In vitro studies on the modification of low-dose hyper-radiosensitivity in prostate cancer cells by incubation with genistein and estradiol

<p>Abstract</p> <p>Background</p> <p>As the majority of prostate cancers (PC) express estrogen receptors, we evaluated the combination of radiation and estrogenic stimulation (estrogen and genistein) on the radiosensitivity of PC cells in vitro.</p> <p>Methods</p> <p>PC cells LNCaP (androgen-sensitive) and PC-3 (androgen-independent) were evaluated. Estrogen receptor (ER) expression was analyzed by means of immunostaining. Cells were incubated in FCS-free media with genistein 10 μM and estradiol 10 μM 24 h before irradiation and up to 24 h after irradiation. Clonogenic survival, cell cycle changes, and expression of p21 were assessed.</p> <p>Results</p> <p>LNCaP expressed both ER-α and ER-β, PC-3 did not. Incubation of LNCaP and PC-3 with genistein resulted in a significant reduction of clonogenic survival. Incubation with estradiol exhibited in low concentrations (0.01 μM) stimulatory effects, while higher concentrations did not influence survival. Both genistein 10 μM and estradiol 10 μM increased low-dose hyper-radiosensitivity [HRS] in LNCaP, while hormonal incubation abolished HRS in PC-3. In LNCaP cells hormonal stimulation inhibited p21 induction after irradiation with 4 Gy. In PC-3 cells, the proportion of cells in G2/M was increased after irradiation with 4 Gy.</p> <p>Conclusion</p> <p>We found an increased HRS to low irradiation doses after incubation with estradiol or genistein in ER-α and ER-β positive LNCaP cells. This is of high clinical interest, as this tumor model reflects a locally advanced, androgen dependent PC. In contrast, in ER-α and ER-β negative PC-3 cells we observed an abolishing of the HRS to low irradiation doses by hormonal stimulation. The effects of both tested compounds on survival were ER and p53 independent. Since genistein and estradiol effects in both cell lines were comparable, neither ER- nor p53-expression seemed to play a role in the linked signalling. Nevertheless both compounds targeted the same molecular switch. To identify the underlying molecular mechanisms, further studies are needed.</p

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation

Purpose : To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. Materials and methods : Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200 kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell beta -galactosidase (beta -gal) staining at pH 6.0; and the secretion of transforming growth factor-beta (TGF-beta1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. Results: A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041 +/- 0.015 to 0.63 +/- 0.05. Radiation-induced clonal differentiation as well as the secretion of TGF-beta1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. Conclusions : In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes

Regional transient portal ischemia and irradiation as preparative regimen for hepatocyte transplantation.

Hepatocyte transplantation is regarded as a promising option to correct hereditary metabolic liver disease. This study describes a novel method involving regional transient portal ischemia (RTPI) in combination with hepatic irradiation (IR) as a preparative regimen for hepatocyte transplantation. The right lobules of rat livers (45% of liver mass) were subjected to RTPI of 30-120 min. Liver specimens and serum samples were analyzed for transaminase levels, DNA damage, apoptosis, and proliferation. Repopulation experiments involved livers of dipeptidylpeptidase IV (DPPIV)-deficient rats preconditioned with RTPI (60-90 min) either with or without prior partial hepatic IR (25 Gy). After reperfusion intervals of 1 and 24 h, 12 million wild-type (DPPIV positive) hepatocytes were transplanted into recipient livers via the spleen. RTPI of 60-90 min caused limited hepatic injury through necrosis and induced a distinct regenerative response in the host liver. Twelve weeks following transplantation, small clusters of donor hepatocytes were detected within the portal areas. Quantitative analysis revealed limited engraftment of 0.79% to 2.95%, whereas control animals (sham OP) exhibited 4.16% (determined as relative activity of DPPIV when compared to wild-type liver). Repopulation was significantly enhanced (21.43%) when IR was performed prior to RTPI, optimum preconditioning settings being 90 min of ischemia and 1 h of reperfusion before transplantation. We demonstrate that RTPI alone is disadvantageous to donor cell engraftment, whereas the combination of IR with RTPI comprises an effective preparative regimen for liver repopulation. The method described clearly has potential for clinical application

Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII

Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes’ Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes’ systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00395-013-0385-6) contains supplementary material, which is available to authorized users