By continuing to browse this site you agree to us using cookies as described in About Cookies
Wiley Online Library is migrating to a new platform powered by Atypon, the leading provider of scholarly publishing platforms. The new Wiley Online Library will be migrated over the weekend of March 17 & 18. You should not experience any issues or loss of access during this time. For more information, please visit our migration page: http://www.wileyactual.com/WOLMigration/
Hideyuki Akaza md, Department of Urology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Email: email@example.com
Objectives: The antitumor effect of bee honey against bladder cancer was examined in vitro and in vivo. Methods: Three human bladder cancer cell lines (T24, 253J and RT4) and one murine bladder cancer cell line (MBT-2) were used in these experiments. In an in vitro study, the antitumor activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, 5-Bromodeoxyuridine (BrdU) labeling index and flowcytometry (FCM). In the in vivo study, cancer cells were implanted subcutaneously in the abdomens of mice, and the effects were assessed by the tumor growth.
Results:In vitro studies revealed significant inhibition of the proliferation of T24 and MBT-2 cell lines by 1–25% honey and of RT4 and 253J cell lines by 6–25% honey. BrdU labeling index was significantly lower. FCM showed lower S-phase fraction, as well as absence of aneuploidy compared with control cells. In the in vivo studies, intralesional injection of 6 and 12% honey as well as oral ingestion of honey significantly inhibited tumor growth.
Conclusion: Bee honey is an effective agent for inhibiting the growth of T24, RT4, 253J and MBT-2 bladder cancer cell lines in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation models. Our results are promising, and further research is needed to clarify the mechanisms of the antitumor activity of honey.
Urothelial cell carcinoma of the bladder is the second most common malignancy of the genito-urinary tract. In Japan, the incidence of bladder cancer was estimated to be 12.4 per 100 000 in men and 3.5 per 100 000 in women. Previous studies have demonstrated that intravesical instillation of antitumor agents is effective for the prevention of bladder cancer recurrence. However, intravesical chemotherapy is sometimes associated with adverse symptoms such as hematuria and vesical pain. In addition, some authors have suggested that the long-term prognosis following postoperative chemotherapy is no better than after transurethral resection alone.1 Intravesical instillation of Bacillus Calmette-Guérin and other agents has also been reported to be effective, but tumor progression was observed during therapy in some patients, and there was a high incidence of bladder irritation and fever, as well as occasional more serious adverse reactions.2
Honey is thought to exhibit a broad spectrum of therapeutic properties including antibacterial, antifungal, cytostatic and anti-inflammatory activity.3 Honey has been used for the treatment of Fournier's gangrene, abdominal wound disruption, gastric ulcers,4 gastroenteritis and burns,5 and for the storage of skin grafts.6 Recent studies by Gribel and Pashiniski indicated that honey possessed moderate antitumor and pronounced antimetastatic effects in five different strains of rat and mouse tumors.7 Furthermore, honey potentiated the antitumor activity of chemotherapeutic drugs such as 5-fluorouracil and cyclophosphamide.8 Honey contains many biologically active compounds including caffeic acid, caffeic acid phenethyl ester and flavonoid glycones. These compounds have been proved to have an inhibitory effect on tumor cell proliferation and transformation by the down regulation of many cellular enzymatic pathways including protein tyrosine kinase, cycloxygenase and ornithine decarboxylase pathways.9 We studied the antitumor activity of diluted unfractionated bee honey against bladder cancer cell lines in vitro and in vivo.
In vitro studies
Pure unfractionated honey was purchased from Manitoba (Tokyo, Japan) and diluted with RPMI-1640 medium to prepare concentrations of 1 to 25% to be used in the in vitro studies. The osmolarity and pH of the honey used are shown in Table 1.
Table 1. pH value and osmolarity of agents used in the in vitro studies
Human bladder cancer (T24, RT4, 253J) and murine bladder cancer (MBT-2) cells were maintained as monolayer cultures in RPMI-1640 supplemented with 10% fetal calf serum, 1% glutamine and 1% each penicillin and streptomycin. The cells were cultured in a 96-well tissue culture plates for 24 h. The culture medium was then aspirated and a 1, 3, 6, 12 or 25% dilution of honey in RPMI-1640 was added. The plates were maintained at 37°C in 5% CO2 in humidified air for 24 h and then examined by phase contrast microscopy.
After 24 h incubation of T24, RT4, 253J and MBT-2 cells in various dilutions of honey (1–25%) in 96-well tissue culture plates, 10 µL of MTT reagent (1 mg/mL) was added to each well, and the plates were incubated for 3–4 h. The medium was then aspirated, and 100 µL DMSO (100%) was added. The highly colored DMSO-soluble Formazan product was assessed spectrophotometrically employing a microplate reader.
The early DNA breaks during apoptosis were assessed by the TUNEL assay using the TumorTACS In Situ Apoptosis Detection kit (R & D Systems, Boston, USA). The cells were allowed to grow and adhere to the cover slides, and then a 6 or 12% concentration of honey was added. Twenty-four hours later, the TUNEL assay was performed according to the manufacturer's protocol. The apoptotic index was calculated from 10 randomly chosen high power fields (400X). The apoptotic index was defined as the percentage of apoptotic cells in relation to the total number of cells.
Cell proliferation in bladder cancer cell lines
Confluent and adherent bladder cancer cells were treated with 12 or 6% honey for 24 h. BrdU (Sigma, Tokyo, Japan) was diluted in RPMI 1640 and added to the cells for 8 h. Cells were collected and fixed and the BrdU was detected immunohistochemically using anti-BrdU antibody (Sigma). Labeling indices (%) were assessed in a total of 1000-cell (× 400) in control or honey-treated cells.
One million bladder cancer cells were treated with 12 or 6% honey for 24 h, trypsinized, suspended in phosphate buffer solution, then fixed in 70% ethanol. The cell suspension was centrifuged, 1 mL RNAase A (1 mg/mL) was added to the pellet for 45 min. The cell suspension was centrifuged, 1 mL of propidium iodide (20 µg/mL) was added to the pellet, then cell sorting was performed using FACSCalibur System (Becton Dickinson, Boston, USA) and the histograms were evaluated using Cell Cycle Analysis Software (Modfit LT).
In vivo studies
Inbred 4-week-old female C3H/He mice were housed at 22–24°C in 50% humidity with a 12-h light/12-h dark cycle.
The N-[4-(5nitro-2-furyl)-2 thiazolyl] formamide (FANFT)-induced mouse bladder tumor cell line (MBT-2) was employed. The abdomen was shaved and 5 × 105 cells/0.1 mL of single-cell suspension was injected into the abdominal wall of the mice using a 26-gauge needle.
The MBT-2 cell suspension was implanted in 100 animals simultaneously. When the tumor volume reached 100–150 mm3, animals were randomly divided into five groups: group I received intralesional (IL) injection of 12% honey, group II intralesional 6% honey, group III intralesional 0.9% physiologic saline, group IV 50% honey in drinking water orally, and group V received no treatment as the control. Intralesional 0.1 mL honey or saline injection was performed twice weekly for 3 weeks. Diluted honey in drinking water was available for 6 h every other day for 3 weeks with an average consumption of 1.5 mL per mouse every other day (Fig. 1).
Evaluation of tumor volume
The experiment was performed twice and the tumor volume and body weight were measured twice a week. Tumor volume was calculated by the following formula:
where D1 was the largest dimension and D2 the smallest dimension. Four weeks after randomization, the animals were sacrificed. Tumors were measured, dissected, weighed and fixed in 10% buffered formaldehyde and then processed for paraffin embedding.
Non-parametric analysis by the Mann–Whitney U-test was used to compare the effect among the treated groups and the Student t-test to evaluate the BrdU labeling indices and S-phase fraction among control and treated cells.
In vitro studies
The assay was repeated five times; there was a concentration-dependant antitumor effect in bladder cancer cell lines. T24 and MBT-2 cells showed a significant retardation in cell number with the increase in honey concentration from 1 to 25% (P < 0.01). RT4 and 253J growth was also retarded by 6 to 25% concentration of honey (P < 0.01). We observed that the antineoplastic activity of honey is concentration- and cell-type dependant (Fig. 2).
T24 cells stained by the TUNEL method and visualized by light microscopy are shown in Fig. 3. Dark brown staining indicates that apoptotic DNA breaks occurred in these cells. The tumor apoptotic index in the control cover slips was 2 ± 0.75% (average ± SD), while apoptosis rates were 23 ± 1.0% in the 6% honey-treated cells and 17 ± 1.1% in the 12% in the honey-treated cells (Fig. 3).
Cell proliferation in bladder cancer cell lines
The total BrdU index in the 12 and 6% honey-treated cells showed significantly less proliferation than in the untreated cells (P < 0.001) (Fig. 4).
Measurement of the DNA content of the examined bladder cancer cell lines revealed a significant low S-phase fraction in cells treated with 12 or 6% honey for 24 h compared with control cells (P < 0.001). The untreated RT4 bladder cancer cell lines showed aneuploidy of 25% and the T24 cell lines showed 16% aneuploidy. This aneuploidy status completely disappeared after 24 h incubation in 12 or 6% honey concentration (Fig. 5).
In vivo studies
There was a significant difference between the final tumor volume or weight (P < 0.05) in the honey-treated groups (IL 12%, IL 6% and oral 50%) and the control group. This tumor growth-inhibitory effect was shown to be significant from the middle of the experimental period in the 6% IL honey group while other groups still failed to exhibit this significant effect (Fig. 6). There was also a significant difference between the final tumor volume (P < 0.05) in the honey-treated groups (IL 6% and oral 50%) compared to the IL saline group. The difference between the final tumor volume or weight in the IL saline group and the control group was not significant. The experiment was performed twice. In the first study, RPMI-1640 was used as the intralesional injection agent and revealed overall similar results. There was no significant difference in the average body weight of all mice regardless of their group, denoting the absence of systemic toxicity for either honey or saline administration. Histopathological examination of the dissected tumors showed tumor cell masses with areas of necrosis and inflammatory cellular infiltrate without precise differences between the examined groups.
In recent years, considerable efforts have been made to identify naturally occurring and related synthetic agents that could prevent the development and recurrence of cancer. Cancer chemoprevention therefore has emerged as an important subject that, in addition to providing a practical approach to identifying potentially useful novel agents as inhibitors of cancer development, offers opportunities to study the mechanism of carcinogenesis. Previously, several naturally occurring compounds such as phenols, indoles, inositol-6-phosphate, aromatic isothiocyanates and dithiolethiones were shown to inhibit several types of cancer, including cancer of the colon, in laboratory animal models.10 Oral administration of Lactobacillus casei has been demonstrated to suppress tumor growth and thus prolong survival in animals with experimental bladder tumors and prevent recurrence in a post-resection tumor recurrence model.11
The objective of our in vitro study was to investigate the antineoplastic activity of a range concentrations of honey against human and murine bladder cancer cell lines. In vitro study showed that diluted honey affected the growth patterns of some bladder cancer cells. The effect was observed to be dependant on the cell line and concentration of honey used. Apoptosis was shown by TUNEL assay in the T24 cell line, and, although the apoptotic index elicited was low (17–23%), it suggests one of the ways by which honey induces cellular death. Marked cellular and nuclear changes suggestive of apoptosis were encountered in other cancer cell lines investigated. Previous studies had shown the growth promoting effect of honey on fibroblasts, hence accelerating wound healing,5 histopathologic assessment of the stored skin grafts showed no deleterious effects on normal epithelial or connective tissue elements.6
Cellular proliferation markers have both a diagnostic and prognostic value in bladder cancer, showing higher indices with more aggressive tumors. In our studies, treatment of cancer cells with honey resulted in a significant lowering of the BrdU labeling index compared to control cells, supporting our hypothesis about the antineoplastic effect of honey.
Measurement of the DNA content of bladder cancer cell lines gives the DNA histogram from which the cell ploidy and the components of the cell cycle can be derived, including the S-phase fraction. Both ploidy and S-phase fractions have a prognostic significance in bladder cancer. Treatment of bladder cancer cell lines with honey resulted in a significant reduction in the S-phase fraction that matches the BrdU labeling studies, and the accumulation of large cell population behind the G1 peak might be an indicator for apoptosis that has been proved by the TUNEL assay. The disappearance of aneuploid cell population after treatment with honey might indicate a specific antitumor effect against the more aggressive tumor cells.
The in vivo studies showed the significant growth inhibitory effect of IL and oral honey administration on the subcutaneously implanted bladder cancer model. This effect began as early as mid-experiment in the 6% honey-treated group, yet the 12% and oral honey-treated groups showed this effect at the end of the experiment. We introduced the IL saline group to alleviate the effect of intralesional manipulation, and we showed a significant tumor inhibition in 6% and oral honey-treated groups but not the 12% honey-treated group as compared with the IL saline group. The in vitro studies showed a higher apoptotic index and a lower S-phase fraction in cells treated with 6% honey than 12%. These findings might explain the greater efficacy of 6% honey.
The hyperosmolarity of bee honey was corrected by dilution so that the concentrations used (6 and 12%) are comparatively similar to the routinely used antibiotic Sulperazon (Pfizer, USA). The same principle was applied in the choice of RPMI-1640 as the solvent because of its ability to buffer much of the acidity of the bee honey. Thus, the acidity and hyperosmolarity of bee honey alone cannot explain the drastic inhibitory effect on tumors. We chose pure unfractionated honey for two reasons: (i) the synergism between the different chemical constituents in honey has been reported previously; and (ii) there are a number of volatile compounds reported to be abundant in bee products that might be lost during simple fractionation methods. Honey contains a variety of compounds including caffeic acid, benzoic acid and esters, substituted phenolic acids and esters, flavonoid glycones, and beeswax.12,13 Some of the observed biological activities of honey may be traced to its chemical constituents.14,15 Wattenburg et al. demonstrated that dietary administration of hydroxycinnamates (constituents of honey) significantly inhibited benzopyrene-induced neoplasia of the forestomach in mice.16 Caffeic acid (3,4-dihydroxycinnamic acid) ester derivatives, which are present in honey at levels of 20–25%, are thought to exhibit a broad spectrum of activities that possibly include tumor inhibition.9,12 Several cellular components that have been associated with cell proliferation, such as polyamines and polyamine synthetic enzyme activities including ornithine decarboxylase, are present at high levels in proliferating normal and neoplastic cells.13,14 In addition, many kinases, such as tyrosine protein kinase (TPK), mediate proliferative as well as metabolic signals in the cells. Eicosanoids, the metabolites of arachidonic acid through the lipoxygenase and cycloxygenase pathways, exerts a variety of biological activities.14,15 The mechanism of the antitumor effect shown in this study is unclear, but it may be related to the inhibitory effect of caffeic acid esters and flavonoid glycones on TPK, lipoxygenase, and cycloxygenase pathways metabolites.
In conclusion, our results indicate that the administration of diluted unfractionated honey is considerably effective against a murine bladder cancer implantation model and some human and murine bladder cancer cell lines in vitro. Further similar studies are required to elucidate the exact mechanism of the action of honey against bladder cancer and other systems malignancies. Our next study will employ the N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder cancer model to investigate the possible antitumor effect of honey when instilled intravesically against a superficial bladder tumor.
We would like to thank Mrs Risa Kawamoto and Mrs Taeko Asano of the Urology laboratory, Institute of Clinical Medicine, University of Tsukuba for their technical help.