Abstract B113: GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis.

Abstract

Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.

K313, a novel benzoxazole derivative, exhibits anti‐inflammatory properties via inhibiting GSK3β activity in LPS‐induced RAW264.7 macrophages

“…In recent years, glycogen synthase kinase‐3 beta (GSK‐3β), a multifunctional serine/threonine kinase, has attracted significant attention for its regulation of innate inflammatory processes . GSK‐3β‐mediated inflammation has been linked to number of diseases, such as diabetes, heart disease, neurodegenerative diseases, and cancer . Many studies have demonstrated that GSK‐3β may be a potential treatment target of anti‐inflammatory drugs for use in the clinic .…”

“…7 GSK-3β-mediated inflammation has been linked to number of diseases, such as diabetes, 8 heart disease, 9 neurodegenerative diseases, 10 and cancer. 11 Many studies have demonstrated that GSK-3β may be a potential treatment target of anti-inflammatory drugs for use in the clinic. 12 For example, lithium chloride (LiCl), a GSK-3β inhibitor, is used to treat bipolar disorder and Alzheimer disease, 13 SB415286 is used to treat neuronal apoptosis, 14 and hymenialdisine is used to treat inflammation.…”