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| Status |
Public on Jul 02, 2019 |
| Title |
Analysis of transcriptome, selected intracellular signaling pathways, proliferation and apoptosis of LNCaP cells exposed to high leptin concentration |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
The androgen-sensitive LNCaP line of prostate cancer cells is one of the most commonly used cells in oncological research. These include, but are not limited to, research into the influence of leptin (LEP) on prostate cancer. However, the results of studies on the effects of this cytokine on the proliferation and apoptosis of LNCaP cells are controversial. Therefore, we performed studies on the effects of high LEP concentration (1x10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools
Most ontological groups are associated with proliferation and apoptosis (7 groups), immune response (6) and extracellular matrix (2). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.
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| Overall design |
LNCaP - the human prostate carcinoma cell line [LNCaP clone FGC (ATCC® CRL-1740DTM)] was purchased from ATCC (American Type Culture Collection, Manassas, Rockville, MD, USA). LNCaP cells were cultivated in the RPMI Medium 1640 (1X), supplemented with GlutaMAX, HEPES (all from Gibco, Life technologies, Carlsbad, California, USA) and antibiotics (Antibiotic/antimycotic Sigma Aldrich, Saint Louis, MO 63103, USA). Cells were grown in the 75 cm2 flask (NUNC EasyFlask with Nunclon surface, Thermo Fisher Scientific) at 37°C in a humidified atmosphere of 5% CO2. The culture medium was changed every 2 days. When cells reached approximately 80% confluence (about 7-8 days of cultivation), they were subcultured into 6-well plates (Nunc, Thermo Fisher Scientific, approximately 343 000 cells per one well – 9.6 cm2), to determine the effect of LEP on LNCaP transcriptome modulation. The applied experimental protocol was as follows: during the first 48 h of cultivation, the cells grew in a standard medium mentioned above. For the next 24 h, the cells were grown in starvation medium (FBS free). Afterwards, the cells were cultivated for 48 h, in a starvation medium supplemented with LEP (Recombinant Human Leptin, PeproTech, Germany) at the following concentrations: 0 (control), 1x10-6 M. After the mentioned period cell supernatants were collected and stored at - 80°C for further analyses.
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| Contributor(s) |
Szyszka M, Tyczewska M, Jopek K, Malendowicz LK, Rucinski M |
| Citation(s) |
31671654 |
| Submission date |
Jul 01, 2019 |
| Last update date |
Dec 09, 2019 |
| Contact name |
Marcin Rucinski |
| E-mail(s) |
marcinruc@ump.edu.pl
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| Organization name |
Poznan University of Medical Science
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| Department |
Histology and Embriology
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| Street address |
Swiecicki 6
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| City |
Poznan |
| ZIP/Postal code |
60-781 |
| Country |
Poland |
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| Platforms (1) |
| GPL13667 |
[HG-U219] Affymetrix Human Genome U219 Array |
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| Samples (4)
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| GSM3913285 |
LnCap Cells - control, biological rep1 |
| GSM3913286 |
LnCap Cells - control, biological rep2 |
| GSM3913287 |
LnCap Cells - LEP treated, biological rep1 |
| GSM3913288 |
LnCap Cells - LEP treated, biological rep2 |
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| Relations |
| BioProject |
PRJNA552046 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE133616_RAW.tar |
8.1 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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