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    • Prior to cellular uptake studies

    2021-10-16

    Prior to cellular uptake studies of Gemcitabine synthesis , docking simulation was performed (A). The result suggested this complex was capable of binding in the cavity of an outward open XylE. The orientation of the sugar moiety in the docked complex was similar to that of the glucose unit bound in the crystal structure, and the same residues (Gln168, Asn294 and Trp392) were identified in the hydrogen-bonding interactions ( and A). Moreover, all hydroxy groups of ribose were directly involved in the H-bonds interaction with XylE. Next, the cellular uptake mechanism of compound was evaluated in A549 cell line which had high level of GLUT1 expression. Cell uptake of compound (the aglycone of compound ) in A549 was not detected by HPLC, possibly due to the low level of uptake (). On contrast, the ribose-based compound shown 60.3% cell uptake rate (), suggested that the presence of ribose increased the cellular uptake. Considering -glucose was the main substrate of glucose transporter, it should compete with and inhibit the glucose transporter-mediated uptake of compound . Different concentrations of -glucose were used to detect their effect on cell uptake. The result suggested that -glucose exhibited a weak inhibitory effect on the uptake of compound , but still with dose-dependent (B). Furthermore, the GLUT1 inhibitor 4,6-Oethylidene-α--glucose (EDG) was used to monitored its effect on cell uptake. The result was presented in C. A 30% reduction in cell uptake of was measured in the presence of 50mM EDG, and with the increase of EDG concentration (to 100mM), a 43% decrease in the cellular uptake was observed. The inhibition of -glucose on cell uptake, as that of EDG showed that GLUT1 played an important role in cell uptake of ribose-modified compound in A549 cell line.
    Introduction The Hippo signaling pathway is an evolutionarily conserved regulator of organ size during development and a potent tumor suppressor in adults [1], [2], [3], [4]. To date, the molecular mechanism responsible for its anti-proliferative and pro-apoptotic activities remains poorly understood. In mammals, the Hippo pathway comprises a central kinase cassette (CKCA) and consists of the MST1/2 serine/threonine kinase, LATS1/2 protein kinase, and two scaffold proteins, SAV1 and MOB1A/1B. CKCA activity inhibits the formation of a complex between the TEAD transcription factors and the YAP/TAZ effectors in a LATS1/2-dependent manner [5], [6]. YAP/TAZ activity is repressed by LATS-dependent phosphorylation, which promotes the association of YAP/TAZ with the 14-3-3 protein and subsequently sequesters the complex in the cytosol [7]. Several reports demonstrated ubiquitin-mediated proteolysis of YAP/TAZ as a consequence of this phosphorylation [3], [8]. In mammals, CKCA is regulated by several upstream components. While the KIBRA-WILLIN-NF2 complex activates CKCA by an unknown mechanism, the apicobasal polarity proteins (such as SCRIB) activate CKCA and G-protein-coupled receptors, bypassing the MST1 and MST2 interaction with LATS1 and LATS2. Several other proteins (for instance AMOT, ZO, and PTPN14) directly regulate the activities of the YAP/TAZ Hippo signaling effectors by sequestration of the complex to the plasma membrane [9], [10], [11]. Actin filaments can regulate Hippo signaling in cancer cells in a Rho-dependent manner [12]. Furthermore, activation of the Hippo pathway by protein kinase A was recently discovered [13]. Therefore, the regulation of the Hippo signaling pathway represents a promising way to effective cancer treatment [14]. The oncoprotein C-MYC acts as a key developmental transcription factor and is strongly involved in the regulation of cellular metabolism and growth [15]. C-MYC is also an important factor responsible for the metabolic reprogramming of cancer cells known as the Warburg effect [16], [17], which primarily depends on the activity of downstream glycolytic components including pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate dehydrogenase kinase (PDK), and monocarboxylate transporter (MCT1). Activity of these downstream glycolytic components blocks the transport of pyruvate into mitochondria and drives the conversion of pyruvate to lactate. This process supports the synthesis of important biomolecules, while simultaneously decreasing the oxidative stress resulting from mitochondrial metabolism. Targeting the activities of C-MYC at various cellular levels has proven useful during the treatments of diverse types of cancer [18], [19].