Isoliquiritigen Enhances the Antitumour Activity and Decreases the Genotoxic Effect of Cyclophosphamide

The aim of this study was to evaluate the antitumour activities and genotoxic effects of isoliquiritigenin (ISL) combined with cyclophosphamide (CP) in vitro and in vivo. U14 cells were treated with either of ISL (5–25 μg/mL) or CP (0.25–1.25 mg/mL) alone or with combination of ISL (5–25 μg/mL) and CP (1.0 mg/mL) for 48 h. The proliferation inhibitory effect in vitro was evaluated by MTT and colony formation assays. KM mice bearing U14 mouse cervical cancer cells were used to estimate the antitumour activity in vivo. The genotoxic activity in bone marrow polychromatic erythrocytes was assayed by frequency of micronuclei. The DNA damage in peripheral white blood cells was assayed by single cell gel electrophoresis. The results showed that ISL enhanced antitumour activity of CP in vitro and in vivo, and decreased the micronucleus formation in polychromatic erythrocytes and DNA strand breaks in white blood cells in a dose-dependent way.


Introduction
Cancer is one of the major causes of mortality in humans throughout the World. According to a report dealing with the incidence and mortality of cancer in the World, about 12.7 million cancer cases and 7.6 million cancer deaths were estimated to have occurred in 2008; of these, 56% of the cases and 64% of the deaths occurred in the economically developing world [1]. In China, cancer has become the leading cause of deaths among urban and rural residents. The high rate of malignancy of these neoplasms is associated with rapid proliferation, numerous early metastases and high resistance to conventional treatments involving surgery, chemotherapy and radiation. Although preventive measures like chemotherapy are very useful, these often result in manifestation of chronic side effects [2,3]. Cyclophosphamide (CP), also known as cytophosphane, is a nitrogen mustard alkylating agent. It has been used to treat leukemia [4], breast cancer [5], small cell lung cancer [6], cervical cancer [7], non-Hodgkin's lymphoma [8] and so on. However, it has severe and life-threatening adverse effects. Some reports indicated that CP could cause temporary or (more rarely) permanent sterility [9] and produce chromosome damage [10], micronuclei [11], sister chromatid exchanges and DNA strand breaks in many kinds of mouse cells [12]. Therefore, it is desirable to find a compound that can decrease the genotoxic effects of cyclophosphamide without having any negative effect on its antitumour activity.
Licorice (Glycyrrhiza uralensis) has been used for more than four millennia as a flavoring agent in foods, beverages, and tobacco, and to treat individuals with gastric or duodenal ulcers [13], sore throats, coughs, bronchitis, arthritis [14], and allergies [15]. Moreover, many studies have revealed that several licorice derived compounds, i.e., glycyrrhizin, isoliquiritigenin (ISL, Figure 1), licochalcone, and glabridin, have a variety of pharmaceutical effects [16][17][18][19]. In addition, ISL, a dietary flavonoid, has been evaluated in terms of its antioxidative effects [20], antiplatelet aggregation effects [21], and estrogenic properties [22]. In particular, ISL can suppress proliferation and induce apoptosis in many kinds of cancer cells, including human promyelocytic cell line [23], human glioma cells [24], human uterine leiomyoma cells [25], colon cancer cells [26], human prostate cancer [27], and human hepatoma cells [28]. ISL also suppresses migration and invasion in human breast cancer cells [29] and mouse renal cell carcinoma [30]. The results from our lab showed that ISL was able to induce monocytic differentiation in HL-60 human leukemia cells [31,32] or B16F0 mouse melanoma cells [33], suppress proliferation and induce apoptosis in HeLa human cervical carcinoma cells [34,35] and SKOV-3 human ovarian carcinoma cells [36], we also found that ISL and CP have synergistic action in inhibition of proliferation of U14 cells [37]. However, useful information about the pharmacological and genotoxic effects of ISL combined with chemotherapeutic agents is scarce. In this study, the effects of ISL on the antitumour acitivity and genotoxic effect of CP were investigated.

High Concentration of ISL Significantly Enhances CP-Induced Inhibition of U14 Cells Proliferation in Vitro
As shown in Figure 2, after treatment by CP (0, 0.25, 0.5, 0.75, 1.0, 1.25 mg/mL) or ISL (5, 10, 15, 20 and 25 μg/mL), respectively, for 48 h, a significant concentration-dependent reduction in cell viability was observed, the proliferation rate of 1.25 mg/mL CP-treated U14 cells decreased by 68.68%, 25 μg/mL ISL decreased by 61.57%. In view of the significant proliferation inhibition of U14 cells induced by CP, we chose the concentration of 1.0 mg/mL for the subsequent in vitro assays. The inhibition rate of co-treatment with 1.0 mg/mL CP and low concentration of ISL (5, 10, 15 μg/mL) is lower than that of 1.0 mg/mL CP alone, however the combination of ISL (20 μg/mL) significantly enhances CP-induced inhibition of U14 cells proliferation, the percentage of viable cells decreased from 47.00% (after treatment with CP 1.0 mg/mL) to 27.64% (treatment with the combination of 1.0 mg/mL CP and 20 μg/mL ISL). To further confirm the potentiation of co-treatment, the optimal combination of ISL 20 μg/mL and CP 1.0 mg/mL was used to investigate the effect on suppression of long-term colony formation. Exposure of U14 with ISL (20 μg/mL) and CP (1.0 mg/mL) resulted in a greater inhibition of colony formation than each agent alone (Figure 3), the colony-forming rate of cells exposed to ISL and CP was decreased by 70.64% compared with the control and 25.41% compared with CP alone.

Co-Treatment with ISL and CP Synergestically Decreases the Tumour Growth in Vivo
To further explore the possible synergistic effects of ISL with conventional chemotherapeutic drug, CP, mouse cervical cancer U14 cells were transplanted subcutaneously into KM mice. As shown in Figure 4, co-treatment with ISL (20 mg/kg) and CP (40 mg/kg) significantly inhibited the growth of cervical cancer U14 cells, the inhibition rate reached 65.66%, while the inhibition rate was 43.55% and 35.57% when treated with ISL(20 mg/kg) or CP (40 mg/kg) alone respectively ( Figure 4).

ISL Inhibits the Micronuclei Yield Induced by CP
CP alone significantly increases micronucleus formation in polychromatic erythrocytes ( Figure 5), approximately 11 times higher than that of control group, while ISL alone has no influence on micronuclei. Pretreatment with ISL partially blocked CP-induced micronuclei in a dose-dependent way. The inhibition rate of ISL (20 mg/kg) treatment on micronuclei reached 41.4% ( Figure 5).

ISL Inhibits the DNA Damage Induced by CP
As shown in Figure 6, CP alone significantly increased the DNA damage detected by SCGE, as shown by the significant increase of olive tail moment; ISL alone had no influence on DNA, and when co-treated with combination of ISL and CP, the olive tail moment decreased in a dose-dependent manner ( Figure 6).

Discussion
Improvements in the therapy of malignant disease during the past two decades have allowed long-term survival and cure in many patients. However, in recent years secondary malignancies have been increasingly recognized as an important late complication after chemo-and radiotherapy [38,39]. Some reports indicate that a few secondary malignant cases are related to cyclophosphamide (CP) [40][41][42] and that CP could induce the formation of urinary bladder tumours in rats [43], while still being used extensively for its efficacy on primary tumours, as the first-line chemotherapy agent against breast cancer, small cell lung cancer, cervical cancer and non-Hodgkin's lymphoma. It has been shown that CP could produce chromosome damage, micronuclei, sister chromatid exchanges and DNA strand breaks in many kinds of mouse cells [10,44,45]. It is obvious that all above indices are related with carcinogenesis. Therefore, it is necessary to find a compound that can decrease the genotoxic effects of CP without having any negative effect on its antitumour activity.
ISL, a dietary flavonoid, exhibits antitumor activities, including the capability to suppresses proliferation and induce apoptosis in human cervical carcinoma cells [35], to suppresses migration and invasion of human breast cancer cells [29], and to induce monocytic differentiation in HL-60 cells [31]. Previous studies indicated that flavonoids may protect the cells from toxicity induced by CP [46]. ISL has been found to counteract the side effects of cisplatin therapy in cancer patients [47] or to reduce pulmonary metastasis, without any weight loss or leukocytopenia [30]. However, limited information is available concerning the antitumour activity and genotoxic effects of ISL combined with CP. In this study, the antitumour activity and genotoxic effect of ISL combined with intraperitoneal injection of cyclophosphamide was investigated. A novel finding obtained from this study is that a combination of ISL and CP significantly inhibited the U14 tumour growth in vitro and in vivo, and the inhibition ratio reached 82.61% and 65.66% respectively. More important, ISL partially decreased CP-induced micronuclei formation, and dose-dependently (5, 10, 20 mg/kg) inhibited CP-induced DNA strand breaks, making ISL may be a candidate to decrease the genotoxic effects of chemotherapy with CP.

Animal Preparation
SPF KM female mice, aged 5-7 weeks and weighed from 18 to 22 g, were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China. The mice were acclimatized to laboratory conditions (22 ± 3 °C and 60% humidity) for 7 days, with a commercial standard mouse cube diet (Shihezi University Laboratory Animal Center, Xinjiang, China) and water ad libitum. After acclimatization, the mice were randomly divided into control and treated groups.

Cell Culture
Mouse cervical cancer U14 cells were obtained from China Center for Type Culture Collection (CCTCC), Wuhan, China. The cells were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO 2 . Cells were split every three days and were diluted one day before each experiment.

Colony Formation Assay
For this assay, cells were seeded at 500 cells per 100 mm culture dish and allowed to attach overnight [49]. The cells were treated with ISL (20 μg/mL), CP (1.0 mg/mL) or their combination and maintained under standard cell culture conditions at 37 °C and 5% CO 2 in a humid environment. After 8 days, the dishes were washed twice in PBS, fixed with methanol, stained with Giemsa dye (Sigma), washed with PBS and air dried. Colonies exhibiting a minimum of 50 viable cells were counted. Colony plating efficiency was calculated to be the number of viable plated cells, and was expressed as a percentage of inoculated cells. The percent of colonies was calculated using the number of colonies formed in treatment divided by number of colonies formed in control group.

Micronucleus Formation Assay
After acclimation, eighty female mice were randomly assigned to the eight groups as above mentioned. ISL was administered orally for three consecutive days. After the last administration, a single dose of CP 40 mg/kg body weight was injected intraperitoneally while the vehicle alone (20 mL/kg) was injected as the control. Blood (about 50 μL) was obtained 24 h after CP treatment from murine tail tips by a small incision and immediately mixed with heparin sodium (20 μL). The animals were then sacrificed by cervical dislocation. Bone marrow smears and staining were done following the procedure described by Tinwell [50]. Femoral bone marrow was flushed out by using 1% sodium citrate solution at 37 °C. The marrow was homogenized and centrifuged at 1,000 g for 5 min. The supernatant was decanted and the pellet was homogenized with the residual fluid and add 2 mL PBS buffer. After harvesting and washing, cells were stained with 50 µg/mL acridine orange and visualized with a Carl Zeiss fluorescence microscope with the corresponding filters (470-490 nm excitation and 515 nm emission wavelengths). For each animal, 1,000 polychromatic erythrocytes were examined by utilizing a number of fields, and the number of micronucleated polychromatic erythrocytes was recorded. Every individual experiment was performed in triplicate.

Comet Assay
The alkaline version of the comet assay (single cell gel electrophoresis, SCGE) was performed according to the protocol developed by Wang et al. [51]. Blood cells/heparin mixtures (7 µL) were embedded in LMP agarose (93 µL, 0.75 g/100 mL PBS) and the resulting mixture was spread over a precoated microscope slide (1.5 g/100 mL agarose). A cover glass was then gently placed over the slide and put at 4 °C for 5 min to allow gel solidification. After removal of the coverslips, the cells were lysed at 4 °C for at least one h in a freshly prepared, ice-cold solution of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma Base, 1% Na-lauryl sarcosinate, pH 10, and 1% Triton X-100, and 10% fresh DMSO added. Shortly after lysis, the slides were placed on a horizontal electrophoresis unit. Then they were exposed to alkali (300 mM NaOH, 1 mM Na2EDTA, pH ± 13) at 4 °C for 20 min, to allow DNA unwinding. Electrophoresis was performed at 300 mA and 25 V (0.90 V/cm) at 4 °C for 15 min. The slides were then neutralized (Tris 0.4 M, pH 7.5), stained with ethidium bromide (20 mg/mL), and were observed and photographed at 400× magnification using a Carl Zeiss reverse fluorescence microscope equipped with an excitation filter (BP 546/12 nm) and a barrier filter (590 nm). The DNA damage degree was assessed with CASP, a free SCGE analysis system published by Konca et al. [52]. The images of one hundred cells were randomly selected from each slide and the tail moment was measured. The tail moment is positively correlated with the level of DNA breakage in a cell. The mean value of the tail moment in a particular sample was taken as an index of DNA damage in this sample.

Statistical Analysis
The results were expressed as mean ± S.D. and analyzed by one-way analysis of variance (ANOVA). The values with p < 0.05 were considered as statistically significant. The analyses were carried out using the Origin 8.0 software (Origin Lab Corporation, Northampton, MA, USA).

Conclusions
In conclusion, our studies indicated that ISL significantly inhibits the genotoxic effects induced by CP, and enhances the antitumor activity of CP both in vitro and in vivo. Therefore, ISL, is a powerful candidate drug, to decrease the toxic effects and to enhance the therapeutic effects of the chemotherapy with cyclophosphamide.