretreatment with PRL markedly reduced the number of cells with double-strand breaks caused by cisplatin alone. (B) Cells were treated with PRL (100 ng/ml) for 24 h followed by 800 ng/ml cisplatin for 8 or 24 h. DNA was isolated and platinum (Pt) content was analyzed by inductively coupled plasma mass spectroscopy. * designates significant difference (P < 0.05) compared with the corresponding cisplatin treatment.
Fig. 5.
Fig. 5.
PRL antagonizes cisplatin cytotoxicity by increasing GST activity. (A) MDA-MB-468 cells were treated with probenecid (10 μM) or ethacrynic acid (10 μM) for 1 h before PRL (100 ng/ml) for 24 h. This was followed by treatment with 800 ng/ml cisplatin for 24 h. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl tetrazolium bromide assay. Each value is a mean ± SEM of six replicates. * designates significant difference (P < 0.05) compared with control, and ** designates significant difference from cisplatin treatment. (B) Cells were treated with AG490 (10 μM) or U0126 (10 μM) for 1 h before PRL (100 ng/ml) for 24 h. GST activity was determined spectrophotometrically by measuring the rate of 1-chloro-2,4,dinitrobenzene conjugation to glutathione. * designates significant difference from control. (C) Cells were treated with ethacrynic acid (EA; 10 μM) for 1 h before addition of PRL (100 ng/ml) for 24 h. Cisplatin (800 ng/ml) was then added for 24 h. DNA was isolated and platinum (Pt) content was analyzed by inductively coupled plasma mass spectroscopy. * designates significant difference compared with control and ** designates significant difference from cisplatin + PRL treatment. (D and E) Cells were treated with 100 ng/ml PRL for 0, 24 or 48 h. Expression of GSTμ and GSTπ were determined by real-time polymerase chain reaction. Values were calculated as fold change over time 0 and represent an average of three separate experiments.
Fig. 6.
Fig. 6.
Model depicting the proposed mechanism by which PRL antagonizes cisplatin-induced apoptosis. Cisplatin (Pt) enters the cell via passive diffusion and enters the nucleus, where it binds to DNA and induces apoptosis. Binding of PRL to its receptor results in its dimerization and association of Jak2 and Shc, and the subsequent signaling via Stat5a/b and ERK1/2 pathways. It is through either, or both, of these signaling pathways that PRL increases transcription of the detoxification enzyme GST. Increased GST activity promotes conjugation of glutathione (GSH) to cisplatin, followed by extrusion of the conjugate from the cell via transporters. Consequently, less cisplatin is available for entry into the nucleus, resulting in decreased apoptosis.
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