GSK3A Is Redundant with GSK3B in Modulating Drug Resistance and Chemotherapy-Induced Necroptosis

<div><p>Glycogen Synthase Kinase-3 alpha (GSK3A) and beta (GSK3B) isoforms are encoded by distinct genes, are 98% identical within their kinase domain and perform similar functions in several settings; however, they are not completely redundant and, depending on the cell type and differentiative status, they also play unique roles. We recently identified a role for GSK3B in drug resistance by demonstrating that its inhibition enables necroptosis in response to chemotherapy in p53-null drug-resistant colon carcinoma cells. We report here that, similarly to GSK3B, also GSK3A silencing/inhibition does not affect cell proliferation or cell cycle but only abolishes growth after treatment with DNA-damaging chemotherapy. In particular, blocking GSK3A impairs DNA repair upon exposure to DNA-damaging drugs. As a consequence, p53-null cells overcome their inability to undergo apoptosis and mount a necroptotic response, characterized by absence of caspase activation and RIP1-independent, PARP-dependent AIF nuclear re-localization. We therefore conclude that GSK3A is redundant with GSK3B in regulating drug-resistance and chemotherapy-induced necroptosis and suggest that inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells to chemotherapy.</p></div

Chemical inhibition abolishes drug resistance of p53-null colon carcinoma cell lines.

<p><b>A</b>, <b>C</b>, <b>E</b> Lysates of HCT116p53KO (<b>A</b>) and SW480 (<b>C</b>) cells were harvested 24hs after treatment with 1 µM 6-bromoindirubin-3′-oxime (BIO) and 20 µM SB216763 (SB2) GSK3 inhibitors; lysates of HT-29 (<b>E</b>) cells were harvested 24 hs after treatment with 2 µM 6-bromoindirubin-3′-oxime (BIO) and 20 µM SB216763 (SB2) GSK3 inhibitors; specificity of the inhibitor for GSK3A was assessed by checking GSK3A activation/inactivation checked by western blot using a mix of pSer21-GSK3A and pSer9-GSK3B antibodies and antibody cross-reacting with both pTyr279-GSK3A and pTyr216-GSK3B. <b>B</b>, <b>D, F</b>) percentage of cell death of HCT116p53KO(<b>B</b>), SW480 (<b>D</b>) and HT-29 (<b>F</b>) treated with 200 µM 5FU in presence and in absence of the indicated inhibitors (72 hrs).</p

GSK3A silencing in p53-null colon carcinoma cell lines does not affect proliferation or cell cycle.

<p><b>A</b>) Western blot on total lysates (30 µg) from HCT116p53KO cells stably infected with retroviral empty (pRS) and GSK3A shRNA-expressing (pRSGSK3A) vectors. <b>B</b>) growth curve of HCT116p53KO-pRS and -pRSGSK3A cells. <b>C</b>) DNA content and percentage of cells in G1, S phase and G2/M of HCT116p53KO-pRS and -pRSGSK3Aas evaluated after PI staning and flow cytometric analysis. <b>D</b>) β-catenin activity as evaluated by reporter assay 48 hrs after co-transfection with a luciferase-encoding vector driven by a β-catenin-dependent promoter. RLU  =  Relative Light Units. encoding vector (pRSGSK3B).</p

p53-null, GSK3B-silenced colon carcinoma cells treated with 5FU die by RIP1-independent necroptosis.

<p><b>A</b>) caspase-3/7 activation in HCT116p53KO-pRS and -pRSGSK3A cells treated with 200 µM 5FU for 72 hrs. HCT116 were used as control. Values indicate the fold increase of enzymatic activity of treated cells relative to the untreated cells arbitrarily set as 1. A representative experiment is shown. <b>B</b>) HCT116p53KO-pRS and -pRSGSK3A treated for 30 hrs with 200 µM 5FU and stained with anti-AIF antibody as well as DAPI. <b>C</b>) percentage of cell death of HCT116p53KO-pRSGSK3A after 72 hs treatment with 200 µM 5FU in presence of Bid inhibitor (20 µM Bi6C9), PARP1 inhibitor (100 µM DiQ), Bi6C9+DiQ or Necrostatin-1 (20 µM Nec1). <b>D</b>) HCT116p53KO-pRSGSK3A treated for 30 hrs with 200 µM 5FU in presence of 100 µM DiQ and stained with anti-AIF antibody as well as DAPI.</p

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury <i>In Vitro</i>

<div><p>Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely <i>Nemo</i> and <i>Tnfaip3</i>, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.</p></div

Atorvastatin significantly decreased circulating leukocytes and activated T-lymphocytes (CD45RO-positive) infiltrates in injured carotids.

<p>The number of circulating WBCs, monocytes and lymphocytes was increased by hypercholesterolemia compared to SD pigs (20215 ±1934/mm³ vs. 11924±1388/mm³, p=0.05, panel I; 600±160/mm³ vs. 180±50/mm³, p=0.04, panel K; and 10200±1160/mm³ vs. 7120±700/mm³, p=0.05, panel M, respectively). This significant difference was abolished by atorvastatin treatment. There was a correlation between circulating WBCs, monocytes, lymphocytes, platelets and the degree of stenosis (r=0.454 p=0.04, J; r=0.710 p=0.01, L; r=0.484 p=0.03, N; r=0.487 p=0.03, P). The CD45RO-positive cell infiltrates in injured carotids was significantly increased in HCD pigs compared to SD ones (58±9 vs. 249± cells/1x10⁷ μm², p=0.05, panels A, B and G) while statin treatment reduced T-lymphocytes infiltration (12±4 vs. 58±9 cells/1x107 μm², p=0.02, panels B, C and G). There were no CD45R0-positive cells in the contralateral uninjured carotid arteries (D-F). CD45R0-positive cells infiltration was positively correlated with the degree of stenosis (r=0.837, p=0.003, H).</p

Changes in <i>Tnfaip3</i> (<i>A20</i>) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells.

<p>Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine <i>Tnfaip3/A20</i> mRNA was quantified by real-time PCR. Data (mean±SD of three independent experiments) are normalized for <i>Gapdh</i> gene and expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, <i>p</i><0.05); [#] indicates a significant difference as compared to untreated cells within the same cell type (ANOVA, <i>p</i><0.05); [a] indicates a significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05).</p

Atorvastatin restored the expression of the protective genes HO-1 and iNOS at the site of vascular injury, enhancing iNOS expression also in contralateral uninjured arteries.

<p>HO-1 expression was reduced in the neointima and injured media of CHOL compared to SD pigs (39±9% vs. 61±3, p=0.04, panels A, B and J, and 38±6 vs. 60±3, p=0.04,panels D, E and J). Atorvastatin treatment restored the expression of HO-1 to levels similar to SD group (62±4% vs. 39±9%, p=0.04, Figure A-C-J; and 63±4 vs. 38±6%, p=0.04, panels D, F and J). There were no differences in contralateral uninjured arteries (G, H, I and J). There was a significant reduction of iNOS expression following vascular injury in the neointima of HCD compared to SD pigs (58±7% vs. 82±2%, p=0.007, panels K, L and T). Atorvastatin treatment significantly restored the expression of iNOS in neointima compared to HCD (90±4% vs. 58±7%, p=0.005, panels K, M and T) and induced a significant expression in the media of injured and contralateral arteries in comparison to HCD and SD (50±2% vs. 39±1%, p=0.05 panels N, O, P and T; 35±3% vs. 18±5%, p=0.05, panels Q, R, S and T).</p

pCX-TRI-2A transfected-cells are protected against TNF-α-induced apoptosis.

<p>Caspase 3/7 activities were determined in all cell groups after 16h (<b>A</b>) or 24h (<b>B</b>) of incubation with 50 ng/ml TNF-α alone or in combination with 20 μM hemin and/or 200 μM ATP. Expression of the three human genes in TG cells significantly reduced the activation of effector caspases 3/7. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, <i>p</i><0.05). [*] indicates significant difference between pCX-TRI-2A and all the other groups within the same treatment (ANOVA, <i>p</i><0.05); [ϕ] indicates a significant difference between single gene-transfected cells and WT/mock within the same treatment (ANOVA, <i>p</i><0.05); [a] indicates significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05); [§] indicates a significant difference as compared to all the other treatments within the same cell type (ANOVA, <i>p</i><0.05).</p

Heme oxygenase-1 and ectonucleotidases functional assays.

<p>(<b>A</b>) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *<i>p</i><0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups.</p