12338
2012
eng
104-110
7
3
article
1
2015-12-17
--
--
Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells
AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation.
METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 x 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on -actin and hypoxia-inducible factor (HIF)-1 mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant).
RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results.
CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.
World Journal of Clinical Oncology
10.5306/wjco.v3.i7.104
PMC3394081
urn:nbn:de:bvb:20-opus-123385
World Journal of Clinical Oncology. 3(7):104-10. doi:10.5306/wjco.v3.i7.104
Harun M. Said
Buelent Polat
Susanne Stein
Mathias Guckenberger
Carsten Hagemann
Adrian Staab
Astrid Katzer
Jelena Anacker
Michael Flentje
Dirk Vordermark
deu
uncontrolled
Strahlentherapie
eng
uncontrolled
brain cancer
eng
uncontrolled
radiotherapy
eng
uncontrolled
human cancer diseases
eng
uncontrolled
Short dsRNA oligonucleotides
eng
uncontrolled
N-Myc down regulated gene 1
Krankheiten
open_access
Klinik und Poliklinik für Strahlentherapie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12338/WJCO-3-104.pdf
9611
2013
eng
article
1
--
--
--
Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci
Background
High expression of constitutive histone γ-H2AX, a sensitive marker of DNA damage, might be indicative of defective DNA repair pathway or genomic instability. 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. This study explores the relationship between the clinical radiosensitivity of tumor patients and the expression/induction of γ-H2AX and 53BP1 in vitro.
Methods
Using immunostaining, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53 BP1 in peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=57) undergoing radiotherapy (RT). Cells from apparently healthy donors (n=12) served as references.
Results
Non-irradiated cells from controls and unselected BC patients exhibited similar baseline levels of DNA damage assessed by γ-H2AX and 53BP1 foci. At the same time, the γ-H2AX assay of in vitro irradiated cells revealed significant differences between the control group and the group of unselected BC patients with respect to the initial (0.5 Gy, 30 min) and residual (2 Gy, 24 h post-radiation) DNA damage. The numbers of 53BP1 foci analyzed in 35 BC patients were significantly higher than in controls only in case of residual DNA damage. A weak correlation was found between residual foci of both proteins tested. In addition, cells from cancer patients with an adverse acute skin reaction (grade 3) to RT showed significantly increased radiation-induced γ-H2AX foci and their protracted disappearance compared to the group of BC patients with normal skin reaction (grade 0–1). The mean number of γ-H2AX foci after 5 clinical fractions was significantly higher than that before RT, especially in clinically radiosensitive patients.
Conclusions
The γ-H2AX assay may have potential for screening individual radiosensitivity of breast cancer patients.
Radiation Oncology
10.1186/1748-717X-8-98
http://www.ro-journal.com/content/8/1/98
urn:nbn:de:bvb:20-opus-96110
In: Radiation Oncology (2013) 8: 98, doi:10.1186/1748-717X-8-98
Cholpon S. Djuzenova
Ines Elsner
Astrid Katzer
Eike Worschech
Luitpold V. Distel
Michael Flentje
Bülent Polat
eng
uncontrolled
DNA damage
eng
uncontrolled
DNA repair
eng
uncontrolled
Peripheral blood lymphocytes
eng
uncontrolled
Radiosensitivity
deu
swd
DNS-Schädigung
deu
swd
DNS-Reparatur
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Förderzeitraum 2013
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/9611/Djuzenova_1748-717X-8-98.pdf
11132
2014
eng
article
1
2015-03-19
--
--
Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status
Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
10.1371/journal.pone.0087052
urn:nbn:de:bvb:20-opus-111322
PLoS ONE 9(1): e87052. doi:10.1371/journal.pone.0087052
Cholpon S. Djuzenova
Simon Memmel
Vladimir L. Sukhorukov
Marcus Höring
Katherine Westerling
Vanessa Fiedler
Astrid Katzer
Georg Krohne
Michael Flentje
eng
uncontrolled
cell membranes
eng
uncontrolled
hypotonic
eng
uncontrolled
capacitance
eng
uncontrolled
isotonic
eng
uncontrolled
microvilli
eng
uncontrolled
membrane characteristics
eng
uncontrolled
membrane proteins
eng
uncontrolled
scanning electron microscopy
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2014
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/11132/026_Sukhorukov_PLoS.pdf
12530
2015
eng
856
15
article
1
2016-01-25
--
--
A prospective study on histone γ-H2AX and 53BP1 foci expression in rectal carcinoma patients: correlation with radiation therapy-induced outcome
Background
The prognostic value of histone γ-H2AX and 53BP1 proteins to predict the radiotherapy (RT) outcome of patients with rectal carcinoma (RC) was evaluated in a prospective study. High expression of the constitutive histone γ-H2AX is indicative of defective DNA repair pathway and/or genomic instability, whereas 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor.
Methods
Using fluorescence microscopy, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53BP1 in peripheral blood mononuclear cells derived from unselected RC patients (n = 53) undergoing neoadjuvant chemo- and RT. Cells from apparently healthy donors (n = 12) served as references.
Results
The γ-H2AX assay of in vitro irradiated lymphocytes revealed significantly higher degree of DNA damage in the group of unselected RC patients with respect to the background, initial (0.5 Gy, 30 min) and residual (0.5 Gy and 2 Gy, 24 h post-radiation) damage compared to the control group. Likewise, the numbers of 53BP1 foci analyzed in the samples from 46 RC patients were significantly higher than in controls except for the background DNA damage. However, both markers were not able to predict tumor stage, gastrointestinal toxicity or tumor regression after curative RT. Interestingly, the mean baseline and induced DNA damage was found to be lower in the group of RC patients with tumor stage IV (n = 7) as compared with the stage III (n = 35). The difference, however, did not reach statistical significance, apparently, because of the limited number of patients.
Conclusions
The study shows higher expression of γ-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome.
BMC Cancer
10.1186/s12885-015-1890-9
urn:nbn:de:bvb:20-opus-125303
BMC Cancer (2015) 15:856. DOI 10.1186/s12885-015-1890-9
Cholpon S. Djuzenova
Marcus Zimmermann
Astrid Katzer
Vanessa Fiedler
Luitpold V. Distel
Martin Gasser
Anna-Maria Waaga-Gasser
Michael Flentje
Bülent Polat
eng
uncontrolled
radiosensitivity
eng
uncontrolled
peripheral blood lymphocytes
eng
uncontrolled
DNA repair
eng
uncontrolled
DNA damage
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Förderzeitraum 2015
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12530/Djuzenova_10_1186_s12885-015-1890-9.pdf
17777
2016
eng
38191-38209
25
7
article
1
2019-03-04
--
--
Dual PI3K-and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule
Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.
Oncotarget
10.18632/oncotarget.9501
urn:nbn:de:bvb:20-opus-177770
Oncotarget 2016, 7:25, 38191-38209. DOI: 10.18632/oncotarget.9501
false
true
Cholpon S. Djuzenova
Vanessa Fiedler
Astrid Katzer
Konstanze Michel
Stefanie Deckert
Heiko Zimmermann
Vladimir L. Sukhorukov
Michael Flentje
eng
uncontrolled
cell cycle arrest
eng
uncontrolled
radiation sensitivity
eng
uncontrolled
histone γH2AX
eng
uncontrolled
DNA damage
eng
uncontrolled
colony survival
Pharmakologie, Therapeutik
open_access
Klinik und Poliklinik für Strahlentherapie
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17777/Djuzenova_Oncotarget.pdf
17071
2017
eng
45298-45310
28
8
article
1
2018-10-30
--
--
Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities
High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
Oncotarget
10.18632/oncotarget.16847
28424411
urn:nbn:de:bvb:20-opus-170719
Oncotarget 2017, Vol. 8, No. 28, 45298-45310. DOI: 10.18632/oncotarget.16847
false
true
Simon Memmel
Dmitri Sisario
Caren Zöller
Vanessa Fiedler
Astrid Katzer
Robin Heiden
Nicholas Becker
Lorenz Eing
Fábio L.R. Ferreira
Heiko Zimmermann
Markus Sauer
Michael Flentje
Vladimir L. Sukhorukov
Cholpon S. Djuzenova
eng
uncontrolled
chemotherapy
eng
uncontrolled
glioblastoma multiforme
eng
uncontrolled
migration
eng
uncontrolled
treatment
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/17071/057_Memmel_ONCOTARGET.pdf
20029
2019
eng
299
19
article
1
2020-03-03
--
--
Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells
Background
Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.
Methods
Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition.
Results
We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy.
Conclusions
Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.
BMC Cancer
10.1186/s12885-019-5517-4
urn:nbn:de:bvb:20-opus-200290
BMC Cancer (2019) 19:299, https://doi.org/10.1186/s12885-019-5517-4
false
true
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Cholpon S. Djuzenova
Vanessa Fiedler
Simon Memmel
Astrid Katzer
Dmitri Sisario
Philippa K. Brosch
Alexander Göhrung
Svenja Frister
Heiko Zimmermann
Michael Flentje
Vladimir L. Sukhorukov
eng
uncontrolled
DNA damage
eng
uncontrolled
glioblastoma multiforme
eng
uncontrolled
histone H2AX
eng
uncontrolled
irradiation
eng
uncontrolled
migration
eng
uncontrolled
mTOR
eng
uncontrolled
PTEN
eng
uncontrolled
p53
eng
uncontrolled
radiation sensitivity
eng
uncontrolled
wound healing
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2019
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/20029/Cholpon_S_Djuzenova_BMCCancer_2019.pdf
26582
2021
eng
21
article
1
2022-04-05
--
--
Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs
Background: Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.
Methods: Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by gamma H2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations.
Results: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (similar to 80% vs. similar to 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells.
Conclusions: The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism.
BMC Cancer
10.1186/s12885-021-08930-1
urn:nbn:de:bvb:20-opus-265826
publish
BMC Cancer (2021) 21:1201. https://doi.org/10.1186/s12885-021-08930-1
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Cholpon S. Djuzenova
Thomas Fischer
Astrid Katzer
Dmitri Sisario
Tessa Korsa
Gudrun Streussloff
Vladimir L. Sukhorukov
Michael Flentje
eng
uncontrolled
DNA damage
eng
uncontrolled
DNA-PK
eng
uncontrolled
Histone gamma H2AX
eng
uncontrolled
p53
eng
uncontrolled
Radiation sensitivity
Medizin und Gesundheit
open_access
Klinik und Poliklinik für Strahlentherapie
Theodor-Boveri-Institut für Biowissenschaften
Förderzeitraum 2021
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/26582/s12885-021-08930-1.pdf