PT45 Cell Research Articles

Structure-activity relationship of trifluoromethyl-containing metallocenes: electrochemistry, lipophilicity, cytotoxicity, and ROS production.

We report the synthesis of trifluoromethylated metallocenes (M=Fe, Ru) and related metal-free compounds for comparison of their biological properties with the aim to establish structure-activity relationships toward the anti-proliferative activity of this compound class. All new compounds were comprehensively characterized by NMR spectroscopy ((1) H, (13) C, (19) F), mass spectrometry, IR spectroscopy, and elemental analysis. A single-crystal X-ray structure was obtained on the Ru derivative, 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3). The cytotoxicity of all compounds was tested on MCF-7, HT-29, and PT-45 cells, and IC50 values as low as 12 μM were observed. Both the metallocene moiety and the hydroxy function are crucial for cytotoxicity. In addition, the activity decreased sharply even if only one trifluoromethyl group was replaced with a methyl group. Electrochemical investigations by cyclic voltammetry revealed that all CF3 -containing compounds are harder to oxidize than the unsubstituted metallocenes. Moreover, log P determination by RP-HPLC showed the fluorinated derivatives to have higher lipophilicity, with log P values up to 4.6. At the same time, the generation of reactive oxygen species (ROS) in Jurkat cells by these compounds was investigated by flow cytometry. Strong ROS production was shown exclusively for the bis-CF3 derivative 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) after 6 and 24 h. Also on the Jurkat cell line, only compound 1 strongly induces necrosis after 24 and 48 h, as shown by annexin V/propidium iodide staining. No induction of apoptosis was observed. We propose that compound 1 is more efficiently incorporated into cancer cells relative to all other derivatives, causing significant induction of oxidative stress within the cell, which ultimately leads to cell death.

Pancreatic-carcinoma-cell-derived pro-angiogenic factors can induce endothelial-cell differentiation of a subset of circulating CD34+ progenitors

BackgroundCD34+ progenitor cells comprise both hematopoietic and endothelial progenitor cells. Recent studies suggest that circulating endothelial progenitor cells are recruited into the angiogenic vascular system of several cancers, including pancreatic carcinoma, and that they correlate with clinical progress. However, whether endothelial progenitor cell mobilization occurs in response to cytokine release by tumor cells is still unclear.MethodsThe chemotactic- and/or differentiating-activities of the poorly-differentiated pancreatic carcinoma cell line PT45, and of the immortal H6c7 cell line, a line of near-normal pancreatic duct epithelial cells, on endothelial progenitor cells were investigated in vitro using circulating CD34+ as model.ResultsThe study showed that Vascular Endothelial Growth Factor produced by PT45 cells and, at lesser extent, by H6c7 cells, predominantly chemoattract peripheral blood CD34+ expressing the type 2 relative receptor. Addition of PT45-conditioned medium to CD34+ cells, cultured under conditions supporting myeloid cell development, diverted the differentiation of a subset of these progenitor cells into cells expressing endothelial cell markers, such as CD146, CD105, VE-cadherin and von Willebrand Factor-related antigen. Moreover, these endothelial-like cells formed capillary networks in vitro, chiefly through the release of Angiopoietin-1 by PT45 cells.ConclusionsThe results demonstrate that pancreatic-carcinoma cells potentially attract circulating endothelial progenitor cells to the tumor site, by releasing high levels of pro-angiogenic factors such as Vascular Endothelial Growth Factor and Angiopoietin-1, and may direct the differentiation of these cell subsets of the CD34+ cell population into endothelial cells; the latter cells may become a component of the newly-formed vessels, contributing to angiogenesis-mediated tumor growth and metastasis.

A Ruthenocene–PNA Bioconjugate — Synthesis, Characterization, Cytotoxicity, and AAS-Detected Cellular Uptake

Labeling of peptide nucleic acids (PNA) with metallocene complexes is explored herein for the modulation of the analytical characteristics, as well as biological properties of PNA. The synthesis of the first ruthenocene-PNA conjugate with a dodecamer, mixed-sequence PNA is described, and its properties are compared to a ferrocene-labeled analogue as well as an acetylated, metal-free derivative. The synthetic characteristics, chemical stability, analytical and thermodynamic properties, and the interaction with cDNA were investigated. Furthermore, the cytotoxicity of the PNA conjugates is determined on HeLa, HepG2, and PT45 cell lines. Finally, the cellular uptake of the metal-containing PNAs was quantified by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS). An unexpectedly high cellular uptake to final concentrations of 4.2 mM was observed upon incubation with 50 μM solutions of the ruthenocene-PNA conjugate. The ruthenocene label was shown to be an excellent label in all respects, which is also more stable than its ferrocene analogue. Because of its high stability, low toxicity, and the lack of a natural background of ruthenium, it is an ideal choice for bioanalytical purposes and possible medicinal and biological applications like, e.g., the development of gene-targeted drugs.

Docosahexaenoic acid inhibits invasion of human RT112 urinary bladder and PT45 pancreatic carcinoma cells via down-modulation of granzyme B expression

Fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFAs) inhibit invasion of some tumor cell types in vitro and in vivo. The mechanisms underlying this activity are unclear. Here, we examined the capability of n-3 PUFA–docosahexaenoic acid (22:6n-3; DHA) to affect the invasiveness of human RT112 urinary bladder and PT45 pancreatic carcinoma cell lines in vitro and the mechanism underlying this activity; n-6 PUFA–arachidonic acid (20:4n-6; AA) served as control. We showed that, in contrast to AA, 25, 50 and 100μM DHA significantly inhibited in a dose-dependent manner the invasion through Matrigel of both RT112 and PT45 cells. Then, we analyzed whether the serine proteinase granzyme B (GrB), originally known as cytotoxic molecule of lymphocytes and recently also characterized for its extracellular functions such as invasion promotion of bladder cancer cells, might be involved in the invasion inhibitory activity exerted by DHA. We demonstrated that, accordingly to RT112 bladder cancer cells, PT45 cells expressed GrB and GrB promoted their invasion, since stealth RNA interference-mediated down-regulation of GrB dramatically suppressed PT45 cell invasion. Notably, we also showed that, in contrast to AA, 25, 50 and 100μM DHA induced a dose-dependent down-modulation of GrB expression in both RT112 and PT45 cells. In conclusion, DHA can reduce the invasive phenotype of bladder and pancreatic carcinoma cells, and we provide the first evidence for a possible causative role of GrB in DHA-induced inhibition of cancer cell invasion. The potential use of fish oil as adjuvant antibladder and antipancreatic cancer agent may be suggested.

Abstract 630: Responses of human pancreatic cancer cells to treatment with insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor NVP-AEW541 alone and in combination with anti-EGFR mAb ICR62 or cytotoxic drugs

Abstract Aberrant expression and activation of growth factor receptor signalling pathways including epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-IR) have been reported in a wide range of epithelial cancers and have been associated with increased cell proliferation, reduced apoptosis, invasion and metastasis. Of the growth factor receptor inhibitors, the EGFR inhibitor erlotinib has been approved for the treatment of pancreatic cancer. However, its overall therapeutic efficacy can be of short duration. In some studies, signalling with the IGF-IR has been associated with resistance to therapy with the EGFR inhibitors. In this study, using the Sulforhodamine B colorimetric assay, we investigated the sensitivity of a panel of human pancreatic cancer cell lines (PT-45, AsPC1, PANC1, MiaPaca2, BxPC3, Capan1 and FA6) to treatment with IGF-IR tyrosine kinase inhibitor NVP-AEW541 alone or in combination with anti-EGFR monoclonal antibody (mAb) ICR62 and cytotoxic agents (i.e. 5-FU, doxycycline and gemcitabine). We also investigated the association between the expression levels of IGF-IR and EGFR in these tumour cells, determined by flow cytometry, and their responses to treatment with NVP-AEW541 and/or ICR62. At concentrations above 5μM, NVP-AEW541 inhibited completely the growth of most of the pancreatic cancer cell lines with IC50 values ranging from 342nM (FA6) to 2.73μM (PT45). At maximum concentration of 150nM used in this study, ICR62 did not have any effect on the growth of human pancreatic tumour cell lines. In addition, treatment with a combination of ICR62 and NVP-AEW541 did not enhance the inhibitory effect of the single agent in pancreatic cancer cells. Interestingly, treatment with a combination of NVP-AEW541 and gemcitabine was found to be synergistic for AsPC1 and PANC1 cells, additive for BxPC3, MiaPaca2 and PT-45 cells, but antagonistic for FA6 and CAPAN1 cells. We also examined the cell cycle distribution of BxPC3 cells following treatment with NVP-AEW541 and found an increase in the population of cells in sub-G1 and G0/G1 phases. In contrast, gemcitabine treatment of BxPC3 cells was accompanied by an increase in the populations of cells in both the sub-G1 and S phases of the cell cycle. Taken together, our results suggest that dual targeting of the EGFR and IGF-IR in pancreatic cancer cells by a combination of ICR62 and NVP-AEW541 is not superior to treatment with the single agent in vitro. Further investigations in vivo on the anti-tumour activity of the NVP-AEW541 in combination with ICR62 and cytotoxic drugs in pancreatic cancer are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 630. doi:10.1158/1538-7445.AM2011-630

Expression and functional activity of fibroblast growth factors and their receptors in human pancreatic cancer

We have analysed expression of the first 7 members of the family of heparin-binding fibroblast growth factor (FGFs) and their 4 high-affinity receptors (FGFRs) in human pancreatic carcinoma cell lines, both at the mRNA and protein levels. In cell lines expressing FGFRs, 2 typical patterns were observed: (i) expression of FGFR-I, -3 or -4 along with the expression of at least one FGF; (ii) co-expression of FGFR-3 and FGFR-4 in the absence of FGF expression. Using RT-PCR, transcripts representing multiple isoforms of both extracellular and intracellular domains of FGFR-I were detected in the cell line PT45. A novel extracellular domain variant of FGFR-I was predicted to encode the first immunoglobulin loop in a potentially secreted form. Protein expression of the splice variants of FGFR-I was confirmed by immunoprecipitation with specific antibodies in radiolabelled ligand cross-linking experiments. The type I carboxyl end and the alpha subtype extracellular domain were detected in the PANC-I cell line, while the type I carboxyl terminus and the gamma subtype extracellular domain were expressed in the PT45 cell line. Expression of FGF-2 in PT45 was also detected by immunoprecipitation using 3 different anti-FGF-2 antibodies. Apart from the 18-kDa product, higher molecular weight isoforms, namely 22- and 23-kDa isoforms, were expressed. In an assay of anchorage-independent growth, exogenous FGF-2 stimulated a maximum 15-fold and 10-fold increase in colony formation by the cell lines MIA PACA-2 and PANC-I respectively. Treatment of monolayer cultures of the same cell lines did not promote growth. However, a specific neutralising antibody against FGF-2 reduced cell proliferation of MIA PACA-2 cells by 50%.