Etoposide VP a semisynthetic podophyllotoxin derivative agen

Etoposide (VP-16), a semisynthetic podophyllotoxin derivative agent, is widely used as a chemotherapic treatment for many cancers (Hande, 1998). VP-16 has been known that its activity is mediated by the interaction with topoisomerase II (an ATP-dependent nuclear enzyme) which regulates DNA topology and induces of protein-cross-linked DNA strand breaks and sister chromatid exchanges (Bromberg et al., 2003, Chatterjee et al., 1990). Accumulating evidence reported that treatment with etoposide not only induces apoptosis in various culture cancer cells, but also widely used in different protocols in pancreatic cancer (Arlt et al., 2001, Iwasa et al., 2010, Sakamoto et al., 2009). Moreover, etoposide also activates the intracellular response-mediated signals such as mitochondrial dysfunction, reactive oxygen species (ROS) production, activation of protein kinase C (PKC) δ, Akt and p53 leading to cell death (Jamil et al., 2015, Marengo et al., 2011, Oh et al., 2007, Zhang and Huang, 2013). Thus, it is possible that etoposide can induce several cellular responses, beside apoptosis, in cancer sc9 resistant to this drug or in non-cancerous viable cells, especially in pancreatic β-cells (the vulnerable to chemicals-induced injuries). A number of stimuli are known to activate members of the mitogen-activated protein kinase (MAPK) family, which mainly consists of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase (Kyosseva, 2004). These proteins are important mediators of intracellular signals and play a key role in the regulation of many processes including survival and apoptosis (Chang and Karin, 2001). Therefore, MAPKs-dependent signal transduction is required for physiological adaption, but inappropriate MAPKs signaling contribute to the development of several interdependent pathological traits and disease processes (Gehart et al., 2010, Kim and Choi, 2010). Recently, a number of studies reported that toxic insults (such as cytokine or heavy metals) contribute to onset pancreatic β-cell injuries leading to the progression of DM, which is correlated with MAPKs-regulated pathways (Akerfeldt et al., 2008, Chang et al., 2013, Yang et al., 2016). Furthermore, glycogen synthase kinase-3 (GSK-3), an ubiquitous serine/threonine kinase that is present in two isoforms: α and β, has been demonstrated as a kinase for nearly 50 different proteins in the variety of pathways which mediates many fundamental processes (including metabolism, cell structure, proliferation and differentiation, and apoptosis) and implicates in the many of diseases development (such as Alzheimer's disease, cancer, and DM)(Grimes and Jope, 2001, Jope et al., 2007). It has been indicated that GSK-3 was found to be an important component of the intrinsic apoptotic responses occurring by DNA damage, mitochondrial toxins, hypoxia/ischemia, and many other insults (Beurel and Jope, 2006). Growing evidence are also reported a central role of GSK-3 in regulating stimulus-induced mammalian cell apoptosis, particularly in pancreatic β-cells contributing to the reduction in the cell mass and proliferation, the impairment of insulin signaling and insulin action, and the development of DM (Lee and Kim, 2007, Liu et al., 2008, Spokoini et al., 2010).
Materials and methods
Results
Discussion Etoposide (VP-16) is an antitumor drug used as a single agent or in association with other chemotherapeutic drugs for hematopoietic and solid tumors such as pancreatic cancer (Iwasa et al., 2010, O'Dwyer et al., 1985, Sakamoto et al., 2009). However, etoposide is also caused severe side effects (such as induced myocardial ischemia or hypotension) and known to be cytotoxicity in various cells including fibroblasts, germ cells, neuroepithelial cells, and mouse mesenchymal cells (Lizama et al., 2011, Nam et al., 2006, Yun et al., 2009). Therefore, etoposide not only can trigger therapeutic responses in pancreatic cancer cells, but also induces cytotoxic effect in pancreatic β-cells (a non-cancerous viable cells). Although some studies reported the activation of apoptosis-related signals in etoposide-caused cell death, the toxicological effects and detailed mechanisms underlying etoposide-induced β-cell death are mostly not understood. The present study for the first time found that etoposide induces cytotoxicity and apoptotic characteristics, including the increase in sub-G1 hypodiploid cells population, apoptotic cells, and caspase-3 activity. Furthermore, mitochondria is a very sensitive organelle to toxic insults, and mitochondrial dysfunction has been demonstrated to a crucial role in regulating mammalian cell apoptosis (Chang et al., 2013, Chen et al., 2013). Depending upon the death stimulus, alteration happens in mitochondrial physiology and ultra-structure eventually leading to the change in permeability of the mitochondrial transition pore and membrane potential and the release of apoptogenic proteins (Lu et al., 2014, Rasola and Bernardi, 2007). Mitochondria function is regulated by Bcl-2 family proteins, which can cause the mitochondrial Ca release dysfunction and the loss of MMP by the high ratio of pro-apoptotic (such as Bax and Bak) to anti-apoptotic (Bcl-2 and BclXL) members, resulting in the cytochrome c release from the intermembrane space of mitochondria to the cytosol carry out the process of apoptosis (Adams and Cory, 2007, Lu et al., 2014). Custodio et al. (2001) findings showed that etoposide can cause the extensive swelling of mitochondria, leading to the depolarization of MMP in rat liver mitochondria, putatively as a consequence of mitochondrial permeability transition (MPT) induction. These results suggested that the induction of apoptosis and/or cellular ATP depletion as consequence of etoposide-induced MPT maybe explain the cytostatic activity of this drug and side-effects in normal cells observed in vivo. In this study, we observed that etoposide-treated RIN-m5F cells cause a significant induce the loss of MMP and a marked increase in cytochrome c release from the mitochondria into the cytosol, which accompanied with the decrease in Bcl-2 and the increase in Bax mRNA and protein expressions, leading to the promotion of caspase proteases (caspase-3/-6/-7/-9) activation and apoptosis in RIN-m5F cells. These results a mitochondria-dependent pathway is involved in etoposide-induced β-cell apoptosis.