Hesperidin Inhibits Cyclophosphamide-Induced Tumor Growth Delay in Mice

Abstract

Hesperidin is a natural compound that has chemoprotective effects in tumor cell lines and protective effects against hematotoxicity induced by cyclophosphamide. The aim of this study was to evaluate the effect of hesperidin on the antitumor effect of cyclophosphamide in tumor-bearing mice. Administration of hesperidin reduced the leukopenia induced by cyclophosphamide in normal mice. White blood cell counts were increased in mice treated with hesperidin at a dose 200 mg/kg prior to cyclophosphamide injection. This significant protective effect was observed at 4 and 7 days after cyclophosphamide injection. Coadministration of hesperidin with cyclophosphamide in colon carcinoma (CT-26)–bearing mice was found to significantly inhibit cyclophosphamide-induced tumor growth delay. Tumor-bearing mice treated with hesperidin had increased tumor development compared with control animals that did not receive any treatment. These results show that hesperidin interacts with cyclophosphamide to inhibit its antitumor effect. In this study, estrogen receptor was negative for the development of CT-26 tumor. These results imply that fruits containing hesperidin, such as citrus, might have side effects on the efficacy of cyclophosphamide in the treatment of patients with colon cancer.

Introduction

Chemotherapy is one of the main methods of cancer therapy. Most of the antitumor drugs used in medicine have cytotoxic effects on normal cells, which lead to side effects. Cyclophosphamide (CP), one of the most widely drugs in chemotherapy, is a cytotoxic alkylating drug with a high therapeutic index and is effective against a variety of cancers. ¹ Although CP is effective for the treatment of cancer, it induces a wide range of adverse side effects and toxicity, such as nausea, vomiting, and hematopoietic toxicity, that restrict the use of this drug in clinic. ¹ Therefore, it is necessary to search for compounds that can reduce the harmful side effects of anticancer drugs in normal tissues.

Flavonoids are a family of polyphenolic compounds found in fruits and vegetables and are common natural products ingested by humans. Flavonoids have diverse biological properties, including antibacterial, antiviral, anticancer, immunostimulatory, and antioxidant effects. ² Hesperidin belongs to a class of flavonoids called flavonones and is found mainly in citrus fruits. Recently, we showed that hesperidin has powerful protective effects on DNA damage induced by genotoxic agents in mice. Specifically, it reduced the frequency of micronuclei induced by gamma irradiation and cyclophosphamide in mice.^3,4 Hesperidin significantly reduced the toxicity of CP in bone marrow cells. ⁴ Additionally, ingestion of hesperidin reduced genotoxicity induced by gamma irradiation in human lymphocytes. ⁵ Hesperidin has been shown to have many biological effects, including anti-inflammatory, antimicrobial, anticarcinogenic, and antioxidant effects. ⁶ Hesperidin, in combination with a flavone called diosmin, is used in Europe as Daflon for the treatment of chronic venous insufficiency. ⁷ This drug is used safely in humans for chronic diseases. However, with regard to the beneficial protective effects of hesperidin, one question remains: Can it protect and have beneficial effects on normal cells without interfering with the effects of chemotherapy on tumor cells?

For the reasons discussed above, it is worthwhile to examine the important biological effects of hesperidin on the effects induced by CP in tumor tissue. In this investigation, the in vivo protective activity of hesperidin on hematotoxicity induced by cyclophosphamide has been investigated in tumor-bearing mice.

Materials and Methods

Animals

Male NMRI and BALB/c mice weighing 25 ± 3 g were purchased from the Pasteur Institute. Mice were housed under approved conditions in university animal facilities and given standard mouse pellet and water ad libitum. All the animals were kept under controlled lighting conditions (light:dark, 12:12 hours) and temperature. This study with animal experiments was approved by the Research Committee of the Mazandaran University of Medical Sciences.

Chemicals

Cyclophosphamide was obtained from Asta Medica AG (Frankfurt, Germany). Hesperidin was purchased from Sigma Chemical Co. (Buchs, Switzerland). All other chemicals were obtained from Merck Company (Darmstadt, Germany).

Hematological Analysis

Male NMRI mice were divided into 4 groups: group I received distilled water as control, groups II and III received CP intraperitoneally at a dose of 25 mg/kg, and groups III and IV received hesperidin by gavage at a dose of 200 mg/kg in distilled water. This dose of hesperidin was selected to show optimum protection according to our previous study. ⁴ CP was given on day 5 after the administration of hesperidin and was continued until day 14.

Hematological analysis was performed on blood taken from the tail vein. Two hundred microliters of blood were added into EDTA and mixed with normal saline to 0.5 mL. Blood counts were carried out using a Coulter counter (Sysmex K21, Kobe, Japan). White blood cells (WBC) were determined in all groups at regular intervals between days 0, 4, 7, 10, and 14 after the administration of CP.

Cell Culture

The mouse colon cell line CT-26 was purchased from the Iranian Pasture Institute (Tehran, Iran), Code C532. Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and streptomycin/penicillin in a humidified atmosphere of 5% CO₂ at 37°C.

Tumor Inoculation and Drug Treatment

Subconfluent CT-26 cells were trypsinized and suspended in RPMI 1640 (without fetal bovine serum). The cell suspension (2 × 10⁵ in 0.1 mL medium) was injected intradermally into the right flank of BALB/c mice. A single solid tumor was grown and developed in the intradermal area. The tumor size was measured with a Vernier caliper every 2 days. The tumor volume (V) was calculated using the a, b, and c diameter values of the tumor according to the formula V = abc × π/6.

The hesperidin treatment was started after the tumor size reached about 800 mm³ at day 15. Tumor-bearing mice were divided into 4 groups: group I received distilled water as a control, groups II and III received CP at a dose of 25 mg/kg (intraperitoneally), and groups III and IV received hesperidin by oral gavage at a dose of 200 mg/kg in distilled water. A total of 32 mice were used with 8 mice in each group. The tumor size and weight were measured in all mice.

Immunohistochemistry

Tumors were removed from mice and fixed. After fixation, tumor slides were prepared at 5 µm. Slides were stained with a mouse monoclonal antibody against the estrogen receptor (Dako Co, Glostrup, Denmark), and was staining with hematoxylin and eosin. The sections were assessed for staining intensity. A block of human breast tissue was used a positive control for estrogen receptor.

Statistical Analysis

The data are presented as means ± SD. One-way analysis of variance and Tukey’s HSD (honestly significant difference) test were used for multiple comparisons of data. A probability value of .05 was designated to denote significance.

Results

Effects of Hesperidin on Leukocytes Treated With Cyclophosphamide

After a single dose of CP, the WBC count was significantly decreased on days 4, 7, 10, and 14. Severe leukopenia was observed after CP administration on day 4 (3.6 ± 0.83 × 10³/µL with CP treatment vs 11.18 ± 1.23 × 10³/µL in the controls, P < .001) as shown in Figure 1. The trend of reduced WBC counts continued until at least 14 days after treatment. Animals received 200 mg/kg of hesperidin daily for 5 days prior to CP treatment, and it was continued for 14 days. Leukocyte counts were increased in animals treated with hesperidin and CP on days 4 and 7 as compared with the group treated with CP alone (P < .001 and P < .05; Figure 1). The increased WBC counts were not significant in the hesperidin + CP group versus CP group comparison on days 10 and 14. The survival rate of animals treated with CP was not increased by hesperidin administration. All mice died on day 13 in the CP and hesperidin + CP groups.

Figure 1.

Effect of hesperidin (HES) and/or cyclophosphamide (CP) on total while blood cell (WBC) counts in normal NMRI male mice (n = 10 mice). P < .001 for CP-treated versus CP + HES–treated groups at day 4; P < .05 for CP-treated versus CP + HES–treated groups at day 7

Effects of Hesperidin and/or Cyclophosphamide on Tumor Volume

Experimental CT-26 tumors were induced in BALB/c mice. The growth rates of the tumors were investigated by measuring their volume and weight. The volume of tumors increased with time from 7 days after inoculation. The tumor volumes were the same in all 4 groups (Figure 2). Tumor volumes increased in a time-dependent manner in mice treated with hesperidin and in control mice. A daily injection of CP inhibited tumor growth in the CP and hesperidin + CP groups, although the suppressive effect of the CP alone group was more than that of the HES + CP group (Figures 2 and 3; P < .05). No significant tumor growth was observed in mice treated with CP, but mice treated with HES before and continuously with CP treatment had more tumor growth compared with CP treatment alone.

Figure 2.

Effect of the interaction of hesperidin (HES) with cyclophosphamide (CP) on growth of CT-26 colon carcinoma cells implanted into BALB/c mice. The hesperidin gavages were started after development of tumor on day 5. CP injection was started on day 8 (4 days after HES gavage)

Figure 3.

Effects of hesperidin (HES) on cyclophosphamide (CP)-induced tumor growth delay at 20 days after tumor development in tumor-bearing mice. P < .05 for control versus HES-treated group; P < .05 for CP-treated versus CP + HES–treated groups

Immunohistochemistry was performed for evaluation of possible high expressed estrogen receptor in tumor tissue in this study. In the immunohistochemistry experiment, no staining was observed in samples from mice colon tumors, but human breast tissue showed positive staining (Figure 4).

Figure 4.

Estrogen receptor staining (immunohistochemistry) of colon carcinoma section derived from (A) tumor-bearing mice or (B) human breast tissue as a positive control (magnification, ×400)

Discussion

The results here showed a partially protective effect of hesperidin on leukopenia induced by CP in mice. This protective effect was observed only at 4 and 7 days after CP injection. However, hesperidin did not elicit any beneficial effect on tumor growth and size in tumor-bearing mice coadministered with CP, a fact that will be discussed here in more detail.

Cyclophosphamide is an alkylation agent with an active metabolite that leads to DNA cross-linking. It has been widely used for treatment of cancer patients although it has several side effects, mainly bone marrow toxicity and severe infection. ⁸ In this study, CP treatment demonstrated a significant reducing effect on WBC counts in mice 4 days after CP injection. Other studies have shown that mice treated with CP had a similar suppression of bone marrow cells and reduction of WBC counts 3 and 5 days after CP treatment^8,9 and that these effects resulted in severe leukopenia and infection. We have demonstrated in several studies that herbal medicines, such as hawthorn and citrus, reduced genotoxicity and toxicity induced by CP in mice bone marrow.^10,11 Although another study showed that flavonoids such as Daflon, which contains diosmin and hesperidin, prevented hematological toxicity induced by chemotherapeutic agents in normal rats, that study did not evaluate the effects of these flavonoids on the efficacy of antitumor agents in a tumor-bearing animal model. ⁸ Our results confirmed the protective effects of hesperidin on leukopenia induced by CP in normal mice.

In our experiments, we observed interference between CP and hesperidin. Hesperidin reduced the antitumor effects of CP in terms of tumor size and development. Tumor growth was observed with coadministration of CP and hesperidin but not with the CP treatment alone group. Because CP was administered by intrapertoneal injection and there would not be intestinal interaction between CP and hesperidin, we can hypothesize a mechanism. CP is a prodrug, meaning it must be converted to active alkylating mustard in the body by the liver. CP is activated by hepatic cytochrome P450 (CYP) isoenzymes, to form 4-hydroxycyclophosphamide, which enters the blood and is transported to tumor cells by erythrocytes. ¹ Because CP is activated by CYP-catalyzed metabolites, drug interactions could modulate the pharmacokinetics by inhibition of a relevant CYP. ¹ Hesperidin has been shown to significantly affect the pharmacokinetics of several drugs, such as diltiazem and verapamil, by inhibition of CYP isoenzymes.^12-15 P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp or Pgp) also known as multidrug-resistant protein 1 is a glycoprotein that in humans has several families. It plays a large role in distribution and elimination of many clinically important therapeutic substances. P-gp and CYP3A could act synergistically in intestinal first-pass metabolism. If one drug is a substrate of both P-gp and CYP3A4 (both found in close proximity in the intestinal wall), and a second drug is added that is an inhibitor of both P-gp and CYP 3A4, then the first drug will be allowed in increased amounts. The effect of hesperidin and its metabolite (hesperitin) was studied on P-glycoprotein, and cytochrome P450 in the liver. Hesperidin and other flavonoids such as naringin were showed strong inhibitory effects on CYP3A4 and other CYP enzymes^15,16 Hesperidin significantly enhanced the oral bioavailability of diltiazem in rats. The authors proposed that it be considered that hesperidin increased the intestinal absorption and reduced the first-pass metabolism of diltiazem in the intestine and in the liver via an inhibition of cytochrome P450 3A or P-glycoprotein. ¹³ It is possible that activation of CP by CYP is inhibited by hesperidin and that CP is not metabolized to an active agent to exert its therapeutic effects.

The development of tumors was increased with coadministration of CP and hesperidin as compare to CP alone group. Another hypothesis related to the effects of CP + HES on tumor growth is that apoptosis is inhibited by blocking reactive oxygen species (ROS) formation. ¹⁷ 4-Hydroxycyclophosphamide is the active metabolite of CP and can produce ROS and cause oxidative DNA damage. The production of ROS is important for induction of apoptosis and death of tumor cells.^17-20 Hesperidin has antioxidant activity and may partially inhibit the apoptosis induced by CP in tumor cells.

In our results, the development of tumors was greater with hesperidin treatment than with control tumor-bearing mice that did not receive any treatment. Previous studies showed that hesperidin had chemopreventive effects and also anticancer effects. Administration of citrus unshiu juice, which contains high amounts of hesperidin and cryptoxanthin, inhibited azoxymethane-induced rat colon carcinoma. ²¹ Dietary hesperidin during the initiation or postinitiation phase effectively inhibited mouse bladder and colon carcinogenesis induced by N-butyl–N-(4-hydroxybutyl) nitrosamine.^7,22 The authors proposed that the chemopreventive effects of hesperidin were related to its antioxidant activity.^7,22

Our results are not consistent with the data discussed above. To explain this apparent paradox, one hypothesis is that the hormone receptor status of tumor can explain this effect. Other studies have established that genistein, an isoflavone compound found in dietary supplements, can bind to the estrogen receptor and stimulate estrogen-dependent breast cancer. Because hesperidin has a 2-phenyl benzopyrane ring with a similar chemical structure to the genistein oxy-benzopyrane ring, ⁶ it may have a similar effect as genistein on the development of tumor in mice. In our experiments, estrogen receptor was negative in mouse colon carcinoma, so the hypothesis discussed here is not consistent with our results. This study has several limitations, including the fact that metabolites of CP and hesperidin concentration in plasma were not determined.

Our results showed that coadministration of hesperidin with cyclophosphamide reduced the antitumor effect of CP in colon cancer-bearing mice and that tumor volume was increased with the hesperidin treatment as compared with the control group. It is possible that an interaction between hesperidin and CP is responsible for this effect, in which hesperidin reduced metabolism CP by liver for producing active CP metabolites. Based on these results, patients with colon cancer who are receiving CP should exclude any hesperidin-containing fruits from their diet. Because several fruits, mainly in the citrus family, have high amounts of hesperidin, observing this caution may be important for treatment efficacy.