Hesperidin suppressed metastasis, angiogenesis and tumour growth in Balb/c mice model of breast cancer

Abstract Considering the unfavourable response of breast cancer (BC) to treatment, we assessed the therapeutic potential hesperidin in mice bearing 4T1 BC tumours. Anti‐tumour effects were assessed by measuring pathologic complete response (pCR), survival analysis, immunohistochemistry for E‐cadherin, VEGF, MMP9, MMP2 and Ki‐67, serum measurement of IFNγ and IL‐4, and gene expression analysis of CD105, VEGFa, VEGFR2 and COX2. Survival of tumour‐bearing mice was the highest in mice receiving a combination of hesperidin and doxorubicin (Dox) (80%) compared to the normal saline (43%), hesperidin 5 (54%), 10 (55.5%), 10 (60.5%) and 40 (66%) mg/kg, and 10 mg/kg Dox‐treated (73%) groups (p < 0.0001 for all). Compared to the normal saline group, there was a significant elevation in IFNγ level in the animals receiving 20 (p = 0.0026) and 40 (p < 0.001) mg/kg hesperidin, 10 mg/kg Dox (p < 0.001), and combined hesperidin (20 mg/kg) and Dox (10 mg/kg) (p < 0.001). A significant reduction in the gene expression of CD 105 (p = 0.0106), VEGFa (p < 0.0001), VEGFR2 (p < 0.0001), and Cox2 (p = 0.034) and a significant higher pCR score (p = 0.006) were noticed in mice treated with 10 mg/kg Dox + 20 mg/kg hesperidin compared to those treated with 10 mg/kg Dox alone. Immunohistochemical staining showed significant reductions in Ki‐67 (p < 0.001) and VEGF (p < 0.001) and a significant elevation in E‐cadherin (p = 0.005) in the 10 mg/kg Dox + 20 mg/kg treatment group than in 10 mg/kg Dox alone group. Hesperidin can be considered as a potentially suitable anti‐cancer agent for BC that can synergize with other chemotherapeutics.


| INTRODUC TI ON
Among other malignancies, women are more likely to develop tumours in their breasts, and breast cancer (BC) accounts for 33% of all women's cancers and is responsible for about 20% of cancer-related deaths (the second leading cause) among females. [1][2][3] This group of tumours show great heterogeneity in terms of response to therapy and biological specifications. In terms of histopathology, BC tumours are primarily categorized based on the expression of receptors for progesterone (PR), oestrogen (ER) and ERBB2 (also known as Her2).
The classification of BC has not stopped on histopathology, extending toward molecular features and genomics and transcriptomics signatures, according to which at least 10 subtypes of BC have been characterized. 4 Around 15% of all BC cases show negative expression for all PR, ER and Her2 (i.e. triple negative) and are known to have an aggressive behaviour, a poor response to conventional treatments, and an adverse immunologic profile with a high rate of lymphocytic infiltration, as an indicator of metastasis. 5 Similar to many cancers, multiple triggering causative agents are considered to play a role in BC development from advanced age and intrinsic predisposing parameters to environmental triggers. Drug resistance is a rising issue in BC and is a main cause of patient death. 6,7 Surgery and radiotherapy that are used to eradicate localized tumours are ineffective in preventing metastases. 8,9 Although chemotherapy is one of the most usual therapeutic methods, due to the lack of selective cytotoxicity, this treatment is associated with many side effects. On the other hand, many cancers are resistant to chemotherapy; therefore, it is essential to find new treatment strategies with fewer side effects. 1,10 Herbal compounds with antioxidant activity have acquired great attention in the field of cancer therapy. 11 The role of natural compounds available in the diet, especially flavonoids, in inhibiting carcinogenesis and cancer treatment has been highlighted. 11 Flavonoids are bioactive compounds that include about 60% of the polyphenolic compounds found in plants and are abundantly found in fruits, vegetables, seeds, nuts and beverages such as tea. 12 In addition to antioxidant, anti-inflammatory, anti-hypertensive and anti-allergic effects, flavonoids have anticancer properties through interference in the three stages of carcinogenesis. 13,14 Hesperetin-7-O-rutinoside, or shortly hesperidin, as the most abundant flavonoid in citrus fruits, is a non-toxic and non-allergic flavanone glycoside with no adverse side effects. 13,14 In recent years, hesperidin's biological and pharmacological effects have been studied. 15,16 Studies show the influential role of hesperidin in improving cardiovascular function and reducing blood lipids and inflammatory markers. Also, various studies have confirmed hesperidin's antibacterial, antifungal, and antiviral role. In addition, hesperidin has anti-cancer properties. [13][14][15][16][17] Experiments have suggested anti-cancer effects for hesperidin, which are supposed to be mediated through inhibiting carcinogenesis, as observed in skin and bladder cancers in animal models, as well as through suppressing tumour growth and proliferation and promoting programmed cellular death (e.g. in colon, breast and prostate cancer cells). 16,18,19 Here, we created subcutaneous breast tumours in Balb/c mice and then injected different doses of hesperidin to assess its tumour growth inhibitory effects.

| Experimental groups and treatments
The seven study groups included normal saline (200 mL), hes-

| Cell proliferation assay
The 4T1 cells plus mouse lymphocytes and mouse stem cells were cultured in RPMI in a 96-cell plate (1.2 × 10 2 cells in each well).
In order to evaluate the cytotoxicity of hesperidin, the concentrations of 100, 50, 25, 12.5, 6.25 and 3 μg/mL were obtained by the serial dilution method. Hesperidin (Sigma-Aldrich) with a concentration of 1 M was prepared in dimethyl sulfoxide (DMSO, Merck, Germany), which as a stock solution was used to prepare target concentrations for 24-, 48-and 72-h treatments for the MTT assay. The highest amount of DMSO was always kept below 0.25%, including in the control group. After 24, 48 and 72 h of treatment, the MTT reagent [3-(4, 5-dimethylthiazol-2-yl)-2,5-di phenyltetrazoliumbromide], which is converted into a formazan product by living cells, was added for 4 h, and then absorbance at 570 nm was read.

| Measurement of serum cytokines
Centrifugation (3000 rpm) for 20 min was used to separate sera, which were frozen (−70°C) until use. Serum IFNγ and IL4 concentrations were measured by commercial ELISA kits (Bioas-say Technology). The substrate, o-phenylenediamine, which is converted by horse radish peroxidase to a colour substance, was added, and absorbance at 492 nm was recorded using an Anthos 2020 microplate reader (Anthos, Wals, Austria). All measurements in serum were performed in duplicates, and intra-and inter-assay CVs for IFNγ and IL4 were <10% and <12%, respectively.

| Tumour size and body weight
On day 14, the mice were started to be weighed every 2 days until day 36. Tumour dimensions (L: length and W: width) were determined at 2-day intervals in parallel with weight measurements. Tumour volume calculation was as follows:

| Haematoxylin and eosin staining
Resected tumour sections were immersed in formalin and embedded in paraffin. Tissue blocks with 5μm thickness were prepared, stained with haematoxylin and eosin (H&E), and visualized under a light microscope. The pathologic complete response (pCR) scoring system, which is based on the rates of pleomorphism, mitosis, necrosis and residual tumour cells, was employed to determine response to treatment. The categories of pCR were as no response (R = 0, reflecting no evident reduction in the quantity of tumour cells), partial-weak response (R = 1, fibrosis of tumour cells = 30%), partial-moderate response (R = 2, fibrosis of tumour cells = 70%) and complete response (R = 3, no residual tumour cells).

| Gene expression measurement
Tumour content of RNA was extracted using Trizol (GeneAll, Korea), which was subjected to quality and quantity assessments using agarose gel visualization and measuring the A260/A280 ratio (NanoDrop 1000 Spectrophotometer, Wilmington, DE, USA). Complementary DNA was generated using Two-Strand Synthesis kit (General, Korea), and gene expression analysis was performed using real-time PCR. Each PCR cycle included a 10-s denaturation phase at 95°C, followed by a 10-s annealing step at 55-62°C and a 30-s extension phase at 72°C. Each PCR reaction contained template cDNA (1 μL), specific pairs of primers (1 μL), deionized water (10.5 μL) and SYBR® Green Master Mix-Plus (Yektatajhiz, Iran) (12.5 μL) performed in Corbett RG6000 thermocycler (Australia). GAPDH was employed as the internal control gene, and the Rotor-Gene 6000 Series Software was used to calculate the cycling threshold (CT) ( Table 1). The CT (Cq) obtained was adjusted according to the respective value obtained for the internal control gene F I G U R E 1 Classification of mice in the study groups.

| Statistical analysis
Means ± standard errors (SEM) with 95% confidence intervals were used to describe the results using GraphPad Prism 5 software (La Jolla, CA) for Windows. One-way analysis of variance, followed by the Tukey's post-hoc test, at a significant level of p value ≤0.05, was used to compare groups.

| Ethical considerations
Standard ethical protocols in working with laboratory animals were followed to ensure minimal harm and inconvenience to animals.
Euthanizing was performed after taking the mice away from other animals. In addition, the methods were reviewed and approved by the university's ethical committee for working with laboratory animals.

| Body weight and tumour volume changes
Body weight was measured in 2-day intervals during the study. Average body weight did not significantly change in the study groups during the study and body weight changes were comparable in animals in different groups during the study ( Figure

| Cell viability
After 72-h of treatment with hesperidin, the survival of tumour cells fell below 50%, showing a significant difference compared to 24-and 48-h treatment groups (p = 0.008). Lymphocyte count, as an indicator of metastasis, was higher than in the 24-h treatment group than in other two groups. Figure 4 and Table 2 show the IC50 values obtained for hesperidin at different times. Another noteworthy point is that none of the groups reached survival below 50% up to a concentration of 200 μg/mL of hesperidin. Moreover, the number of stem cells in the 24-h group was more than in the other two groups. Also, the decrease in the number of stem cells in the 72-h treatment group was statistically significant compared to other groups (p = 0.0032).

TA B L E 1
The sequences of the forward and reverse primers used to amplify target genes by real-time PCR.

| Hesperidin effects on inflammatory cytokines
As shown in Figure Figure 7 shows the expression levels for the genes analysed.

| VEGFa
The expression of VEGFa gene significantly decreased in Dox-treated animals compared to normal saline group, and hesperidin-treated mice at the doses of 5 and 10 mg/kg (p < 0.0001 for all comparisons) but not compared to animals receiving 20 mg/kg hesperidin. On the other hand, the expression of this gene was significantly lower in the 40 mg/kg hesperidin (p = 0.0004) and combined hesperidin + Dox (p < 0.0001) compared to Dox-treated group ( Figure 7A).

| VEGFR2
The downregulation of the VEGFR2 gene in the 10 mg/kg Doxtreated group was statistically significant compared to the normal saline, and 5 and 10 mg/kg hesperidin groups (p < 0.0001 for all comparisons) but it was comparable with the 20 and 40 mg/kg hesperidin groups. Also, the expression of this gene was significantly lower in the combined treatment group compared to the group receiving Dox alone (p < 0.0001, Figure 7B).  Figure 7C).

| CD105
Regarding the expression of CD 105, a significant reduction was observed in mice treated with 10 mg/kg Dox (p < 0.0001) compared to normal saline group and 10 mg/kg hesperidin group (p < 0.0001); however, this reduction was comparable between Dox-treated and hesperidin-treated (5, 20, and 40 mg/kg) animals. The downregulation of CD105 was statistically significant in the combination therapy group (i.e. Dox + hesperidin) compared to Dox-treated animals (p < 0.0106, Figure 7D).

| Pathologic analysis of 4T1 mammary tumours
As shown in Figures 8 and 9A, compared to the normal saline- the combinational treatment of hesperidin + Dox delivered a significantly higher pCR score than the Dox group alone (p = 0.006) and normal saline (p < 0.001) (Figures 8 and 9A).

| Findings of IHC
The results of immunohistochemical staining have been shown in

| MMP2
Compared to the normal saline group, the groups treated with hes-

| Adverse effects of hesperidin on main tissues
Tissue sections were obtained from the liver (n = 11, five from hesperidin-treated and six from normal saline-treated), lungs (n = 8, three from hesperidin-treated and five from normal salinetreated) and kidneys (n = 9, four from hesperidin-treated and five from normal saline-treated) ( Table 3). The results showed that hesperidin treatment led to slight necrosis, oedema, haemorrhage and hydropic degeneration compared to the animals treated with normal saline.

| DISCUSS ION
Hesperidin has been reported to have various therapeutic activities, including cardioprotective, 23 neuroprotective, 24 hypolipidemic, 25 anticancer, 26 antidiabetic, 27 antimicrobial, anti-inflammatory and antioxidant activities 28 that directly reflect the therapeutic potential of hesperidin in treating numerous medical conditions such as cancer. [29][30][31] Our findings showed that hesperidin boosted survival rate and decreased tumour sizes in 4T1-tumour bearing mice. In line with our results, decreased mean growth and volume of the MG-63 tumour were reported after 2 weeks of treatment with hesperidin, and this in vivo inhibitory effect was concentration-and time-dependent. 13 In DMBA-induced rat models of breast cancer, hesperidin (200 mg/ kg) was shown to increase survival, prevent tumour development, and reduce tumour volume. 32 We noticed that the group treated with hesperidin (20 mg/ kg) + Dox (10 mg/kg) had increased concentration of IFNγ and decreased concentration of IL-4i n serum. IFNγ is essential for the effective stimulation of cellular immunity and subsequently stimulating the antitumor immune response. A similar study by Patel et al. 32 in mice with breast cancer found that hesperidin F I G U R E 7 Real-time PCR experiment to determine the expression of a number of genes involved in angiogenesis and inflammation in Balb/c mice carrying 4T1 breast tumours after receiving different doses of hesperidin. Values represent mean ± standard error (SEM) of six measurements. * and ^: p < 0.05; ** and ^^: p < 0.01; ^^^: p < 0.001. The signs * and ^ represent in comparison with the normal saline and doxorubicin groups, respectively.
(200 mg/kg) increased IFNγ and decreased IL-4, which decreased the growth of cancer cells. 33 In the 4T1 mammary tumour tissues harvested from hesperidintreated mice, we observed a significant downregulation in the VEGFa, VEGFR2, CD110 and COX2 genes. CD105 is a strong indicator of neovascularization in human malignancies, 34 and VEGFa roles as a stimulator for the proliferation and migration of endothelial cells and a suppressor of apoptosis. Furthermore, VEGFa increases vascular permeability 35 and COX-2 stimulates the activity of cancer stem cells and enhances apoptotic resistance, proliferation and angiogenesis. 36 In a similar study in a mouse model of breast cancer, hesperidin was reported to suppress VEGF production and tumour growth and to reduce microvessel density through suppressing the expression of VEGF, VEGFR2 and NFATc3 proteins. 37 In addition, the inhibitory effects of hesperidin on COX-2 in kidney cancer (100 mg/kg) 38 and lung cancer (25 mg/kg) 39 have previously been reported. The main difference between these studies was the effective dose of hesperidin, which was much higher than in the current study, indicating that hesperidin can exert equally significant effects at relatively lower doses as well.
We here demonstrated that hesperidin at different doses led to an increase in pCR and the ratio of cancerous cells undergoing cell death but these effects were inferior compared to doxorubicin, where the most prominent effects were observed when the combination of hesperidin and doxorubicin was used compared to the normal saline and doxorubicin alone groups.
We also observed that VEGF expression decreased following the administration of different doses of hesperidin, but this effect was less prominent in doxorubicin alone treatment group. However, combined treatment with doxorubicin and hesperidin had more prominent reducing effects than the normal saline and doxorubicin alone treatment groups. In different types of cancer, including breast cancer, VEGF seems to be involved in angiogenesis. 40  HER2, and Ras 1 (Rac1) proteins. 49 In a similar study in mice bearing breast tumours, hesperidin significantly inhibited VEGF production and angiogenesis and suppressed tumour growth via modulating the expression of VEGF, VEGFR2 and NFATc3. 37 In another study investigating the expression of VEGFR2 after treatment with hesperidin in a mouse model of glioma, it was shown that hesperidin (20 mg/ kg) could significantly inhibit the HIF-1a/VEGF/VEGFR2 pathway. 20 Ki-67 and E-cadherin are cancer prognostic and diagnostic factors related to cell proliferation. 41,50 In the current study, IHC was  53 and the AKT and NF-κB signalling pathways. 42 Patel et al. 32 reported that the combination of doxorubicin and hesperidin could inhibit the growth of breast cancer cells in female rats via reducing Ki67 expression compared to the animals treated with DMBA. 32 It is noteworthy that subcutaneous models have shortcomings compared to orthotopic models in terms of heterogeneity, tumour growth condition and tumour progression. 54,55 Further studies should include an orthotopic 4T1 model, and possibly an orthotopic human xenograft model in immunocompromised mice.

| CON CLUS ION
Hesperidin showed significant anti-angiogenesis and antiproliferative effects in mice bearing 4T1 breast cancer, suggesting this compound as a potential treatment either with or without standard treatment (doxorubicin) to be used in future clinical research. The role of hesperidin as an inhibitor of angiogenesis, tumour growth and metastasis was confirmed in this study. It is Infiltrative cells 0 (0%) 0 (0%) TA B L E 3 Liver, lung and kidney histopathology in healthy mice treated with hesperidin or normal saline.
suggested to consider clinical studies to investigate the effects of this agent in humans. Data curation (equal); project administration (equal).

ACK N O WLE D G E M ENTS
This work was financially supported by a grant from Mazandaran University of Medical Sciences (Grant No: 7105 and 7106).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.