Suppression of mTOR pathway and induction of autophagy-dependent cell death by cabergoline

Abstract

Cabergoline (CAB), the first-line drug for treatment of prolactinomas, is effective in suppressing prolactin hypersecretion, reducing tumor size, and restoring gonadal function. However, mechanisms for CAB-mediated tumor shrinkage are largely unknown. Here we report a novel cytotoxic mechanism for CAB. CAB induced formation of autophagosome in rat pituitary tumor MMQ and GH3 cells at the early stage through inhibiting mTOR pathway, resulting in higher conversion rates of LC3-I to LC3-II, GFP-LC3 aggregation, and increased autophagosome formation. Interestingly, CAB treatment augmented lysosome acidification and resulted in impaired proteolytic degradation within autolysosomes. This blocked the autophagic flux, leading to the accumulation of p62 aggregation and undigested autolysosomes. Knockdown of ATG7, ATG5, or Becn1, could significantly rescue the CAB-mediated cell death of MMQ cells (p < 0.05). CAB-induced autophagy and blockade of autophagy flux participated in antitumoral action in vivo. In conclusion, our study provides evidence that CAB concomitantly induces autophagy and inhibits the autophagic flux, leading to autophagy-dependent cell death. These findings elucidate novel mechanisms for CAB action.

Keywords: autophagic cell death; autophagic flux; autophagy; cabergoline; prolactinoma.

Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1. CAB induces both apoptosis and non-apoptosis cell death

A–B. Cell survival was determined by the MTS assay. C. MMQ and GH3 cells were treated with CAB as indicated for apoptosis assay by Annexin V-FTIC and PI double staining. D–E. MMQ and GH3 cells were treated with CAB as indicated, and total proteins analyzed by Western blot using antibodies against caspase-3, PARP and GAPDH. F. MMQ and GH3 cells were treated with CAB in the presence or absence of Z-VAD-FMK at different concentrations for 48 hours, and cell survival was determined by the MTS. G. Transmission electron microscopy (TEM) images with enlargements; boxes showed autophagic vacuoles in MMQ cells treated with CAB for 48 h, and a cell death morphology accompanied by large-scale autophagic vacuoles in the cytoplasm with the absence of chromatin condensation.

Figure 2. CAB suppresses the AKT/mTOR pathway

A. CAB-treated GH3 and MMQ cellular lysates were blotted with antibodies recognizing p-mTOR (Ser 2448), mTOR, p-4EBP1 (Thr37/46), 4EBP1, p-p70s6k(Thr389) and p70s6k. A mouse anti-GAPDH antibody was utilized as a loading control. Blots are representative of three independent experiments. B. Immunoblots analysis of p-mTOR, p-4EBP1 and GAPDH in MMQ cells treated CAB with or without rapamycin. C. MMQ cells were treated with CAB in the presence or absence of rapamycin (100 nM) for 24 hours, and cell survival was determined by the MTS.

Figure 3. CAB induces autophagy

A. Immunoblot analysis of LC3-I and LC3-II in GH3 and MMQ cells with or without CAB treatment at different time points. B. CAB treatment at 24 hours induced punctuative distribution of membrane-associated lipidated LC3-II in GH3-GFP-LC3 (left panels) and MMQ-GFP-LC3 (right panels) cells, observed with a confocal microscope. Scale bar represents 10 μm. Histogram shows the LC3 puncta from multiple experiments counting a total of 50 cells (mean ± SD). C. Electron micrographs of GH3 and MMQ cells with or without 24 h CAB treatment. Enlarged images of CAB treated cell (bottom parts) indicate autolysosomes (right). Histogram shows the autolysosome structures from multiple experiments counting a total of 50 cells (mean ± SD).

Figure 4. CAB blocks autophagic flux

A. Immunoblotof p62 in MMQ (left panel) or GH3 (right panel) cells with or without CAB treatment at different time points. B. Analysis of p62 mRNA levels in MMQ (left panel) or GH3 (right panel) cells with or without CAB treatment at different time points.

Figure 5. CAB impairs lysosomal degradation within autolysosomes

A. CAB-treated MMQ (GFP-LC3) cells stained with Lysotracker. Scale bar: 5 μm. B. Following CAB treatment for 24 h, cells were loaded with 1 μM Lysosensor Green and examined with confocal microscopy. Scale bar: 20 μm. C. Samples from B were analyzed by flow cytometry. D. MMQ cells treated with DMSO, CAB, BafA1, or CAB+BafA1were loaded with 1 μM Lysosensor Green for 24 h and analyzed by flow cytometry. E. MMQ cells treated with DMSO, CAB, BafA1, or CAB+BafA1 assayed for cell proliferation in 24 h. F. Immunoblots analysis of PARP in MMQ cells treated with DMSO, CAB, BafA1, or CAB+BafA1 for 6 h and 24 h. ***p < 0.001.

Figure 6. Silencing of autophagic key genes rescues CAB-induced cell death

A–D. MMQ cells were transfected with control (Ctrl) or with ATG7 siRNA for three days before treating with CAB or DMSO, followed by immunoblot analysis for LC3, p62, PARP and β-tubulin (A) Histogram shows the average number of autolysosome seen in electron microscope (B), cell proliferation assay (C), and Annexin-V/PI staining assay (D). E–F. MMQ cells were transfected with control (Ctrl) or with ATG5 or Becn1 siRNA for three days before treating with CAB or DMSO, followed by cell proliferation assay. At the end of incubation, the cell survival rates were determined by CellTiter-Glo®luminescent cell viability assay and the cell survival rate was calculated. Results were reported as the mean ± SD of three independent experiments performed in four replicates. **p < 0.01, ***p < 0.001.

Figure 7. The growth-suppressive effect of CAB on MMQ cells in vivo

A–C. CAB treatment inhibited the in vivo tumor growth of MMQ cells. The representative images for xenograft tumor on the nude mouse are shown in Figure 7A, and the tumor weight and tumor growth curve is shown in Figure 7B and 7C, respectively (n = 5). D. Immunoblot analyses showing p-4EBP1, p62, LC3 and β-tubulin in tumor samples. The protein on behalf of the average of the five tumor tissues.