Quantitative proteomic analysis of global effect of LLL12 on U87 cell's proteome: An insight into the molecular mechanism of LLL12.

Glioblastoma multiforme (GBM) is one of the most devastating and dreadful WHO grade IV brain tumors associated with poor survival rate and limited therapeutics. Signal transducer and activator of transcription factor 3 (STAT3) is persistently active in several cancers, including gliomas, and STAT3 inhibitors hold great promise for treatment of glioma. LLL12, a curcumin derivative, inhibits STAT3 functions, thereby reduces growth of GBM. However, the global effects of targeting STAT3 using LLL12 have not been studied well. To shed light on this aspect, we performed quantitative proteomic analyses using differential in-gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantitation (iTRAQ) as well as label-free mass spectrometric analysis with 0.5μM (IC50) concentration of LLL12. Through this approach, we identified a total dataset of 1012 proteins with 1% FDR, of which 143 proteins were differentially expressed associated with various cellular functions. Results suggest that LLL12 influences central cellular metabolism and cytoskeletal proteins, in addition to its apoptosis inducing and anti-angiogenic activities, which altogether contribute to its anti-tumorigenic function. Interestingly, triose phosphate isomerase (TPI), phosphoglycerate mutase 1 (PGAM1), adaptor molecule (CRK2), protein DJ-1 (PARK7) and basic transcription factor 3 (BTF3) were found to be down-regulated and can be studied further to understand their therapeutic potential in gliomas. TPI1 and PGAM1 protein expressions were validated using immunoblot. Conclusively, our results suggest the therapeutic potential of LLL12 and it can be investigated further for a significant role in glioma treatment. BIOLOGICAL SIGNIFICANCE: LLL12 holds great promise for therapeutic development in gliomas with constitutive expression of STAT3. This study investigated the global effect of LLL12 on the proteome of U87 glioma cells using complementary proteomic approaches, and our findings suggest that LLL12 influences central metabolism, translation, transport processes, and cytoskeleton of a cell in addition to its anti-angiogenic and apoptosis inducing functions which altogether contributes to anti-tumorigenic activity of LLL12. This study leads to the identification of several proteins which may serve as prognostic or predictive markers in GBM. We identified TPI1, PGAM1, CRK and BTF3 as potential therapeutic targets and further investigations on these candidates may facilitate therapeutic development.