Abstract
Objective To investigate plasma steroid hormone levels in postmenopausal breast cancer patients with and without adjuvant endocrine therapy and in healthy postmenopausal women.
Methods ...Steroid hormone levels in postmenopausal breast cancer patients treated with aromatase inhibitors (n = 32) were compared with breast cancer patients treated with tamoxifen (n = 34), breast cancer patients without adjuvant endocrine therapy (n = 15), and healthy postmenopausal women (n = 56). Pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone (DHEA), androstenedione, total testosterone, dihydrotestosterone, estrone and estradiol were measured using liquid chromatography-tandem mass spectrometry. Sex hormone binding globulin was measured by solid-phase chemiluminescent immunometric assays, and the free androgen index was calculated.
Results Aromatase inhibitor users did not differ in dihydrotestosterone, total testosterone, androstenedione, DHEA, or free androgen index levels from healthy controls or untreated breast cancer patients. The highest total testosterone levels were found in tamoxifen-treated women, who had significantly higher plasma concentrations than both women treated with aromatase inhibitors and breast cancer patients without adjuvant treatment. Concentrations of cortisol and cortisone were significantly greater in aromatase inhibitor users as well as tamoxifen users, in comparison with healthy controls and untreated breast cancer patients. Aromatase inhibitor users had lower estrone and estradiol plasma concentrations than all other groups.
Conclusion Adjuvant treatment with aromatase inhibitors or tamoxifen was associated with increased cortisol and cortisone plasma concentrations as well as decreased estradiol concentrations. Androgen levels were elevated in tamoxifen-treated women but not in aromatase inhibitor users.
Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high ...efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.
The anti-tumour drug treosulfan (L-threitol 1,4-bismethanesulphonate, Ovastat) is a prodrug for epoxy compounds by converting non-enzymatically to L-diepoxybutane via the corresponding monoepoxide ...under physiological conditions. The present study supports the hypothesis that this conversion of treosulfan is required for cytotoxicity in vitro. DNA alkylation and interstrand cross-linking of plasmid DNA is observed after treosulfan treatment, but this is again produced via the epoxide species. Alkylation occurs at guanine bases with a sequence selectivity similar to other alkylating agents such as the nitrogen mustards. In treosulfan-treated K562 cells, cross-links form slowly, reaching a peak at approximately 24 h. Incubation of K562 cells with preformed epoxides shows faster and more efficient DNA cross-linking.
Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage‐specific transcriptional programs. Structure‐specific recognition protein‐1 ...(SSRP1) is a histone chaperone which is important for chromatin‐associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage‐specific effects by increasing expression of adipocyte‐specific genes and decreasing the expression of osteoblast‐specific genes. Consistent with a role in controlling lineage specification, transcriptome‐wide RNA‐sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active β‐catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. Stem Cells 2016;34:1369–1376
Treosulphan has recently demonstrated antileukaemic activity and potent haematopoietic stem cell toxicity. Dose-escalated treosulphan (3 x 12 or 3 x 14 g/m2) combined with cyclophosphamide (Cy) was ...chosen for a new preparative regimen before allogeneic haematopoietic stem cell transplantation in 18 patients (median age 44, range 19-64 years) with haematological malignancies, considered ineligible for other myeloablative preparative regimens. Pharmacokinetic studies demonstrated rapid treosulphan plasma clearance and a dose-dependent increase of its maximum plasma concentrations and area under the concentration-time curves. Rapid and sustained white blood cell and platelet recovery and full donor chimerism was attained in all evaluable patients. Nonhaematological regimen-related CTC grades 3-4 adverse events were transient and predominantly consisted of cardiac (28%), gastrointestinal (39%), and hepatic (39%) toxicities. The 1-year nonrelapse mortality was 22%. Principal causes of transplant-related lethal events were infections in three of four affected patients. Only one patient died from regimen-related cardiac toxicity. The 1-year relapse estimate is 22%, overall and progression-free survival estimates are 67 and 56%, respectively. In conclusion, this new treosulphan and Cy combination is an effective, comparatively well-tolerated myeloablative preparative regimen even in patients with an increased risk for regimen-related toxic complications.
Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a ...highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimer’s disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7(−/−) fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.
Treosulfan (L-threitol-1,4-bis-methanesulphonate; Ovastat®) is a bifunctional alkylating drug indicated for the treatment of advanced ovarian carcinoma. Recent data revealed immunosuppressive ...characteristics and substantial haematopoietic stem cell toxicity after repeated dosing of mice. Therefore, treosulfan is considered to be an alternative conditioning agent to busulfan (for example) administered prior to allogeneic/autologous stem cell transplantation of patients with haematological malignancies. An antineoplastic activity for treosulfan has been previously shown in preclinical models of melanoma, breast, lung and renal-cell carcinomas. Here, in vivo antileukaemic activity of treosulfan is compared with the activity of equitoxic doses of cyclophosphamide or busulfan for the first time using human acute lymphoblastic leukaemia (ALL)-models of paediatric origin xenotransplanted into non-obese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Treosulfan treatment achieved an optimum treated to control (T/C) value of 159% (survival time) against B-ALL-SCID 7 and a T/C value of 0% (tumour growth) against T-ALL-SCID 4 and proB-ALL-SCID 19, respectively. Complete regression of established subcutaneously (s.c.) growing nodules of ALL-SCID 4 and 19 was obvious and long-term survivors without tumour re-growth were observed. Equitoxic doses of busulfan (ALL-SCID 4, 7, 19) or cyclophosphamide (ALL-SCID 19) were less effective with regard to the numbers of complete regressions and the number of cured animals. Side-effects included myelotoxicity and a small reduction in body weight, but these were tolerable. Treosulfan can be considered a highly active antileukaemic drug whose corresponding clinical value is to be tested in appropriate protocols with leukaemic patients.
Purpose
5′ adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely ...defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor.
Methods
High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation.
Results
We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux.
Conclusions
The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.
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