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Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines

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Abstract

Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR−) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the EBR-induced apoptosis only in LNCaP (AR+) cells. In a similar way, MDL 72,527, the specific PAO and SMO inhibitor, co-treatment with EBR during 24 h, reduced the formation of cleaved fragments of PARP in LNCaP (AR+) cells.

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Abbreviations

AR:

Androgen receptor

AZI:

Antizyme inhibitor

BAK1:

BRI1-associated receptor kinase

BRI1:

Brassinosteroid insensitive 1

BR:

Brassinosteroid

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DiOC6:

3,3′-Dihexyloxacarbocyanine iodide

DMSO:

Dimethyl sulfoxide

EBR:

Epibrassinolide

HPLC:

High-pressure liquid chromatography

HRP:

Horseradish peroxidase

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

OAZ:

ODC antizyme

ODC:

Ornithine decarboxylase

PA:

Polyamine

PAO:

Polyamine oxidase

PBS:

Phosphate-buffered saline

PVDF:

Polyvinyldifluoride

ROS:

Reactive oxygen species

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SSAT:

Spermidine/spermine N 1-acetyltransferase

TBS-T:

Tris-buffered saline with Tween-20

Δψm:

Mitochondrial membrane potential

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Acknowledgments

This work was partially supported by Istanbul University Research Projects Center (Project no: 20958), Istanbul Kultur University Scientific Projects Support Center; Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) (EA); Istituto Superiore di Sanità “Project Italy–USA” (EA); funds MIUR-PRIN (Cofin) (EA) and Istituto Pasteur Fondazione Cenci Bolognetti (EA).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Istanbul Kultur University, Atakoy Campus, Bakirkoy, 34156, Istanbul, Turkey

    Pinar Obakan, Elif Damla Arisan & Narçin Palavan-Unsal

  2. Department of Technology and Health, National Institute of Health, Rome, Italy

    Annarica Calcabrini

  3. Istituto Pasteur Fondazione Cenci Bolognetti and Department of Biochemical Sciences “A. Rossi Fanelli”, CNR Institute Biology and Molecular Pathology, SAPIENZA University of Rome, Piazzale A. Moro 5, 00185, Rome, Italy

    Enzo Agostinelli

  4. Department of Biology, Istanbul University, Vezneciler, 34134, Istanbul, Turkey

    Şehnaz Bolkent

Authors
  1. Pinar Obakan
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  2. Elif Damla Arisan
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  3. Annarica Calcabrini
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  4. Enzo Agostinelli
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  5. Şehnaz Bolkent
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  6. Narçin Palavan-Unsal
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Corresponding author

Correspondence to Narçin Palavan-Unsal.

Electronic supplementary material

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726_2013_1574_MOESM1_ESM.pdf

EBR did not modulate cell cycle distribution and induce apoptosis in PNT1a prostate epithelial cells (a) PNT1a cells were seeded into a 6-well plate and treated with EBR (25 μM) for 24 h. After ethanol fixation and PI (propidium iodide) staining, samples were analyzed by flow cytometry. Cell cycle distribution of was determined by CellQuest software. Data shown are obtained from a representative experiment (b) Total proteins were isolated from PNT1a cells; the expression profiles of Apaf-1 and cleaved caspase-9 were determined by immunoblotting following 24 h EBR treatment while PARP expression was analyzed at 24, 48 and 72 h. β-actin was used as a loading control. (PDF 102 kb)

726_2013_1574_MOESM2_ESM.pdf

SSAT and PAO silencing determination with qRT-PCR. Cells were seeded in 6 well plates and treated with EBR for 24 h. Total RNA was isolated and cDNA synthesis was perfomed. To determine the efficiency of selected concentration of siRNA to silence (a) SSAT or (b) PAO, qRT-PCR with appropriate primers was performed. (PDF 62 kb)

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Obakan, P., Arisan, E.D., Calcabrini, A. et al. Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines. Amino Acids 46, 553–564 (2014). https://doi.org/10.1007/s00726-013-1574-1

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