Proline oxidase silencing inhibits p53-dependent apoptosis in MCF-7 breast cancer cells.

Proline oxidase (POX) is mitochondrial proline-degrading enzyme of dual apoptosis/survival function. POX expression and proline availability are considered an underlying mechanism for differential POX functions. The mechanism for POX-dependent regulation of cell death/survival was studied in wild-type (MCF-7WT) and shRNA POX-silenced breast cancer cells (MCF-7iPOX). Proline concentration and proteomic analyses were determined by LC/MS/QTOF and LC/MS/ORBITRA, respectively. Inhibition of collagen biosynthesis (proline utilizing process) by 2-methoxyestradiol (2ME) contributed to induction of apoptosis in MCF-7WT cells, as detected by increase in the expression of active caspase-3, -9 and p53. The process was not shown in MCF-7iPOX. In MCF-7iPOX cells prolidase activity and expression as well as proline concentration were drastically increased, compared to MCF-7WT cells. Down-regulation of p53 in MCF-7iPOX cells was corroborated by proteomic analysis showing decrease in the expression of p53-related proteins. The mechanism for down-regulation of p53 expression in MCF-7iPOX cells was found at the level of p53-PEPD complex formation that was counteracted by hydrogen peroxide treatment. In this study, we found that silencing POX modulate pro-survival phenotype of MCF-7 cells and suggest that the mechanism of this process undergoes through down-regulation of p53-dependent signaling.

Evaluation of the Anticancer Activities of Novel Transition Metal Complexes with Berenil and Nitroimidazole.

Novel transition metal complexes (Au, Pd, Pt) with berenil and 2-(1-methyl-5-nitroimidazol-2-yl)ethanol were obtained through two-step synthesis. The cytotoxicity assay against MCF-7 and MDA-MB-231 breast cancer cells revealed that novel platinum and palladium complexes cause a reduction on the viability of MCF-7 and MDA-MB-231 breast cancer cells to a greater extent than cisplatin. The complexes showed lower cytotoxicity on normal MCF-10A human breast epithelial cells than on tumor cells. Furthermore, we observed that these complexes selectively concentrate in tumor cell mitochondria due to the characteristic for these cells increased membrane potential that may explain their increased proapoptotic activity. The activity of the synthesized compounds against topoisomerase type IIα and their increased impact on DNA defragmentation also were documented. The novel complexes also induced autophagosome changes and inhibited tumor growth in xenograft models (established using breast cancer cells).

Estimates of Medication Expenditure for Ischemic Heart Disease Accompanying Chronic Obstructive Pulmonary Disease.

Ischemic heart disease (IHD) is a frequent accompaniment of chronic obstructive pulmonary disease (COPD). Co-occurrence of these two diseases is associated with many risk factors, difficulties in implementing appropriate therapies, numerous complications, and high spending for treatment. All these elements significantly reduce the quality of life of patients. The aim of this study was to estimate the expenditure for medications involved with IHD pharmacotherapy in the course of COPD. This retrospective study was based on the review of medical files of 57 patients, 27 women and 30 men, diagnosed with IHD, according to the severity classification, in the course of COPD which was staged according to the GOLD criteria. We found a considerable increase in per capita per year retail spending for drugs. The spending increased with the severity class of IHD; from 27.41 EUR in Class I to 142.30 EUR in Class IV. This spending did not include the treatment cost for the basic disease, i.e., COPD. A high individual cost burden was decreased by a discounting intervention of the National Health Fund. Despite a relatively high drug expenditure, we consider the treatment being cost-effective since we noticed a reduction in the classical risk factors for IHD, related to metabolic disturbances and lifestyle features, as soon as 2 months after treatment initiation. This study confirms that heart disease accompanying COPD is a frequent occurrence, generating high costs of treatment, which relates to the severity of this comorbidity.

Erythropoietin Intensifies the Proapoptotic Activity of LFM-A13 in Cells and in a Mouse Model of Colorectal Cancer.

The Bruton’s tyrosine kinase (BTK) inhibitor LFM-A13 has been widely employed as an antileukemic agent, but applications in solid cancer have been found recently. The compound promotes apoptosis, has an antiproliferative effect, and increases cancer cell sensitivity to chemotherapy drugs. We decided to assess the impact of the simultaneous use of erythropoietin (Epo) and LFM-A13 on signal transduction in colon DLD-1 and HT-29 cells, as well as in tumor xenografts. The induction of apoptosis by Epo and LFM-A-13 in the cells was confirmed by phosphatidylserine externalization, loss of mitochondrial membrane potential, and modulation of the expression of apoptotic protein BAX and antiapoptotic protein BCL-2 in colon adenocarcinoma cells. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13 in order to evaluate the degree of tumor regression. The simultaneous use of Epo and LFM-A13 severely inhibited cell growth, activated apoptosis, and also inhibited tumor growth in xenografts. The addition of Epo to LFM-A13 intensified the antiproliferative effect of LFM-A13, confirmed by the loss of mitochondrial membrane potential and the accumulation of apoptotic colon cancer cells with externalized phosphatidylserine (PS). These preclinical results suggest that the combination of Epo and LFM-A13 has a high proapoptotic activity and should be tested in the clinic for the treatment of solid tumors such as colon cancer.

The effect of estrogen on prolidase-dependent regulation of HIF-1α expression in breast cancer cells.

The role of estrogen in breast cancer progression and activation of prolidase activity and HIF-1α led us to study the effect of estrogen on nuclear HIF-1α expression in breast cancer estrogen-dependent MCF-7 and estrogen-independent MDA-MB-231 cells. We have found that in MCF-7 cells (expressing α and ß estrogen receptor) cultured without estrogen receptor activator (phenol red, estradiol), HIF-1α was down-regulated, compared to the cells cultured with estrogen receptor activator. This effect was not observed in MDA-MB-231 cells (expressing only ß estrogen receptor), suggesting that α estrogen receptor is involved in down-regulation of HIF-1α. However, in MDA-MB-231 cells (expressing high prolidase activity) cultured in the presence of prolidase substrates, Gly-Pro or Gly-HyPro, HIF-1α expression was induced in a dose-dependent manner, independently of estrogen receptor activation. In MCF-7 cells (with constitutively low prolidase activity) the effect of studied iminodipeptides on HIF-1α expression was much less pronounced but it was estrogen-dependent, showing importance of prolidase activity in mechanism of this process. The data were supported by confocal microscopy bio-imaging of HIF-1α in nucleus of MCF-7 and MDA-MB-231 cells that were cultured in the presence and absence of estrogen activator and prolidase substrates. It suggests that estrogen receptor may represent important therapeutic target in pharmacotherapy of estrogen receptor positive breast cancer, while ECM degradation enzymes, including prolidase may represent target in pharmacotherapy of estrogen receptor negative breast cancers.

Troglitazone-Induced PRODH/POX-Dependent Apoptosis Occurs in the Absence of Estradiol or ERß in ER-Negative Breast Cancer Cells.

The impact of estradiol on troglitazone (TGZ)-induced proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied in wild-type and PRODH/POX-silenced estrogen receptor (ER) dependent MCF-7 cells and ER-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, prolidase activity evaluated by colorimetric method, ROS production was measured by fluorescence assay. Protein expression was determined by Western blot and proline concentration by LC/MS analysis. PRODH/POX degrades proline yielding reactive oxygen species (ROS). Estrogens stimulate collagen biosynthesis utilizing free proline and limiting its availability for PRODH/POX-dependent apoptosis. TGZ cytotoxicity was highly pronounced in wild-type MDA-MB-231 cells cultured in medium without estradiol or in the cells cultured in medium with estradiol but deprived of ERß (by ICI-dependent degradation), while in PRODH/POX-silenced cells the process was not affected. The TGZ cytotoxicity was accompanied by increase in PRODH/POX expression, ROS production, expression of cleaved caspase-3, caspase-9 and PARP, inhibition of collagen biosynthesis, prolidase activity and decrease in intracellular proline concentration. The phenomena were not observed in PRODH/POX-silenced cells. The data suggest that TGZ-induced apoptosis in MDA-MB-231 cells cultured in medium without estradiol or deprived of ERß is mediated by PRODH/POX and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis. It suggests that combined TGZ and antiestrogen treatment could be considered in experimental therapy of estrogen receptor negative breast cancers.

Ceragenin CSA-13 as free molecules and attached to magnetic nanoparticle surfaces induce caspase-dependent apoptosis in human breast cancer cells via disruption of cell oxidative balance.

Natural antimicrobial peptides and ceragenins, as non-peptide amphipathic mimics, have been proposed as anti-cancer agents. To date, it has been confirmed that cathelicidin LL-37 and ceragenin CSA-13, both in free form and immobilized on the surface of magnetic nanoparticles (MNP@LL-37, MNP@CSA-13) induce apoptosis in colon cancer cells. Nevertheless, the question remains whether ceragenins, as synthetic analogs of LL-37 peptide and mimicking a number of its properties, act as antineoplastic agents in breast cancer cells, where LL-37 peptide stimulates oncogenesis. Considering potential anticancer activity, we determined whether CSA-13 and MNP@CSA-13 might be effective against breast cancer cells. Our study provides evidence that both CSA-13 and MNP@CSA-13 decreased viability and inhibit proliferation of MCF-7 and MDA-MB-231 cells despite the protumorigenic properties of LL-37 peptide. Flow cytometry-based analyses revealed that ceragenin treatment results in increases in dead and PI-negative/low-viability cells, which was associated with glutathione (GSH) depletion and increased reactive oxygen species (ROS) generation followed by mitochondrial membrane depolarization, caspase activation, and DNA fragmentation. These findings demonstrate that both CSA-13 and MNP@CSA-13 cause disruption of the oxidative balance of cancer cells. This novel mechanism of ceragenin-mediated eradication of cancer cells suggest that these agents may be developed as a possible treatment of breast cancer.

αIIbß3-integrin Ligands: Abciximab and Eptifibatide as Proapoptotic Factors in MCF-7 Human Breast Cancer Cells.

Integrin receptors are considered to be the key factors in carcinogenesis. αIIbß3-Integrin (GP IIb/IIIa) is the main glycoprotein of the surface of platelets, its presence has also been noted on the certain cancer cell lines. The molecular mechanism of its action in cancer cells remains unknown. This study presents effects of two αIIbß3-inhibitors: Abciximab and Eptifibatide on apoptosis, expression of proline oxidase (POX), signaling molecules ERK 1/2, transcription factor NF-κB and HIF-1α, vascular endothelial growth factor (VEGF) as well as DNA biosynthesis, collagen biosynthesis and prolidase activity in MCF-7 breast cancer cells. Both ligands induced apoptosis, however we found significant differences in molecular mechanism of action between tested αIIbß3-inhibitors. These differences include expression of POX, HIF-1α, NF-κB,VEGF and collagen biosynthesis. Eptifibatide presented stronger proapoptotic activity in MCF-7 cells than Abciximab. Results of this study suggest that Eptifibatide may be considered as a novel candidate for development of new anticancer therapy.

Magnetic nanoparticles enhance the anticancer activity of cathelicidin LL-37 peptide against colon cancer cells.

The pleiotropic activity of human cathelicidin LL-37 peptide includes an ability to suppress development of colon cancer cells. We hypothesized that the anticancer activity of LL-37 would improve when attached to the surface of magnetic nanoparticles (MNPs). Using colon cancer culture (DLD-1 cells and HT-29 cells), we evaluated the effects of MNPs, LL-37 peptide, its synthetic analog ceragenin CSA-13, and two novel nanosystems, ie, MNP@LL-37 and MNP@CSA-13, on cancer cell viability and apoptosis. Treatment of cancer cells with the LL-37 peptide linked to MNPs (MNP@LL-37) caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free LL-37 peptide. Additionally, we observed a strong ability of ceragenin CSA-13 and MNP@CSA-13 to induce apoptosis of DLD-1 cells. We found that both nanosystems were successfully internalized by HT-29 cells, and cathelicidin LL-37 and ceragenin CSA-13 might play a key role as novel homing molecules. These results indicate that the previously described anticancer activity of LL-37 peptide against colon cancer cells might be significantly improved using a theranostic approach.

Prolidase-dependent regulation of TGF ß (corrected) and TGF ß receptor expressions in human skin fibroblasts.

Transforming growth factor beta 1 (TGF ß1) is a protein that in most cells control proliferation and differentiation. One of the best characterized functions of TGF ß1 is stimulation of collagen biosynthesis that may lead to tissue fibrosis. Several reports suggest that prolidase, through regulation of expression of growth factors and transcription factors, e.g. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1 α) may be important in many physiologic and pathophysiologic processes like: wound healing, inflammation and angiogenesis. We found that inhibitors of prolidase activity (N-benzyloxycarbonyl-l-proline, Cbz-Pro and phosphoenolopyruvate, PEP) induced decrease in TGF ß1 and its receptor expressions. On the other hand, products of prolidase catalytic activity, proline (Pro) and hydroxyproline (HyPro) induced increase in the amount of TGF ß1 and TGF ß receptors. Simultaneously, inhibitors of prolidase induced down-regulation of expression of the phospho-AKT. An addition of Pro or HyPro to the cells induced increase in the expression of phospho-AKT. An important transcription factor involved in signal induced by TGF ß receptor is mammalian target of rapamycin (mTOR). We found that prolidase inhibitors induced decrease in the expression of phospho-mTOR, while Pro or HyPro counteracted the effect. Rapamycin (pharmacological inhibitor of mTOR) resulted in decrease in prolidase activity. The down-regulation of phospho-mTOR by rapamycin contributed to down-regulation of prolidase activity suggesting its important role in prolidase-dependent function. It seems, that products of prolidase activity, Pro or HyPro may act as an interface between mTOR and phospho-mTOR in regulation of numerous TGF ß receptor-dependent functions.