Ciprofloxacin inhibits proliferation and synergistic effect against hepatocellular carcinoma cancer lines with cisplatin

Combination therapy has generated significant interest in the clinical setting since it increases the therapeutic potential of anticancer drugs. The potential therapeutic benefits in combination of cisplatin and ciprofloxacin have not been fully explored in hepatocellular carcinoma cancer therapy. In the results to be presented here, cellular viability of Hep G2 and Bel-7402 was determined by the 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay and the assessment of synergism, additivity or antagonism was carried out by the median effect analysis. Mechanism of cytotoxicity of the drugs was probed by reverse transcriptase-polymerase chain reaction (RT-PCR). Cellular DNA breaks were evaluated by comet assay. In addition, the effect of the drugs on topoisomerases was also investigated. The results show that ciprofloxacin could affect viability of Hep G2 and Bel-7402 lines, its combination with cisplatin resulted in mixed interactions; synergistic and additive effect. Further investigation indicated that the synergistic effect or additive was correlated to Fas involved apoptotic path, but the effects in combination on topoisomerases were not evident. The results obtained in this study suggest that the therapeutic benefit of cisplatin anti-hepatocellular carcinoma (HCC) may be influenced by ciprofloxacin.


INTRODUCTION
Hepatocellular carcinoma (HCC) represents the sixth most common malignancy and the third most common cause of cancer related death worldwide and accounts for as many as one million deaths annually (Varela et al., 2003).Surgical resection, local ablation therapies, and liver transplantation are regarded as potentially curative treatment modalities for HCC patients depending on the size and number of tumors.However, surgical prognosis for many patients with HCC is not favorable due to a higher likelihood of intrahepatic and remote recurrences (Yu et al., 2012).The effective treatment of this kind of disease in clinics is of higher priority.
Ciprofloxacin and its derivatives belong to the class of 4-fluoroquinolone antibiotics which are commonly used in the therapy of many bacterial infections (Koziel et al., 2010).It has been shown that the fluoroquinolones (FQs) target the bacterial enzyme DNA gyrase and also stabilize DNA strand breaks created by DNA gyrase and topoisomerase IV, but the FQs have lesser affinity for the eukaryotic DNA gyrase homologue, topoisomerase II (topo II).Normally, at concentrations higher than normally achieved in blood, inhibition of topo II can occur resulting in the formation of stabilized cleavage complexes and ultimately the production of DNA double-strand breaks (Burden et al., 1998).In addition to the antibacterial activity of ciprofloxacin, well documented evidences have shown that it has substantial antiproliferative activity against various cancer cells, such as bladder, colorectal, human prostate cancer, osteosarcoma and leukemic cell lines in vitro (Aranha et al., 2002a;Aranha et al., 2002b;Herold et al., 2002;Miclau et al., 1998;Theodore et al., 1998;Somekh et al., 1989).Aranha et al. also observed that ciprofloxacin could inhibit growth of human hepatocellular carcinoma cell line Hep G2 at higher doses, similar inhibition against Bel-7402 cell line was found in our laboratory.However, in order for ciprofloxacin to act as an anticancer agent, it has to be used at a much higher concentration than those used for the treatment of infectious diseases (Smart et al., 2008).At high concentrations (usually 200-300 μg/ml), ciprofloxacin can effectively induce apoptosis of bladder carcinoma cells and lead to cell cycle arrest at the S/G2 stage (Aranha et al., 2000).Prompted by this, it is worthwhile to try ciprofloxacin in anticancer therapy.
Cisplatin is one of the first metal-based anticancer drugs discovered in the early 19th century and it is also of the most useful antineoplastic agents.Cisplatin mediates its anticancer effect by covalently binding to DNA so as to form adducts that interferes with transcription and DNA replication, and thereby triggers programmed cell death (Victoria et al., 2007).Clinical use of cisplatin determined that many patients with different types of cancer have been successfully treated, including sarcoma cancers, cancers of soft tissue, bones, muscles, and blood vessels.Such cancers received better prognosis and therefore became less life threatening these days (Rosenberg, 1980;Desoize et al., 2002).However the side-effects of cisplatin, such as nephrotoxicity, neurotoxicity and hearing loss were limited to widely use in clinics (Tsang et al., 2009;Shah et al., 2009).To reduce the side-effects of cisplatin, dose-reducing is one of options by different drug combination.Although cisplatin has been used in combined therapy, its combination with ciprofloxacin in vitro was not evaluated.In work to be presented here, we investigated whether ciprofloxacin works in synergy with cisplatin and the possible molecular mechanism as well as its potential application in clinics.

Cell culture and treatment
Bel-7402 and Hep G2 human hepatoma (Y-S Biotechnology Inc., Shanghai, China) were propagated continuously in RPMI 1640 cell culture.Cells were grown at 37°C in RPMI 1640 medium supple-mented with 10% freshly inactived fetal calf serum , 100 units/ ml penicillin G and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 and 95% air.The cells harvested from exponentialphase of growing were used for biological evaluations.

Cytotoxicity assay
The stock solutions used in these studies were 1 mg/ml cisplatin (in physiological saline) and 20 mM ciprofloxacin (in H2O).Bel-7402( or Hep G2) cells in exponential-phase were washed three times with phosphate buffered saline (PBS), followed by detaching with 0.25% trypsin and centrifugation.The cells were re-suspended in RPMI-1640 and were seeded (5×10 3 /well) into 96-well plate.The cells were incubated in a humidified atmosphere of 5% CO2 for at least 6 h after which the drugs to be evaluated were added at final concentrations 1000, 500, 250, 125, 60, 30 and 15 μM for ciprofloxacin or 40,30,18,15,12,9,6 and 3 μM for cisplatin and incubated for 48 h at 37°C.After 48 h, 10 µl MTT solution (1 mg/ml) was added to each well, followed by further incubation of 4 h.Following incubation, the cell culture was removed by aspiration and 100 μl dimethyl sulfoxide (DMSO) was added in each well to dissolve the formazan crystals.The measurement of absorbances of the solution that was related to the number of live cells was performed on a microplate reader (MK3, Thermo Scientific) at 492 nm.Percent growth inhibition was defined as percent absorbance inhibition within appropriate absorbance in each cell line.The same assay was done in triplet.

Cytotoxicity studies with simultaneous drug combinations
After an initial pre-screening assay to evaluate single drug IC50, dose range was chosen to cover the concentrations below and above the IC50 of each drug to explore the potential interaction between the individual drugs in the given cell lines.The exposure of ciprofloxacin and cisplatin simultaneously to the cell lines were investigated at non-equipotent drug ratios.In the experiments, concentration of ciprofloxacin was at 30, 60 and 120 μM, respectively.Meanwhile the tests at constant equipotent ratio (molar ratio of ciprofloxacin/cisplatin = 5) were conducted too.The cell viability in different combination was determined using same procedure as in individual drugs.

Median effect plot analyses
The combined effects of two drugs in terms of synergism, summation, or antagonism were analyzed by the median effect plot (Chou et al., 1984;Chou et al., 2010).If the observed effect of two drugs acting simultaneously is larger or smaller than that calculated from the product expression, it is assumed that synergism or antagonism, respectively, has occurred.The procedure was performed by plotting the dose-effect curves for each drug and different combinations of the drugs using the "median effect" equation 1. Where, D is the dose, Dm is the dose required for 50% effect, fa is the fraction affected by the drug(s), fu is the fraction unaffected, and m is a coefficient signifying the sigmoidicity of the dose-effect curve.
The dose-effect curve was plotted using a logarithmic conversion of equation 1 and the values of m and Dm were determined.Based on the slope of the dose-effect curves, it can be determined whether the agents have exclusive effects or mutually nonexclusive effects.Combination indices (CI) was then determined using the equation 2, below: Where, (Dx)1 and (Dx)2 in the denominators are the doses (or concentrations) of D1 (drug 1) and D2 (drug 2) alone that gives x% inhibition whereas (D)1 and (D)2 in the numerators are the doses of D1 and D2 in combination that also inhibits x% (that is, isoeffective).If the drugs are mutually exclusive, then α is 0; if mutually nonexclusive, then α is 1.CI < 1 shows synergism, whereas CI = 1 indicates an additive effect and CI > 1 implies antagonism.

Preparation of nuclear extracts
Crude nuclear extract was prepared according to the published procedure (Ewhm et al., 1995) with some modification.Briefly, cells were detached using trypsin followed by centrifugation and washing twice with cold nucleus buffer (NB) (150 mM sodium chloride, 1 mM potassium dihydrogen phosphate, 5 mM magnesium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiotreitol and 1 mM phenylmethanesulfonyl fluoride (PMSF), pH 6.4) at 4°C.The supernatant was discarded and the cell pellet was re-suspended in 300 μl NB with an 0.3% Triton X-100 and placed on ice for 10 min, then transferred into a glass Dounce homogenizer with ten up-anddown strokes using a loose-fitting pestle.The suspension solution was centrifuged at 150 g for 10 min at 4°C, the pellet washed with Triton X-l00-free cold NB. 150 μl of cold NB solution containing 0.35 M NaCl was added to the pellet and allowed to stand 30 min at 4°C in order to extract the nuclear proteins.The supernatant was obtained by centrifugation at 10,000 g for 10 min at 4°C.The protein concentration was determined using the Bradford method.

Topoisomerase I activity assay.
To evaluate the effect of the individual or combined drug on DNA topoisomerase I (topo I) activity, the topo I assay was conducted.Briefly, nuclear extract (0.4 μg) treated by the drugs was added to the topo I reaction buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol and 0.4 μg supercoiled DNA plasmid (pMD18 @ -T) at a final volume of 20 μl and incubated at 37°C for 30 min.Following incubation, the reaction was terminated by adding 5 μl of stopping buffer (10% SDS, 0.025% bromophenol blue and 5% glycerol).The reaction products were analyzed by electrophoresis on 1% agarose gel using a TBE buffer (89 mM Tris-HCl, 89 mM boric acid and 62 mM EDTA) at 45 V for 3 h, stained by ethidium bromide (0.5 μg/ml) and photographed using a short wavelength UV lamp on Tocan 360 gel scanner (Shanghai Tiancheng Technology Inc, China).

DNA topoisomerase II assay
Relaxation activity of DNA topo II was determined by measuring the conversion of supercoiled pMD18 @ -T plasmid DNA to its relaxed form as described in topo I activity assay except in the assay in the presence of ATP (Ting et al., 2003;Oksuzoglu et al., 2008;Emine et al., 2008).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
To explore the underlying mechanism, especially at early phase, the Hep G2 cells were treated with 1/4 or 1/2 IC50 dose and a short time period incubation.The RT-PCR was conducted to determine the changes of apoptotic genes, such as Fas, caspase-3 and caspase-8 at mRNA level.The basal gene expression of caspase and Fas were set as control in untreated cells and was measured after 12 h incubation with different concentrations of ciprofloxacin, cisplatin and their combinations.Total RNA was extracted from the cells using Trizol reagent (Sangon, Shanghai, China) according to the Manufacturer's protocol.Three micrograms of total RNA were used for reverse transcription in a total volume of 20 μl with the M-MLV reverse transcriptase (Rnase H -) system (LifeFeng biological technology corporation, Shanghai, China).Aliquots of 2 μl cDNA were subsequently amplified in a total volume of 25 μl using the 2xTaq PCR kit (LifeFeng biological technology corporation, Shanghai, China) following conditions recommended by the manufacturer.The sense and antisense primer ( primers were synthesized by Shanghai generay bioengineering corporation company in the study, Shanghai, China) for beta actin were 5'-ACACTGTGCCCATCTACGAGG-3'and 5'-CGGACTCGTCATACTCCTGCT-3'(516 bp) that were used as an internal control; the sense and antisense primers for Fas were 5'-GGATCAAGGCACCTACCA-3' and 5'-GAACGCCCTCCTCAACAA -3'(289 bp); the sense and antisense primers for caspase 3, 5'-GAAGCGAATCAATGGACTCTGG-3' and 5'-ACATCACGCATCAATTCCACAA-3'(241bp); the sense and antisense primers for caspase 8, 5'-AAGTTCCTGAGCCTGGACTACAT-3', 5'-ATTTGAGCCCTGCCTGGTGTCT-3'(227bp) respectively.The cycling conditions: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min, and a final extension of 72°C for 10 min.PCR products were separated on the 1.5% agarose gel viewed by DNAgreen staining.These data were acquired with Tocan 360 gel imager (version 3.2.1 software).

Comet assay
The single-cell gel electrophoresis (comet assay) was adapted from the method of Singh et al. (1988).Bel-7402 cells with (half IC50) or without treatment of the drugs were harvested by centrifugation at 1000 rpm and then embedded in 0.5% low-melting-point agarose at a final concentration of 10 4 cells/ml.20 μl of this cellular suspension was then spread onto duplicate frosted slides that had previously been covered with 1% normal-melting-point agarose as a basal layer.Slides were allowed to solidify for 10 min at 4°C before being placed in lysis buffer for 1 h (2.5 M NaCl, 0.1 methylene diamine tetraacetic acid, 0.01 M Tris, 1% Triton X-100, 10% dimethyl sulphoxide, pH 10).After lysis, the slides were transferred into alkaline buffer for 40 min (0.001 M EDTA, 0.3 M NaOH, pH > 13) to allow the DNA to unwind before migration at 0.66 V/cm and 300 mA for 30 min.All these steps were performed in the dark.After neutralisation in 0.4 M Tris-HCl pH 7.4, slides were stored at 4°C for further analysis within 24 h.Before analysis, the slides were stained with ethidium bromide (20 μg/ml) and covered with a coverslip.The photography of the stained slides was taken on fluorescent microscopy.

Statistical analysis
The data were analyzed using the Sigmaplot version 10.0.Statistical comparisons among groups were performed by Student's t-test.P values indicate level of significance compared to CI =1.0.Degree of interaction is defined as significant at p<0.05.

Single-drug cytotoxicity studies
The dose-response curves of cisplatin and ciprofloxacin determined against Bel-7402 and Hep G2 cells are depicted in Figure 1.As shown in Figure 1, both drugs (ciprofloxacin and cisplatin) had significant growth inhibition in the hepatocellular carcinoma cell lines and exhibited dose-dependence compared to cells untreated by the drug.Interestingly, ciprofloxacin inhibited proliferation of Bel-7402 and Hep G2 cell lines at higher concentration (＞100 μM).The IC 50 values were acquired based on the median-effect plot after 48 h of ciprofloxacin treatment.These were 187 ± 11.3 and 290 ± 18.4 μM for Bel-7402 and for Hep G2 cells, respectively.The IC 50 values after 48 h treatment of cisplatin were 9.4 ± 1.4 and 18.9 ± 2.4 μM for Bel-7402 and Hep G2 cell, respectively.

Median effect analysis of combined effect
The different combinations of ciprofloxacin with cisplatin were applied to treat the cell lines, and the changes of combination indices (CIs) values calculated based on method of Chou and Talalay (2010) are shown in Figures 2 and 3. Figure 2 shows the plots of combination indices versus cisplatin concentration at known concentration of ciprofloxacin when the drugs were expose to Bel-7402 cell line.As shown in Figure 2A, the presence 30 μM of ciprofloxacin displays two kinds of interactions namely; synergistic (P < 0.05, four combinations) and additive effects (P < 0.05, five combinations).With increasing concentration of ciprofloxacin (Figures 2B and 2C), the antagonistic effect was displayed, one in 60 μM ciprofloxacin (P < 0.05, CI = 2.58 >1), three in 120 μM ciprofloxacin (P < 0.05, CI = 5.4, 4.1 and 1.9 >1).In the experiment of constant molar ratio of ciprofloxacin/ cisplatin (Figure 2D), the CI values indicated synergistic effects at lower concentration (P < 0.05, four combinations at CI (mean) = 0.3471, 0.3568, 0.4825 and 0.7170 <1) and additive effects (P > 0.05, three combinations at CI (mean) = 0.7950, 0.7209 and 0.8258) in the combinations (Figure 2D).For Hep G2 cells, in the presence of 30 ~120 μM ciprofloxacin, there were additive effects between the two drugs (all P > 0.05), but at constant molar ratio, the synergistic and additive (last two combinations, P > 0.05) effects can be seen in Figure 3.The results demonstrated synergistic effect could be achieved at lower molar ratio of ciprofloxacin/cisplatin for both cell lines.And the slight difference between the HCC cell lines can also be seen.

Effect of the drugs in individual or in combination on topoisomerase catalytic activity
The effect of ciprofloxacin, cisplatin and their combination on expression regulation of topo I and II are shown in Figure 4.As shown in Figure 4, the different DNA cleavage pattern was also included in Figure 4. Some differences were obvious, the DNA topo I activities of drug treated nucleic extracts were stronger than that of control, but no significant differences were observed between the drug treated groups.However, DNA top II in the nucleic extract in the combination was different from among individuals and there were two more relaxed DNA.

Evaluation of DNA fragmentation in vitro by Comet assay
Ciprofloxacin (IC 50 = 187 μM) and cisplatin (IC 50 = 9.4 μM) had definite antitumor activity against Bel-7402 cell line and caused the cellular DNA break in a time and dose dependent manner.Figure 5 shows the DNA fragmentation of Bel-7402 cells after exposure to individual drug at half IC 50 or in combination for 48 h.Compared to control (non-drug treatment), both cisplatin and ciprofloxacin led to the fragmentation of genetic materials of the cells at different degrees, ~30% DNA damaged for cisplatin, ~50% for ciprofloxacin and ~100% for in combination.From comet assay, it could be seen that normal comet images account for more than 90%, ~2% cells undergoing apoptosis that has a small head and a spread tail (Figure 5) (Huang et al., 2010).In the combination group, the synergistic effect between drugs was evident for highest percentage of cellular DNA breaks.

Effect of ciprofloxacin and cisplatin on caspases and Fas
The effects of the drugs investigated in individual or combined on the genes regulation are presented in Figure 6.As shown in Figure 6b, the Fas gene expression was significantly increased in the drug(s) treated group compared to control.And higher gene expression in combined than that in individual could be seen.In contrast to Fas gene expression, the response of caspase 3 and caspase 8 was irregular (Figure 6a), the changes of caspase 3 was not significant, but caspases 8 showed a down-regulation trend.The differences in apoptotic molecular response further indicated the induced cytotoxicity by the drugs was time and dose dependent.

DISCUSSION
Although the cisplatin-induced cytotoxicity and nephrotoxicity have been very well studied in both clinical and animal studies, hepatotoxicity has not been well documented.To reduce the side effects of cisplatin, a large number of studies have been focused on the ways for prevention of cisplatin side effects by supplementing with preventive agents (Liao et al., 2008).This study aimed to explore their optimal combination of ciprofloxacin used along with cisplatin to achieve higher therapy index and decrease the hepatotoxicity and nephrotoxicity of cisplatin.And their non-overlapping toxicities could also provide the basis for the investigation  The DNA relaxation assay carried out in the presence of supercoiled pMD18 @ -T DNA and nucleic extracts from the drug treated or untreated cells.For topo I assay: Lane 1 was DNA marker; lane 6 was pMD18 @ -T DNA alone; lanes 2-5 were control, cisplatin, ciprofloxacine and combination, respectively; for topo II assay, 1 mM ATP was added in the reaction buffer, lanes 7-10 were corresponding to the nucleic extract from control, cisplatin, ciprofloxacine and combination group.Topo I activity was getting stronger than that of control; the supercoiled, relaxed and nicked circular DNA are indicated by I, II and III.   of a potential therapeutic concurrent against HCC cell lines in vitro.To probe the potential interaction of the drugs, the cytotoxicity of ciprofloxacin against HCC cell lines were evaluated, indicating that ciprofloxacin exhibits definite anti-proliferation at higher concentration, which was slightly different from that reported (Herold et al., 2002).This difference derived from two groups was due to the different incubation time: in our experiments, 48 h was chosen, but in latter 18 or 24 h exposure to the ciprofloxacin were used.This also demonstrates that the cytotoxicity of ciprofloxacin is both dose and time dependent.Meanwhile, the CIs were calculated using the median-effect analysis of Chou and Talalay (2010).Since the median-effect plots obtained for the drugs individually were not parallel (different slopes, data not shown), exclusivity of the drug interaction effects could not be specified (Ting et al., 2003), but only the ones obtained under the second assumption were considered in our study.This has been reported as a more conservative criterion to determine the type of interaction effects since the addition of a third term in Equation 2 slightly increases the CI value (Ana et al., 2009).As shown in Figure 2, when ciprofloxacin fixed, a varied cisplatin were applied to inhibit growth of Bel-7402 cells, a mixed manner, synergistic, additive and antagonistic effect occurred.With increase of ciprofloxacin, interaction between them was gradually changed, which can be reflected by combination indices.In the presence of 30 μM ciprofloxacin, the CI value being less 1 was more, and CI increased with increase of ciprofloxacin.When a constant ratio of ciprofloxacin/cisplatin (< 5) was used, there were more CI values less one.The information indicated the interactions displayed between the drugs were a dose and ratio dependent.Similar results were obtained when the drugs combined were applied on Hep G2 cells (Figure 3).The synergistic and additive effects were predominated under the studied condition.A slight difference between the two cell lines was due to the difference in cellular sensitivity exposure to the drugs.It was clearly shown that ciprofloxacin had a definite function to influence therapeutic index of cisplatin in the current study.Dose reduction of cisplatin in clinic will be a good option when same therapeutic index was achieved by addition of less toxic supplements, such as ciprofloxacin.Although, ciprofloxacin has significant cytotoxicity at higher dose, its side-effect is much less compared to cisplatin.For HCC patients, especially liver partial resection, administration of ciprofloxacin could be beneficial because the lower dose cisplatin applied could reduce its nephrotoxicity and neurotoxicity.
DNA topo I and II are essential nuclear enzymes that modulate DNA topology during multiple cellular processes, they play a crucial role in chromosome structure, condensation/decondensation and segregation during mitosis (Wang, 1996).Effects of cisplatin and ciprofloxacin on DNA topo I expression and their activities were also evaluated in our study.In general, tumor cells with high topo I activity are sensitive to topo I inhibitors (Andoh et al., 1987;Barry et al., 1990).Beyond our expectation, the DNA topo I activity of nucleic extract after 48 h exposure of cisplatin was stronger than that of control, showing a up-regulation of DNA topo I (Figure 4), which was not in agreement with the previously published results by Keisuke et al. (2004).Their research found the topo I activity in the given cell lines began to decrease at ~1-2 h after cisplatin exposure, but gradually recovered after 4-5 h.The up-regulation of topo I activity after cisplatin exposure may reflect the mechanism of cellular repair.However, the relationship between the effect of cisplatin on topo I activity and its sensitivity to topo I inhibitors is still unclear (Keisuke et al., 2004).
It has been demonstrated that futile attempts to repair cisplatin-induced DNA damage may result in the triggering of apoptosis (Miguel et al., 2003).And the effects of cisplatin on caspases and Fas of cancer cells have well been documented in many literature, however, the information revealed were mostly from treatment by after 48 h incubation.How the apoptotic molecules in early phase respond to the treatment of ciprofloxacin and cisplatin was paid less attention.We investigated the changes of the apoptotic molecules in the early phase after 12 h treatment of the drugs in individual or in combined model, indicating the Fas gene can be upregulated when treatment of Hep G2 cells with the drugs in individual or in combined.It was clearly shown that synergistic and additive effects on Fas gene expression in the combined treatment, indicating the induction of apoptosis by cisplatin and ciprofloxacin were both involved in Fas-mediate signal path.It has been observed that addition of ciprofloxacin did not result in any difference in Fas expression compared to that of control Jurkat T cells (Jun et al., 2003), but ciprofloxacin effected on Fas gene expression of malignant cells was less reported.In our study, it was clear that ciprofloxacin induced an up-regulated Fas gene expression, which might indicate the cytotoxic effects of ciprofloxacin on Hep G2 cell was involved in Fas/FasL death path instead of inhibition of topo II.Recently E reported heat shock factor 1 is a transcription factor of Fas gene (E et al., 2010), whether the up-regulated Fas gene expression by ciprofloxacin was due to recruitment of heat shock factor 1 or other transcription promoter is not clear.Cisplatin induced cell death may result from both Fas receptor dependent pathway and mitochondriadependent pathway (Ame´lie et al., 2010).We presume that the two drugs exhibited a synergistic and additive effect came from their regulation action on Fas gene expression.
In conclusion, ciprofloxacin promotes cisplatin-evoked Hep G2 cancer cell apoptosis via up-regulated Fas gene expression synergistically.Different molar ratio of ciprofloxacin/cisplatin may have a different interaction pattern, suggesting that any concurrent supplementation of ciprofloxacin during the course of cisplatin-based chemotherapy could be beneficial for patients.The mechanism of ciprofloxacin induced the up-regulated Fas gene expression via recruitment of heat shot factor 1 or other transcription factor was not clear, a further study to probe the underlying path was required.

Figure 1 .
Figure 1.Effectiveness of different concentrations of cisplatin and ciprofloxacin on cell growth in the Bel-7402 and Hep G2 cell lines, as described in MTT assay in methods: a, Growth inhibition of cisplatin; b, ciprofloxacin growth inhibition in the cell lines.Each value represents the mean ± SE of three independent experiments.

Figure 2 .
Figure 2. Combination index plots for Bel-7402 cells exposed to cisplatin and ciprofloxacin.Combination index (CI) versus cisplatin concentration plots obtained from median-effect analysis of Chou-Talalay.The effects of ciprofloxacin concentration on CI are indicated in the figure panel is shown in the figure.CI < 1, = 1 and > 1 indicates synergistic, additive and antagonistic effect, respectively(constant ratio, last two P > 0.05; 30 μM cip: all P > 0.05; 60 μM, all P > 0.05; 120 μM, all P > 0.05).The data were derived from HepG2 cell line.

Figure 4 .
Figure 4.The DNA relaxation assay carried out in the presence of supercoiled pMD18 @ -T DNA and nucleic extracts from the drug treated or untreated cells.For topo I assay: Lane 1 was DNA marker; lane 6 was pMD18 @ -T DNA alone; lanes 2-5 were control, cisplatin, ciprofloxacine and combination, respectively; for topo II assay, 1 mM ATP was added in the reaction buffer, lanes 7-10 were corresponding to the nucleic extract from control, cisplatin, ciprofloxacine and combination group.Topo I activity was getting stronger than that of control; the supercoiled, relaxed and nicked circular DNA are indicated by I, II and III.

Figure 5 .
Figure 5.The apoptotic cells were detected by comet assay method, as described in materials and methods section.After cell electrophoresis and staining with EB, bel-7402 cells were photographed by fluorescent microscopy: a, Control; b, cisplatin; c, ciprofloxacin; d, combination.