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Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence.

Liu Y, Hawkins OE, Su Y, Vilgelm AE, Sobolik T, Thu YM, Kantrow S, Splittgerber RC, Short S, Amiri KI, Ecsedy JA, Sosman JA, Kelley MC, Richmond A - EMBO Mol Med (2012)

Bottom Line: Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP).Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP.Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA.

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Drug-induced senescence initiates SASP and activates NF-κBHs294T cells were pre-treated with 1 µM MLN8237 or vehicle control for 5 days. After treatment, cytokine secretion into the medium was assayed by cytokine array.Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 or 5 µM MLN8237 for 5 days and the levels of p-p65 (S536) and p65 were analysed by Western blot. Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 µM MLN8237 for 3 days or 5 days, and the level of IκB-α was analysed by Western blot.NF-κB reporter-stable Hs294T cells were treated with 1 µM MLN8237 for 5 days. After treatment, NF-κB transcriptional activity was measured by luciferase assay and the results were normalized by cell number. Data indicate mean values ± SD (n = 3) from a representative experiment performed three times.Hs294T cells were treated with 1 or 5 µM MLN8237 for 5 days. Levels of phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), total ERK, phospho-p38 MAPK (p-p38), total p38, phospho-STAT3 (p-STAT3), total STAT3 and GAPDH were measured by Western blot.Conditioned medium from senescent Hs294T cells was added to the bottom wells in 96 transwell cell-plates. 200 µl (106 cells/ml) of dHL60 cells (human promyelocytic leukcmia cells differentiated along a neutrophil cell lineage) were seeded in the chemotaxis chamber. The chamber was incubated at 37°C 5% CO2 for 1 h, then the transmigrated cells were counted by haemocytometer. Data indicate mean values ± SD (n = 4).
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fig04: Drug-induced senescence initiates SASP and activates NF-κBHs294T cells were pre-treated with 1 µM MLN8237 or vehicle control for 5 days. After treatment, cytokine secretion into the medium was assayed by cytokine array.Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 or 5 µM MLN8237 for 5 days and the levels of p-p65 (S536) and p65 were analysed by Western blot. Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 µM MLN8237 for 3 days or 5 days, and the level of IκB-α was analysed by Western blot.NF-κB reporter-stable Hs294T cells were treated with 1 µM MLN8237 for 5 days. After treatment, NF-κB transcriptional activity was measured by luciferase assay and the results were normalized by cell number. Data indicate mean values ± SD (n = 3) from a representative experiment performed three times.Hs294T cells were treated with 1 or 5 µM MLN8237 for 5 days. Levels of phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), total ERK, phospho-p38 MAPK (p-p38), total p38, phospho-STAT3 (p-STAT3), total STAT3 and GAPDH were measured by Western blot.Conditioned medium from senescent Hs294T cells was added to the bottom wells in 96 transwell cell-plates. 200 µl (106 cells/ml) of dHL60 cells (human promyelocytic leukcmia cells differentiated along a neutrophil cell lineage) were seeded in the chemotaxis chamber. The chamber was incubated at 37°C 5% CO2 for 1 h, then the transmigrated cells were counted by haemocytometer. Data indicate mean values ± SD (n = 4).

Mentions: To investigate whether therapy-induced senescence alters the SASP in melanoma cells, we examined the levels of several cytokines and chemokines secreted into the media of MLN8237-treated melanoma cells by cytokine array (Supporting Information Table S2). The results demonstrated that IL-6, IL-7, IL-10, GM-CSF, IL-8, RANTES, GRO and GRO-α were upregulated in response to drug treatment (Fig 4A). We then further tested the levels of IL-6 and IL-8 by ELISA in four melanoma cell lines (Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28) treated with MLN8237 or vehicle and confirmed that both IL-6 (Supporting Information Fig S11A) and IL-8 (Supporting Information Fig S11B) were increased following MLN8237 treatment.


Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence.

Liu Y, Hawkins OE, Su Y, Vilgelm AE, Sobolik T, Thu YM, Kantrow S, Splittgerber RC, Short S, Amiri KI, Ecsedy JA, Sosman JA, Kelley MC, Richmond A - EMBO Mol Med (2012)

Drug-induced senescence initiates SASP and activates NF-κBHs294T cells were pre-treated with 1 µM MLN8237 or vehicle control for 5 days. After treatment, cytokine secretion into the medium was assayed by cytokine array.Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 or 5 µM MLN8237 for 5 days and the levels of p-p65 (S536) and p65 were analysed by Western blot. Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 µM MLN8237 for 3 days or 5 days, and the level of IκB-α was analysed by Western blot.NF-κB reporter-stable Hs294T cells were treated with 1 µM MLN8237 for 5 days. After treatment, NF-κB transcriptional activity was measured by luciferase assay and the results were normalized by cell number. Data indicate mean values ± SD (n = 3) from a representative experiment performed three times.Hs294T cells were treated with 1 or 5 µM MLN8237 for 5 days. Levels of phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), total ERK, phospho-p38 MAPK (p-p38), total p38, phospho-STAT3 (p-STAT3), total STAT3 and GAPDH were measured by Western blot.Conditioned medium from senescent Hs294T cells was added to the bottom wells in 96 transwell cell-plates. 200 µl (106 cells/ml) of dHL60 cells (human promyelocytic leukcmia cells differentiated along a neutrophil cell lineage) were seeded in the chemotaxis chamber. The chamber was incubated at 37°C 5% CO2 for 1 h, then the transmigrated cells were counted by haemocytometer. Data indicate mean values ± SD (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569660&req=5

fig04: Drug-induced senescence initiates SASP and activates NF-κBHs294T cells were pre-treated with 1 µM MLN8237 or vehicle control for 5 days. After treatment, cytokine secretion into the medium was assayed by cytokine array.Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 or 5 µM MLN8237 for 5 days and the levels of p-p65 (S536) and p65 were analysed by Western blot. Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28 cells were treated with 1 µM MLN8237 for 3 days or 5 days, and the level of IκB-α was analysed by Western blot.NF-κB reporter-stable Hs294T cells were treated with 1 µM MLN8237 for 5 days. After treatment, NF-κB transcriptional activity was measured by luciferase assay and the results were normalized by cell number. Data indicate mean values ± SD (n = 3) from a representative experiment performed three times.Hs294T cells were treated with 1 or 5 µM MLN8237 for 5 days. Levels of phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), total ERK, phospho-p38 MAPK (p-p38), total p38, phospho-STAT3 (p-STAT3), total STAT3 and GAPDH were measured by Western blot.Conditioned medium from senescent Hs294T cells was added to the bottom wells in 96 transwell cell-plates. 200 µl (106 cells/ml) of dHL60 cells (human promyelocytic leukcmia cells differentiated along a neutrophil cell lineage) were seeded in the chemotaxis chamber. The chamber was incubated at 37°C 5% CO2 for 1 h, then the transmigrated cells were counted by haemocytometer. Data indicate mean values ± SD (n = 4).
Mentions: To investigate whether therapy-induced senescence alters the SASP in melanoma cells, we examined the levels of several cytokines and chemokines secreted into the media of MLN8237-treated melanoma cells by cytokine array (Supporting Information Table S2). The results demonstrated that IL-6, IL-7, IL-10, GM-CSF, IL-8, RANTES, GRO and GRO-α were upregulated in response to drug treatment (Fig 4A). We then further tested the levels of IL-6 and IL-8 by ELISA in four melanoma cell lines (Hs294T, SK-Mel-2, SK-Mel-5 and SK-Mel-28) treated with MLN8237 or vehicle and confirmed that both IL-6 (Supporting Information Fig S11A) and IL-8 (Supporting Information Fig S11B) were increased following MLN8237 treatment.

Bottom Line: Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP).Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP.Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN, USA.

Show MeSH
Related in: MedlinePlus