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Autor     Shuchen Gu, Natalia Papadopoulou, Eva-Maria Gehring, Omaima Nasir, Konstantinos Dimas, Shefalee K Bhavsar, Michael Föller, Konstantinos Alevizopoulos, Florian Lang, Christos Stournaras
Titel    Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo
Zeitschrift    Molecular Cancer
Verlag    Springer
Ausgabe    8
Datum    1. Dezember 2009
Seiten    114ff
Anmerkung    1. Shg stellt auf Seite 61 seiner Dissertation den Artikel explizit in den Zusammenhang mit seiner Diplomarbeit. Wörtlich heißt es:
"Previously, it has been reported that activation of mAR with non permeable testosterone derivatives induced pro-apoptotic responses [Gu S Diploma Thesis, Gu S, et al. 2009]."
Also besteht an den Stellen, wo sich Material aus Gu et al (2009) wiederfinden lässt, die Möglichkeit, dass es sich hierbei um Inhalte handelt, die bereits im Rahmen der Diplomarbeit präsentiert wurden. Die mathematisch-naturwissenschaftliche Fakultät der Universität Tübingen verlangt für die Zulassung zum Promotionsverfahren, dass Kandidaten/Kandidatinnen mit ihrer Unterschrift bestätigen: "Die vorgelegte Dissertation ist noch nicht ganz oder teilweise veröffentlicht worden (gemeint ist: als Dissertation) und sie ist noch nicht ganz oder teilweise als Dissertation oder sonstige Prüfungsarbeit eingereicht worden." (vgl. Erklärungen zum Antrag auf Zulassung zum Promotionsverfahren) bzw. andernfalls "eine Erklärung beifügen, wann und wo, in welchem Fach und mit welchem Ergebnis" dies bereits geschehen ist.

2. Im Artikel selbst findet sich eine explizite Aufschlüsselung der "Authors' contributions". Dort heißt es:
"SG and NP carried out the mAR staining in colon tumors and cell lines, the analysis of actin and tubulin reorganization and the pro-apoptotic responses. EMG carried out the binding studies. ON performed the animal experiments. KD prepared the mouse xenografts. SKB carried out the flutamide and cytochalasin B control experiments. MF participated in the design of the study and performed the statistical analysis. KA participated in the design of the study and drafting of the manuscript. FL participated in the coordination of the study and evaluation of the results. CS conceived of the study, participated in the design coordination and drafting of the manuscript. All authors read and approved the final manuscript."
Ausdrücklich wird also die Formulierung des Manuskripts den Autoren Konstantinos Alevizopoulos und Christos Stournaras zugeschrieben. Shg selbst hat demnach nicht zu den Formulierungen des Artikels beigetragen, diese lediglich "gelesen und genehmigt". Sämtliche in der Dissertation von Shg wiederzufindende Passagen aus Gu et al (2009) stammen also nicht von Shg und sind somit, wo nicht explizit auf den Artikel verwiesen wird, analog zu ungekennzeichneten Übernahmen aus einem von anderen Autoren verfassten Aufsatz zu werten.

Zum Zwecke der Dokumentation werden die Seitenzahlen des verlinkten PDF Files verwendet.
DOI    10.1186/1476-4598-8-114
URL    http://www.molecular-cancer.com/content/8/1/114

Literaturverz.   

ja
Fußnoten    ja
Fragmente    23


Fragmente der Quelle:
[1.] Shg/Fragment 017 01 - Diskussion
Zuletzt bearbeitet: 2014-11-02 01:26:47 Hindemith
Fragment, Gesichtet, Gu et al 2009, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Shg

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Untersuchte Arbeit:
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Quelle: Gu et al 2009
Seite(n): 1-2, Zeilen: 1: li.Sp.1-2 - re.Sp. 1-3 - 2: li.Sp. 1-9
1.1.2. Membrane Androgen Receptor

Scientific evidence accumulated in recent year’s [sic] points to the existence of membrane androgen receptors (mARs), triggering rapid, non-genomic signals. Although the exact molecular identity of mAR still remains unknown, non-genomic androgen actions manifested within minutes have been reported in various cell types including macrophages and T cells [Benten WP, et al. 1999; Benten WP, et al. 1999], LNCaP [Kampa M, et al. 2002; Wang Z, et al. 2008], T47D [Kampa M, et al. 2005], MCF7 [Kallergi G, et al. 2007], DU145 [Hatzoglou A, et al. 2005; Papadopoulou N, et al. 2008a; Papadopoulou N, et al 2008b], C6 [Gatson JW, et al. 2006], PC12 [Alexaki VI, et al. 2006] or VSMC cells [Somjen D, et al 2004]. These effects are clearly different from those manifested upon activation of the intracellular androgen receptors (iARs) mediating genomic androgen signals resulting in receptor dimerization, nuclear translocation and subsequent activation of androgen-specific target genes.


Alexaki VI, Charalampopoulos I, Kampa M, Nifli AP, Hatzoglou A, Gravanis A, Castanas E. (2006). Activation of membrane estrogen receptors induce pro-survival kinases. J Steroid Biochem Mol Biol. 98:97-110.

Benten WP, et al. (1999a). Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol Biol Cell10: 3113-3123.

Benten WP, et al.(1999b). Functional testosterone receptors in plasma membranes of T cells. Faseb J, 13:123-133.

Gatson JW, Kaur P, Singh M. (2006). Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology 147:2028-2034.

Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, et al. (2005). Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab, 90: 893-903.

Kallergi G, Agelaki S, Markomanolaki H, Georgoulias V, Stournaras C. (2007). Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells. Cell Physiol Biochem, 20:977-986.

Kampa M, Papakonstanti EA, Hatzoglou A, Stathopoulos EN, Stournaras C, Castanas E. (2002). The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton. Faseb J, 16:1429-1431.

Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E. (2006) Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther, 5:1342-1351.

Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C. (2008 a). Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res, 314: 3162-3174.

Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C. (2008 b). Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc. 7:88.

Somjen D, Kohen F, Gayer B, Kulik T, Knoll E, Stern N. (2004). Role of putative membrane receptors in the effect of androgens on human vascular cell growth. J Endocrinol. 180:97-106.

Wang Z, Liu L, Hou J, Wen D, Yan C, Pu J, Ouyang J, Pan H. (2008). Rapid membrane effect of testosterone in LNCaP cells. Urol Int.81(3):353-9

[Seite 1]

Introduction

Scientific evidence accumulated in recent years points to the existence of membrane androgen receptors (mAR), triggering rapid, non-genomic signals. Although the exact molecular identity of mAR still remains unknown, nongenomic androgen actions manifested within minutes

[Seite 2]

have been reported in various cell types including macrophages and T cells [1,2], LNCaP [3,4], T47D [5], MCF7 [6], DU145 [7-9], C6 [10], PC12 [11] or VSMC cells [12]. These effects are clearly different from those manifested upon activation of the intracellular androgen receptors (iAR) mediating genomic androgen signals resulting in receptor dimerization, nuclear translocation and subsequent activation of androgen-specific target genes (reviewed in [13]).


1. Benten WP, Lieberherr M, Stamm O, Wrehlke C, Guo Z, Wunderlich F: Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol Biol Cell 1999, 10:3113-3123.

2. Benten WP, Lieberherr M, Giese G, Wrehlke C, Stamm O, Sekeris CE, Mossmann H, Wunderlich F: Functional testosterone receptors in plasma membranes of T cells. Faseb J 1999, 13:123-133.

3. Kampa M, Papakonstanti EA, Hatzoglou A, Stathopoulos EN, Stournaras C, Castanas E: The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton. Faseb J 2002, 16:1429-1431.

4. Wang Z, Liu L, Hou J, Wen D, Yan C, Pu J, Ouyang J, Pan H: Rapid membrane effect of testosterone in LNCaP cells. Urol Int 2008, 81(3):353-359.

5. Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E: Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther 2006, 5:1342-1351.

6. Kallergi G, Agelaki S, Markomanolaki H, Georgoulias V, Stournaras C: Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells. Cell Physiol Biochem 2007, 20:977-986.

7. Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, Gravanis A, Castanas E: Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab 2005, 90:893-903.

8. Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C: Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res 2008, 314:3162-3174.

9. Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C: Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc 2008, 7:88.

10. Gatson JW, Kaur P, Singh M: Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology 2006, 147:2028-2034.

11. Alexaki VI, Charalampopoulos I, Kampa M, Nifli AP, Hatzoglou A, Gravanis A, Castanas E: Activation of membrane estrogen receptors induce pro-survival kinases. J Steroid Biochem Mol Biol 2006, 98:97-110.

12. Somjen D, Kohen F, Gayer B, Kulik T, Knoll E, Stern N: Role of putative membrane receptors in the effect of androgens on human vascular cell growth. J Endocrinol 2004, 180:97-106.

13. Heinlein CA, Chang C: Androgen receptor in prostate cancer. Endocr Rev 2004, 25:276-308.

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Übernahme eines Fremdtextes (inkl. aller dort zu findenden Literaturreferenzen) ohne jede Kennzeichnung.

Sichter
(Graf Isolan), SleepyHollow02

[2.] Shg/Fragment 020 01 - Diskussion
Zuletzt bearbeitet: 2014-11-02 15:35:54 Graf Isolan
BauernOpfer, Fragment, Gesichtet, Gu et al 2009, SMWFragment, Schutzlevel sysop, Shg

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Untersuchte Arbeit:
Seite: 20, Zeilen: 1-8, 10-21
Quelle: Gu et al 2009
Seite(n): 2, Zeilen: li.Sp. 10-19, 24-32, 41-53 - re.Sp. 1-
[Taken together, these studies clearly established that functional mARs trigger strong anti-tumorigenic effects in prostate and breast cancer cells, implying a] potential role of mAR as a novel target for the development of selective cancer treatments [Papadopoulou et al., 2009]. It was shown that mAR activation resulted in actin reorganization regulated by distinct mechanisms involving small GTPases’ specific signaling cascades. [Papadopoulou N, et al 2008b] Furthermore, it was shown that mAR activation induced potent apoptotic regression of prostate cancer cells in vitro [Papadopoulou N, et al 2008a] and in mouse xenografts in vivo and suppressed cell growth and motility. [Hatzoglou A, et al 2005, Kampa et al 2006] [...] However, it remained elusive whether mARs were also expressed in other tumors and whether their activation could result in the induction of anti-tumorigenic effects similar to the ones described in prostate and breast cancer cells.

In my diploma thesis, by using either colon cancer tissues isolated from mice xenograft tumors or two established colon cancer cell lines (Caco2 and HCT116 cells), the expression and function of functional role of mAR has been analyzed. As a result, testosterone binding sites were expressed in the membrane of colon cancer cells and qualify as bona fide membrane androgen receptors as assessed by radioligand binding studies, Scatchard analysis and displacement assays. The activation of those receptors with non permeable testosterone derivatives induced pro-apoptotic responses. [Gu S, et al. 2009].


Gu S, Papadopoulou N, Gehring EM, Nasir O, Dimas K, Bhavsar SK, Foller M, Alevizopoulos K, Lang F, Stournaras C. (2009) Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo. Mol Cancer. 8:114.

Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, et al. (2005). Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab, 90: 893-903.

Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E. (2006) Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther, 5:1342-1351.

Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C. (2008 a). Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res, 314: 3162-3174.

Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C. (2008 b). Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc. 7:88.

Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C. (2009). Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life, 61(1): 56-61.

The mAR-dependent signaling was recently characterized in detail in prostate and breast cancer cell lines (reviewed in [14-17]). Using non-permeable androgen derivatives that do not bind to iAR, it was shown that mAR activation resulted in actin reorganization regulated by mechanisms involving small GTPases [8,18]. Furthermore, it was shown that mAR activation induced profound apoptotic regression of prostate cancer cells in vitro and in mouse xenografts in vivo [7,19] and suppressed cell growth and motility [6,19]. [...]

Taken together, these studies clearly established that functional mARs trigger strong anti-tumorigenic effects, implying a potential role of mAR as a novel target for the development of selective cancer treatments (reviewed in [17]). However, it remained elusive whether mARs are also expressed in other tumors and whether their activation could result in the induction of anti-tumorigenic effects similar to the ones described in prostate and breast cancer cells. [...] Since the membrane androgen receptor, in contrast to the classical intracellular androgen receptor, induces tumor regression in target tissues (reviewed in [17]), we sought to determine the expression and functional status of mAR in colon cancer. To this end, we used colon cancer tissues isolated from mice xenograft tumors and from two established colon cancer cell lines (Caco2 and HCT116 cells). As a result, testosterone binding sites were expressed in the membrane of colon cancer cells and qualify as bona fide membrane androgen receptors as assessed by radioligand binding studies, Scatchard analysis and displacement assays. The activation of those receptors with nonpermeable testosterone derivatives triggered rapid and profound actin and tubulin cytoskeleton reorganization and induced pro-apoptotic responses.


6. Kallergi G, Agelaki S, Markomanolaki H, Georgoulias V, Stournaras C: Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells. Cell Physiol Biochem 2007, 20:977-986.

7. Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, Gravanis A, Castanas E: Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab 2005, 90:893-903.

8. Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C: Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res 2008, 314:3162-3174.

9. Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C: Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc 2008, 7:88.

14. Kampa M, Pelekanou V, Castanas E: Membrane-initiated steroid action in breast and prostate cancer. Steroids 2008, 73(9-10):953-960.

15. Michels G, Hoppe UC: Rapid actions of androgens Front Neuroendocrinol 2008, 29(2):182-198.

16. Foradori CD, Weiser MJ, Handa RJ: Non-genomic actions of androgens. Front Neuroendocrinol 2008, 29(2):169-181.

17. Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C: Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life 2009, 61(1):56-61.

18. Papakonstanti EA, Kampa M, Castanas E, Stournaras C: A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors. Mol Endocrinol 2003, 17:870-881.

19. Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E: Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther 2006, 5:1342-1351.

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Übernahme von Formulierungen eines Fremdtextes (inkl. aller dort zu findenden Literaturreferenzen) ohne jede Kennzeichnung.

Durch den Vergleich ergibt sich auch (implizit), dass Shg die an dieser Stelle in Gu et al (2009) beschriebenen Untersuchungen als Inhalt ihrer "Diploma thesis" ansieht.

Sichter
(Graf Isolan), SleepyHollow02

[3.] Shg/Fragment 041 01 - Diskussion
Zuletzt bearbeitet: 2014-11-02 13:08:15 Singulus
BauernOpfer, Fragment, Gesichtet, Gu et al 2009, SMWFragment, Schutzlevel sysop, Shg

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Quelle: Gu et al 2009
Seite(n): 2, 4, Zeilen: 2: re.Sp. 9-24, 28-36; 4: re.Sp. 7-12
3.2 Methods

3.2.1. Cell culture

The Caco2 human colon cancer cell lines and IEC06 non transformed intestinal cells were obtained from the American Type Culture Collection (Manassas, VA) and were studied between passages 60 and 70. CACO2 at 20,000/ml were cultured in DEME medium supplemented with 20% fetal bovine serum in culture flasks in a CO2 incubator at 37°C. Based on previous titration experiments [Gu et al., 2009] we have used throughout this study a 10-7 M testosterone-HAS concentration for mAR stimulation.

3.2.2. Preparation of steroid solution

Before each experiment testosterone-3- (O-carboxymethyl) oxime-Human Serum Albumin, referred to as testosterone-HSA (or Testo-HSA), DHT and estradiol, were dissolved in serum-free culture medium at a final concentration of 10-5 M. This stock solution was incubated for 30 min at room temperature with 0.3% charcoal and 0.03% dextran, centrifuged at 3000 x g and passed through a 0.45 μm filter to remove any potential contamination with free steroid. Testosterone-HSA, estradiol and DHT solutions were used at a final concentration of 10-7 M throughout all studies. If not otherwise stated all treatments and incubations with steroids including apoptosis assays were performed in serum-containing medium. Testosterone-HSA-FITC or control HSA-FITC constructs were generated by conjugating Testosterone-HSA or HSA with FITC using standard techniques.

3.2.3. In vivo animal experiment

Colon carcinoma was generated as described previously (Wang et al., 2004). In a first series of experiments, 7-week old Balb/c mice (both male and female) were divided into two groups, A (n=5) and B (n=7). Both groups [underwent carcinogenic treatment.]


[Seite 2]

Materials and methods

Cell cultures

The Caco2 and HCT116 human colon cancer cell lines and the non transformed intestinal IEC06 cells were obtained from the American Type Culture Collection (Manassas, VA) and were studied between passages 55 and 70.

Preparation of steroid solution

Before each experiment testosterone-3-(O-carboxymethyl) oxime human serum albumin, (testosterone-HSA or Testo-HSA; Sigma) was dissolved in serum-free culture medium at a final concentration of 10-5 M. This stock solution was incubated for 30 min at room temperature with 0.3% charcoal and 0.03% dextran, centrifuged at 3,000 x g and passed through a 0.45 μm filter to remove any potential contamination with free steroid. This is highly important for the interpretation of the results to disconnect any possible intracellular testosterone- and/or iAR-interference with the effects mainly induced by the mAR activation. Testosterone HSA, estradiol and dihydrotestosterone (DHT) (Sigma) solutions were used at a final concentration of 10-7 M throughout the study unless otherwise mentioned. All treatments and incubations with steroids including apoptosis assays were performed in serum-containing medium. Testosterone-HSA-FITC or control HSA-FITC constructs were generated by conjugating Testosterone-HSA or HSA with FITC (Sigma) using standard techniques.

[Seite 4]

Induction of colon carcinoma

Colon carcinoma was generated as described previously [26]. In a first series of experiments, 7-week old Balb/c mice (both male and female) were divided into two groups, A (n = 5) and B (n = 7). Both groups underwent carcinogenic treatment.


26. Wang JG, Wang DF, Lv BJ, Si JM: A novel mouse model for colitis-associated colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sulfate sodium. World J Gastroenterol 2004, 10:2958-2962.

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Übernahme eines Fremdtextes ohne jede Kennzeichnung.

Ferner: aus dem "10-7" der Vorlage wird bei Shg in der Einleitung "10-7".

Eine Referenz für "(Wang et al., 2004)" fehlt im Literaturverzeichnis von Shg.

Sichter
(Graf Isolan), SleepyHollow02

[4.] Shg/Fragment 042 01 - Diskussion
Zuletzt bearbeitet: 2014-11-02 01:24:59 Hindemith
Fragment, Gesichtet, Gu et al 2009, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Shg

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Bearbeiter
Graf Isolan
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Untersuchte Arbeit:
Seite: 42, Zeilen: 1-23
Quelle: Gu et al 2009
Seite(n): 4, Zeilen: li.Sp. 47-49 - re.Sp. 1-6, 12 ff.
At the age of 9 weeks animals were subjected to three cycles of alternating administration of distilled water containing 30 g/L synthetic dextran sulfate sodium (DSS; molecular mass 5000 Da; Wako Pure Chemical Industries, Led. Japan) for 7 days followed by distilled water for subsequent 14 days after intraperitoneal pretreatment with 20 mg/kg 1, 2-dimethylhydrazine (DMH; Sigma-Aldrich Corp. St.Louis.MO.USA). Group B mice received in addition to the carcinogenic treatment 5 mg/kg testosterone-HSA subcutaneously injected three times per week throughout the study period. All mice were sacrificed at the age of 20 weeks. After death, the entire colorectum from the colorectal junction to the anal verge was examined. Fresh specimens were placed in liquid nitrogen and subsequently stored at -80°C for further analysis. Then, the colon was opened longitudinally, washed with PBS, and divided into three portions (proximal, middle and distal). After macroscopic inspection the colon was fixed in a 40% g/L formaldehyde buffer solution (pH.7.4).

In APC mice, animal experiments were carried out in mice of either sex with mutated apc resulting in spontaneous colon tumor development (APCMin/+) obtained from the Jackson Laboratory (USA). The animals were housed under controlled environmental conditions (22-24°C, 50-70% humidity and a 12-h light/dark cycle). Throughout the study the mice had free access to standard pelleted food (C1000, Altromin, Lage, Germany) and tap water. All animal experiments were conducted according to the German law for the care and welfare of animals and were approved by local authorities.

Experiments were carried out on 7-week old wild-type Balb/c mice of either sex. The animals were housed under controlled environmental conditions (22-24°C, 50-70% humidity and a 12 h light/dark cycle). Throughout the study the mice had free access to standard pelleted food (C1310, Altromin, Heidenau, Germany) and tap water. All animal experiments were conducted according to the German law for the care and welfare of animals and were approved by local authorities

[...]

[...] At the age of 9 weeks animals were subjected to three cycles of alternating administration of distilled water containing 30 g/L synthetic dextran sulfate sodium (DSS; molelucar [sic] mass 5000 Da; Wako Pure Chemical Industries, Led. Japan) for 7 days followed by distilled water for subsequent 14 days after intraperitoneal pretreatment with 20 mg/kg 1, 2-dimethylhydrazine (DMH; Sigma-Aldrich Corp. St.Louis. MO. USA). Group B mice received in addition to the carcinogenic treatment 5 mg/kg testosterone-HSA subcutaneously injected three times per week throughout the study period. All mice were sacrificed at the age of 20 weeks. After death, the entire colorectum from the colorectal junction to the anal verge was examined. Fresh specimens were placed in liquid nitrogen and subsequently stored at -80°C for further analysis. Then, the colon was opened longitudinally, washed with PBS, and divided into three portions (proximal, middle and distal). After macroscopic inspection the colon was fixed in a 40% g/L formaldehyde buffer solution (pH.7.4).

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Übernahme eines Fremdtextes ohne jede Kennzeichnung.

Sichter
(Graf Isolan), SleepyHollow02

[5.] Shg/Fragment 043 02 - Diskussion
Zuletzt bearbeitet: 2014-11-03 19:55:26 Graf Isolan
BauernOpfer, Fragment, Gesichtet, Gu et al 2009, SMWFragment, Schutzlevel sysop, Shg

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3.2.4. Immunofluorescence analysis and confocal laser scanning microscopy

For testosterone-HSA-FITC staining, 5-μm-thick frozen tissue sections from the Balb/c or APC mouse tumors were fixed with 4% PFA for 15 min and incubated with 5% BSA/1x PBS/0.3% Triton for 1 hour at room temperature. After two washes with PBS 1.5% FBS specimens were exposed to testosterone-HSA-FITC (10-7 M, Sigma) for 1h at room temperature. Nuclei were stained with DRAQ-5 dye (1:1000, Biostatus, Leicestershire, UK) for 10 min at room temperature.

For direct fluorescence microscopy of F-actin, cells were fixed with 3 % paraformaldehyde in PBS for 30 min, permeabilized with 0.5 % Triton X-100 in PBS (10 min) and incubated with rhodamine-phalloidin (Molecular Probes, Eugene, OR, 1:100 dilution) for 40 min in the dark. For indirect immunofluorescence staining, cells were incubated for 2h at room temperature with mouse monoclonal anti-tubulin (Cell signaling, 1: 1000 dilution). Secondary FITC-conjugated rabbit anti-mouse IgG (Invitrogen) was used in a 1: 200 dilution. Nuclei were stained with DRAQ5™ (Biostatus Limited). Slides were mounted using the ProLang® Gold Antifade reagent (Invitrogen).

Immunofluorescence analysis and confocal laser scanning microscopy

Cells were cultured on glass cover slips with testosterone- HSA-FITC or control HSA-FITC using the concentrations and the incubation periods indicated in the figure legends. For testosterone-HSA-FITC staining, cells or specimens were washed twice with PBS containing 1.5% FBS for 1.5 min and incubated for 1 h with 1% BSA in PBS at room temperature. After two washes with PBS/1.5% FBS cells were exposed to 10-7 M testosterone-HSA-FITC, while control cells were incubated with 4 × 10-7 M HSA-FITC for 1 h at room temperature. Nuclei were stained with DRAQ5™ (Biostatus Limited) or TO-PRO-3 (Invitrogen). After two washes with PBS/1.5% FBS and fixation with 0.5% paraformaldehyde for 30 min cells were washed twice with PBS/1.5% FBS for 3 min and mounted with slow anti-fade. For direct fluorescence microscopy of F-actin, cells were fixed with 3% paraformaldehyde in PBS for 30 min, permeabilized with 0.5% Triton X-100 in PBS (10 min) and incubated with rhodamine-phalloidin (Molecular Probes, Eugene, OR, 1:100 dilution) for 40 min in the dark. For indirect immunofluorescence staining cells were incubated for 2 h at room temperature with mouse monoclonal anti-tubulin (Cell signaling, 1: 1000 dilution). Secondary FITC-conjugated rabbit anti-mouse IgG (Invitrogen) was used at a 1:200 dilution. Nuclei were stained with DRAQ5™ (Biostatus Limited). Slides were mounted using the ProLang® Gold Antifade reagent (Invitrogen).

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Insbesondere im zweiten Absatz liegt die Übernahme eines Fremdtextes ohne jede Kennzeichnung vor.

Ferner: aus dem "10-7" der Vorlage wird bei Shg "10-7".

Gu et al 2009 has been mentioned once on page 41 with respect only to "previous titration experiments".

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3.2.10. TUNEL assay

The colonic cancer tissue was cut to 8 μm frozen sections and subsequently fixed in 4 % paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were permeabilized in a solution of 0.1 % Triton X-100 in sodium citrate for 2 min. Samples, washed with PBS, were then incubated in the TUNEL reaction mix for 1 h at 37oC, according to the manufacturer’s instructions (Roche, Germany). Nuclei were stained with DRAQ5™ (Biostatus Limited). Sections were analyzed with a confocal laser scanning microscope (Carl Zeiss).

3.2.11. APOPercentage apoptosis assay

Caco2 cells were cultured in 96-well plates for the APOPercentage apoptosis assay (Biocolor Ltd., Belfast, Ireland). In the presence or absence of 10-6 M anastrozole (Sigma), they were stimulated or not with 10-6 M TAC for 24 hours in serum containing medium. Untreated cells cultured in serum free medium were used as positive control for the apoptotic response.

APOPercentage apoptosis assay Caco2 cells (in RPMI 1640, supplemented with 25 mM HEPES, 2 mM L-Glutamine and 10% FBS) were cultured in 96-well plates for the APOPercentage apoptosis assay (Biocolor Ltd., Belfast, Ireland). In the presence or absence of 10-7 M flutamide, cytochalasin B (Sigma) and DEVD-fmk, they were stimulated or not for 24 h in serumcontaining medium with 10-7 M of the following steroids: testosterone-HSA, dihydrotestosterone (DHT) and estradiol (E2). Untreated cells cultured in serum-free medium were used as positive control for apoptosis.

TUNEL assay The colonic cancer tissue was cut to 8 μm frozen sections from mouse colon tumors and subsequently fixed in 4% paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were permeabilized in a solution of 0.1% Triton X-100 in sodium citrate for 2 min. Samples were washed with PBS and incubated in the TUNEL reaction mix for 1 h at 37°C, according to the manufacturer's instructions (Roche, Germany). Nuclei were stained with DRAQ5™ (Biostatus Limited). Sections were analysed by confocal microscopy.

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3.2.12. Statistical analysis

Data are provided as means ± SEM; n represents the number of independent experiments. Data were tested for significance using unpaired student’s t-test when two-sample means were tested. Differences were considered statistically significant when p-values were < 0.05. All statistical analysis was performed with GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, www.graphpad.com.

Statistical analysis Data are provided as means ± SEM, n represents the number of independent experiments. Data were tested for significance using unpaired student's t-test, when twosample means were tested. Differences were considered statistically significant when p-values were < 0.05. All statistical analysis was performed with GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, http://www.graphpad.com. Molecular Cancer 2009, 8:114 http://www.molecular-cancer.com/content/8/1/114
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4. RESULTS

4.1 mAR expression in colon cancer cell lines

In my diplom thisis, while analyzing in vivo mAR expression in paraffin blocks generated from xenograft tumor tissues of various origins, we have noticed significant mAR expression in colon cancer xenograft specimens. In line with these findings mAR expression was subsequently detected by confocal laser scanning microscopy using the fluorescent testosterone-HSA-FITC conjugate in cultured HCT116- (Figure5 e,f), or in Caco2-colon cells (Figure 5a,b) while HSA-FITC labeled CaCo2 or HCT116 cells showed no apparent staining (Figures 5 c, d, g, h). These results indicate that mAR in expressed in colon cancer cell lines. Interestingly, mAR staining could not be detected in the non-transformed intestinal cell line IEC06 (Fig. 6A). These staining experiments and the fact that testosterone-HSA-FITC is an impermeable conjugate disclosed mAR expression preferentially in colon cancer cell lines and tumors. In addition, mAR could be also detected in iAR silenced of Caco2 cells by using testosterone-HSA-FITC. These results imply that the molecular identity of mAR is probably not identical with iAR (Fig. 6B)

Figure 5 Membrane staining of mAR in Caco2 and HCT 116 colon cancer cells

Results

mAR expression in specimens of colon tumors and colon cancer cell lines While analyzing paraffin blocks generated from in vivo xenograft tumor tissues of various origins, we noticed significant mAR expression in colon tumors. Specifically, using testosterone-HSA-FITC fluorescent conjugates we detected specific, FITC-related fluorescence in membrane specimens of colon xenograft tumors generated from wild type HCT116 cells (WCL2) (Fig. 1a,a') or HCT116 p53-/- cells (MCL3) (Fig. 1d,d'). Conversely, no apparent staining could be identified in control tissues labeled with HSA-FITC (Fig. 1c,f). Although the apparent visualization of mAR staining in tissue preparations is restricted by technical limitations, in cultured HCT116- (Fig. 1h) or in Caco2-colon cells (Fig. 2A a,b) the membrane staining of mARs was obvious by confocal laser scanning microscopy using the fluorescent testosterone-HSA-FITC conjugate. No apparent staining was evident in HSA-FITC-labeled HCT116 or Caco2 cells (Fig. 1j, 2Ac,d). Interestingly, mAR staining could not be detected in the membrane of the non-transformed intestinal cell line IEC06 (Fig. 2B). These staining experiments and the fact that testosterone- HSA-FITC is an impermeable conjugate disclosed mAR expression preferentially in colon cancer cell lines and tumors.

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Confocal laser scanning microscopic analysis of Caco2 cells (a-d) and HCT 116 cells(e-h) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes, or HSA-FITC, showing no apparent membrane staining. Visualization of nuclei was evident by DRAQ5™ or TO-PRO-3 staining. Magnification, ×100.

A

B

Figure 6 Membrane staining of mAR in IEC 06 cells and iAR silenced Caco2 cells. A) Confocal laser scanning microscopic analysis of IEC 06 cells (a-b) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane. Visualization of nuclei was evident by DRAQ5™ staining. B) Confocal laser scanning microscopic analysis of iAR silenced cells (a-d) stained with testosterone-HSA-FITC, showing membrane fluorescence at the cell membrane. Visualization of nuclei was evident by DRAQ5™ staining.

Membrane staining of mAR in colon tumors and colon cancer cells. Confocal laser scanning microscopic analysis of WCL2 (a-c) and MCL2 (d-f) colon tumor specimens and HCT116 cells (g-j) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes. Control staining with HSA-FITC showed no apparent membrane fluorescence. Visualization of nuclei was evident by DAPI or TO-PRO-3 staining. Magnification, ×100.

Membrane staining and binding assays of mAR in Caco-2 cells. A) Confocal laser scanning microscopic analysis of Caco2 cells (a-d) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes (a, b). No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (c, d). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. B) Confocal laser scanning microscopic analysis of IEC 06 cells (e-f) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane. Visualization of nuclei was evident by DRAQ5™ staining.

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4.2 mAR expression in 2 different colon cancer animal models

The findings provided so far indicate that mAR are expressed in colon cancer cell lines Caco2 and HCT-116 in vitro. [Gu S, et al. 2009] Thus, we aimed to further evaluate the in vivo effects of albumin-conjugated androgens in colon cancer animal models. To this end we first estimated the expression of mAR in colon tumors generated in Balb/c mice and APC mice. As shown in figure 7, using testosterone-HSA-FITC we detected specific, FITC-related fluorescence in membrane specimens of Balb/c mice colon tumors (Fig. 7 A, a, a1), and APC mice colon tumors (Fig.8). No apparent staining could be identified in tissues labeled with HSA-FITC (Fig. 7A, b) and no apparent staining could be found in healthy tissue labeled with testosterone-HSA-FITC (Fig. 7B).

Figure 7: In vivo testosterone-HSA expression in BALB/c mice

A) Confocal laser scanning microscopic analysis of BALB/c colon tumor frozen sections stained with testosterone-HSA-FITC (a, a’), showing specific FITC related fluorescence at the cell membranes. No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (b).

B) Confocal laser scanning microscopic analysis of BALB/c colon tumor and healthy frozen sections stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes of tumor sections.

Membrane staining of mAR in colon tumors and colon cancer cells. Confocal laser scanning microscopic analysis of WCL2 (a-c) and MCL2 (d-f) colon tumor specimens and HCT116 cells (g-j) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes. Control staining with HSA-FITC showed no apparent membrane fluorescence.

A) Confocal laser scanning microscopic analysis of Caco2 cells (a-d) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes (a, b). No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (c, d). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. B) Confocal laser scanning microscopic analysis of IEC 06 cells (e-f) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane.

conjugated androgens in colon cancer animal models. To this end, we first estimated the expression of mAR in colon tumors generated in Balb/c mice. As shown in fig. 7A and fig. 7B, using testosterone-HSA-FITC we detected specific, FITC-related fluorescence in membrane specimens of Balb/c mice colon tumors (Fig. 7Aa, a'), while no apparent staining could be identified in tissues labeled with HSA-FITC (Fig. 7Ab). Interestingly, mAR staining was very low in healthy colon tissue specimens of Balb/c mice (Fig. 7B) further supporting earlier findings pointing to cancer tissue specificity of mAR [31]. Having a clear indication for mAR-expression, we assessed the 12-week tumor incidence of colon tumors generated in Balb/c mice by chemical carcinogenesis (see Materials and Methods) in the presence or absence of continuous testosterone- HSA treatment.

In vivo testosterone-HSA effects on tumor incidence in BALB/c mice. A) Confocal laser scanning microscopic analysis of BALB/c colon tumor frozen sections stained with testosterone-HSA-FITC (a, a'), showing specific FITC related fluorescence at the cell membranes. No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (b). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. (B) Confocal laser scanning microscopic analysis of BALB/c colon tumor and healthy frozen sections stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes of tumor sections (a). Very low membrane fluorescence was shown in healthy colon sections stained with testosterone-HSA-FITC

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Figure 8: In vivo testosterone-HSA expression in APC mice

Confocal laser scanning microscopic analysis of APCMin/+ colon tumor frozen sections stained with testosterone-HSA-FITC, showing specific FITC-related fluorescence at the cell membranes. Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100.

4.3 mAR activation by testosterone-HSA was followed by extensive reduction of tumor incidence in vivo

Having a clear indication for mAR-expression, the 12-week tumor incidence of colon tumors generated in Balb/c mice was assessed by chemical carcinogenesis (see Experimental Procedures) in the presence or absence of continuous testosterone-HSA treatment. The animals used for these studies were divided in two groups comprising 5 and 7 animals. One group (7 animals) was treated subcutaneously (3 times/week, for 12 weeks) with 5mg/kg testosterone-HSA, whereas the other group (5 animals) remained untreated. The results (Figure 9, A) show that testosterone-HSA-treatment produced a clear and significant reduction of tumor incidence by 65%. The histological analysis of tumors by Tunel assay (Figure 9, B) confirmed that apoptotic cells were present in significant numbers predominantly at the tumors of animals treated with testosterone-HSA, while they were significantly less in the non-treated animals. These results collectively show that mAR is a functional target that may be used for the selective elimination of colon cancer cells in vivo.

A) Confocal laser scanning microscopic analysis of Caco2 cells (a-d) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes (a, b). No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (c, d). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. B) Confocal laser scanning microscopic analysis of IEC 06 cells (e-f) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane. Visualization of nuclei was evident by DRAQ5™ staining.

Having a clear indication for mAR-expression, we assessed the 12-week tumor incidence of colon tumors generated in Balb/c mice by chemical carcinogenesis (see Materials and Methods) in the presence or absence of continuous testosterone- HSA treatment. The animals used for these studies were divided in two groups comprising of 5 and 7 animals respectively. One group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5 mg/kg testosterone- HSA, whereas the other group (5 animals) remained untreated. The results (Fig. 7C) show that testosterone- HSA treatment produced a clear and significant reduction of tumor incidence by 65%. The histological analysis of tumors by TUNEL assay confirmed that apoptotic cells were present in significant numbers predominantly in the tumors of animals treated with testosterone- HSA (Fig. 7D, middle panels), while they were significantly less either in the non-treated animals (Fig. 7D, right panels), or in healthy tissues of treated animals (Fig. 7D, left panels). These results collectively show that mAR is a functional and specific target that may be used for the selective elimination of colon cancer cells in vivo.

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Figure 9: In vivo testosterone-HSA effects on tumor incidence in BALB/c mice

A) Arithmetic means ± SEM of colonic tumor incidence in BALB/c mice. Following treatment with the carcinogenic drug 1,2 dimethylhydrozine followed by dextrane sodium sulphate, one group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5mg/kg testosterone-HAS (black bar), whereas the other group (5 animals) remained untreated (white bar). # indicates significant difference between both groups (# P<0.01). B) After treatment, the colonic cancer tissue was cut to 8 μm frozen sections and fragmented DNA was assessed using TUNEL assay according to the manufacturer's instructions. Confocal laser scanning microscopy analyzed samples. Magnification, ×100. To further establish the in vivo role of mAR activation in mice model, in a second series of experiments APC mice have been used. In these experiments, animals were divided in two groups comprising 6 and 4 animals. One group (6 animals) was treated subcutaneously (3 times/week, for 8 weeks) with 5mg/kg testosterone-HSA, whereas the other group (4 animals) remained untreated. As shown in Fig. 10A, testosterone-HSA treatment resulted in a significant reduction of the tumor incidence by 80%. The histological analysis of tumors by TUNEL assay confirmed that apoptotic cells were present in appreciable numbers predominantly in the tumors of animals treated with testosterone-HSA (Fig. 10B, left panels) whereas they were significantly less abundant in the non-treated animals (Fig. 10B, right panels).

The animals used for these studies were divided in two groups comprising of 5 and 7 animals respectively. One group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5 mg/kg testosterone- HSA, whereas the other group (5 animals) remained untreated. The results (Fig. 7C) show that testosterone- HSA treatment produced a clear and significant reduction of tumor incidence by 65%. The histological analysis of tumors by TUNEL assay confirmed that apoptotic cells were present in significant numbers predominantly in the tumors of animals treated with testosterone- HSA (Fig. 7D, middle panels), while they were significantly less either in the non-treated animals (Fig. 7D, right panels), or in healthy tissues of treated animals (Fig. 7D, left panels)

Arithmetic means ± SEM of colonic tumor incidence in BALB/c mice. Following treatment with the carcinogenic drug 1, 2- dimethylhydrazine followed by dextrane sodium sulphate, one group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5 mg/kg testosterone-HAS (black bar), whereas the other group (5 animals) remained untreated (white bar). # indicates significant difference between both groups (# P < 0.01). (D) After treatment, the colonic cancer and healthy tissue was cut to 8 μm frozen sections and fragmented DNA was assessed by TUNEL assay according to the manufacturer's instructions. Confocal laser scanning microscopy analyzed samples. Magnification, ×100.

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4.6 mAR activation triggered rapid actin and tubulin reorganization in colon cancer cells

Cytoskeleton reorganization is a prominent early functional response of various cancer cells to steroid hormones targeting membrane binding sites [Koukouritaki et al., 1997, Kampa et al., 2002, Kampa et al., 2006, Papadopoulou et al., 2008a]. Accordingly, to analyze the functional impact of mAR in colon cancer rapid cytoskeleton modifications was investigated in Caco2 cells upon activation of mAR with testosterone-HSA for various time intervals. Cellular actin cytoskeleton dynamics were initially assessed by appropriate quantitative techniques as described in Papakonstanti et al., 2007. As shown in figure 13A, quantitative immunoblot analysis of Triton X-100 insoluble cytoskeletal pellets and corresponding supernatants revealed a significant decrease of the Triton-soluble (monomeric) to total actin ratio in Caco2 cells treated with 10-7 M testosterone-HAS, indicating actin polymerization. This effect was evident 15 min upon testosterone-HSA treatment; and returned to nearly control levels after 1-2 hours (Fig. 13A). The quantitative data were fully supported by confocal laser scanning microscopic analysis, showing redistribution of microfilamentous structures and formation of stress fibers and filopodia in testosterone-HSA treated cells (Fig. 13B).


Kampa M, Papakonstanti EA, Hatzoglou A, Stathopoulos EN, Stournaras C, Castanas E. (2002). The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton. Faseb J, 16:1429-1431.

Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E. (2006) Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther, 5:1342-1351.

Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C. (2008 a). Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res, 314: 3162-3174.

mAR activation triggered rapid actin and tubulin reorganization in colon cancer cells

Cytoskeleton reorganization is a prominent early functional response of various cancer cells to steroid hormones targeting membrane binding sites [3,8,19,27]. Accordingly to analyze the functional impact of mAR in colon cancer we investigated rapid cytoskeleton modifications in Caco2 cells upon activation of mAR with testosterone- HSA for various time intervals. Cellular actin cytoskeleton dynamics were initially assessed by appropriate quantitative techniques as described in [25]. As shown in fig. 3A, quantitative immunoblot analysis of Triton X-100-insoluble cytoskeletal pellets and correspond-

[Seite 7]

ing supernatants revealed a significant decrease of the Triton-soluble (monomeric) over total actin ratio in Caco2 cells treated with 10-7 M testosterone-HSA, indicating actin polymerization. This effect was evident 15 min upon testosterone-HSA treatment and returned to nearly control levels after 1-2 h (Fig. 3A). The quantitative data were fully supported by confocal laser scanning microscopic analysis showing redistribution of microfilamentous structures and formation of stress fibers and filopodia in testosterone-HSA treated cells (Fig. 3B).


3. Kampa M, Papakonstanti EA, Hatzoglou A, Stathopoulos EN, Stournaras C, Castanas E: The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton. Faseb J 2002, 16:1429-1431.

8. Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C: Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res 2008, 314:3162-3174.

19. Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E: Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther 2006, 5:1342-1351.

27. Koukouritaki S, Margioris A, Gravanis A, Hartig R, Stournaras C: Dexamethasone induces actin polymerization in human endometrial cells without affecting its synthesis. J Cell Biochem 1997, 65:492-500

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Die Quelle Koukouritaki fehlt im Literaturverzeichnis.

Man beachte die Anmerkung zur Quelle Gu et al. (2009)

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Figure 13: Modulation of the dynamic equilibrium G- and Total actin in testosterone-HSA stimulated Caco2 cells.

24h serum starved cells were stimulated with 10 -7 M androgen conjugate for the indicated time points.

A) Total and G- actin were measured by quantitative immunoblot analysis after Triton X-100 subcellular fractionation. Bars present the G/Total actin mean value  SE of four independent duplicate experiments (** P < 0.01).

B) Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei.

Tubulin cytoskeleton reorganization was further analyzed by confocal laser scanning microscopy. A clear redistribution of the microtubular network became evident in cells treated with 10-7 M testosterone-HSA for 15 to 60 minutes (Fig. 14).

We further analyzed tubulin cytoskeleton reorganization by confocal laser scanning microscopy. A clear redistribution of the microtubular network became evident in cells treated with 10-7 M testosterone-HSA for 15 to 60 min (Additional File 1).

FMiogduurleat 3ion of the dynamic equilibrium between G- and Total actin in testosterone-HSA stimulated Caco2 cells Modulation of the dynamic equilibrium between G- and Total actin in testosterone-HSA stimulated Caco2 cells. 24 h serum starved cells were stimulated with 10 -7 M androgen conjugate for the indicated time points. (A) Total and Gactin were measured by quantitative immunoblot analysis after Triton X-100 subcellular fractionation. Bars present the G/Total actin mean value ± SE of four independent duplicate experiments (** P < 0.01). (B) Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei. Confocal laser scanning microscopy analyzed samples. Magnification, ×100.

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Figure 14: Modulation of the dynamic equilibrium rapid tubulin reorganization in testosterone-HSA stimulated Caco2 cells.

Caco2 cells treated or not with 10-7 M testosterone-HSA for different time points were cultured in coverslips, fixed and stained with rabbit anti-α-tubulin. Anti-rabbit-FITC was used as secondary antibody and DRAQ5™ for nuclei staining. Confocal laser scanning microscopy analyzed samples. Magnification, ×100.

Previously, it has been reported that activation of mAR with non permeable testosterone derivatives induced pro-apoptotic responses [Gu S Diploma Thesis, Gu S, et al. 2009]. However, the mechanism regulating the mAR-induced apoptotic responces is still unknown. In recent years, the cross-talk between actin cytoskeleton components and apoptotic signaling has attracted specific interest. Indeed, modifications of actin dynamics seem to be crucial for apoptotic responses [Gourlay CW, et al. 2005, Franklin-Tong VE,et al. 2008]. More recently the functional role of actin reorganization in regulating the pro-apoptotic responses induced by mAR was established in prostate cancer cells [Papadopoulou N, et al. 2008,] Based on these results we assessed the mAR-dependent apoptosis and caspase-3 activation in the presence of anti-actin drugs. As shown in Figures 15A,B, in Caco2 cells pre-[treated with cytochalasin B, at a concentration (10-7M) which blocks actin redistribution without exerting toxic effects [Stournaras et al 1996], the mAR-induced apoptotic response (Fig 15A) and caspase-3 activation (Fig 15B) were abolished.]

To establish the functional role of actin reorganization in regulating the pro-apoptotic responses induced by mAR, as previously reported for various cell systems [8,28,29], we assessed mAR-dependent apoptosis and caspase-3 activation in the presence of antiactin drugs. As shown in Fig. 5A, B, in Caco2 cells pretreated with cytochalasin B, at a concentration (10-7M) which blocks actin redistribution without exerting toxic PFrigou-arpeo p4totic effects of testosterone-HSA in Caco2 cells Pro-apoptotic effects of testosterone-HSA in Caco2 cells.

Rapid tubulin reorganization in testosterone-HSA stimulated Caco2 cells. Caco2 cells treated or not with 10-7 M testosterone-HSA for different time points were cultured in coverslips, fixed and stained with rabbit anti- -tubulin. Anti-rabbit-FITC was used as secondary antibody and DRAQ5™ for nuclei staining. Confocal laser scanning microscopy analyzed samples. Magnification, ×100.

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[As shown in Figures 15A,B, in Caco2 cells pre-] treated with cytochalasin B, at a concentration (10-7M) which blocks actin redistribution without exerting toxic effects [Stournaras et al 1996], the mAR-induced apoptotic response (Fig 15A) and caspase-3 activation (Fig 15B) were abolished. These results indicate that actin redistribution is a mandatory step for the apoptotic response of mAR-stimulated colon cancer cells.

TAC: Testosterone-HSA Cyto B: cytochalasin B

Figure 15: Pro-apoptotic effects of testosterone-HSA, DHT and Estradiol in the absence or presence of inhibitors in Caco2 cells.

A) Quantitative APOPercentage apoptosis assay of testosterone-HSA stimulated Caco2 cells and similar experiments in the presence of cytochalasin B (Cyto B). Cells were exposed to 10-7 M testosterone-HSA for 24 hours and proapoptoric responses were assessed by the APOPercentage apoptosis assay. Equally, cells pre-treated or not with 10-7 M Cyto B or flutamide, were exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods of time served as a positive control for apoptosis. Bars present the mean OD measured at 550 nm. ** P<0, 01, n=4.

B) Cells were pre-treated or not with Cyto B for 1h and then exposed or not to 10-7 M testosterone-HSA for 4h, lysed and incubated with the caspase-3 substrate DEVD conjugated to the chromophore pNA according to the manufacturer's instructions. Caspase-3 activity was measured at 405 nm. ** P<0, 01, n=4.

effects [30], the mAR-induced apoptotic response (Fig. 5A) and caspase-3 activation (Fig. 5B) were abolished. These results indicate that actin redistribution is a mandatory step for the apoptotic response of mAR-stimulated colon cancer cells.

Thus, we aimed to further evaluate the in vivo effects of albumin- PFrigou-arpeo p5totic effects of testosterone-HSA, DHT and estradiol in the absence or presence of inhibitors in Caco2 cells Pro-apoptotic effects of testosterone-HSA, DHT and estradiol in the absence or presence of inhibitors in Caco2 cells. (A) Quantitative APOPercentage apoptosis assay of testosterone-HSA, DHT or estradiol stimulated Caco2 cells and in the presence/absence of cytochalasin B (Cyto B) or flutamide. Cells were exposed to 10-7 M testosterone-HSA, DHT or estradiol for 24 hours and pro-apoptoric responses were assessed by the APOPercentage apoptosis assay. Equally, cells pretreated or not with 10-7 M Cyto B or flutamide, were exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods of time served as a positive control for apoptosis. Bars present the mean OD measured at 550 nm (** P < 0.01, N = 4). (B) Cells were pre-treated or not with Cyto B or flutamide for 1 h and then exposed or not to 10-7 M testosterone- HSA for 4 h, lysed and incubated with the caspase-3 substrate DEVD conjugated to the chromophore pNA according to the manufacturer's instructions. Caspase-3 activity was measured at 405 nm (** P < 0.01, N = 4).

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However, It still remained unknown whether mARs are also expressed in colon cancer and whether their activation could result in the induction of anti-tumorigenic effects similar to those described in other cancer cells.

In the present work we provide experimental evidence that membrane androgen receptors are expressed in colon tumors. Using tissue specimens from colon tumors and established colon tumor cell lines we show here that colon cancer cells express functional mARs. [Gu S, et al. 2009]


Gu S, Papadopoulou N, Gehring EM, Nasir O, Dimas K, Bhavsar SK, Foller M, Alevizopoulos K, Lang F, Stournaras C. (2009) Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo. Mol Cancer. 8:114.

However, it remained elusive whether mARs are also expressed in other tumors and whether their activation could result in the induction of anti-tumorigenic effects similar to the ones described in prostate and breast cancer cells.

In the present work we provide strong experimental evidence that membrane androgen receptors are expressed in colon tumors. Using tissue specimens derived from colon tumors and established colon tumor cell lines we showed that colon cancer cells predominantly express functional mAR, while mAR expression is undetectable in healthy mouse colon tissues or non-transformed intestinal cells.

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Aus dem Diskussionsteil der Arbeit. Quelle ist genannt. Mit übernommen ist der Plural We, der im Quelltext auf zehn Verfasser referenziert.

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[Moreover,] membrane-impermeable testosterone albumin conjugates induce considerable apoptosis via activation of the pro-apoptotic executor caspase-3. [Gu S, et al. 2009] The observed mAR-activated effects are specific and independent from the classical intracellular androgen receptors (iAR), since they were manifested in the presence of the anti-androgen flutamide. In addition, mAR staining could be also detected in iAR silenced Caco2 cells (Fig 6B) and iAR-deficient DU145 human prostate cancer cells [Hatzoglou A, et al. 2005]. All those imply that the molecular identity of mAR is probably not identical with iAR, targeted to the plasma membrane.

5.1 Membrane androgen receptor activation in colon cancer triggers pro-apoptotic responses in vitro and in vivo

The results from my diplom thesis have show that membrane-impermeable testosterone albumin conjugates induced considerable apoptosis via activation of the pro-apoptotic executor caspase-3. Moreover, the results from other studies indicated as well that membrane androgen receptors are predominantly expressed in tumor cells. Activation of these receptors triggers pro-apoptotic responses. One possible rationalization for the expression of those receptors is that tumor cells may compensate mAR-dependent apoptosis by over-expressing anti-apoptotic proteins or other compensatory mechanisms that collectively protect against mAR-dependent pro-apoptotic effects. Previous reports support this assumption: Indeed, iAR deficient DU145 human prostate cancer cells were shown to over-express the pro-survival PI-3K/Akt pathway, which was down-regulated following long-term mAR activation [Papadopoulou N, et al. 2008A]. In addition, the FAK/PI3K pathway was constitutively activated in DU145 cells and mAR activation was unable to further alter the short-term phosphorylation levels of those kinases [Papadopoulou N, et al. 2008], while long term activation induced significant de-phosphorylation [Papadopoulou N, et al. 2008A].


Gu S, Papadopoulou N, Gehring EM, Nasir O, Dimas K, Bhavsar SK, Foller M, Alevizopoulos K, Lang F, Stournaras C. (2009) Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo. Mol Cancer. 8:114.

Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, et al. (2005). Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab, 90: 893-903.

Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C. (2008 a). Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res, 314: 3162-3174.

Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C. (2008 b). Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc. 7:88.

Moreover, membrane-impermeable testosterone albumin conjugates induced a) profound and rapid actin and tubulin reorganization, and b) considerable apoptosis via activation of the pro-apoptotic executor caspase-3. The observed mAR-activated effects were specific for testosterone and testosterone conjugates, since other steroid hormones such as estradiol did not exhibit any pro-apoptotic activity.

(Fig 5A, B), imply that the molecular identity of mAR is probably not identical with iAR, targeted to the plasma membrane.

The results from this and other studies indicated that membrane androgen receptors are predominantly expressed in tumor cells ([31] and Fig. 2B, 7B). In addition, activation of these receptors triggers pro-apoptotic responses. One possible rationalization for the expression of those receptors is that tumor cells may compensate mAR-dependent apoptosis by over-expressing anti-apoptotic proteins or other compensatory mechanisms that collectively protect against mAR-dependent apoptosis. Previous reports support this assumption: Indeed, iARdeficient DU145 human prostate cancer cells were shown to overexpress the pro-survival PI-3K/Akt pathway, which was down-regulated following long-term mAR activation [9]. In addition, the FAK/PI3K pathway was constitutively activated in DU145 cells and mAR activation was unable to further alter the short-term phosphorylation levels of those kinases [8], while long term activation induced significant de-phosphorylation [9].


8. Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C: Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res 2008, 314:3162-3174.

9. Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C: Membrane androgen receptor activation triggers downregulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc 2008, 7:88.

31. Dambaki C, Kogia C, Kampa M, Darivianaki K, Nomikos M, Anezinis P, Theodoropoulos PA, Castanas E, Stathopoulos EN: Membrane testosterone binding sites in prostate carcinoma as a potential new marker and therapeutic target: study in paraffin tissue sections. BMC Cancer 2005, 5:148

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Recent studies using mouse xenografts have shown that a testosterone–albumin conjugate (testosterone-BSA) induced potent apoptotic regression of prostate tumors in vivo [Hatzoglou A, et al. 2005]. In addition, testosterone-BSA was also reported to potentiate the paclitaxel-mediated cytotoxicity both in vitro and in vivo [Kampa M, et al. 2006]. These reports are supported by in vivo experimental findings presented in this work. Indeed, when Balb/c mice were treated with testosterone-HSA and the 12-week tumor incidence of colon tumors was assessed the chemically induced tumors were reduced by 65% in the testosterone-HSA-treated animals. Most probably this effect was due to the apoptotic regression of tumor cells as indicated by the Tunel assay. These results point out clearly that activation of mAR by testosterone-HSA significantly affects the incidence of colon tumors in vivo and they are in line with the previously reported prostate tumor regression in mice [Hatzoglou A, et al. 2005, Kampa M, et al. 2006]. Interestingly, mAR is strongly expressed in tissues derived from p53-deficient xenograft tumors. Since p53 is a frequently inactivated gene in tumors, it is interesting to hypothesize that mAR activation [may result in eradication of p53 tumors in vivo.]

Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, et al. (2005). Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab, 90: 893-903.

Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E. (2006) Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther, 5:1342-1351.

Recent studies using mouse xenografts have shown that a testosterone-albumin conjugate (testosterone-BSA) induced potent apoptotic regression of prostate tumors in vivo [7]. In addition, testosterone-BSA was also reported to potentiate the paclitaxel-mediated cytotoxicity both in vitro and in vivo [19] [...]

Interestingly, the chemically-induced colon tumors were reduced by 65% in the testosterone-HSA-treated animals. Most probably this effect was due to the apoptotic regression of tumor cells as indicated by the TUNEL assay (Fig. 7D, middle panel). These results point out clearly that activation of mAR by testosterone-HSA significantly affects the incidence of colon tumors in vivo. Interestingly, mAR is strongly expressed in tissues derived from p53- deficient xenograft tumors (Fig. 1d, d'). Since p53 is a frequently inactivated gene in tumors, it is interesting to hypothesize that mAR activation may result in eradication of p53 tumors in vivo.


17. Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C: Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life 2009, 61(1):56-61.

19. Kampa M, Kogia C, Theodoropoulos PA, Anezinis P, Charalampopoulos I, Papakonstanti EA, Stathopoulos EN, Hatzoglou A, Stournaras C, Gravanis A, Castanas E: Activation of membrane androgen receptors potentiates the antiproliferative effects of paclitaxel on human prostate cancer cells. Mol Cancer Ther 2006, 5:1342-1351.

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[Since p53 is a frequently inactivated gene in tumors, it is interesting to hypothesize that mAR activation] may result in eradication of p53 tumors in vivo. Notwithstanding the above, and despite the fact that additional experiments are required for the detailed evaluation of mAR-dependent biological effects in colon cancer, our data support the recently postulated notion [Papadopoulou N, et al. 2009] that mAR may represent a novel specific tumor target.

Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C. (2009). Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life, 61(1): 56-61.

Since p53 is a frequently inactivated gene in tumors, it is interesting to hypothesize that mAR activation may result in eradication of p53 tumors in vivo. [...]

Despite the fact that additional experiments are required for the detailed evaluation of mARdependent biological effects in colon cancer, our findings fully enforce the potential significance of the recently postulated notion [17] that mAR may represent a novel and specific tumor target.


17. Papadopoulou N, Papakonstanti EA, Kallergi G, Alevizopoulos K, Stournaras C: Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life 2009, 61(1):56-61

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5.2 Membrane androgen receptor activation blocks migration

The connection between actin cytoskeleton components and androgen signaling has attracted specific interest in recent years [Ting HJ, et al. 2008]. Actin dynamics seem to be crucial for apoptotic responses [Gourlay CW, et al.2005, Franklin-Tong VE, et al.2008]. The findings in our present work further underscored the key role of actin cytoskeleton rearrangements in regulating apoptosis. Indeed, it was clearly shown that actin (and tubulin) reorganization represent major early events following mAR activation by testosterone-HSA. Moreover, early blockade of actin rearrangement by depolymerizing drugs e.g. cytochalasin B, virtually abrogated the pro-apoptotic responses (Fig. 15A, B). The involvement of the early actin rearrangement in mediating the late apoptotic responses was addressed in earlier studies in prostate cancer cells. In these studies it was shown that inhibition of either up-stream or down-stream signals regulating early actin polymerization blocked the late activation of NFkB and FasL signaling [Papadopoulou N, et al. 2008A]. Although this pro-apoptotic signaling was not addressed in the present study we hypothesise that the actin reorganization is an early functional step in the pro-apoptotic response. These findings, which are in close agreement with similar results reported recently in prostate cancer cells treated with testosterone albumin conjugates [Papadopoulou N, et al. 2008 and 2008A], further emphasize the functional cross-talk between cytoskeleton rearrangements and regulation of apoptosis [Gourlay CW, et al.2005, Franklin-Tong VE, et al.2008].


Gourlay CW, Ayscough KR. (2005).The actin cytoskeleton: a key regulator of apoptosis and ageing? Nat. Rev. Mol. Cell. Biol, 6(7):6583-589.

Franklin-Tong VE, Gourlay CW. (2008). A role for actin in regulating apoptosis/programmed cell death: evidence spanning yeast, plants and animals. Biochem J. 413(3):389-404.

Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C. (2008 a). Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res, 314: 3162-3174.

Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C. (2008 b). Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc. 7:88.

The connection between actin cytoskeleton components and androgen signaling has attracted specific interest in recent years (for a review see [36]). Actin dynamics seem to be crucial for apoptotic responses [28,29]. The findings in our present work further underscored the key role of actin cytoskeleton rearrangements in regulating apoptosis. Indeed, it was clearly shown that actin (and tubulin) reorganization represent major early events following mAR activation by testosterone-HSA. Moreover, early blockade of actin rearrangement by depolymerizing drugs e.g. cytochalasin B, virtually abrogated the pro-apoptotic responses (Fig. 5A, B). The involvement of the early actin rearrangement in mediating the late apoptotic responses was addressed in earlier studies in prostate cancer cells. In these studies it was shown that inhibition of either upstream or down-stream signals regulating early actin polymerization blocked the late activation of NF-κB and FasL signaling [9]. Although the pro-apoptotic signaling was not addressed in the present study we hypothesize that the actin reorganization is an early functional step in the pro-apoptotic response. These findings, which are in close agreement with similar results reported recently in prostate cancer cells treated with testosterone albumin conjugates [8,9], further emphasize the functional crosstalk between cytoskeleton rearrangements and regulation of apoptosis [28,29].

8. Papadopoulou N, Charalampopoulos I, Alevizopoulos K, Gravanis A, Stournaras C: Rho/ROCK/Actin signaling regualtes membrane androgen receptor induced apoptosis in prostate cancer cells. Exp Cell Res 2008, 314:3162-3174.

9. Papadopoulou N, Charalampopoulos I, Anagnostopoulou V, Konstantinidis G, Föller M, Gravanis A, Alevizopoulos K, Lang F, Stournaras C: Membrane androgen receptor activation triggers downregulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU-145 prostate cancer cells. Mol Canc 2008, 7:88

28. Gourlay CW, Ayscough KR: The actin cytoskeleton: a key regulator of apoptosis and ageing? Nat Rev Mol Cell Biol 2005, 6(7):6583-589.

29. Franklin-Tong VE, Gourlay CW: A role for actin in regulating apoptosis/programmed cell death: evidence spanning yeast, plants and animals. Biochem J 2008, 413(3):389-404.

36. Ting HJ, Chang C: Actin associated proteins function as androgen receptor coregulators: an implication of androgen receptor's roles in skeletal muscle. J Steroid Biochem Mol Biol 2008, 111(3-5):157-163

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Die Quelle Ting hat Shg zwar zitiert, aber vergessen im Quellenverzeichnis aufzulisten.

Man beachte die Anmerkung zur Quelle Gu et al. (2009)

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6. Conclusions

In conclusion, the results presented here add a clear and significant piece of evidence on the potential anti-tumorigenic role of membrane androgen receptors.

They indicate that

  • The functional mAR is expressed not only in hormone-dependent tumors but also in colon tumors.
  • mAR conjugates also induced rapid actin and tubulin reorganization.
  • The activation through steroid albumin conjugates induces potent pro-apoptotic responses regulated by cytoskeletal rearrangements.

[...]

These receptors may represent specific targets for the development of novel drugs since their activation drastically regress tumor growth and tumor incidence in vivo

In conclusion, the results presented here add a clear and significant piece of evidence to the potential anti-tumorigenic role of membrane androgen receptors. They indicate that a) functional mAR are expressed not only in hormone- dependent tumors but also in colon tumors, b) their activation through steroid albumin conjugates induces potent pro-apoptotic responses regulated by cytoskeletal rearrangements, and c) these receptors may represent specific targets for the development of novel Molecular Cancer 2009, 8:114 http://www.molecular-cancer.com/content/8/1/114 Page 13 of 14 (page number not for citation purposes) drugs, since their activation drastically regresses tumor growth and tumor incidence in vivo. Additional experiments are now required for the identification of the molecular identity of these receptors.
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Substantial parts of the Conclusion seem to have been published two years before the thesis.

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Figure 19: Pro-apoptotic effects of testosterone-HSA in the absence or presence of inhibitors in Caco2 cells

Quantitative APOPercentage apoptosis assay of TAC stimulated Caco2 cells and similar experiments in the presence of anastrozole. Cells were exposed or not to 10-7 M testosterone-HSA for 24 hours and proapoptoric responses were assessed by the APOPercentage apoptosis assay. Equally, cells pre-treated or not with 10-6 M anastrozole was exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods of time served as a positive control for apoptosis. Bars present the mean OD measured at 550 nm. *** P<0,001, n=3.

Thus, we aimed to further evaluate the in vivo effects of albumin- PFrigou-arpeo p5totic effects of testosterone-HSA, DHT and estradiol in the absence or presence of inhibitors in Caco2 cells Pro-apoptotic effects of testosterone-HSA, DHT and estradiol in the absence or presence of inhibitors in Caco2 cells. (A) Quantitative APOPercentage apoptosis assay of testosterone-HSA, DHT or estradiol stimulated Caco2 cells and in the presence/absence of cytochalasin B (Cyto B) or flutamide. Cells were exposed to 10-7 M testosterone-HSA, DHT or estradiol for 24 hours and pro-apoptoric responses were assessed by the APOPercentage apoptosis assay. Equally, cells pretreated or not with 10-7 M Cyto B or flutamide, were exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods of time served as a positive control for apoptosis. Bars present the mean OD measured at 550 nm (** P < 0.01, N = 4). (B) Cells were pre-treated or not with Cyto B or flutamide for 1 h and then exposed or not to 10-7 M testosterone- HSA for 4 h, lysed and incubated with the caspase-3 substrate DEVD conjugated to the chromophore pNA according to the manufacturer's instructions.
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