Kamila Domińska

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation.

Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3 μm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.

Effect of an angiotensin II type 1 receptor blocker on caveolin-1 expression in prostate cancer cells

Introduction Caveolin-1, the major structural protein of caveolae, interacts directly with the AT1 receptor. The biological functions of caveolin-1 in cancer are compound, multifaceted, and depend on cell type, tumour grade and cancer stage. The AT1-R-caveolin complex in caveolae may coordinate angiotensin II (Ang II) induced signalling. The aim of this study was to determine the effect of the angiotensin II receptor type 1 blocker candesartan on caveolin expression in human metastatic prostate adenocarcinoma cells PC-3. Material and methods WST-1 and BrdU assays were used as indicators of cell viability and proliferation after angiotensin II and/or candesartan stimulation. Real-time RT–PCR and western blot were used to study the effect of Ang II and/or candesartan on the expression of Cav-1 and AT1-R in PC-3 cells Results We found that the expression of caveolin-1 mRNA in the PC-3 cells treated with CV was significantly decreased in comparison with the control (2.9 ±0.17, 4.7 ±0.6, p < 0.05), whereas a higher caveolin-1 mRNA expression was observed in those after Ang II treatment (6.0 ±0.43, 4.7 ±0.6, p < 0.05). Protein analysis indicate that the expression of caveolin-1 protein in the PC-3 cells treated with candesartan was significantly decreased when compared with the control (0.69 ±0.05, 1.6 ±0.12, p < 0.05), whereas higher caveolin-1 protein expression was observed after Ang II treatment (2.5 ±0.20, 1.6 ±0.12, p < 0.05). Conclusions These results provide new information on the action of candesartan and may improve the knowledge about AT1 receptor inhibitors, which can be potentially useful in prostate cancer therapy.

A comparison of the effects of Angiotensin IV on androgen-dependent and androgen-independent prostate cancer cell lines

Introduction: Angiotensin IV is one of the biologically active peptides of the renin–angiotensin system. Limited data suggests that this hexapeptide could contribute to cancer development and/or progression. Materials and methods: Using the MTT reduction assay as an indicator of cell viability, and the bromodeoxyuridine incorporation assay as an indicator of cell proliferation, the influence of Angiotensin IV was evaluated on two human prostate cancer lines: androgen-dependent (LNCaP) and androgen-independent (DU-145). The potential effect of Angiotensin IV classic angiotensin receptors was examined by using the selective antagonists losartan and PD123319. Finally, the changes in expression levels of AT1 and AT2 receptors were compared, before and after angiotensin treatment. Results: Angiotensin IV caused significant changes in cell viability and proliferation in LNCaP cells but not in DU-145. It was found that AT2 receptor blocker (PD123319) was able to diminish the suppressor effect of Angiotensin IV on bromodeoxyuridine incorporation into the DNA of androgen-dependent prostate cancer cells. Simultaneously, it was reported that Angiotensin IV is the factor that modulates the density of AT1 and AT2 receptors in prostate cancer cells. Conclusions: These findings suggested that Angiotensin IV can modulate tumour cell proliferation in the early stage of androgen-dependent prostate cancer. The effect might be promoted by the change of the angiotensin receptor level.

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

PURPOSE Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. METHODS The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. RESULTS We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). CONCLUSIONS Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.

Influence of myocardial infarction on changes in the expression of angiotensin type 1 receptor in the rat prostate

Angiotensin II (AngII) is the biologically active peptide of the renin-angiotensin system (RAS). Tissue- based, local RAS has been identified in the prostate, testis, epididymis and coagulating glands. Experimental and clinical studies have consistently shown that myocardial infarction (MI) is associated with activation of the systemic RAS with increased concentration of angiotensin peptides in the blood and changes in expression of angiotensin receptors (AT). Changes in angiotensin receptors in the renal and cardiovascular system after MI are well recognized, but the effects of MI influence on changes in other tissue like the prostate gland are unknown. In the present study, we investigated the effect of myocardial infarction on angiotensin receptor protein and mRNA expression in the rat prostate gland. MI model was established in Wistar rats by ligating the left coronary artery (modified Selye method). The levels of AT1a-b and AT2 receptor mRNAs and proteins were measured in the rat prostate. Our study demonstrates tissue-specific changes in AT1a-b and AT2 receptor expression after myocardial infarction. The results show that MI has a strong influence on the expression of angiotensin receptor type AT1 in the prostate at the protein and mRNA level.

Angiotensin modulates human mammary epithelial cell motility

Introduction: Angiotensin II is an effector peptide showing multiple physiological effects, such as regulation of vascular tone, tissue growth and remodelling. Postlactational involution of mammary gland involves changes such as high matrix metalloproteinase activity and release of bioactive fragments of fibronectin and laminin, which may be directly regulated by angiotensin II. The aim of the present study was to evaluate the influence of angiotensin II on proliferation, viability and motility of normal human mammary epithelial cells (184A1 cell line) and to determine the role of angiotensin II receptors in these processes. Materials and methods: Real-time reverse transcription-PCR, western blot and gelatin zymography were used to study the effect of angiotensin II on the expression of angiotensin receptors and matrix metalloproteinases in 184A1 cells. WST-1, AlamarBlue and BrdU assays were used as indicators of cell viability and proliferation after angiotensin II stimulation. Boyden chamber assays and monolayer wound migration assay were used to evaluate in vitro the changes in cell adhesion, migration and invasion. Results: Angiotensin II increased motility of the 184A1 cells and the ability of wound closure. Modifications in cell–substrate adhesion systems and increased secretion and activity of matrix metalloproteinases were also observed. The effect of angiotensin II was abolished by blocking angiotensin type 1 receptor with specific inhibitors candesartan and losartan. Conclusions: The results indicate that angiotensin II modulates cell behaviour via AT1-R and stimulates secretion of MMP-2 by human mammary epithelial cells.

Coexpression of CAV-1, AT1-R and FOXM1 in prostate and breast cancer and normal cell lines and their influence on metastatic properties

The aim of this study was to evaluate the coexpression of caveolin-1 (CAV-1), angiotensin II type 1 receptor (AT1-R) and forkhead box Ml (FOXM1) in prostate and breast cancer cell lines, in comparison with normal cell lines. CAV-1, AT1-R and FOXM1 expression was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis in the prostate cancer cell lines PC3, DU145 and LNCaP; prostate normal cell line PNT1A; breast cancer cell lines MCF-7 and MDA-MB-231; and the normal breast cell line 184A1. A correlation between the expression levels of the investigated genes and their metastatic properties was determined by the Spearmans rank test (P<0.05) and Aspin-Welsch t-test, respectively. In prostate cell lines, a significant correlation was noted between CAV-1 and AT1-R expression and between FOXM1 and CAV-1 expression. A correlation between the expression levels of the investigated genes and their metastatic potential was also observed, with relatively high expression of all the investigated genes in the normal prostate cell line PNT1A. In comparison to prostate cancer cell lines, an adverse dependency between CAV-1, AT1-R, FOXM1 expression and metastatic potential was observed in the breast cancer cell lines. Relatively high expression of all tested genes was observed in the normal breast cell line 184A1, which was decreasing respectively with increasing metastatic potential of breast cancer cell lines. The results obtained here indicate that CAV-1, FOXM1 and AT1-R may be potential markers of tumorigenesis in certain types of cancer in vitro.

A common effect of angiotensin II and relaxin 2 on the PNT1A normal prostate epithelial cell line

The prostate gland is a part of the male reproductive tract which produces both angiotensin II (Ang II) and relaxin 2 (RLN2). The present study analyzes the effect of both these peptide hormones at concentration 10−8M on viability, proliferation, adhesion, migration, and invasion of normal prostate epithelial cells (PNT1A). Improved survival in two- and three-dimensional cell cultures was noted as well as visual changes in colony size and structure in Geltrex™. Stimulatory influence on cell viability of each peptide applied single was lower than in combination. Enhanced survival of PNT1A cells appears to be associated with increased BCL2/BAX messenger RNA (mRNA) expression ratio. Modulation of cell spreading and cell-extracellular matrix adhesion dynamics were also altered as an influence of tested hormone application. However, long-term Ang II and RLN2 effects may lead to an increase of normal prostate cell migration and invasion abilities. Moreover, gelatin zymography revealed that both gelatinases A and B were augmented by Ang II treatment, whereas RLN2 significantly stimulated only MMP-9 secretion. These results support the hypothesis that deregulation of locally secreted peptide hormones such as Ang II and RLN2 may take part in the development of certain cancers, including prostate cancer. Moreover, the observed ability of relaxin 2 to act as a regulator of mRNA expression levels not only LGR7 but also classic angiotensin receptors suggested that renin-angiotensin system and relaxin family peptide system are functionally linked.

Regulation of mRNA gene expression of members of the NF-κB transcription factor gene family by angiotensin II and relaxin 2 in normal and cancer prostate cell lines

An increasing number of researchers are focusing on the influence of local peptide hormones such as angiotensin II (Ang II) and relaxin 2 (RLN2) in the regulation of inflammation and carcinogenesis. The interaction between the renin‑angiotensin system (RAS) and relaxin family peptide system (RFPS) is known to influence the proliferation, adhesion and migration of normal and cancer prostate cell lines. The aim of the present study was to evaluate changes in the expression of nuclear factor‑κB subunit 1 (NFKB1), nuclear factor‑κB subunit 2 (NFKB2), REL proto‑oncogene nuclear factor‑κB p65 subunit (REL), RELA proto‑oncogene nuclear factor‑κB subunit (RELA) and RELB proto‑oncogene nuclear factor‑κB subunit (RELB) mRNA caused by Ang II and RLN2. The members of NF‑kB family are involved in many processes associated with cancer development and metastasis. Reverse transcription‑quantitative polymerase chain reaction analysis identified that both peptide hormones have an influence on the relative expression of nuclear factor‑κB. Following treatment with either peptide, NFKB1 expression was downregulated in all prostate cancer cell lines (LNCaP, DU‑145 and PC3), but not in normal epithelial cells (PNT1A). Conversely, RELB mRNA was enhanced only in non‑cancerous prostate cells. RELA expression was strongly stimulated in the most aggressive cell line, whereas REL mRNA was unchanged. In many cases, the effect was strictly dependent on the cell line and/or the type of peptide: Ang II increased expression of both RELA and REL genes in the androgen‑dependent cell line while RLN2 enhanced NFKB2 and RELA mRNA in androgen‑independent cells (DU‑145). Further research is needed to understand the regulation of NF‑κB family members by key renin‑angiotensin system and RFPS peptides in prostate cancer cells; however, prostate carcinogenesis appears to be influenced by the balance between the cross‑regulation of nuclear factor‑κB (NF‑κB) and androgen receptor pathways by Ang II and relaxin 2.

Anticancer activity of some polyamine derivatives on human prostate and breast cancer cell lines

The aim of this study was to expand our knowledge about anticancer activity of some polyamine derivatives with quinoline or chromane as terminal moieties. Tested compounds were evaluated in vitro towards metastatic human prostate adenocarcinoma (PC3), human carcinoma (DU145) and mammary gland adenocarcinoma (MCF7) cell lines. Cell viability was estimated on the basis of mitochondrial metabolic activity using water-soluble tetrazolium WST1 to establish effective concentrations of the tested compounds under experimental conditions. Cytotoxic potential of polyamine derivatives was determined by the measurement of lactate dehydrogenase activity released from damaged cells, changes in mitochondrial membrane potential, the cell cycle distribution analysis and apoptosis assay. It was revealed that the tested polyamine derivatives differed markedly in their antiproliferative activity. Bischromane derivative 5a exhibited a rather cytostatic than cytotoxic effect on the tested cells, whereas quinoline derivative 3a caused changes in cell membrane integrity, inhibited cell cycle progression, as well as induced apoptosis of prostate and breast cancer cells which suggest its potential application in cancer therapy.