Abstract
Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.
Highlights
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumour in adults, with poor prognosis as only 28.4% of patients survive one year and 3.4% survive to year five [1,2,3]
The membranes were blocked for 1 h at room temperature (RT) in 5% BSA (Sigma-Aldrich) in Tris buffered saline (TBS) with 0.001% Tween20 (TBS-T), followed by overnight incubation at 4 ◦C with the following primary antibodies for the cell lysates: anti-PAD2, anti-PAD3, anti-PAD4, anti-prohibitin, anti-citH3-r2-r8-r17, F95 (1/5000; [45]), while for Extracellular vesicles (EVs) characterization the EV-specific markers CD63 and Flot-1 were used (1/1000 in TBS-T)
For the first time a modulatory effect of pan-Peptidylarginine deiminases (PADs)-inhibitor Cl-amidine is shown on EV release and EV cargo in GBM cells
Summary
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumour in adults, with poor prognosis as only 28.4% of patients survive one year and 3.4% survive to year five [1,2,3]. Changes in deimination of histone H3 and prohibitin (PHB) were validated in response to treatment with PAD-inhibitor Cl-amidine, as well as in combination with chemotherapy using TMZ. Prohibitin Protein and Post-Translational Deimination Levels Change after 1 h Cl-Amidine Treatment and in Combinatory Treatment with TMZ in GBM Cells. After 1 h treatment with Cl-amidine, total PHB levels were reduced by 5–52% in LN18 and by 3–18% in LN229 cells (Figure 3C). Prohibitin Protein and Post-Translational Deimination Levels Change after 1 h Cl-Amidine Treatment and t. R) rPepHreBsenptsrroeltaetiivne dleenvseitlosmaetrrye croemdpuarcededto iβn-acLtiNn, 18 and LN229 after 1 h treatmwheinchtwwasituhsedCals-tahme inidteirnnael.lo(aDdin)gPcoonsttr-otl.ranslational deimination of PHB is reduced in GBM cells following 1 h Cl-amidine treatment. After 1 h combinatory treatment with Cl-amidine and TMZ, citH3 levels were reduced by 19–62% in LN18 and 2–31% in LN229 cells, compared to TMZ treatment alone (Figure 6A,B).
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