Ciprofloxacin decreases survival in HT-29 cells via the induction of TGF-beta1 secretion and enhances the anti-proliferative effect of 5-fluorouracil

Abstract

Background and purpose: Fluoroquinolones are potent anti-microbial agents with multiple effects on host cells and tissues. Previous studies have highlighted their pro-apoptotic effect on human cancer cells and an immunoregulatory role in animal models of inflammatory bowel disease. We examined the effect of ciprofloxacin on proliferation, cell cycle and apoptosis of HT-29 cells, a human colonic epithelial cell line sensitive to transforming growth factor (TGF)-beta1-mediated growth inhibition and its role in TGF-beta1 production. We also examined the effect of ciprofloxacin on proliferation of HT-29 cells in combination with 5-fluorouracil (5-FU), a well-established pro-apoptotic agent.

Experimental approach: Using subconfluent cultures of HT-29 and Caco-2 cells, we studied the effect of ciprofloxacin, TGF-beta1 and 5-FU on proliferation, apoptosis, necrosis and cell cycle. The effect of ciprofloxacin on TGF-beta1 mRNA expression and production was studied in RNA extracts and cell culture supernatants respectively, using confluent cultures.

Key results: Ciprofloxacin decreased proliferation of HT-29 cells in a concentration- and time-dependent manner. This was mediated by accumulation of HT-29 cells into the S-phase but without any effect on apoptosis or necrosis. Additionally, ciprofloxacin enhanced the antiproliferative effect of 5-FU. Interestingly, ciprofloxacin was found to up-regulate TGF-beta1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-beta1 was blocked. Confirming this mechanism further, ciprofloxacin had no effect on Caco-2, a human colonic epithelial cell line that lacks functional TGF-beta1 receptors.

Conclusions and implications: We demonstrate a novel anti-proliferative and immunoregulatory effect of ciprofloxacin on human intestinal epithelial cells mediated via TGF-beta1.

Figure 1

Ciprofloxacin suppresses HT-29 cell proliferation by inducing cell cycle arrest. (A) MTT assessed proliferation of HT-29 cells after treatment with 0–100 µg·mL⁻¹ of ciprofloxacin for 3 and 6 days, as described in Methods. (B) MTT assessed proliferation of HT-29 cells treated with ciprofloxacin for 6 days, which was followed by removal of ciprofloxacin and subsequent 2 days treatment with vehicle; ×10, ×50 and ×100 indicate initial concentrations of ciprofloxacin (*P < 0.05, **P < 0.01 different from control values). (C) Apoptosis of HT-29 cells after treatment with vehicle (Ctr) or ciprofloxacin (50 µg·mL⁻¹) evaluated by 7-AAD and annexin-V staining. (D) Effects of ciprofloxacin on cell cycle assessed by propidium iodide (PI) staining. HT-29 cells were treated for 2 days and 4 days with vehicle (CTR1, CTR2) or 50 µg·mL⁻¹ of ciprofloxacin (CIP1, CIP2). Two representative graphs from six independent experiments are shown. 7-AAD, 7-amino-actinomycin D; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide.

Figure 2

Ciprofloxacin-induced cell cycle arrest is mediated through TGF-β1. (A) MTT assessed proliferation of HT-29 cells after treatment with 0–100 ng·mL⁻¹ of TGF-β1 for 3 days. (B) TGF-β1 production by HT-29 cells after 24 and 48 h treatment with vehicle (0) or ciprofloxacin (100 µg·mL⁻¹) evaluated by elisa in cell culture supernatants. (C) Expression of TGF-β1 mRNA by HT-29 cells. Total RNA was extracted from HT-29 cells after treatment with ciprofloxacin (100 µg·mL⁻¹) for 1 to 6 h and multiplex RT-PCR for TGF-β1 and GAPDH was performed. Upper panel: representative PCR blot of mRNA expression for TGF-β1 and GAPDH. Lower panel: the ratio of the integrated density of TGF-β1 divided by that of GAPDH, measured by densitometry analysis, is expressed as a percentage of the maximum TGF-β1 mRNA expression after stimulation with ciprofloxacin (100 µg·mL⁻¹). One experiment representative of three independent experiments with similar results is shown. (D) MTT assessed proliferation of HT-29 cells after treatment with ciprofloxacin (100 µg·mL⁻¹) for 6 days in the presence of 10 µg·mL⁻¹ of neutralizing anti-human-TGF-β1 (LAP) antibody or 10 µg·mL⁻¹ of purified mouse IgG2a isotype control (*P < 0.05, **P < 0.01, ***P < 0.001). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide; RT-PCR, reverse transcription polymerase chain reaction; TGF, transforming growth factor.

Figure 3

Ciprofloxacin has no effect on TGF-β1-resistant Caco2 cells. (A) MTT assessed proliferation of Caco-2 cells after treatment with TGF-β1 for 3 days. (B) MTT assessed proliferation of HT-29 and Caco-2 cells after treatment with ciprofloxacin for 6 days (*P < 0.05, **P < 0.01 different from control values). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide; TGF, transforming growth factor.

Figure 4

Ciprofloxacin augments the anti-proliferative effect of 5-FU. MTT assessed proliferation of HT-29 cells after treatment for 6 days with ciprofloxacin (100 µg·mL⁻¹) added alone or in combination with 1 mmol·L⁻¹ of 5-FU. (*P < 0.05, ***P < 0.001 different from control values, ^§P < 0.05 different from 5-FU treatment). 5-FU, 5-fluorouracil; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide.