Cancer Res Treat.  2017 Apr;49(2):358-373. 10.4143/crt.2016.017.

Inhibition of SKP2 Sensitizes Bromocriptine-Induced Apoptosis in Human Prolactinoma Cells

• Huang J
• Zhang F
• Jiang L
• Hu G
• Sun W
• Zhang C
• Ding X

• KMID: 2378108
• DOI: http://doi.org/10.4143/crt.2016.017

Abstract

PURPOSE
Prolactinoma (prolactin-secreting pituitary adenoma) is one of the most common estrogen-related functional pituitary tumors. As an agonist of the dopamine D2 receptor, bromocriptine is used widely to inhibit prolactinoma progression. On the other hand, it is not always effective in clinical application. Although a dopamine D2 receptor deficiency contributes to the impaired efficiency of bromocriptine therapy to some extent, it is unknown whether there some other underlying mechanisms leading to bromocriptine resistance in prolactinoma treatment. That is the main point addressed in this project.
MATERIALS AND METHODS
Human prolactinoma samples were used to analyze the S-phase kinase associated protein 2 (SKP2) expression level. Nutlin-3/adriamycin/cisplatin-treated GH3 and MMQ cells were used to analyze apoptosis in SKP2 overexpression or knockdown cells. SKP2 expression and the interaction partners of SKP2 were also detected after a bromocriptine treatment in 293T. Apoptosis was analyzed in C25 and bromocriptine-treated GH3 cells.
RESULTS
Compared to normal pituitary samples, most prolactinoma samples exhibit higher levels of SKP2 expression, which could inhibit apoptosis in a p53-dependent manner. In addition, the bromocriptine treatment prolonged the half-life of SKP2 and resulted in SKP2 overexpression to a greater extent, which in turn compromised its pro-apoptotic effect. As a result, the bromocriptine treatment combined with C25 (a SKP2 inhibitor) led to the maximal apoptosis of human prolactinoma cells.
CONCLUSION
These findings indicated that SKP2 inhibition sensitized the prolactinoma cells to bromocriptine and helped promote apoptosis. Moreover, a combined treatment of bromocriptine and C25 may contribute to the maximal apoptosis of human prolactinoma cells.

Keyword

Apoptosis; Bromocriptine; Prolactinoma; SKP2; C25

MeSH Terms

Apoptosis*
Bromocriptine
Half-Life
Hand
Humans*
Pituitary Neoplasms
Prolactinoma*
Receptors, Dopamine D2
S-Phase Kinase-Associated Proteins
Bromocriptine
Receptors, Dopamine D2
S-Phase Kinase-Associated Proteins

Figure

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  Fig. 1. SKP2 was up-regulated in pituitary prolactinoma. (A) Real-time RT-PCR analysis for SKP2 expression in normal and different types of adenomatous pituitary tissues as indicated. (B) Representative western blotting showing protein expression of the SKP2 in pituitary adenoma samples. Samples with the two highest SKP2 gene expressions in Fig. 1A of each group were selected for protein analysis. (C) Immunohistochemistry of PRL and normal pituitary adenoma samples to detect the level of SKP2. SKP2, S-phase kinase associated protein 2; RT-PCR, reverse transcription polymerase chain reaction; PRL, prolactin; GH, growth hormone; ACTH, adreno-cortico-tropic-hormone; NFPA, non-functioning pituitary adenoma.

• 

  Fig. 2. SKP2 overexpression suppresses apoptosis through p53 in GH3 cells. (A) GH3 cells infected with a lentiviral vector stably expressing the rat SKP2-inducible system. SKP2 was induced by 1 μg/mL doxycycline for 24 hours, and cells were then lysed and collected for immunoblotting analysis. (B, C). Twenty-four hours after inducing SKP2, the cells were treated with 5 μM nutlin-3 for 48 hours, and then lysed and collected either for immunoblotting analysis using the antibodies as indicated (B) or for quantitative real-time PCR analysis (C). (D) Twenty-four hours after inducing SKP2 expression, a reporter containing a synthetic p53-binding site (p53-luc) or reporter containing a mutation on the p53-binding site (p53MUT-luc) was transfected into the GH3 cells, together with a control reporter containing renilla luciferase. Twelve hours after transfection, the cells were treated with different concentrations of nutlin-3, as indicated for 48 hours, and the cells were then lysed and collected for luciferase activity analysis. (E) Twenty-four hours after SKP2 induction, the cells were treated with 10 μM nutlin-3 for 48 hours; apoptosis was determined and is shown by measuring the relative caspase 3/7 activity. SKP2, S-phase kinase associated protein 2; PCR, polymerase chain reaction; Dox, doxycycline. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

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  Fig. 3. Skp2 knockdown synergizes with DNA damage induction agents to promote apoptosis in PRL-secreting pituitary cells. (A, B) Twenty-four hours after doxycycline addition, the GH3 cells were treated with 1 μg/mL adriamycin for 48 hours followed by either immunoblotting analysis using the antibodies as indicated (A) or real-time PCR analysis (B). (C) Twenty-four hours after SKP2 induction, the cells were treated with 1 μg/mL adriamycin or 5 μg/mL cisplatin for 48 hours in GH3 cells, followed by relative caspase 3/7 activity determination. (D) A control or Skp2 siRNA was transfected into the wild-type GH3 cells and stable p53 knockdown GH3 cells. Forty-eight hours after transfection, the cells were lysed and collected to analyze SKP2 expression by immunoblotting. (E, F) Twenty-four hours after transfection of the control or Skp2 siRNA, the cells were treated with 1 μg/mL adriamycin or 5 μg/mL cisplatin for 48 hours in the GH3 cells or MMQ cells (E), or indicated adriamycin concentration in wild-type GH3 cells and stable p53 knockdown GH3 cells (F), followed by a determination of the relative caspase 3/7 activity. SKP2, S-phase kinase associated protein 2; PRL, prolactin; PCR, polymerase chain reaction; Dox, doxycycline; DMSO, dimethyl sulfoxide; si-CTR, control siRNA. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

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  Fig. 4. Bromocriptine stabilizes SKP2 expression by inhibiting SKP2 ubiquitination and degradation. (A, B) SKP2 expression in GH3 cells treated with or without 20 μM bromocriptine for 24 hours, the cells were collected and subjected to immunoblotting (A) or real-time PCR analysis (B). (C) The GH3 cells were transfected with Flag-SKP2. Twenty-four hours after transfection, the cells were treated with 10 μg/mL CHX and 20 μM bromocriptine for the indicated times. The cells were then lysed and subjected to immunoblotting analysis. The SKP2 levels from three independent assays were quantified by densitometry and the measurements were normalized to the start point (0 hour) to calculate the percentage changes following stimulation (C, lower panel). (D, E) the GH3 cells were transfected with Flag-SKP2 and Myc-Cul1 (D) or Flag-SKP2 and Myc-Cdh1 (E). Twenty-four hours after transfection, the cells were treated with 10 μM MG-132 and different concentration of bromocriptine, as indicated for 24 hours, and then cells were subjected to co-immunoprecipitation analysis. (F) The GH3 cells were transfected with Flag-SKP2 and HA-Ub. Twenty-four hours after transfection, the cells were treated with 10 μM MG-132 and 20 μM bromocriptine for 24 hours, and cells were then subjected to co-immunoprecipitation analysis. SKP2, S-phase kinase associated protein 2; CTR, control; PCR, polymerase chain reaction; CHX, cycloheximide; DMSO, dimethyl sulfoxide; HA-Ub, HA-ubiquitin. **p < 0.01, ***p < 0.001; ns, no significance.

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  Fig. 5. SKP2 inhibition sensitizes bromocriptine-induced apoptosis in GH3 cells. (A) GH3 cells were treated with 20 μM bromocriptine or 20 μM C25, as indicated for 48 hours. The cells were then lysed and collected for immunoblotting analysis using the indicated antibodies. (B) GH3 cells were treated with 20 μM C25 and different concentrations of bromocriptine, as indicated, for 48 hours. The cells were then lysed and collected for apoptosis determination by annexin-V staining and flow cytometry. (C) The GH3 cells were treated as the same in panel B, and cells were then subjected to determine the number of viable cells. (D) Twenty-four hours after SKP2 induction, the GH3 cells were subjected to 20 μM bromocriptine for 48 hours. The cell lysates were used for immunoblotting analysis using the indicated antibodies. (E) Twenty-four hours after SKP2 induction, the GH3 cells were exposed to the indicated bromocriptine concentrations for a further 48 hours, and cells were then lysed and collected for apoptosis determination by annexin-V staining and flow cytometry. (F) GH3 cells were treated as the same in panel F, and then subjected to a cell viability assay kit to determine the number of viable cells. SKP2, S-phase kinase associated protein 2; Cyt C, cytochrome C; PARP, poly(ADP-ribose) polymerase; DMSO, dimethyl sulfoxide; Dox, doxycycline. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 6. Schematic model. Schematic model showing how SKP2 overexpression compromised the bromocriptine-induced apoptosis by inhibiting the p53 pathway and the inhibition of SKP2 by C25 sensitizes the bromocriptine-induced apoptosis in PRL-secreting pituitary cells. SKP2, S-phase kinase associated protein 2; PRL, prolactin.

Reference

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