Sensitization of sarcoma cells towards doxorubicin

Doxorubizin ist eines der effektivsten Chemotherapeutika und wird bei einer Vielzahl von Tumoren eingesetzt. Obwohl Doxorubizin auch bei der Therapie von Rhabdomyosarkomen Anwendung findet, ist sein Einsatz bei dieser Tumorentität umstritten. Im Rahmen dieser Arbeit sollte das Ansprechen von Sarkomzellen auf Doxorubizin gesteigert werden. Hierfür wurde Doxorubizin in Kombination mit Medikamenten eingesetzt, die laut Literatur die TOP2a-Expression modulieren können. Getestet wurde zudem der PI3Kinase/mTOR-Inhibitor PI103, da er die antitumorale Wirkung von Doxorubizin in Glio- und Neuroblastomzellen verstärken kann. Anhand von Proliferations- und Apoptosemessungen konnte gezeigt werden, dass PI103 Rhabdomyosarkomzellen gegenüber Doxorubizin-induzierten antiproliferativen und proapototischen Effekten sensitiviert. Der kooperative proapototische Effekt wurde daraufhin sorgfältiger untersucht. Zunächst scheint dieser Effekt unabhängig von der TOP2a zu sein. Auch spielt die durch PI103 hervorgerufene Hemmung von mTOR eine untergeordnete Rolle. Mittels qRT-PCR-Analysen konnte jedoch gezeigt werden, dass PI103 die Expression der Effluxtransporter MDR1 und MRP1 hemmt. Dies resultiert in einer intrazellulären Akkumulation von Doxorubizin. Diese Akkumulation ist jedoch nicht die alleinige Ursache des kooperativen proapoptotischen Effekts. Vielmehr kooperieren Doxorubizin und PI103 in der Aktivierung des proapoptotischen Proteins BAX, in der Freisetzung von Cytochrom c aus den Mitochondrien, und in der Aktivierung der Caspase 3. Eine Aktivierung der Caspase 3 kann auch in einem ersten in vivo- Experiment an transplantierten Rhabdomyosarkomzellen gezeigt werden. Zusammengefasst zeigen die Daten, dass die durch PI103 hervorgerufene Sensitivierung von Sarkomzellen gegenüber den proapoptotischen Effekten von Doxorubizin über die Komponenten des mitochondrialen Apoptosewegs zustande kommt

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

<div><p>We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of <em>MDR1</em> and <em>MRP1</em>, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.</p> </div

PI103 sensitizes RD cells to DOX-induced apoptosis.

<p><b>A)</b> Levels of phospho-Akt, Akt, phospho-S6 ribosomal protein and S6 ribosomal protein were measured by Western blot analyses in RD, TP5014 and HT1080 cells after treatment with 1 µM or 3 µM PI103 as indicated. <b>B)</b> Upper panel: Proliferation was estimated by BrdU incorporation after treatment with 0.5 µM DOX and/or 3 µM PI103. Middle panel: Apoptosis was analyzed by FACS after treatment with 1 µM DOX and/or 3 µM PI103 or solvent. Data represent mean+SEM of at least three independent experiments performed in triplicates. Comparisons were made with ANOVA/Tukey’s testing. *<i>P</i><0.05 compared to cells treated with solvent; #<i>P</i><0.05 compared to cells treated with either drug alone. Lower panel: Protein levels of phospho-Akt, Akt and caspase 3 in cells treated with 1 µM DOX and/or 3 µM PI103 or respective solvents.</p

Inhibition of mTOR does not sensitize RD cells to DOX-induced apoptosis.

<p><b>A) and B)</b> RD cells were treated for 24h with 1 µM DOX and/or 1 µM everolimus (ever; respective left panels) or with 1 µM DOX and/or 20 µM LY294002 (respective right panels) or solvent. <b>A)</b> Annexin V positive RD cells were analysed by FACS. Data represent mean+SEM of two independent experiments performed in duplicates. n.s. not significant by Students <i>t</i>-test. <b>B)</b> Western blot analyses.</p

GDC-0941 inhibits tumor growth and increases DOX-mediated effects on caspase 3 activity.

<p><b>A)</b> Inhibition of RD tumor growth (in %) in nude mice treated with 1.2 mg/kg DOX (i.p. every third day for 22 days; n = 19), 75 mg/kg GDC-0941 (orally every day for 22 days; n = 9), the combination of both drugs (n = 12) at the time points indicated. Vehicle-treated animals served a controls (n = 18). <b>B)</b> Caspase 3 positive cells (in %) in tumors of nude mice isolated after 22 days of treatment with DOX and/or GDC-0941 or solvent. Data represent mean+SEM. *<i>P</i><0.05 by ANOVA/Tukey’s testing.</p

PI103-induced DOX accumulation does not account for cooperative proapoptotic effects in RD cells.

<p><b>A)</b> Intracellular DOX autofluorescence after treatment with 1 µM DOX (red line), 3 µM PI103 (black line), 2 µM DOX (blue line), 1 µM DOX plus 3 µM PI103 (green line) or solvent (orange line) at 530 nm. The data are expressed as the percentage of the maximum (max) number of positive cells. <b>B)</b> Expression levels of <i>MDR1</i> (left panel) and <i>MRP1</i> (middle panel) after treatment with 3 µM PI103 in relation to solvent treated cells (set = 1). Data represent mean+SEM of at least five independent experiments performed in triplicates. *<i>P</i><0.05 by Students <i>t</i>-test. Protein level of MRP1 after treatment with 3 µM PI103 or the solvent (right panel). <b>C)</b> Annexin V positive RD cells were analysed after treatment with 1 µM DOX, 3 µM PI103, 1 µM DOX plus 3 µM PI103, 2 µM DOX or the solvent. Data represent mean+SEM. Comparisons were made with Students <i>t</i>-test. <b>D)</b> Cells were treated as in C and caspase 3 was assessed by Western blot analyses. <b>E)</b> Annexin V positive RD cells (left panel) and intracellular DOX autofluorescence (right panel) after a 12-hours pretreatment (preinc.) with 3 µM PI103 and subsequent addition of 1 µM DOX for additional 24 h. Annexin V data represent mean+SEM. Comparisons were made with Students <i>t</i>-test.</p

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACFA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal AGFA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NEI reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis