Perspectives

4.6.1 ER β in PCa treatment

The investigation of the role of ER β in PCa development and progression is limited by the accuracy of ER β expression analysis. Many supposed specific antibodies are available, but still no consensus about the expression of ER β in PCa could be found (Christoforou et al.

2014; Dey et al. 2014). In the present study, contradictory results between ER β mRNA and protein expression were found. Therefore, better analysis tools to estimate ER β expression are needed. Only when reliable data about the expression status of ER β in PCa could be generated, reliable studies about ER β-mediated signaling could be made.

Another major issue during the investigation of the role of ERs in PCa was associated with the TRAMP mouse model. As described above, TRAMP mice developed SGCa with high incidences. SGCa in humans are very rare (Tarjan et al. 2009) and in TRAMP mice SGCa occurs as a side effect of the SV40 T/t antigene overexpression (Tani et al. 2005). However, the massive growth of SGCa resulted in the earlier termination of the experiments. Other PCa mouse models, i.e. the PTEN mouse model could reduce undesired side effects and therefore offers the possibility to analyze the prostate and prostate cancer development over longer time periods.

Most studies claimed that prostate cancer cells exhibit only low ER β expression (Dey et al.

2014; Christoforou et al. 2014). The epigenetic silencing of ER β expression during PCa progression is one argument for the identification of ER β as a tumor suppressor in PCa (Stettner et al. 2007). Important studies on the role of ER β in PCa therefore use artificial overexpression of ER β together with ER β agonist treatment to show how ER β activation impairs signaling in PCa cells (Dey et al. 2014). Since overexpression experiments are susceptible for artificial effects and moreover not applicable in patients, other methods to induce ER β re-expression are needed. It could be shown that treatment with DNA methlytransferase inhibitors and histone deacetylase inhibitors induces the expression of ER β in advanced PCa (Walton et al. 2008). In this way, the induced re-expression of ER β in PCa is sufficient to reduce PCa proliferation and induce apoptosis (Stettner et al. 2007;

Walton et al. 2008). Therefore, it would be interesting if a combined treatment with DNA methlytransferase inhibitors and histone deacetylase inhibitors together with ER β agonists could further improve treatment effects.

The molecular signaling mediating 8VE2-induced treatment effects seems to be ER β-independent. However, the exact mechanism could not be fully elucidated in the present study. It should be considered that the whole mRNA-sequencing analysis did not give information about RNA without polyadenylation (in consequence of the usage of oligo-dT primers for library generation). Therefore, we cannot make assertions about the potential involvement of those regulatory RNAs. Questions concerning the role of the cholesterol synthesis pathway and AR signaling remain unsolved. It could be shown that several enzymes involved in the cholesterol synthesis pathway are overexpressed upon 8β-VE2 treatment and that this effect is not induced by inhibition of P450scc. However, as described above the cholesterol synthesis pathway is involved in PCa progression and therefore, the deregulation of cholesterol synthesis pathway upon 8β-VE2 treatment might be an important factor. It is unclear, whether the upregulation of the cholesterol synthesis pathway is a direct effect, induced by loss of feedback inhibition because cholesterol is depleted or a consequence of induction of other pathways. Indeed, the cholesterol synthesis pathway is known to be regulated by PI3K and AR signaling in PCa (Krycer, Brown 2013). To get more insights into the processes a measurement of cholesterol and steroid hormones in the cells and the medium could be performed. If cholesterol concentration is high, this would indicate that cholesterol synthesis is used to promote survival in 8β-VE2-treated cells. Low cholesterol concentration would indicate either cholesterol or cholesterol precursor depletion or inhibition of another enzyme involved in the synthesis process.

The observation that 8β-VE2 treatment reduced cell survival and induced apoptosis in the AR-positive cell line VCaP but not in AR-negative PC3 cells indicates that the 8β-VE2-mediated effects rely on AR activation. Similar effects were observed for HE3235, which was also only effective in AR-positive cancer cells (Trauger et al. 2009). Performing further experiments with HE3235 could help to elucidate which mechanisms are induced. In order to prove that AR activation is essential for 8β-VE2-induced effects, PC3 cells with artificial AR expression should be treated with 8β-VE2 and cell survival, apoptosis induction and gene expression should be investigated. Furthermore, the results from the present study suggest that the inhibition of an alternative AR-mediated survival pathway could be the underlying mechanism mediating 8β-VE2 treatment effects. Here, Wnt/β-catenin signaling and EGF/EGFR signaling were reported to be potential mediators of the alternative survival pathway. Investigation of this hypothesis should be performed by combined treatment of VCaP cells with 8β-VE2 and Wnt, β-catenin or EGFR inhibitors. The effects of Wnt, β-catenin or EGFR inhibition on cell survival, proliferation and gene expression should be investigated.

4.6.2 Amygdalin in PCa treatment

This study showed that the alternative cancer drug amygdalin potentially induces anti-cancerous effects. However, it became clear that one of the main obstacles in amygdalin

treatment is that it is still not fully understood how amygdalin is metabolized and how it

It needs to be addressed if and how amygdalin selectively can affect tumor cells. The present study described that β-glucosidase and rhodanase expression alone could not explain the supposed tumor cell specificity. Moon et al. (2015) reported that rather increased enzyme activity than increased expression leads to tumor cell selective effects of amygdalin. To investigate if amygdalin can potentially be metabolized in PCa cells, an in vitro study could be performed, i.e. cyanide concentration could be measured in medium of amygdalin-treated PCa and fibroblast cells. Furthermore, the inhibition of telomerase upon amygdalin treatment was demonstrated (Moon et al. 2015). The experiments performed by Moon et al. (2015) described effects on different cancer cell lines including lung cancer, breast cancer and glioblastoma. Therefore, it would be interesting to investigate if the effect described by Moon et al. (2015) also occurs in PCa cells. Inhibition of telomerase activity should induce telomere shortening and thus could be responsible for the induction of senescence. Shortened telomeres could be detected in vitro and in vivo by FISH analysis. Incubation with telomere specific probes should give information about telomere size. Moreover, the expression of PARP, p21 and p53 should be investigated to elucidate the mechanism of senescence and necrosis induction. Analysis of the expression of β1 and β4 integrin could reveal information about the molecular signaling events underlying the reduced invasive potential of PCa cells upon amygdalin treatment. The degradation of protein quality during amygdalin treatment hinders such investigations in vitro and in vivo. The induction of necrosis furthermore impedes signaling analysis in prostate tumor tissue. Therefore, it would be reasonable to terminate in vivo experiments at an earlier time point, because in non-advanced prostate tumors necrosis might not be progressed, and therefore, differences in cell signaling could be detected. However, adequate timing is mandatory since in vitro analysis showed that amygdalin induces effects only after longer periods of treatment. To further elucidate the underlying mechanisms of amygdalin action in the TRAMP-FVB mouse model, in vivo analysis of induction of senescence in prostate tumors should be performed. This could give information whether necrosis is induced as a consequence of oxidative stress in senescent tumor cells or mediated by a senescence-independent mechanism.