Western blotting and flow cytometry was employed
Western blotting and flow cytometry was employed to assess the DNA-PK inhibition of LTU28 and LTU31 in combination with radiation. It has been previously reported that phosphorylation of DNA-PKcs at the Thr2609 cluster plays an important role in DSB repair and resistance to radiation (Ding et al., 2003, Reddy et al., 2004, Nagasawa et al., 2011). Another in vivo study showed autophosphorylation of Ser2056 is also a reliable indicator of DNA-PK activation and that radiation induced DNA-PKcs autophosphorylation plays a major role in DSB repair through NHEJ. (Chen et al., 2005, Chen et al., 2007). Our results showed that LTU28 + 6Gy inhibited DNA-PK phosphorylation at Ser2056 and Thr2609 in both cell lines.
The action of LTU28 and LTU31 on AKT phosphorylation, one of the downstream targets of DNA-PK was also analysed. Our results showed that LTU28 + 6Gy inhibited AKT1 phosphorylation at Ser473 and Thr308 residues though not completely. Previous studies have reported the involvement of various signalling pathways other than DNA-PKcs in the activation of AKT (Brazil and Hemmings, 2001, Sarbassov et al., 2005, Guertin et al., 2006). We are hypothesizing that other modes of activation would be involved in failure to completely inhibit AKT phosphorylation by LTU28 after exposure to radiation.
The effect of LTU28 and LTU31 in combination with radiation on the activation of BRCA1, one of the key enzymes that facilitates DNA-DSB repair through HR pathway was also analysed. Our results showed that LTU31 in combination with radiation completely inhibited the phosphorylation of BRCA1. The effect of a BRCA1 inhibitor that radiosensitized breast cancer MM-102 synthesis to radiation and inhibited DSB repair through HR has been reported earlier (Pessetto, Yan, Bessho, & Natarajan, 2012). We are proposing that LTU31 when used in combination with radiation is inhibiting BRCA1 activation and thereby inhibits the HR pathway.
Conflict of interest
Acknowledgements The authors would like to thank the staff of Peter MacCallum Bendigo Radiotherapy Centre for their support and technical assistance for irradiation of cell lines. Suraj Radhamani was a recipient of Latrobe University Postgraduate Research scholarship (LTUPS) and Full fee Research scholarship (LTUFFRS), with additional research funding by La Trobe University.
Introduction One of the aging-associated metabolic changes is the loss of mitochondrial content and function in tissues such as skeletal muscle (Barazzoni et al., 2000, Petersen et al., 2003, Short et al., 2005). Since mitochondria convert nutrients to energy and heat, the mitochondrial decline is relevant to aging-associated decline in metabolic rate and exercise capacity. Lee et al. (2010) reported that the rise of mitochondrial reactive oxygen species (ROS) with aging causes a decline in mitochondrial function in skeletal muscle. This can lead to metabolic dysfunctions such as insulin resistance, which can lead to chronic diseases such as type 2 diabetes. Accumulating evidence indicates that the aging-associated decline in skeletal muscle activity of AMP-activated protein kinase (AMPK), a key regulator of mitochondrial function and energy balance (Hardie, 2007), plays an important role in the metabolic decline associated with aging (Koonen et al., 2010, Lee et al., 2010, Qiang et al., 2007, Reznick et al., 2007). AMPK has numerous functions, including stimulation of glucose uptake, fat oxidation, energy production, scavenging of oxygen radicals, and mitochondrial biogenesis (Ruderman et al., 2013). Increased AMPK activity decreases visceral fat (Narkar et al., 2008) and increases mitochondrial biogenesis and energy production in skeletal muscle, resulting in improved physical fitness (Zong et al., 2002). On the other hand, AMPK deficiency in skeletal muscle leads to mitochondrial loss, impaired glucose uptake, and exercise intolerance (O’Neill et al., 2011). Metformin, the most commonly used type 2 diabetes drug, acts in part by AMPK activation (Zhou et al., 2001). However, the molecular mechanism by which aging decreases AMPK activity in skeletal muscle is poorly understood.