The lysine-acetylation status of PDHA1 has been shown to be able to modulate its activity by impacting the affinity with its upstream regulator, PDK1 and PDP137, so theres a great chance that deacetylation of PDHA1 by SIRT2 in our experiment could dissociate PDK from PDHA1s active site to inhibit its phosphorylation and synergize with DCA. which enabled the two inhibitors to synergize with DCA to further trigger PDHA1. Besides, a AMPK-ROS feed-forward loop was notably activated after the combined treatments compared with mono-therapy. Our results indicate that this combination of DCA and SIRT2 inhibitor may provide a encouraging therapeutic strategy to effectively kill malignancy cells. Keywords: Sodium dichloroacetic acid (DCA), SIRT2, warburg effect, PDHA1, drug synergy Introduction Sirtuins (SIRT1-7) are a class of enzymes with nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacylase function.1 Among these seven users, SIRT2 has been shown to regulate multiple cellular processes including cell motility, cell proliferation and survival, cell-cycle progression, apoptosis, lipid synthesis, fatty acid oxidation, glucogenesis and oxidative stress.2-5 Because of the wide range of its functions, desire for SIRT2 as a potential target leads to the development and utilization of various specific inhibitors.6-10 Among them, AGK211,12 and Sirtinol13,14 are two selective and potent inhibitors which have therapeutic value for malignancy intervention. Sirtinol was identified as an inhibitor of silent information regulator (Sir2) family of proteins in a high throughput phenotypic screening of cells.8 Later researches revealed its anticancer potential in multiple cancer cells, including MCF-7 and H1299 cells.15 Moreover, Sirtinol enhanced chemo-sensitivity to camptothecin and cisplatin in PC3, DU145 and HeLa cells.14,16 AGK2 was originally reported to rescue alpha-synuclein-mediated toxicity in models of Parkinsons disease.9 Subsequent studies disclosed that AGK2 also achieved neuroprotection in cellular and invertebrate models of Huntingtons disease (HD).17 In addition to its neuroprotective effect, AGK2 was shown to have anti-cancer effects in cervical cancer cells12,18 and glioma cells.19 Within the past few decades, drug combination therapy has been intensively analyzed in oncology and other complex disease areas, as this strategy has the potential to improve treatment response, minimize or delay development of resistance and reduce dose and toxicity.20 There is evidence revealing the link between SIRT2 expression and poor prognosis in non-small cell lung cancer,21 as well as its role in the response of LGR3 the tumor to chemotherapy.22,23 On these bases, we believe that inhibiting SIRT2 pharmacologically by Sirtinol and AGK2 has the potential to enhance sensitivity of current small molecular drugs. In this study, we combined SIRT2 inhibitor Sirtinol, with a panel of small molecular anticancer brokers and we found that Dichloroacetate acid (DCA), a pyruvate dehydrogenase kinase inhibitor, could combine with Sirtinol/AGK2 to produce a synergistic therapeutic benefit. Further, we recognized that this drug combination cooperates to activate PDHA1, shift the metabolism to OXPHOS, VE-821 enhance ROS generation and activate AMPK signaling. These results indicate that this combination of DCA with Sirtinol and AGK2 may provide a encouraging therapeutic approach for NSCLC. Results Combination of Sirtinol/AGK2 with DCA prospects to synergistic killing of non-small cell lung malignancy cells To identify the small molecular anticancer brokers that would be more effective at killing malignancy cells by combining with Sirtinol, A549 cells seeded in 96-well plates were treated with a panel of compounds (5-FU, cisplatin, Irinotecan, Paclitaxel, Erlotinib, Oxaliplatin, Etoposide, Gefitinib, 2DG, DCA, Metformine) with Sirtinol for 72h and cell viability was determined by CCK-8 assay. The results show VE-821 that Sirtinol could enhance therapeutic effects of several drugs to numerous extent, with DCA having the most dramatic combinational effect (Physique 1A). DCA is usually a generic drug VE-821 with low price which has been utilized for human treatments for more than 30 years and has the ability to penetrate most tissues after oral administration. Therefore, we carried out further research focusing on DCA and SIRT2 inhibitor. The co-treatment of DCA with Sirtinol or AGK2 effectively decreased survival by 80C90% consistently in both H1299.