Matthias B. Stope

Antiestrogens Induce Transforming Growth Factor β–Mediated Immunosuppression in Breast Cancer

Antiestrogens are universally used to treat estrogen receptor--positive breast cancer, but relapses occur commonly due to the development of drug resistance. The ability of antiestrogen to induce transforming growth factor beta (TGFbeta) in breast cancer cells may be relevant to the emergence of resistance, not only at the level of cell autonomous effects of TGFbeta on cancer progression but also at the level of its effects on the host immune system. To evaluate the potential role of tumor-derived, antiestrogen-induced TGFbeta as an immune suppressor, we established in vitro mixed lymphocyte tumor reactions (MLTR) using MCF-7 cells and peripheral blood mononuclear cells (PBMC), as well as tumor tissue and autologous tumor infiltrating lymphocytes (TIL) obtained from primary breast cancer biopsies. In allogeneic MLTR, antiestrogen-treated MCF-7 cells caused downregulation of the effector molecules granzyme B, perforin, and Fas ligand in CD8(+) T cells, and suppressed the generation of cytotoxic effector cells in a TGFbeta-dependent manner. Furthermore, we documented induction of regulatory T cells in CD4(+) T cells, based on Foxp3 expression and T-cell activation in cocultures. In autologous MLTR, antiestrogen treatment gave rise to enhanced Foxp3 expression of TIL/PBMC and decreased the number of apoptotic tumor cells. These effects were reversed by addition of a TGFbeta neutralizing antibody. Our findings offer evidence that antiestrogen induces immunosuppression in the tumor microenvironment, through a TGFbeta-dependent mechanism that may contribute to the development of antiestrogen resistance in breast cancer.

Cold Atmospheric Plasma Treatment Induces Anti-Proliferative Effects in Prostate Cancer Cells by Redox and Apoptotic Signaling Pathways

One of the promising possibilities of the clinical application of cold plasma, so-called cold atmospheric plasma (CAP), is its application on malignant cells and cancer tissue using its anti-neoplastic effects, primarily through the delivery of reactive oxygen and nitrogen species (ROS, RNS). In this study, we investigated the impact of CAP on cellular proliferation and consecutive molecular response mechanisms in established prostate cancer (PC) cell lines. PC cells showed a significantly reduced cell growth following CAP treatment as a result of both an immediate increase of intracellular peroxide levels and through the induction of apoptosis indicated by annexin V assay, TUNEL assay, and the evaluation of changes in nuclear morphology. Notably, co-administration of N-acetylcysteine (NAC) completely neutralized CAP effects by NAC uptake and rapid conversion to glutathione (GSH). Vitamin C could not counteract the CAP induced effects on cell growth. In summary, relatively short treatments with CAP of 10 seconds were sufficient to induce a significant inhibition of cancer proliferation, as observed for the first time in urogenital cancer. Therefore, it is important to understand the mode of CAP related cell death and clarify and optimize CAP as cancer therapy. Increased levels of peroxides can alter redox-regulated signaling pathways and can lead to growth arrest and apoptosis. We assume that the general intracellular redox homeostasis, especially the levels of cellular GSH and peroxidases such as peroxiredoxins affect the outcome of the CAP treatment.

Investigating the Mutagenicity of a Cold Argon-Plasma Jet in an HET-MN Model.

Objective So-called cold physical plasmas for biomedical applications generate reactive oxygen and nitrogen species and the latter can trigger DNA damage at high concentrations. Therefore, the mutagenic risks of a certified atmospheric pressure argon plasma jet (kINPen MED) and its predecessor model (kINPen 09) were assessed. Methods Inner egg membranes of fertilized chicken eggs received a single treatment with either the kINPen 09 (1.5, 2.0, or 2.5 min) or the kINPen MED (3, 4, 5, or 10 min). After three days of incubation, blood smears (panoptic May-Grünwald-Giemsa stain) were performed, and 1000 erythrocytes per egg were evaluated for the presence of polychromatic and normochromic nuclear staining as well as nuclear aberrations and binucleated cells (hen’s egg test for micronuclei induction, HET-MN). At the same time, the embryo mortality was documented. For each experiment, positive controls (cyclophosphamide and methotrexate) and negative controls (NaCl-solution, argon gas) were included. Additionally, the antioxidant potential of the blood plasma was assessed by ascorbic acid oxidation assay after treatment. Results For both plasma sources, there was no evidence of genotoxicity, although at the longest plasma exposure time of 10 min the mortality of the embryos exceeded 40%. The antioxidant potential in the egg’s blood plasma was not significantly reduced immediately (p = 0.32) or 1 h (p = 0.19) post exposure to cold plasma. Conclusion The longest plasma treatment time with the kINPen MED was 5–10 fold above the recommended limit for treatment of chronic wounds in clinics. We did not find mutagenic effects for any plasma treatment time using the either kINPen 09 or kINPen MED. The data provided with the current study seem to confirm the lack of a genotoxic potential suggesting that a veterinary or clinical application of these argon plasma jets does not pose mutagenic risks.

Estrogen receptor α attenuates transforming growth factor-β signaling in breast cancer cells independent from agonistic and antagonistic ligands

To investigate a presumed crosstalk between estrogen receptor α (ERα) and the TGF-β signaling pathway in breast cancer, we analyzed the TGF-β-induced expression of the plasminogen activator inhibitor 1 (PAI-1) gene in ER-positive MCF-7 cells. After siRNA-mediated knock-down of endogenous ERα, the transcription level of PAI-1 was upregulated, pointing to an attenuation of TGF-β signaling by the presence of ERα. We verified these findings by a vice versa approach using a primary ER-negative cell model transiently overexpressing either ERα or ERβ. We found that ERα, but not ERβ, led to a strong inhibition of the TGF-β1 signal, monitored by TGF-β reporter assays. This attenuation was completely independent of receptor stimulation by β-estradiol (E2) or inhibition by the pure antagonist ICI 182.780 (ICI). Our results indicate a permanent repression of PAI-1 by ERα and suggest a ligand-independent crosstalk between ERα and TGF-β signaling in breast cancer cells.

Immediate and transient phosphorylation of the heat shock protein 27 initiates chemoresistance in prostate cancer cells

Drug resistance minimizes the effects of prostate cancer (PC) chemotherapy with docetaxel and is generally considered to be associated with the expression of heat shock protein (HSP) 27 including various cytoprotective pathways. In the present study, we investigated the effects of HSP27 phosphorylation on PC cell growth underlying docetaxel treatment. Cell counting revealed significantly reduced cell growth during docetaxel treatment as a result of both activation of mitogen-activated protein kinase p38 (MAPK p38) and protein kinase D1 (PKD1), and, most importantly, the overexpression of the phosphorylation-mimicking mutant HSP27-3D. Further analysis revealed a docetaxel-dependent induction of HSP27 accompanied by an initial phosphorylation and rapid dephosphorylation of the protein. Based on the data, we can conclude that phosphorylation of HSP27 protein is a crucial mechanism in the initiation of chemoresistance in PC. Moreover, the results indicate a key impact of HSP27 on viability and proliferation of PC cells underlying anticancer therapy. The protective function depends on the initial phosphorylation status of HSP27 and represents a putative co-therapeutic target to prevent chemoresistance during docetaxel therapy.

Cytochrome P450 17A1 inhibitor abiraterone attenuates cellular growth of prostate cancer cells independently from androgen receptor signaling by modulation of oncogenic and apoptotic pathways

Abiraterone provides significant survival advantages in prostate cancer (PC), however, the current understanding of the molecular mechanisms of abiraterone is still limited. Therefore, the abiraterone impact on androgen receptor (AR)-positive LNCaP and AR-negative PC-3 cells was assessed by cellular and molecular analyses. The present study demonstrated, that abiraterone treatment significantly decreased cell growth, AR expression, and AR activity of AR-positive LNCaP cells. Notably, AR-negative PC-3 cells exhibited comparable reductions in cellular proliferation, associated with DNA fragmentation and pro-apoptotic modulation of p21, caspase-3, survivin, and transforming growth factor β (TGFβ). Our observations suggest that the attenuation of AR signaling is not the only rationale to explain the abiraterone anticancer activity. Abiraterone efficacy may play a more global role in PC progression control than originally hypothesized. In this regard, abiraterone is not only a promising drug for treatment of AR-negative PC stages, even more, abiraterone may represent an alternative for treatment of other malignancies besides prostate cancer.

Cardiolipin composition correlates with prostate cancer cell proliferation

Prostate cancer (PC) is the second most diagnosed cancer in men. It has been recognized that diet can play a crucial role in PC genesis and progression. In this context, free fatty acids are considered as modulators of cell proliferation. Recently, a relationship between the composition of the mitochondrial phospholipid cardiolipin (CL) and cell proliferation has been discussed. The aim of this study was to analyse the interrelationship between CL composition and the proliferation of prostate cells by exposing PC-3 tumour cells to different fatty acids and by analysing the CL composition in prostate tissue from PC patients after prostatectomy. Among the applied fatty acids, palmitic acid was found to stimulate proliferation of PC-3 cells, whereas oleic acid (OA) had an inhibiting effect. The lipidomic analysis of CL revealed that fatty acids supplied to PC-3 cells were incorporated into CL molecules. Further, the CL content of palmitoleic acid (C16:1) exclusively correlated with the proliferation of PC-3 cells. The CL composition significantly differed between tumour and normal prostate tissue from PC patients. In five out of six patients, the CL content of palmitoleic acid was higher in tumour prostate tissue in comparison to normal prostate tissue. Our data illustrate that the composition of CL can be easily modified by the fatty acid environment of cells. OA was most effective in decreasing the amount of palmitoleic acid within the CL molecules and deceleration of PC-3 cell proliferation. In conclusion, a diet rich in OA might be beneficial in protecting from rapid proliferation of PC cells.

MicroRNA-1 properties in cancer regulatory networks and tumor biology

Short non-coding microRNAs have been identified to orchestrate crucial mechanisms in cancer progression and treatment resistance. MicroRNAs are involved in posttranscriptional modulation of gene expression and therefore represent promising targets for anticancer therapy. As mircoRNA-1 (miR-1) exerted to be predominantly downregulated in the majority of examined tumors, miR-1 is classified to be a tumor suppressor with high potential to diminish tumor development and therapy resistance. Here we review the complex functionality of miR-1 in tumor biology.

Jump in the fire — heat shock proteins and their impact on ovarian cancer therapy

Ovarian cancer (OC) is a major problem in gynecological oncology. Options for diagnosis and treatment of advanced stages and thus for patient prognosis have not been improved substantially over the past decades. Heat shock proteins (HSP) are characterized as stress-induced molecular chaperones performing cell survival factor functions. In cancer cells, various crucial and clinically important cell responses are vitally influenced and modulated by HSPs, e.g., cell growth and treatment resistance. Despite the limited knowledge on HSPs in OC progression, their roles as biomarkers, prognostic factors and their drug target properties appears promising for future clinical applications and therapeutic approaches.

Telomeres and Telomerase in Hematopoietic Dysfunction: Prognostic Implications and Pharmacological Interventions

Leukocyte telomere length (TL) has been suggested as a marker of biological age in healthy individuals, but can also reflect inherited and acquired hematopoietic dysfunctions or indicate an increased turnover of the hematopoietic stem and progenitor cell compartment. In addition, TL is able to predict the response rate of tyrosine kinase inhibitor therapy in chronic myeloid leukemia (CML), indicates clinical outcomes in chronic lymphocytic leukemia (CLL), and can be used as screening tool for genetic sequencing of selected genes in patients with inherited bone marrow failure syndromes (BMFS). In tumor cells and clonal hematopoietic disorders, telomeres are continuously stabilized by reactivation of telomerase, which can selectively be targeted by telomerase-specific therapy. The use of the telomerase inhibitor Imetelstat in patients with essential thrombocythmia or myelofibrosis as well as the use of dendritic cell-based telomerase vaccination in AML patients with complete remissions are promising examples for anti-telomerase targeted strategies in hematologic malignancies. In contrast, the elevation in telomerase levels through treatment with androgens has become an exciting clinical intervention for patients with BMFS. Here, we review recent developments, which highlight the impact of telomeres and telomerase targeted therapies in hematologic dysfunctions.