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19 ungesichtete Fragmente: Plagiat

[1.] Shg/Fragment 013 14 - Diskussion
Bearbeitet: 29. October 2014, 18:44 (Kybot)
Erstellt: 24. October 2014, 13:39 SleepyHollow02
BauernOpfer, Fragment, SMWFragment, Schutzlevel, Shg, Zhu Kyprianou 2008, ZuSichten

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Quelle: Zhu Kyprianou 2008
Seite(n): 841, Zeilen: abstract
The iAR activated target genes are an array of growth factor genes, e.g. epidermal growth factor (EGF); fibroblast growth factor (FGF); Insulin-like growth factor 1(IGF1); vascular endothelial growth factor (VEGF); transforming growth factor-β (TGFβ). The ability of iAR to cross-talk with key growth factor [signaling events toward the regulation of cell cycle, apoptosis, and differentiation outcomes in prostate cancer cells has been established.] The ability of AR to cross-talk with key growth factor signaling events toward the regulation of cell cycle, apoptosis, and differentiation outcomes in prostate cancer cells is established. In this paper, we review the functional interaction between AR and an array of growth factor signal transduction events (including epidermal growth factor; fibroblast growth factor; IGF1; vascular endothelial growth factor; transforming growth factor-b) in prostate tumors.

Androgen-induced prostate epithelial cell proliferation is regulated by an indirect pathway involving paracrine mediators produced by stromal cells, such as insulin-like growth factor (IGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF; Cunha & Donjacour 1989, Byrne et al. 1996).

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(SleepyHollow02)

[2.] Shg/Fragment 014 02 - Diskussion
Bearbeitet: 14. December 2014, 08:24 (SleepyHollow02)
Erstellt: 24. October 2014, 13:51 SleepyHollow02
BauernOpfer, Fragment, SMWFragment, Schutzlevel, Shg, Zhu Kyprianou 2008, ZuSichten

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Seite(n): 843, Zeilen: figure 1
IGF, FGF, VEGF, and TGFβ secreted by the prostate stromal cells activate their receptors and interact with iAR signal axis. In prostate epithelial cells, the androgenic signal engages secreted VEGF and TGFβ which affect the prostate tumor microenvironment by inducing angiogenesis, stromal cell growth and differentiation. EGF signaling encounters iAR signal in a tight control of multiple pathways. Growth factor signaling may proceed via iAR signal and regulate the downstream effectors of iAR regulating key cellular processes including proliferation, differentiation, apoptosis, and survival of prostate cancer cells. [Meng-Lei Zhu, Natasha Kyprianou. 2008] IGF, FGF, VEGF, and TGFB secreted by the prostate stromal cells activate their receptors and interact with AR signal axis. In prostate epithelial cells, the androgenic signal engages secreted VEGF and TGFB which affects the prostate tumor microenvironment by inducing angiogenesis and stromal cell growth and differentiation. EGF signaling encounters AR signal in a tight control of multiple pathways. Growth factor signaling may proceed via AR signal and regulate the downstream effectors of AR regulating key cellular processes including proliferation, differentiation, apoptosis, and survival of prostate cancer cells.
Anmerkungen

Praktisch wörtliche Übernahme, die aus der Quellenangabe am Ende nicht hervorgeht.

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(SleepyHollow02)

[3.] Shg/Fragment 015 01 - Diskussion
Bearbeitet: 29. October 2014, 18:44 (Kybot)
Erstellt: 25. October 2014, 05:53 SleepyHollow02
BauernOpfer, Foradori et al 2008, Fragment, SMWFragment, Schutzlevel, Shg, ZuSichten

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[It has to take the additional time for] mRNA to be translated into proteins and for those proteins to be processed and induce measurable responses. The cellular responses are changes in free intracellular calcium, and activations of second messenger pathways. The second criterion is membrane mediated. The response should include embedding membrane or associating receptors or binding proteins. The action can be induced even when the steroid is conjugated to molecules which prohibit it from translocation to the nucleus when bound to a receptor. The most common example is the use of testosterone (T) conjugated to large molecules such as bovine serum albumin (BSA). The last is lacking transcription/ translation machinery activation. Experiments using cell lines either lacking the necessary machinery for a genomic response or identifying androgen effects, they are insensitive to inhibitors of transcription and translation. All this demonstrated that certain steroid responses can be elicited in systems where gene transcription or protein synthesis is was unlikely or impossible. [C.D.Foradori, et al. 2008] However, it then takes additional time for mRNA to be translated into proteins and for the proteins to be processed and induce measurable responses. Typically, cellular responses, which meet this requirement, are changes in free intracellular calcium, and activation of second messenger pathways. 2) Membrane mediated: the response may involve membrane embedded or associated receptors or binding proteins, and with an action that can be induced even when the steroid is conjugated to molecules that prohibit it from entering deep into the cytoplasm or from translocating to the nucleus when bound to a receptor. The most common example is the use of testosterone (T) conjugated to large molecules such as bovine serum albumin (BSA). 3) Lacking transcription/translation machinery activation: experiments using either cell lines that lack the necessary machinery for a genomic response or identify androgen effects which are insensitive to inhibitors of transcription and translation, thereby demonstrate that certain steroid responses can be elicited in systems where gene transcription or protein synthesis is unlikely or impossible.
Anmerkungen

might be considered "no verdict" as well since the source is given and it is not a literal copy.

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(SleepyHollow02)

[4.] Shg/Fragment 074 26 - Diskussion
Bearbeitet: 14. December 2014, 08:25 (SleepyHollow02)
Erstellt: 1. November 2014, 13:03 SleepyHollow02
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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.

Anmerkungen

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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(SleepyHollow02)

[5.] Shg/Fragment 075 01 - Diskussion
Bearbeitet: 1. November 2014, 13:12 (SleepyHollow02)
Erstellt: 1. November 2014, 13:12 SleepyHollow02
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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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[6.] Shg/Fragment 076 17 - Diskussion
Bearbeitet: 2. November 2014, 08:09 (SleepyHollow02)
Erstellt: 1. November 2014, 13:18 SleepyHollow02
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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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(SleepyHollow02)

[7.] Shg/Fragment 077 01 - Diskussion
Bearbeitet: 1. November 2014, 13:23 (SleepyHollow02)
Erstellt: 1. November 2014, 13:23 SleepyHollow02
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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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(SleepyHollow02)

[8.] Shg/Fragment 048 10 - Diskussion
Bearbeitet: 2. November 2014, 13:17 (SleepyHollow02)
Erstellt: 2. November 2014, 13:17 SleepyHollow02
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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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[9.] Shg/Fragment 049 02 - Diskussion
Bearbeitet: 2. November 2014, 13:21 (SleepyHollow02)
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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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[10.] Shg/Fragment 050 01 - Diskussion
Bearbeitet: 2. November 2014, 13:24 (SleepyHollow02)
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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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[11.] Shg/Fragment 051 01 - Diskussion
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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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[12.] Shg/Fragment 052 01 - Diskussion
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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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[13.] Shg/Fragment 053 01 - Diskussion
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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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[14.] Shg/Fragment 054 01 - Diskussion
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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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[15.] Shg/Fragment 082 01 - Diskussion
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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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[16.] Shg/Fragment 66 01 - Diskussion
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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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[17.] Shg/Fragment 060 01 - Diskussion
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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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[18.] Shg/Fragment 061 01 - Diskussion
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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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[19.] Shg/Fragment 062 01 - Diskussion
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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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