Anti-proliferative effects of tricyclodecan-9-yl-xanthogenate (D609) involve ceramide and cell cycle inhibition

Abstract

Tricyclodecan-9-yl-xanthogenate (D609) inhibits phosphatidylcholine (PC)-phospholipase C (PLC) and/or sphingomyelin (SM) synthase (SMS). Inhibiting SMS can increase ceramide levels, which can inhibit cell proliferation. Here, we examined how individual inflammatory and glia cell proliferation is altered by D609. Treatment with 100-μM D609 significantly attenuated the proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC(1) astrocytes, without affecting cell viability. D609 significantly inhibited BrdU incorporation in BV-2 microglia and caused accumulation of cells in G(1) phase with decreased number of cells in the S phase. D609 treatment for 2 h significantly increased ceramide levels in BV-2 microglia, which, following a media change, returned to control levels 22 h later. This suggests that the effect of D609 may be mediated, at least in part, through ceramide increase via SMS inhibition. Western blots demonstrated that 2-h treatment of BV-2 microglia with D609 increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21 and down-regulated phospho-retinoblastoma (Rb), both of which returned to basal levels 22 h after removal of D609. Exogenous C8-ceramide also inhibited BV-2 microglia proliferation without loss of viability and decreased BrdU incorporation, supporting the involvement of ceramide in D609-mediated cell cycle arrest. Our current data suggest that D609 may offer benefit after stroke (Adibhatla and Hatcher, Mol Neurobiol 41:206-217, 2010) through ceramide-mediated cell cycle arrest, thus restricting glial cell proliferation.

Fig. 1

D609 activates caspase-3 in a dose- and time-dependent manner. BV-2 cells treated with 20 μM etoposide for 24 h were used as a positive control for caspase-3 activation (53, 54). (A): Western blot showing dose-response for D609 treatment of BV-2 cells for induction of cleaved caspase-3. Cells were incubated with 50, 100 and 200 μM D609 for 2 h, media was changed to remove D609 and cells were harvested after incubation for additional 22 h. Controls were incubated without D609. Lanes: 1) positive control (+), 2) untreated control (−), 3) 50 μM D609, 4) 100 μM D609, 5) 200 μM D609. (B): Western blot showing effect of different treatment durations of BV-2 with 100 μM D609 on caspase-3 cleavage. BV-2 cells were treated with 100 μM D609 for 2 h, 8 h, 16 h and 24 h and harvested at the end of each respective treatment time. Lanes: 1) untreated control (−), 2) 2 h D609, 3) 8 h D609, 4) 16 h D609, 5) 24 h D609, 6) positive control (+).

Fig. 2

D609 inhibited proliferation of (A) BV-2 microglia, (B) N9 microglia, (C) RAW 264.7 macrophages and (D) DITNC₁ astrocytes. Cell lines were first plated and allowed to adhere overnight. At the start of treatment, parallel dishes of cells were harvested and counted to determine the initial cell number. Cells were treated with 100 μM D609 for 2 h, followed by a media change. The media was centrifuged to recover any non-adherent cells, which were returned to the respective dishes. Cells were counted using a hemocytometer and viability was determined by trypan blue exclusion following 22 h incubation without D609. Viability was greater than 90% in all cells. * p < 0.05 and ** p < 0.01 compared with controls by unpaired t-test (n=3/group).

Fig. 3

D609 inhibited BrdU incorporation in BV-2 cells. (A): BrdU uptake in BV-2 controls and cells treated with D609. Cells were plated in 96-well plates and allowed to attach overnight, then treated with 100 μM D609 for 2 h. The media was then replaced with fresh media containing BrdU and cells were incubated for 2 h. Cells were labeled with anti-BrdU antibody followed by DAPI counterstaining, and imaged. Blue: DAPI, green: BrdU. Original magnification 200x. (B): Quantification of BrdU-positive cells as a percentage of total cells. *** p < 0.001 compared with controls by unpaired t-test.

Fig. 4

Effect of D609 treatment on cell cycle progression in BV-2 cells. (A): Representative DNA histogram from control and D609 treatment groups. BV-2 cells were treated with 100 μM D609 for 2 h followed by 2 h in media without D609, then stained with propidium iodide. DNA content of the samples were analyzed by flow cytometry. (B): Quantification of the percentage of cells in each phase of cell cycle. 2 h D609 followed by 2 h in the media without D609 has arrested the cells in the G₁ phase with a smaller percentage of cells entering into the S phase. * p < 0.05 compared with controls by unpaired t-test; from 3 independent experiments.

Fig. 5

D609 increased ceramide levels and induced p21 expression in BV-2 cells. BV-2 cells were treated with 100 μM D609 for 2 h, then media was changed and cultures were incubated for an additional 2 h or 22 h without D609. (A): D609 increased ceramide levels in BV-2, which remained elevated for 2 h after removal of the agent and normalized to control levels after 22 h. ** p < 0.01 compared with controls or D609 2 h + 22 h w/o by ANOVA with Bonferroni’s post-test; n=4 per group from 2 independent experiments. Ceramide forms consisted mainly of palmitic (C16-ceramide), lignoceric (C24-ceramide) and nervonic (C24:1-ceramide). (B): Western blots demonstrating induction of p21 and down-regulation of pRb following D609 treatment. Lanes: 1) untreated control, 2) 100 μM D609 2 hr, 3) 100 μM D609 2 hr + 2 hr w/o, 4) 100 μM D609 2 hr + 22 hr w/o.

Fig. 6

Exogenous C8-ceramide decreased cell proliferation in BV-2 cells. (A) 30 μM C8-ceramide was added to BV-2 cultures and cell counts and viability were determined after 24 h. ** p < 0.01 compared with controls by unpaired t-test. (B) Uptake of C8-ceramide and endogenous ceramide levels in BV-2 after 2 h and 24 h treatment with 30 μM C8-ceramide. ** p < 0.01 compared with controls by ANOVA with Bonferroni’s post-test.

Fig. 7

Exogenous C8-ceramide inhibited BrdU incorporation in BV-2 cells. (A): Representative images of BrdU uptake in BV-2 controls and cells treated with exogenous C8-ceramide. Cells were plated in 96-well plates and allowed to attach overnight, then treated with 30 μM C8-ceramide for 6 h. The media was then replaced with fresh media containing BrdU and cells were incubated for 2 h. Cells were labeled with anti-BrdU antibody followed by DAPI counterstaining, and imaged. Blue: DAPI, green: BrdU. Original magnification 200x. (B): Quantification of BrdU-positive cells as a percentage of total cells. Two sets of images were captured from 4 wells each from controls and C8-ceramide treated. *** p < 0.001 compared with controls by unpaired t-test.