Apoptosis Mitochondrial activation

The study of the cytotoxicity of doxorubicin, a red coloured molecule for which cytotoxic effects involve at least three mechanisms (necrosis/apoptosis, mitochondrial activity and oxidative stress) connected in some way or another, is a prime example to demonstrate the relevance of fluorimetric methods. 

Numerous studies have demonstrated that degradation products of (3-carotene exhibit deleterious effects in cellular systems (Alija et al., 2004, 2006 Hurst et al., 2005 Salerno et al., 2005 Siems et al., 2003). A mixture of (3-carotene degradation products exerts pro-apoptotic effects and cytotoxicity to human neutrophils (Salerno et al., 2005 Siems et al., 2003), and enhances the geno-toxic effects of oxidative stress in primary rat hepatocytes (Alija et al., 2004, 2006), as well as dramatically reduces mitochondrial activity in a human leukaemic cell line, K562, and RPE 28 SV4 cell line derived from stably transformed fetal human retinal pigmented epithelial cells (Hurst et al., 2005). As a result of degradation or enzymatic cleavage of (3-carotene, retinoids are formed, which are powerful modulators of cell proliferation, differentiation, and apoptosis (Blomhoff and Blomhoff, 2006). 

Intrinsic (mitochondrial) pathway of caspase activation is initiated by the permeabilization of the mitochondrial outer membrane by proapoptotic members of the Bcl-2 family, resulting in a release of cytochrome c and other proteins from the intermembrane space of mitochondria into the cytosol. Cytochrome c translocation to the cytosol may follow a number of possible mechanisms. However, once in the cytosol, cytochrome c binds to apoptosis protease activating factor (Apaf-1) and in the presence of dATP or ATP facilitates Apaf-1 oligomerization and the recruitment of procaspase-9. The formation of this caspase-activating complex, termed the apoptosome, results in the activation of procaspase-9, and this in turn cleaves and activates the effector caspase-3 and -7. Activated effector caspases cleave key substrates in the cell and produce the cellular and biochemical events characteristic for apoptosis [33-35]. 

It had long been known that apoptosis and activation of caspase are coupled with mitochondrial permeabilization. However, until 1996, the molecular link between mitochondria and caspase activation had not been known. Dr. Wang s group at the University of Texas isolated activators of caspase 3 by using classical fractionation methods in 1996-1997. One protein that was required for activation of caspase 3 was a 15-kD protein with pink color, which was cytochrome c. This finding is rather surprising because cytochrome c is localized in mitochondria and has a well-established role as an electron carrier. Cytochrome c was later shown to be released... 

Li, P., Nijhawan, D., and Wang, X. Mitochondrial activation of apoptosis. Cell 116, S57-S59. Newmeyer, D. D., and Ferguson-Miller, S. Mitochondria Releasing power for life and unleashing the machineries of death. Cell 112, 481 190, 2003. 

MK-2 induced apoptosis to a much lesser extent than GG. The major difference between MK-2 and GG is the u,(J>-unsaluraled ketone structure present in MK-2 but not in GG. The induction of cell death by MK-2 or GG was not coupled with radical generation. It has recently been reported that the induction of apoptosis by MK-4 in human ovary cancer cells is mediated by oxidative stress in mitochondria [70]. Since autophagy plays an important role in reducing mitochondrial damage and reactive oxygen species, there is a possibility that the apoptosis-inducing activity of MK-4 may be derived from the inhibition of autophagy. 

Figure 1. Schematic of the apoplotic signaling pathways. The intrinsic apoptotic pathway consists of the mitochondrial pathway. The extrinsic pathway mediates apoptosis through activation of the death receptors, TNF-o/Fas receptors. Apoptosis is executed by activation of proteases, known as caspascs. ATP is essential for apoptosis. Bel-2 family proteins are apoptosis regulating proteins Bcl-2 inhibits while Bid, Bax, Bad facilitate apoptosis. An interaction between these two apoptotic pathways exists. See text for a more detailed explanation.

Mitochondrion-selective reagents for assessment of mitochondrial activity, localization and abundance, monitoring effects of pharmacological agents, such as anesthetics that alter mitochondrial function. Important role in apoptosis... 

The second phase, proposed by Moghimi et al., was said to occur 24 h after exposure, and consisted of a loss of mitochondrial membrane potential which was revealed by translocation of phosphatidylserine as a consequence of PEI-induced channel formation. This led to release of the pro-apoptotic cytochrome c and subsequent activation of caspases-3 triggering apoptosis. Mitochondrial-mediated apoptotic events induced by polycations have also been reported in cell lines treated with high MW PLL in free form, DNA polyplexes and PAMAM dendrimers. In the study by Lee et al., PAMAM dendrimers around 45 nm in size exhibited mitochondrial co-localization, decreased expression of mitochondrial genes and mitochondria membrane... 

Roos et al. demonstrated the induction of p53-dependent apoptosis through p53 transcriptional activation in mES cells. Upon induction of CX-methylguanine by treatment with N-methyl-N-nitro-N-nitrosoguanidine, apoptosis seemed activated via the Fas death pathway as the Fas receptor, which is transcriptionally regulated by p53, was upregulated and caspase-3 and -7 were activated. The lack of cytochrome-C release and the increase of the Bcl-2/Bax fraction, indicative of pa-otection against mitochondrial-mediated apoptosis, demonstrate the inactivity of this latter apwptotic p>athway (Roos et al. 2007). 

Tan-I inhibited cell growth dose dependently at concentrations ranging from 0.5 to 25 pM and induced apoptosis in activated T-HSC/C-6 hepatic stellate cells. Tan-1 increased caspase-3 activation via cytochrome c release and loss of mitochondrial membrane potential [48]. Nizamutdinova and coworkers investigated the effect of Tan-1 on the induction of apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) in vitro. Tan-I inhibited cell proliferation of MCF-7 and MDA-MB-231 cells in a dose- and time-dependent manner and significantly induced apoptosis in MCF-7 and MDA-MB-231 cells. The induction of apoptotic cell death was mediated by the activation of caspase-3 the downregulation in the level of the... 

Cytochrome c-dependent mitochondrial apoptosis involves a number of concerted steps leading to the formation of the apoptosome and the downstream activation of caspase-3 [10,11]. The predominant mechanism is believed to involve the following events (Fig. 4) (1) a transient collapse of the mitochondrial transmembrane potential (2) opening of the permeabiUty transition pore (FTP) (3) release of cytochrome c and procaspase-9 (4) recruitment of Apaf-1 (apoptosis protease activating factor-1) and dATP and assembly of the mitochondrial apoptosome (5) activation of procaspase-9 by the apoptosome and (6) the caspase-9-catalyzed activation of caspase-3, the molecular executor of apoptosis. Amplification through the caspase cascade leads to the downstream... 

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