|Year : 2017 | Volume
| Issue : 6 | Page : 956-960
The effect of ciprofloxacin on the growth of B16F10 melanoma cells
Dalal Fares Jaber, Mary-Ann Nabil Jallad, Alexander Micheal Abdelnoor
Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
|Date of Web Publication||13-Dec-2017|
Prof. Alexander Micheal Abdelnoor
Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Hamra, Beirut
Source of Support: None, Conflict of Interest: None
Objective: The antitumor effect of ciprofloxacin has been widely assessed in-vitro, and positive results have been reported. The aim of this study was to investigate the influence of ciprofloxacin treatment on the growth of B16F10 melanoma cells both in-vitro and in-vivo.
Materials and Methods: Groups of C57BL/6 female mice challenged with B16F10 melanoma cells were kept untreated or were treated with sterile water, intraperitoneal ciprofloxacin, or ciprofloxacin through drinking water for 10 days. The serum levels of vascular endothelial growth factor (VEGF) were measured by ELISA 1 and 3 h after the last dose of ciprofloxacin. Mice were monitored for an additional 10 days for survival assessment. Moreover, B16F10 melanoma cells were cultured in 24-well plates and exposed to different concentrations of ciprofloxacin (10–1000 μg/ml). Viability was determined, after 24 and 48 h, using trypan blue.
Results: The serum levels of VEGF significantly decreased in ciprofloxacin-treated mice when compared to the controls. None of the control mice survived beyond day 8, whereas 16.67% of those treated with ciprofloxacin survived up to 18 days. In addition, the viability of B16F10 melanoma cells, in-vitro, significantly decreased with increasing concentrations of ciprofloxacin after 24 and 48 h.
Conclusion: Ciprofloxacin seems to exhibit antitumor activity both in-vivo and in-vitro. This effect might be explained by several mechanisms such as directly inducing cancer cell death or altering the immune response through the modification of the normal microbiota.
Keywords: B16F10 melanoma cells, cancer, ciprofloxacin, vascular endothelial growth factor
|How to cite this article:|
Jaber DF, Jallad MAN, Abdelnoor AM. The effect of ciprofloxacin on the growth of B16F10 melanoma cells. J Can Res Ther 2017;13:956-60
| > Introduction|| |
Studies related to the association between the use of antibacterial agents and tumorigenesis have been reported. The lack of an association was reported by Wirtz et al. and Sørensen et al., whereas others noted that a number of antibacterial agents were associated with an increased risk of several types of cancer such as breast cancer, prostate cancer, and lung and respiratory cancers.
The quinolones were discovered in 1962 as derivatives of chloroquine, an antimalarial drug. In an attempt to enhance the spectrum of activity and the tissue distribution of the drug, it was fluorinated. This gave birth to the second generation of quinolones known as the fluoroquinolones., Fluoroquinolones function by inhibiting the bacterial DNA gyrase, a topoisomerase II enzyme essential for DNA replication and hence for cell survival. This mode of action is similar to that of some anticancer drugs used and so the effect of fluoroquinolones on cancer cells has been widely investigated and interestingly, fluoroquinolones were reported to have antitumor activity.,,,
Ciprofloxacin, a widely used second-generation quinolone characterized by being an inhibitor of bacterial DNA gyrase, was shown to inhibit topoisomerase II in the mammalian cells.,, It was also reported to induce cell cycle arrest, produce DNA double-strand breaks, and trigger apoptosis of cancer cells in-vitro.,, Based on these findings, the anticancer effects of ciprofloxacin were extensively assessed using various human and murine cancer cell lines. Somekh et al. reported that ciprofloxacin used in 5 times the therapeutic dose caused an inhibition of the clonogenic growth of the K-562 human leukemic cell line. Similar results were reported when ciprofloxacin was tested, in-vitro, against different human transitional cell carcinoma lines,,, human osteosarcoma cells, human and rat colorectal carcinoma cells, hamster ovarian cancer cells, and human lung cancer and human hepatocellular carcinoma cells. Ciprofloxacin exhibited significant time- and dose-dependent cytotoxicity against the examined cells. Moreover, Kamat et al. reported that ciprofloxacin significantly enhanced the cytotoxicity of the anticancer drug doxorubicin. Consequently, these promising results support the hypothesis that ciprofloxacin can serve as an adjuvant therapy for a number of cancers.
Based on these reports, this study was carried out to determine the effect of ciprofloxacin treatment on the growth of B16F10 melanoma cells in-vivo, by comparing the survival rates of ciprofloxacin-treated mice with those of the controls.
Moreover, the effect of ciprofloxacin on tumor angiogenesis was assessed by determining the serum levels of vascular endothelial growth factor (VEGF), a potent angiogenic protein playing a pivotal role in promoting and regulating angiogenesis.,
Finally, the effect of ciprofloxacin on B16F10 melanoma cells was assessed in-vitro, by comparing the viability of antibiotic-treated cells with that of the controls.
| > Materials and Methods|| |
Effect of ciprofloxacin on B16F10 melanoma cells in-vivo
A preparation of B16F10 melanoma cells
The B16F10 melanoma cells, syngeneic with the C57BL/6 mice, were used in this study. These adherent cells were maintained in-vitro as monolayers in complete growth medium (RPMI-1640 supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum, Lonza, B-4800 Verviers, Belgium) and incubated at 37°C in a 5% CO2 incubator (Thermo Scientific, Forma, series II water jacket, CO2 incubator). When needed for administration into mice, the cells were detached using trypsin (2.5% trypsin in 10X in HBSS without calcium or magnesium, Lonza, B-4800 Verviers, Belgium), counted, and then re-suspended in RPMI-1640. Trypan blue was used to determine viability.
Challenge of mice with tumor cells and treatment
All experiments were approved by the Institutional Animal Care and Use Committee at the Faculty of Medicine, American University of Beirut. Thirty-eight C57BL/6 female mice, 6–8 weeks old, were each injected intraperitoneally (IP) with the 105 B16F10 melanoma cells at day 0 and divided into four groups (12 mice in each group). The injection protocol is given in [Table 1]. The control groups were Group 1: Untreated and Group 3: Given sterile water, 0.4 ml/mouse, IP whereas Group 2 was given ciprofloxacin in drinking water (125 mg/L + sucrose 20 g/L) and Group 4 was given ciprofloxacin IP (10 mg/kg body weight), daily for 10 days. The drinking water for Group 2 was changed every 3 days.
Procurement of specimens
At 1 and 3 h after the last dose of ciprofloxacin (day 10), three mice from each group were anesthetized each with a 0.5 mL mixture of 0.12 mL ketamine (50 mg/mL), 0.03 mL xylazine (20 mg/mL), and 0.35 mL pyrogen-free saline. Blood from each group was collected and pooled; serum was separated and used for VEGF quantification.
The remaining mice from each group (six mice/group) were monitored for 10 days to determine the rate of tumor growth and number of survivals. Upon death, the mice were dissected to confirm that death was caused by the tumor.
Vascular endothelial growth factor quantification
VEGF mouse ELISA kit (Abcam, ab100751, USA) was used to determine the levels of VEGF in the mice sera. The procedure was performed according to the manufacturer's protocol.
Effect of ciprofloxacin on B16F10 melanoma cells in-vitro
Culture of melanoma cells in the presence of ciprofloxacin
The B16F10 melanoma cells were seeded in 24-well plates at a density of 2 × 104 cells/500 μl/well and incubated for 24 h at 37°C and 5% CO2. Stock solution of ciprofloxacin (Hikma Pharmaceuticals, Portugal), 2 mg/ml, was used to prepare five different dilutions in complete growth medium (20, 100, 200, 1000, and 2000 μg/ml). Subsequently, 500 μl of each concentration of ciprofloxacin was added to the sample wells. No antimicrobial agent was added to the control wells. All wells were run in duplicate and incubated at 37°C and 5% CO2 and viability was determined after 24 and 48 h.
Determination of cell viability
Trypan blue was used to determine viability. Following an incubation period, the media from all wells were discarded; the cells in each well were washed with 0.5 ml phosphate-buffered saline and then detached using 0.5 ml of 0.05% trypsin. After centrifugation at 900 rpm for 5 min, the cells were re-suspended in 1 ml of medium. A cell suspension from each well was mixed with an equal amount of trypan blue, and viable cells were counted using the Neubauer chamber.
The effect of ciprofloxacin on the B16F10 melanoma cells was determined by comparing the viability of cells in the different concentrations of ciprofloxacin with that in the control wells. Viability was calculated as follow:
where V = viability, Ns = average number of viable cells in the sample well, and Nc = average number of viable cells in the control well.
Whenever applicable, data were expressed as mean ± standard deviation. Mice survival was evaluated by generating Kaplan–Meier survival curves. Statistical analysis was performed using PASW Statistics 18 for Windows and the Student unpaired t-test; P < 0.05 was considered statistically significant.
| > Results|| |
Vascular endothelial growth factor serum levels
In group of mice that received ciprofloxacin in drinking water
When compared to the control group (Group 1), the serum levels of VEGF slightly decreased (P = 0.4647) 1 h after the last ciprofloxacin dose, yet it significantly decreased (P = 0.0027) 3 h after the last ciprofloxacin [Figure 1].
|Figure 1: Vascular endothelial growth factor (pg/ml) levels in Group 1 (control) and Group 2 (ciprofloxacin placed in drinking water). *Statistically significant at P < 0.05 as compared to the control group|
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In group of mice that received ciprofloxacin intraperitoneal
When compared to the control group (Group 3), the serum levels of VEGF significantly decreased (P = 0.0099) 1 h after the last dose of ciprofloxacin. However, a slight decrease (P = 0.1083) was observed 3 h after the last dose of ciprofloxacin [Figure 2].
|Figure 2: Vascular endothelial growth factor (pg/ml) levels in Group 3 (controls) and Group 4 (ciprofloxacin administered intraperitoneally). *Statistically significant at P < 0.05 as compared to the control group|
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Comparison of VEGF serum levels in mice administrated ciprofloxacin in drinking water and intraperitoneal
The serum levels of VEGF significantly decreased, 1 and 3 h after the last dose of ciprofloxacin, in Group 2 that received ciprofloxacin through drinking water (P = 0.0292 and 0.0056, respectively) when compared to the IP ciprofloxacin-treated group (Group 4).
On day 10, 5 out of 6 mice in the groups that received ciprofloxacin IP or through drinking water were dead (16.67% survived). However, none of the mice in the control groups, untreated or given sterile water IP, survived beyond day 8 (0% survived) [Figure 3]. These results were further assessed by generating the Kaplan–Meier survival curves, showing the probability of survival in the given period [Figure 4] and [Figure 5].
|Figure 3: Survival curve for different groups of mice after 10 days monitoring. Group 1 = Mice receiving B16F10 melanoma cells, Group 2 = Mice receiving B16F10 melanoma cells + oral ciprofloxacin, Group 3 = Mice receiving B16F10 melanoma cells + intraperitoneal sterile water, Group 4 = Mice receiving B16F10 melanoma cells + intraperitoneal ciprofloxacin|
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|Figure 4: Kaplan–Meier survival curve for Groups 1 and 2. Group 1 = Mice receiving B16F10 melanoma cells, Group 2 = Mice receiving B16F10 melanoma cells + oral ciprofloxacin|
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|Figure 5: Kaplan–Meier survival curve for Groups 3 and 4. Group 3 = Mice receiving B16F10 melanoma cells + intraperitoneal sterile water, Group 4 = Mice receiving B16F10 melanoma cells + intraperitoneal ciprofloxacin|
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The statistical significance of the results obtained was assessed by determining the P values; P≤ 0.05 was considered statistically significant. The survival results from the groups that received ciprofloxacin, orally or IP, did not show statistical significance when compared to their controls (untreated and given IP sterile water, respectively) despite the fact that the remaining ciprofloxacin-treated mice survived beyond the 18th day of monitoring.
The viability of B16F10 melanoma cells significantly decreased with increasing concentrations of ciprofloxacin; after 24 and 48 h, at a concentration of 10 μg/ml, the viability decreased to 73% (P = 0.0258) and 67% (P = 0.0118), respectively. It kept on significantly decreasing until it reached, at the highest concentration of 1000 μg/ml, 4.5% (P = 0.0002) and 0% (P = 0.0001), after 24 and 48 h, respectively [Figure 6].
|Figure 6: Viability (%) of B16F10 melanoma cells (in-vitro) after treatment with ciprofloxacin. *Statistically significant at P < 0.05 as compared to the control group|
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| > Discussion|| |
After being reported to inhibit mammalian topoisomerase II,,, ciprofloxacin has been extensively tested for its effect on cancer and reported to induce DNA breaks and apoptosis in several cancer cell lines. This study was carried out to determine the effect of ciprofloxacin on B16F10 melanoma cells both in-vivo and in-vitro.
The anticancer effect of ciprofloxacin was assessed in-vivo by determining mice survival rates and VEGF serum levels in mice receiving ciprofloxacin both orally and IP and comparing them to those in mice from corresponding control groups.
When ciprofloxacin was administered orally, the serum levels of VEGF slightly decreased 1 h after the last dose (2.64% decrease), then a significant decrease was noted 3 h after the last dose (71.3% decrease). However, when ciprofloxacin was administered IP, the serum levels of VEGF significantly decreased 1 h after the last dose (25.35% decrease) and only a slight decrease was observed 3 h after the last dose (17.7% decrease). The difference in the serum levels of VEGF between the 1 and 3 h specimens can be explained by the fact that an oral dose takes up to 2 h to reach the maximum serum concentration whereas an IP dose takes >1 h. In compliance with these results, ciprofloxacin treatment resulted in 16.67% survival compared to 0% survival in the untreated groups. Statistical significance was not achieved probably because of the small sample size.
The viability of the B16F10 melanoma cell line decreased with increasing concentrations of ciprofloxacin, in-vitro, which is consistent with the results reported by Kloskowski et al. However, the latter described a lower efficacy of ciprofloxacin at high concentrations (1000 μg/ml) with 20% viability even after 72 and 96 h incubation while, in this study, B16F10 melanoma cells were completely eradicated at 1000 μg/ml of ciprofloxacin after 48 h.
Regarding the effect of ciprofloxacin on B16F10 melanoma cells, the in-vitro results of the present study were not completely concurrent with those in-vivo since statistical significance was not reached when assessing mice survival rate between ciprofloxacin-treated and untreated mice. This discrepancy can be justified by the renal and intestinal elimination of serum ciprofloxacin in-vivo and the fact that oral ciprofloxacin undergoes efflux resulting in a lower bioavailability (50–80%); i.e. in-vivo, the B16F10 melanoma cells were not continuously exposed to the antibacterial agent whereas in-vitro they were.,
| > Conclusion|| |
Ciprofloxacin seems to exhibit antitumor activity both in-vivo and in-vitro. This might have a substantial impact on the clinical practices since the potentially protective effect is achieved even upon using doses within ciprofloxacin's therapeutic window.
Several mechanisms may explain the potentially protective role of this antibacterial agent such as directly inducing cancer cell death or altering the immune response through the modification of the normal microbiota. Yet, extensive investigations are needed in this area to identify the specific mechanism and procure a better understanding of its dynamics.
Financial support and sponsorship
The Lebanese National Council for Scientific Research partially funded this study.
Conflicts of interest
There are no conflicts of interest.
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