The Influence of β-Carotene and Its Liposomal Form on the Expression of EMT Markers and Androgen-Dependent Pathways in Different Prostate Cell Lines
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and Treatment
2.2. Preparation of LP-BCs
2.3. Analysis of Purity and Stability of BC
2.4. Cytotoxicity Assay
2.5. Preparation of Cell Culture Media Supplemented with BC
2.6. Cellular Uptake of BC Measured with HPLC
2.7. Proliferation Assay
2.8. Wound Healing Assay and Migration Assay
2.9. RNA Extraction and Reverse Transcription
2.10. qPCR
2.11. Gene Expression Analysis
2.12. Western Blot Analysis
2.13. Statistical Analysis
3. Results
3.1. Cytotoxicity
3.2. Proliferation of Cells
3.3. Wound Healing/Migration Assay and Zymography
3.4. Western Blot Analysis
3.4.1. Incubation with Empty LPs
3.4.2. Incubation with LP-BCs
3.4.3. Incubation with BC
3.5. RT-qPCR
3.5.1. Influence of Incubation with Empty LPs
3.5.2. Incubation with LP-BCs
3.5.3. Incubation with BC
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Cell Line | 22Rv1 | RWPE | LNCaP | PC-3 | Du145 |
---|---|---|---|---|---|
Histological origin of cells | Human prostate carcinoma epithelial cell line derived from a xenograft, CWR22R | Epithelial cells derived from the peripheral zone of histologically normal adult prostate | Human prostate carcinoma cells | ||
Origin of tissues the cells came from | Xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft | Obtained from a healthy, 54-year-old Caucasian man | Obtained from a metastatic lesion in a lymph node of a 50-year-old Caucasian man | Obtained from a bone metastasis in a 62-year-old man | Obtained from a metastasis to the brain of androgen-unresponsive prostate carcinoma in a 69-year-old man |
Type of growth | Grown as an adherent culture | ||||
Tumorigenicity in mice | Tumorigenic | Not tumorigenic | Tumorigenic | Tumorigenic | Tumorigenic |
Presence of AR and PSA | AR+ PSA+ growth inhibited by androgen withdrawal | AR+ PSA+ AR is upregulated after the exposure to androgens | AR+ PSA+ growth inhibited by androgen withdrawal | AR− PSA− growth independent of androgens | AR− PSA− androgen-independent |
Target of Primary Antibody | Host Species | Dilution (at Application) | Vendor |
---|---|---|---|
β-actin | mouse | 1:12,000 | Sigma Aldrich |
ZO-1 | rabbit | 1:1000 | Cell Signaling Technology |
Snail | rabbit | 1:1000 | Cell Signaling Technology |
Slug | rabbit | 1:1000 | Cell Signaling Technology |
Vimentin | rabbit | 1:1000 | Cell Signaling Technology |
GSK | mouse | 1:1000 | Cell Signaling Technology |
Twist | mouse | 1:1000 | Sigma Aldrich |
ZEB | rabbit | 1:1000 | Cell Signaling Technology |
N-cadherin | mouse | 1:1000 | Cell Signaling Technology |
E-cadherin | mouse | 1:1000 | Cell Signaling Technology |
SREB | rabbit | 1:500 | AB Clonal |
LDL-R | rabbit | 1:500 | AB Clonal |
AR | rabbit | 1:1000 | Cell Signaling Technology |
NF-κB | rabbit | 1:500 | AB Clonal |
LXR | rabbit | 1:500 | AB Clonal |
Akt | mouse | 1:500 | BD Transduction Laboratories |
Cell Line | Time of Incubation | Cytotoxicity (% of Dead Cells) | |||||||
---|---|---|---|---|---|---|---|---|---|
1.5 μM BC | 3.0 μM BC | 5.0 μM BC | 7.5 μM BC | 15.0 µM BC | 30.0 μM BC | LPs | 3.0 μM LP-BCs | ||
LNCaP | 24 h | 0.34 | 0.44 | 0.91 | 1.40 | 2.50 | 6.40 | 0.24 | 0.45 |
48 h | 0.39 | 0.33 | 1.10 | 1.90 | 0.90 | 5.90 | 0.47 | 0.28 | |
PC-3 | 24 h | 0.20 | 0.30 | 0.35 | 0.50 | 4.50 | 12.50 | 0.45 | 0.34 |
48 h | 0.25 | 0.40 | 0.55 | 0.80 | 5.80 | 12.95 | 0.45 | 1.50 | |
Du145 | 24 h | 0.15 | 0.60 | 1.50 | 2.00 | 6.00 | 7.55 | 0.95 | 2.10 |
48 h | 0.35 | 0.95 | 1.85 | 1.90 | 5.39 | 6.55 | 1.80 | 2.90 | |
22Rv1 | 24 h | 0.15 | 0.85 | 1.55 | 7.55 | 8.00 | 9.55 | 0.55 | 0.65 |
48 h | 0.65 | 2.55 | 3.35 | 7.85 | 8.20 | 8.65 | 0.75 | 0.45 |
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Dulińska-Litewka, J.; Dykas, K.; Boznański, S.; Hałubiec, P.; Kaczor-Kamińska, M.; Zagajewski, J.; Bohn, T.; Wątor, G. The Influence of β-Carotene and Its Liposomal Form on the Expression of EMT Markers and Androgen-Dependent Pathways in Different Prostate Cell Lines. Antioxidants 2024, 13, 902. https://doi.org/10.3390/antiox13080902
Dulińska-Litewka J, Dykas K, Boznański S, Hałubiec P, Kaczor-Kamińska M, Zagajewski J, Bohn T, Wątor G. The Influence of β-Carotene and Its Liposomal Form on the Expression of EMT Markers and Androgen-Dependent Pathways in Different Prostate Cell Lines. Antioxidants. 2024; 13(8):902. https://doi.org/10.3390/antiox13080902
Chicago/Turabian StyleDulińska-Litewka, Joanna, Kacper Dykas, Stanisław Boznański, Przemysław Hałubiec, Marta Kaczor-Kamińska, Jacek Zagajewski, Torsten Bohn, and Gracjan Wątor. 2024. "The Influence of β-Carotene and Its Liposomal Form on the Expression of EMT Markers and Androgen-Dependent Pathways in Different Prostate Cell Lines" Antioxidants 13, no. 8: 902. https://doi.org/10.3390/antiox13080902