br Materials and Methods A human skin SCC
Materials and Methods A431 (human skin SCC) and HT-297.T (human AK) cell lines were purchased from ATCC (Manassas, VA) and were maintained in DMEM supplemented with 10% fetal calf serum, 4 mM L-Gln, 1 mM sodium pyruvate, 100 U/mL penicillin, and 1 μg/mL streptomycin. Human HK1 and HK2 plasmids were purchased from Addgene (Cambridge, MA) and were purified from Escherichia coli. VDAC1 was purified from sheep liver mitochondria as described (Arbel et al., 2012). Human skin tissue microarray (SK208 and SK 805) were purchased from US Biomax Inc (Rockville, MD), which attests that all human materials was obtained under institutional approval of experiments and with written informed patient consent.
Conflict of Interest All authors, except EL and MH, are employees at Vidac Pharma, Ltd, Israel. EL is an employee at Patho-Logica Ltd, Israel and was not a paid consultant. MH is a stock holder at Vidac Pharma and the inventor of Comp-1.
Introduction In the heart, ischemic preconditioning (IPC) is a process whereby repeated brief episodes of ischemia/reperfusion (I/R) protect the heart from injury during a subsequent prolonged I/R episode . Although much research has been devoted to this phenomenon and its other variants, including pharmacologic preconditioning (PPC) and ischemic post-conditioning (IPoC), the underlying mechanisms of cardioprotection remain elusive. Common to all of these cardioprotective strategies is activation of a signaling cascade called the Reperfusion Injury Salvage Kinase (RISK) pathway involving phosphatidylinositol-3-kinase (PI3K), Akt, glycogen synthase kinase 3 beta (GSK3β) and other glatiramer acetate  (Fig. 1). In addition, cardioprotective signaling can also be transmitted via the Survivor Activating Factor Enhancement (SAFE) pathway involving the activation of tumor necrosis factor alpha (TNFα) and signal transducer and activator of transcription-3 (STAT-3) , , , , which may crosstalk with the RISK pathway. Although the details are still fuzzy, these signals appear to converge ultimately on the mitochondrion , and avert cell death by inhibiting mitochondrial permeability transition pore (mPTP) opening in the inner mitochondrial membrane. Of note, activation of the RISK pathway depends on a modest burst of reactive oxygen species (ROS) production during the preconditioning ischemia, because if ROS scavengers are present during this period, cardioprotection is abolished . When the RISK pathway is activated by pre- or post-conditioning, the massive ROS burst that typically occurs after prolonged I/R in the absence of pre- or post-conditioning is markedly attenuated . This may be a major factor in averting the mPTP opening and cell death, since oxidative stress is one of the major factors promoting mPTP opening . The finding that post-conditioning confers almost equivalent cardioprotection as pre-conditioning suggests that RISK pathway activation at any time point up and during early reperfusion is the main requirement for effective cardioprotection . Once activated, the cardioprotected state has two phases. The early phase, attributed to acute post-translational modification of target proteins by the RISK pathway, lasts about 1–2h. Cardioprotection then dissipates, but returns again within 12–24h, and lasts for another 48–72h. This late phase is related to gene reprogramming and new protein synthesis triggered by RISK pathway activation . How do hexokinases (HKs) fit into this picture? During low-flow ischemia or anoxia, glucose metabolism becomes the major source for ATP production via anaerobic glycolysis, and HKs mediate the first step in this process, namely the conversion of glucose to glucose-6-phosphate (G6P). G6P is a hub metabolite that can be directed to a number of catabolic or anabolic fates (Fig. 2). The main catabolic fate is glycolysis, which first generates ATP anaerobically via conversion to pyruvate and lactate, and then aerobically by mitochondrial oxidation of pyruvate and lactate. The main anabolic fates of G6P are two-fold: glycogen synthesis to store energy for deferred use, and the pentose phosphate shunt to generate ribose-5-phosphate (R5P). The conversion of G6P to R5P is a major source of cytoplasmic NADPH generation which is critical for maintaining antioxidant function by regenerating reduced glutathione (GSH) from oxidized glutathione (GSSG). R5P is also a precursor for synthesis of nucleotides, amino acids and fatty acids. Small amounts of G6P are also used by the hexosamine pathway for O-GlyNAtion of proteins, and some of the G6P directed to pyruvate is used in the TCA cycle for amino acid and fatty acid synthesis via anaplerosis.