Imidazole-thiazolidinone inhibits oesophageal cancer cell proliferation via induction of apoptosis and cell cycle arrest at S phase

Purpose: To investigate the effect of imidazole-thiazolidinone on oesophageal cancer (OC) cell proliferation, and the mechanism of action involved.Methods: Human OC cells (HCE-6 and KYSE-1170) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin solution at 37 ˚C for 24 h in a humidified atmosphere of 5 % CO2 and 95 % air. After attaining 60 - 70 % confluency, the cells were treated with serum-free medium and graded concentrations of imidazolethiazolidinone (up to 160 μM) for 24 h. Normal cell culture without imidazole-thiazolidinone served as control. Cells in logarithmic growth phase were selected and used in this study. Cell proliferation and apoptosis were assessed using 3 (4,5 dimethyl thiazol 2 yl) 2,5 diphenyl 2H tetrazolium bromide (MTT), and flow cytometric assays, respectively. The levels of expression of apoptosis-related proteins were determined using Western blotting.Results: Treatment of HCE-6 and KYSE-1170 cells with imidazole-thiazolidinone for 48 h led to significant and dose-dependent reduction in their proliferation, as well as significant and dosedependent increase in the number of apoptotic cells (p < 0.05). Light microscopy revealed significantreduction in HCE-6 cell count, detached cells, reduced cell size and irregular cytoplasmic vacuoles. Imidazole-thiazolidinone treatment significantly and dose-dependently decreased HCE-6 and KYSE-1170 cell migration, and arrested HCE-6 cell cycle at S phase (p < 0.05). In HCE-6 cells, imidazolethiazolidinone treatment significantly and dose-dependently upregulated the expressions of cleaved caspase-3/8/9 and bax, but down-regulated bcl-2 expression significantly and dose-dependently (p < 0.05). However, metalloproteinases 2 and 9 (MMP-2 and MMP-9) expressions in HCE-6 and KYSE-1170 cells were significantly and dose-dependently down-regulated by imidazole-thiazolidinone treatment (p < 0.05).Conclusion: The results obtained in this study suggest that imidazole-thiazolidinone suppresses OC cell proliferation via induction of apoptosis and arrest of cell cycle at S phase. Keywords: Imidazole-thiazolidinone, Oesophageal cancer, Metastasis, Cell cycle arrest, Apoptosis

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Moxifloxacin Enhances the Antiproliferative and Apoptotic Effects of VP-16 but Inhibits Its Proinflammatory Effects in THP-1 and Jurkat Cells.

Blood ◽

10.1182/blood.v106.11.4290.4290 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4290-4290

Author(s):

Ina Fabian ◽

Debby Haite ◽

Avital Levitov ◽

Drora Halperin ◽

Itamar Shalit

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Arrest ◽

Mammalian Cells ◽

Caspase 3 ◽

S Phase ◽

Jurkat Cells ◽

Cycle Arrest ◽

Topo Ii ◽

Number Of Cells

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.

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Interaction between the p53 genotype of lung cancer and response to mTOR inhibitor combined with irradiation therapy

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e22028 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e22028-e22028

Author(s):

Y. Nagata ◽

T. Tojo ◽

K. Ohnishi ◽

A. Takahashi ◽

T. Ohnishi ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Survival ◽

Cell Cycle Arrest ◽

Mtor Inhibitor ◽

P53 Gene ◽

S Phase ◽

Cycle Arrest ◽

Irradiation Therapy

e22028 Background: Frequent activation of the PI3K/Akt/mTOR pathway and aberrations of tumor suppressor gene p53 are associated with therapeutic resistance in lung cancer. Nevertheless, the possibility of the variants of p53 genotype to affect response to mTOR inhibitor combined with irradiation therapy remains still unclear. Methods: Human non-small lung cancer cell line H1299 with p53 null genotype, was transfected with wild type or mutated p53 gene (H1299/wtp53 (WT), H1299/mp53 (MT)). Both cell survival and cell proliferation were estimated by colony formation assay to assess differences between WT and MT in sensitivity to rapamycin and ability of rapamycin to enhance radiation sensitivity. Cells were treated according to the individual study; DMSO (control), rapamycin (100 nM for 1 hour), irradiation (IR) (increasing doses), combination (RR) (rapamycin followed by irradiation). Changes in the cell cycle were also analyzed by flow cytometry. Results: Rapamycin decreased cell survival only in WT (P < 0.01, vs. control). MT was resistant to rapamycin exhibiting slightly inhibited cell proliferation. Compared with IR, RR with no less than 6 Gy radiation enhanced inhibitory effects on both cell survival and proliferation independent of p53 genotype (P < 0.01 in WT and P < 0.01 in MT, respectively), that indicating additive interaction. Cell cycle analysis demonstrated rapamycin induced G1 cell cycle arrest in both types of cells compared with controls (P < 0.01 in WT and P < 0.05 in MT, respectively) at 24 hours after treatment. Enhancement of G1 arrest by RR was observed in both WT (P < 0.01, vs. IR) and MT (P < 0.01, vs. IR) at the same time point. In addition, RR caused a greater reduction of cells in S phase than that of IR regardless of p53 gene status (P < 0.01 in WT and P < 0.01 in MT, respectively). Conclusions: Rapamycin is an effective radiosensitizer in a p53 independent manner, in which increased G1 cell cycle arrest and decrease in S phase cells are responsible for increased growth inhibitory effect. It will enable us to provide more appropriate treatment to patients with mutated p53 gene. No significant financial relationships to disclose.

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