Amygdalin treatment induces senescence in vitro

We could show that the agent amygdalin induces senescence in the murine primary prostate cancer cell lines T244 and 2E. Upon amygdalin treatment, the cells cease proliferation without induction of apoptosis or cytotoxic cell death. Together with the time- and dose-dependent changes of cell morphology and the induction of SA β-gal expression clear evidence of senescence induction was found.

Cellular senescence is an irreversible arrest of proliferation. Senescent cells stay metabolic active and maintain cellular signaling, especially release of immunogenic cytokines (Campisi 2013). Senescent cells show morphological changes (Bayreuther et al. 1988; Cho et al.

2004) and SA β-gal expression (Dimri et al. 1995). SA β-gal staining is based on the increased expression of lysosomal β-galactosidase in senescent cells and is used as a senescence marker (Lee et al. 2006). Different causes for induction of senescence are known. Replicative senescence, also known as Hayflick limit, occurs after a cell line-specific number of cell cycles due to telomere shortening (Hayflick, Moorhead 1961). Telomeres, the DNA-protein structures that cap the ends of linear chromosomes, shorten with every cell division (Allsopp et al. 1995). Telomere shortening is associated with chromosomal instability (Murnane 2012). To prevent instability and the associated risk of cancer development p53-mediated senescence is induced (Rodier et al. 2005). Accelerated senescence is induced by other factors, e.g. DNA damage, induction of DSB or mitotic catastrophe (Nakamura et al.

2008; Robles, Adami 1998). Oxidative stress or strong mitogenic activation driven by oncogenes can also induce accelerated senescence (Robles, Adami 1998; Nakamura et al.

2008; Barascu et al. 2012; Parrinello et al. 2003; Blagosklonny 2003). Furthermore, the activation of tumor suppressor genes such as p53, p21 or pRB (retinoblastoma protein) can induce and mediate senescence (Adams 2009; Roninson 2003). The secretion of immunogenic cytokines by senescent cells induces immune recognition and clearance and thereby prevents malignant degeneration (Freund et al. 2010). During their degeneration cancer cells shut down pathways that are responsible for proliferation inhibition and cell death induction. Therefore, the induction of replicative senescence as well as accelerated senescence is often circumvented in cancer cells (Dimri 2005). Moreover, it is hypothesized that the physiological function of accelerated senescence is the blockade of oncogenic degeneration. It could be shown that inhibition of senescence induction facilitates malignant degeneration of cells (Boehm, Hahn 2005).

Therefore, senescence induction is desired in cancer treatment and prevention (Collado, Serrano 2010; Prieur, Peeper 2008). Stop of proliferation of cancer cells by induction of cellular senescence was shown to be very effective in cancer treatment and prevention (Chen et al. 2005; Dimri 2005). Chen et al. (2005) reported that the induction of senescence can inhibit malignant degeneration of prostate in PTEN-deficient mice. In prostate cancer, senescence was identified, next to apoptosis and mitotic catastrophe, to be induced by classical anti-cancer treatment e.g. docetaxel (Schwarze et al. 2005). Also ADT was reported to induce senescence in PCa cell lines (Burton et al. 2013). Moon et al. (2015) reported that amygdalin treatment decreases telomerase activity in cancer cells but not in fibroblast cells.

Telomerase is the enzyme is responsible for the maintenance of telomeres and therefore of essential importance for the control of replication (Greider, Blackburn 1985). Inactivation of telomerase leads to chromosomal instability and therefore potentially induces senescence

(Xu et al. 2015). Moreover, Moon et al. (2015) demonstrate a cancer-specific effect of amygdalin treatment, the mechanism of this specificity could not be identified so far. The present study showed that DSBs and mitotic catastrophe occurs after amygdalin treatment and thus indicates that DNA damage plays a major role in the amygdalin-mediated induction of senescence. DSB as well as mitotic catastrophe can be induced by different causes and through different mechanisms. DSB can be caused by e.g. ionizing radiation, DNA intercalating chemotherapeutics (e.g. cisplatin or melphalan), potassium cyanide or the detergent Triton-X 100 (Mladenov, Iliakis 2011; Vock et al. 1998; Winn et al. 2003; Mladenov, Iliakis 2011). Amygdalin is a cyanide-containing compound, therefore it is likely that the induction of DSBs is mediated by the cyanide ion. Cyanide-containing compounds were described to induce DSBs by inhibition of the mitochondrial enzyme cytochrome oxidase (Pettersen, Cohen 1993). The inhibition of cytochrome oxidase induced a number of events, next to others, the release of reactive oxygen species (ROS) (Way 1984). ROS can bind to DNA and thereby induce DSB (Winn et al. 2003). Furthermore, ROS can directly activate p53 and therefore induces senescence (Singh et al. 2010). The exact mechanism how DSB induce senescence is still under debate. A complex network of DNA damage sensors, apical local kinases, DNA damage mediators, downstream diffusible kinases and other effectors is active and responsible for induction of either transient cell cycle arrest, senescence or apoptosis (Di d'Adda Fagagna 2008).

Mitotic catastrophe results from chromosomal instability (Vitale et al. 2011). Missegregation of chromosomes during mitosis leads to aneuploid daughter cells. Aneuploidy is not unusual in cancer cells, thus in mitotic catastrophe the chromosome integrity is damaged beyond repair. Cell division is terminated and therefore mitotic catastrophe results in either apoptosis or senescence induction (Mansilla et al. 2006; Eom et al. 2005). DNA damage can also result in mitotic catastrophe when cell cycle checkpoints are deregulated (Huang et al. 2005). Here, it is unclear if amygdalin treatment directly induces mitotic catastrophe or if mitotic catastrophe is a consequence of amygdalin-induced DSBs. The inactivation of telomerase as described by Moon et al. (2015) could also result in mitotic catastrophe (Hornsby 2007).

As described above, DSBs as well as mitotic catastrophe can induce either apoptosis or senescence. In the present study senescence was induced. Other studies report cell cycle arrests (Makarevic et al. 2016) or apoptosis induction (Chang et al. 2005) in PCa cells upon amygdalin treatment. Both, cell cycle arrest and apoptosis induction could also be consequences of DNA damage. The severity of damage as well as the molecular profile of cells seems to be responsible for which cell fate is induced, either apoptosis or cell cycle arrest (Di d'Adda Fagagna 2008; Vakifahmetoglu et al. 2008).