The online version of this article (https://doi.org/10.1186/s12885-017-3896-y) contains supplementary material, which is available to authorized users.
The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro.
The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests.
We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness.
Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.
Additional file 1: Full western blots of gels from Fig. 1. (DOXC 243kb)
Additional file 2: Full western blots of gels from Fig. 5. (DOXC 200kb)
Ko CH, Takahashi JS. Molecular components of the mammalian circadian clock. Hum Mol Genet. 2006;15:271–7. CrossRef
Guillaumond F, Dardente H, Giguere V, Cermakian N. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J Biol Rhythm. 2005;20:391–403. CrossRef
Ko ML, Shi L, Tsai JY, Young ME, Neuendorff N, Earnest DJ, Ko GY. Cardiac-specific mutation of clock alters the quantitative measurements of physical activities without changing behavioral circadian rhythms. J Biol Rhythm. 2011;26:412–22. CrossRef
Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nature rev. Cancer. 2009;9:537–49. PubMed
Loesch M, Chen G. The p38 MAPK stress pathway as a tumor suppressor or more? Front. Bioscience. 2008;13:3581–93.
del Barco BI, Nebreda AR. Roles of p38 MAPKs in invasion and metastasis. Biochem Soc Trans. 2012;40:79–84. CrossRef
Yang K, Liu Y, Liu Z, Liu J, Liu X, Chen X, Li C, Zeng Y. p38γ overexpression in gliomas and its role in proliferation and apoptosis. Sci Rep 2013;3:2089.
Demuth T, Reavie LB, Rennert JL, Nakada M, Nakada S, Hoelzinger DB, Beaudry CE, Henrichs AN, Anderson EM, Berens ME. MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther. 2007;6:1212–22. CrossRefPubMed
Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A. 2004;101:5339–46. CrossRefPubMedPubMedCentral
Ramanathan C, Khan SK, Kathale ND, Xu H, Liu AC. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters. J Vis Exp. 2012;67:4234.
Allen G, Rappe J, Earnest DJ, Cassone VM. Oscillating on borrowed time: diffusible signals from immortalized suprachiasmatic nucleus cells regulate circadian rhythmicity in cultured fibroblasts. J Neurosci. 2001;21:7937–43. PubMed
Natarajan SR, Wisnoski DD, Singh SB, Stelmach JE, O'Neill EA, Schwartz CD, Thompson CM, Fitzgerald CE, O'Keefe SJ, Kumar S, et al. p38 MAP kinase inhibitors. Part 1: design and development of a new class of potent and highly selective inhibitors based on 3,4-dihydropyrido[3,2-d]pyrimidone scaffold. Bioorg Med Chem Lett. 2003;13:273–6. CrossRefPubMed
Chansard M, Molyneux P, Nomura K, Harrington ME. Fukuhara C: c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms. Neurosci. 2007;145:812–23. CrossRef
- Inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness
Charles S. Goldsmith
Sam Moon Kim
L. Gerard Toussaint
David J. Earnest
- BioMed Central
Neu im Fachgebiet Onkologie
e.Med Kampagnen-Visual, Mail Icon II