Abstract
We previously reported that the fluoroquinolone moxifloxacin (MXF) inhibits NF-kB, mitogen-activated protein kinase activation and the synthesis of proinflammatory cytokines in activated human monocytic cells (AAC48:1974,2004). Since MXF acts on topoisomerase II (Topo II) in mammalian cells, we investigated its effect in combination with another Topo II inhibitor, VP-16, on cell proliferation (by the MTT method), cell cycle, caspase-3 activity and proinflammatory cytokine release in THP-1 and Jurkat cells. THP-1 cells were incubated for 24 h with 0.5–3 μg/ml VP-16 in the presence or absence of 5–20 μg/ml MXF. VP-16 induced a dose dependent decrease in cell proliferation. An additional 2.5-and 1.6-fold decrease in cell proliferation was observed upon incubation of the cells with 0.5 or 1 μg/ml VP-16 and 20 μg/ml MXF, respectively (up to 69% inhibition). To further elucidate the mechanism of the antiproliferative activity of MXF, its effect on cell cycle progression was investigated. In control cultures 1%, 45%,18% and 36% of cells were in G0, G1, S and G2/M phases at 24 h, respectively. In contrast, in cultures treated with 1 μg/ml VP-16 and VP-16+ 20 μg/ml MXF, the number of cells in G1 decreased to 5.4 and 6.5%, respectively, while the number of cells in S phase increased to 25.5 and 42%, respectively and the number of cells in G2/M cells increased to 60 and 44%, respectively. These data provide evidence for S-G2/M cell cycle arrest induced by VP-16 and that addition of MXF shifted the S-G2/M arrest more towards the S phase. Since the antiproliferative effects of MXF could also be attributed to apoptotic cell death in addition to cell cycle arrest, we investigated the effect of the drugs on apoptosis. Using the fluorogenic assay for caspse-3 activity, we show that incubation of THP-1 cells for 6 h with 1.5 μg/ml VP-16 resulted in 630±120 unit/50μg protein of caspase-3 activity while the combination of 1.5 μg/ml VP-16 and 20 μg/ml MXF enhanced caspase-3 activity up to 1700±340 units/50μg protein (vs.233±107 in control cells), indicating that MXF synergises with VP-16 in activation of caspase-3. In Jurkat cells, the addition of 0.5 or 1 μg/ml VP-16, did not affect cell proliferation while in the presence of 20 μg/ml MXF and 1 μg/ml VP-16 there was a 62% decrease in cell proliferation (p<0.05). Exposure of Jurkat cells to 3 μg/ml VP-16 alone resulted in 504±114 units/50μg protein of caspase-3 activity and the addition of 20μg/ml MXF enhanced caspase-3 activity up to 1676± 259 units/50μg protein (vs 226±113 units/50μg protein in control cells). We further examined pro-inflammatory cytokine secretion upon stimulation of THP-1 cells with VP-16, MXF or their combination. VP-16 alone at 3 μg/ml increased IL-8 and TNF-α secretion from THP-1 cells by 2.5 and 1.8-fold respectively. Addition of MXF (5–20 μg/ml) inhibited the two cytokines secretion by 72–77% and 58–72%, respectively. The above combined data indicate that MXF, at clinically attainable concentrations, demonstrates pronounced synergistic effect with VP-16 as an anti-proliferative agent mainly by enhancing caspase-3 activity and apoptosis. At the same time MXF inhibits the pro-inflammatory effects conferred by VP-16 in the tumor cells studied. The clinical significance of the above anti-proliferative and anti-inflammatory effects of MXF in combination with VP-16 should be further investigated in animal models.
Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast