Melatonin decreases metastasis,primary tumor growth and angiogenesis in a mice model of breast cancer

Abstract

The goal of this study was to mechanistically analyze the effects of pre-treatment or post-treatment melatonin on the metastatic spread in a mice model. Consequently, the effects on the tumor growth, angiogenesis and metastasis were evaluated with immunohistochemical and western blot analysis. 8–10 weeks-old female BALB/c mice (n = 60, 10/group) were used. Liver metastatic cells (4TLM) from 4T1 murine breast carcinoma were previously isolated. Melatonin was administrated either before or after the injection of 4TLM cells into the mammary pad. Tumor and vehicle (%6 ethanol) injections were given to vehicle groups. Tumor group consisted of the mice injected with only 4TLM cells injected to tumor group and no intervention to control group. Necropsies were performed 27 days after injection of 4TLM. Primary tumors and metastatic tissues were removed. Furthermore, changes in lung and liver metastasis and primary tumor growth and angiogenesis were evaluated. In our study neutrophil levels were noted to be increased in peripheral blood of the tumor-bearing mice. Melatonin exerted inhibitory effects on the 4TLM-induced leukocytosis. Melatonin significantly decreased lung and liver metastasis, primary tumor growth and angiogenesis. The results demonstrated that melatonin might have a therapeutic role through reducing systemic inflammatory responses, metastasis, tumor growth and angiogenesis.

Introduction

Preclinical cutting-edge research work has conceptually changed the way we perceive breast cancer. Therefore, because of technological advancements, a wider spectrum of genomic alterations underlying its malignant progression are now being recognized. Genomic and proteomic studies have helped us in the identification of genetic/epigenetic alterations and deregulated signaling pathways that can be targeted pharmaceutically in the settings of metastatic disease. Furthermore, drug resistance is reflected in the molecular heterogeneities of the disease and poses stumbling blocks on the road to an effective clinical development of emerging targeted agents. The goal of this study was to mechanistically analyze the effects of pre-treatment or post-treatment melatonin on the metastatic spread in a mice model investigate the effects of melatonin either given before or after the injection of highly aggressive metastatic breast carcinoma cells in a mice model of breast cancer.

Breast cancer is the second most prevalent cause of cancer death and the incidence has increased rapidly during the last decades. In many developing countries it has become a global public health problem. ¹ Ground-breaking discoveries have enabled us in developing a better understanding of underlying causes, complex etiology and poor response to the treatment. Inflammatory metastatic breast carcinoma is considered to be more aggressive and has a poor prognosis as compared to other types of breast cancer. ²

N-acetyl-5-methoxytryptamine (melatonin) is produced by the pineal gland and other organs, including retina, bone marrow, thymus and airway epithelium. Melatonin was shown to exert oncostatic effects on various models of breast cancer via anti-inflammatory effects, immunomodulation, anti-oxidation, enzyme regulation, regulation of various kinases and transcription factors. ³ Effects of melatonin were analyzed in various in vitro and in vivo studies. These pioneering studies highlighted true potential of melatonin as a promising anticancer agent due to its ability to inhibit the inflammation, tumor growth, motility and proliferation.^3–6 Additionally, melatonin is involved in the regulation of immune functions in tumor microenvironment by stimulating the activities of immune cells, including T and B lymphocytes, monocytes, macrophages, and natural killer cells, and stimulating the several cytokine productions, for example, interferon-γ, interleukin IL-1, IL-2, IL-6, and tumor necrosis factor-α. ³

Angiogenesis is an important process for several physiological events such as cell growth, development, wound healing. ⁷ However, uncontrolled angiogenesis plays a role in many pathological events such as inflammatory diseases, chronic inflammation, tumor growth and tumor metastasis. ⁸ Vascular network is important to the metastatic spread of cancer cells which are dependent on adequate oxygen and nutrients. Several studies have shown that melatonin inhibits angiogenesis with various mechanisms.^9–11

Marked accumulation of CD11b⁺/Gr1⁺ myeloid cells were defined as myeloid-derived suppressor cells (MDSCs). They have been described as important negative regulators of anticancer immune responses and immunosuppressive functions are significantly upregulated in the tumor microenvironment. ¹²

Experimental studies have shown that melatonin regulates the physiology and biology of tumor cells through a variety of mechanisms.^5,13,14 The anticancer efficacy of melatonin has been shown in both experimental and clinical studies. ¹⁵

The aim of the present study was to investigate the effects of melatonin treatment either given before or after inoculation of highly aggressive metastatic breast carcinoma cells inoculation in a mice model of breast cancer. Consequently, we also studied the expressions of angiogenic markers (VEGF-A, VEGFR1 and VEGFR2) in primary tumors mediated by pre- and post-melatonin administration and the responsible immune mechanisms of the tumor microenvironment. Also, this study highlighted the potential role of pre- and post-melatonin administration in the prevention of metastasis in specific distant organs. Additionally, it has been shown that melatonin administration is involved in the regulation of Gr1⁺, CD11b⁺ and CD33 cells immune functions in tumor microenvironment.

Materials and methods

Cell culture

In this study we used 4T1 derived 4TLM (4T1-Liver Metastatic Tumor) cells. 4TLM cell lines were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM-F12) (Gibco; #11320-074, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; #10270, Waltham, MA, USA) and 1% Penicillin-Streptomycin (Thermo Fisher; #15140122, Waltham, MA, USA). The experiments were conducted when cells were 75–80% confluent.

Animals and experimental protocol

8–10 weeks of age and 20–22 g in weight female BALB/c mice ¹⁶ (n = 60 for all groups, 10 mice in each group) were obtained from an animal study laboratory and the experimental protocol was approved by the Animal Care and Use Committee of our faculty. Mice were housed in a controlled environment with a cycle of 12 L:12 D (12 h light: 12 h darkness) with ad libitum access to food.

BALB/c mice were divided into 5 groups were orthotopically injected with 4T1 derived 4TLM (4T1-Liver Metastatic Tumor) tumor cells. Confluent cells were used for orthotopic transplantation and the cells used in this study were syngeneic for BALB/c mice. 4T1 cell line was obtained from the same spontaneously formed breast carcinoma in BALB/c mice. Liver metastasis formed by 4T1 cells was previously described by Erin et al. ¹⁷ and was termed 4TLM. The cell lines were grown in DMEM-F12 supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.02 mM nonessential amino acids. Equal numbers of 4TLM cells in DMEM-F12 (10⁵ cells per mouse) were injected into the right upper mammary gland just beneath the armpit of BALB/c mice under ketamine/xylazine anesthesia (15 mg/kg i.m.). Primary tumors, lung and liver tissues were removed 27 days after injection. The 27-day incubation period was decided based on our experience in previous studies. Organ failure and death occur after the 27^th day.^16,18

Experimental groups

• Melatonin 1 (M1): Melatonin dissolved in 6% concentrated ethanol was administered intraperitoneally at a dose of 10mg/kg/day for 4 weeks before the tumor injection and 30mg/kg/day (M2) for 27 days after the tumor injection.

• Vehicle 1 (V1): Vehicle (6% EtOH) injected to this group for 4 weeks. On 29th day tumor injected, and one day after the tumor injection vehicle (6% EtOH) injected for 27 days.

• Melatonin 2 (M2): On the first day tumor injected to this group. One day after the tumor injection melatonin dissolved in 6% concentrated ethanol was administered intraperitoneally at a dose of 30mg/kg/day for 27 days.

• Vehicle 2 (V2): On the first day tumor injected to this group. One day after the tumor injection vehicle (6% EtOH) injected for 27 days.

• Tumor (T): Only tumor cells injected to this group.

• Control (C): Group with no intervention (Figure 1).

Figure 1.

Schematic representation of material and methods were seen in the picture. Metastatic 4TLM cell line was used in in vivo study.

Melatonin (Sigma; #M5250, St. Louise, MO, USA) dissolved in 6% concentrated ethanol was administrated either before (M1) or after (M2) the injection of 4TLM cells into the mammary pad. Vehicle (6% EtOH) was given to vehicle groups (V1, V2). 10mg/kg and 30mg/kg doses were both used physiologically in former studies.^19,20 Melatonin was divided into two equal doses and administered two times a day (09.⁰⁰ am and 5.⁰⁰ pm just before turning off the light of the animal facility). We have administered two doses since melatonin has been shown to be effective both in the morning and in the evening,^9,13,14 besides we wanted to keep the level of melatonin high constantly.

Tumor measurement

After 7 days of tumor implantation, size of the tumors was measured with a digital caliper (Sigma; #Z136115-1EA, St. Louise, MO, USA) on every second days. Tumor volume was calculated by measuring the tumor length (L) and width (W) using calipers and calculating the tumor area as A = (L × W). In addition, primary tumor weights were measured during the necropsy.

Peripheral blood smears

Peripheral blood smears were obtained under anesthesia before final necropsy and were stained with hematoxylin and eosin. Neutrophil, lymphocyte, and monocyte numbers were counted using 10 slides at 40X magnification from each animal.

Metastasis assay

To evaluate metastasis, lung and liver tissues were examined under the microscope. Specifically, from each tissue sample, 5 sections were taken after every 5 sections and were stained with hematoxylin and eosin to determine the extent of metastasis. For each animal, 20 photographs from 5 different sections of each mouse were randomly taken at 20X magnification; then areas of microscopic metastatic lesions were selected and measured (mm²) with the SPOT Advanced 4.6 software (Diagnostic Instruments, Sterling Heights, MI). Data represent the area of metastasis per area of 20X magnification of tissue visualized on the photograph.

Immunohistochemistry

The tissues of primary tumor, lung and liver were fixed in 10% formalin (Merck; #1040031000, Burlington, MA, USA) and embedded in paraffin. Paraffin sections were deparaffinized and blocked for endogenous peroxidase activity with methanol (Merck; #1.060.092.511, Burlington, MA, USA) containing 3% H₂O₂ (Merck; # 1.08600.1000, Burlington, MA, USA) for 20 minutes. Also, for nonspecific binding with universal blocking reagent (Thermo Fisher Scientific; #TA-125-UB, Waltham, MA, USA) for 7 minutes at room temperature. Primary antibodies (CD11b Abcam; #ab133357; GR1(Ly6G) Abcam; #ab25377; CD33 Abcam; #ab203032; VEGF-A Abcam; #ab51745; VEGFR-1 Abcam; #ab32152; VEGFR-2 Abcam; #ab45010, Cambridge, UK) diluted in dilution buffer were applied overnight at 4°C in a humidified chamber. For negative controls, the primary antibodies were replaced by normal rabbit immunoglobulin (Ig) G serum (Vector Laboratories; #I-1000, Burlingame, CA, USA) at the same concentration. After several washes in phosphate-buffered saline (PBS), sections were incubated with biotinylated secondary antibody (Vector Lab.; #BA-1000) followed by streptavidine peroxidase complex (ScyTek Laboratories; #SHP-125, Logan, UT) incubation for 40 minutes. Samples were rinsed with PBS. Antibody complexes were visualized by incubation with diaminobenzidine chromogen (SigmafastTM; #D4168-50SET St Louis, MO). Sections were counterstained with Mayer hematoxylin (Dako; #CS700, Glostrup, Denmark), dehydrated, mounted and examined by a Zeiss-Axioplan (Carl Zeiss GmbH, Jena, Germany) microscope.

Statistical analysis

Micrographs were taken using SPOT Advanced 4.6 at 20X magnification. All these micrographs were analyzed with Image-J 1.46 (Image Processing and Analysis in Java; US National Institutes of Health, Bethesda, MD; https://imagej.nih.gov/ij/) software by scanning 10 non-overlapping fields in each tissue and expressing the positive areas as a percentage of the total area.

Statistical significance was determined by analysis of variance with the Dunnett posttest by Sigma Stat 3.5 (Systat Software, San Jose, CA) software. Data are shown as means standard error of the mean and were considered statistically significant at p < 0,005.

Western blot analysis

The protein concentration was determined using a standard bicinchoninic acid (BCA) (Thermo Fisher Scientific; #23225, Waltham, MA, USA) assay and 50 mg protein was applied per lane. Before electrophoresis, samples were heated for 5 minutes at 95°C. Samples were then subjected to SDS polyacrylamide gel electrophoresis under standard conditions and then transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad; #1620177, Hercules, CA) in a buffer containing 0.2 mol/L glycine (BioRad; #1610718, Hercules, CA), 25 mM Tris (Fisher Sci.; #77-86-1, Waltham, MA, USA), and 20% methanol (Merck; #1.060.092.511, Burlington, MA, USA) overnight at 4°C. The membranes were blocked for 1 hour with 5% non-fat dry milk (Biorad; 1706404xtu, Hercules, CA, USA) in TBS-T for 1 hour to decrease nonspecific binding. Afterward, the membranes were incubated with primary antibodies against VEGF-A Abcam; #ab51745; VEGFR-1 Abcam; #ab32152; VEGFR-2 Abcam; #ab45010 in 5% nonfat dry milk in TBS-T for 1 hour. The membranes were then incubated with secondary antibody (dilution 1/10,000; Vector Laboratories; #PI-1000, Burlingame, CA, USA) for 1 hour. Immunolabeling was visualized using the chemiluminescence-based SuperSignal CL HRP Substrate System (Pierce; #34080, Rockford, IL, USA), and the membranes were exposed to Hyperfilm (Amersham, #28906835, Piscataway, NJ, USA). After the membrane was stripped using stripping solution (Pierce; #21059), equal loading of proteins in each lane was confirmed by reprobing the membrane with mouse monoclonal GAPDH antibody (Abcam, #ab181602, Cambridge, UK). Image-J (NIH, Bethesda, MD, USA) was used to quantify the density of the western blot bands.

Results

Tumor growth and peripheral blood smear analysis

Tumor growth was measured on every second day and T, V1, V2 group tumors were bigger in size and bulk than those of the M1 and M2 groups. Tumor growth rate of M1 group increased slowly as ed to M2 group until the last week of the experiment (Figure 2A) 6% EtOH as the vehicle, did not alter the tumor growth and peripheral blood smear analysis. In all groups neutrophil levels were higher than the control group (Figure 2B). Neutrophil, lymphocyte, monocyte levels were significantly higher in tumor group when compared to M1, M2 and C groups (p < 0,05) (Figure 2 B–D). There were no differences between the M1 and M2 groups in any parameter.

Figure 2.

Representative figure was shown that tumor growth (A) and peripheral blood smear analysis (B–D). A) Changes in primary tumor growth compared with control, metastatic breast carcinoma cells injected groups and melatonin administration groups. B–D) Statistical analysis of peripheral blood smears. Increased numbers of neutrophils, lymphocytes and monocytes were seen in peripheral blood of mice injected with metastatic breast carcinoma cells compared to melatonin administration groups. *p < 0,05.

Metastasis assay

All the groups injected with 4TLM cells developed macroscopically visible lung and liver metastasis (Figure 3). Lung and liver metastases in all groups mice were demarcated (Figure 3A, B). 6% EtOH was used as a vehicle and did not alter metastasis. Microscopically lung and liver metastases were significantly decreased in melatonin groups. Microscopic lung (*/**p < 0,05) and liver (*/**p < 0,05) metastases were significantly higher number in tumor and vehicle groups as compared to melatonin groups (Figure 3C, D). There were no differences between the M1 and M2 groups.

Figure 3.

Distant metastasis such as lung (A) and liver (B) tissues of tumors were stained with Hematoxylin and Eosin and then demarcated. Microscopic appearance of distant metastasis and the measurement of metastatic lesions using Spot advanced 4.6 program. C, D: Changes in lung and liver metastasis measured as number of metastatic lesions.

Immunohistochemistry for MDSC and angiogenic markers of primary tumors

Primary tumors were strongly expressed of CD11b⁺, CD33 and GR1⁺ which are the markers for MDSC. There were no differences between the tumor group and vehicle groups in any parameter. The expressions were clearly decreased in melatonin groups compared to tumor and vehicle groups (Figure 4A, B). There were no differences between the M1 and M2 groups. This finding clearly indicated that melatonin administration reduced infiltration of CD11b⁺, CD33 and GR1⁺ myeloid cells in primary tumors. Collectively, these results supported the notion that melatonin played critical a role in regulation of MDSC cells in the tumor microenvironment.

Figure 4.

Primary tumors immunohistochemical analysis showed that CD11b, CD33, GR1 (A) and also VEGF-A, VEGFR-1, VEGFR-2 (C) expressions were significantly decreased in melatonin administration groups compared with tumor injected group. A) Metastatic cell lines injected group was strongly expressed in CD11b, CD33 and GR1 expressions (arrows). Scale bars represent 50 μm. B) Image-J analysis of CD11b, CD33 and GR1 expressions in primary tumors. CD11b */**p < 0,05; CD33 and GR1 */**p < 0,005 is a statistically significant difference. C) VEGF-A, VEGFR-1, VEGFR-2 expressions were also increased in tumor injected groups compare that the melatonin administration groups. Scale bars represent 50 μm. D) Image-J analysis of the expressions was seen. */**p < 0,005 is a statistically significant.

To investigate the effect of melatonin on angiogenesis we evaluate the angiogenic markers as VEGF-A, VEGFR-1 and VEGFR-2 expressions. 6% EtOH was used as the vehicle because it did not alter MDSC and angiogenic markers of primary tumors. The expression levels were found to be considerably higher in primary tumor and vehicle groups as compared to melatonin groups (Figure 4C, D). There were no differences between the M1 and M2 groups. Negative control (NC) immunostaining with normal rabbit IgG confirmed the specificity of immunostaining patterns in primary tumors.

Immunohistochemistry for MDSC and angiogenic markers of metastatic liver tissues

CD11b⁺, CD33 and GR1⁺ expressions were clearly decreased in melatonin groups compared to tumor and vehicle groups in metastatic liver tissues (Figure 5A–B). 6% EtOH as the vehicle, didn’t alter MDSC and angiogenic markers of metastatic liver tissues. VEGF-A, VEGFR-1 and VEGFR-2 positive cells were clearly detected in liver metastatic tissues. There were no differences between the tumor group and vehicle groups in any parameter. Interestingly, MDSC and angiogenic markers immunoreactions in the melatonin administration groups were significantly less than the other groups (Figure 5A–D). CD11b⁺, CD33 and GR1⁺ expressions were found in only metastatic side of the liver tissues Negative control (NC) immunostaining with normal rabbit IgG confirmed the specificity of immunostaining patterns in metastatic tissues. As seen in Figure 5B and D, positive cells for the immunohistochemical staining were analyzed by Image J.

Figure 5.

Representative figure was shown that immunohistochemical expression for MDSC and angiogenic markers of metastatic liver tissues. Aggressive metastatic tumors was clearly expressed in MDSC and angiogenic markers of the metastatic area in liver tissues compared to melatonin administration groups (A, C). Scale bars represent 50 μm. B, D: Image-J analysis of the expressions was seen. */**p < 0,05 is a statistically significant.

Western blot analysis

VEGF-A, VEGFR-1 and VEGFR-2 expressions were analyzed by Western blotting from primary tumors in all groups of animals. The blots revealed dense bands for VEGF-A, VEGFR-1 and VEGFR-2 corresponding to 27 kDa, 151 kDa and 150 kDa, respectively. Equivalent amounts of total proteins were loaded per lane as indicated by the immune expression of GAPDH corresponding to 36 kDa. We obtained same results in all 10 samples. 6% EtOH as the vehicle, did not alter Western blot analysis results. The protein level of VEGF-A, VEGFR-1 and VEGFR-2 were significantly lower in melatonin groups than the other groups. In accordance with Image J analysis the antibodies expression was higher in tumor group as compared to melatonin administration groups (Figure 6A–B).

Figure 6.

A) Western blot analysis of VEGF-A, VEGFR-1 and VEGFR-2 expressions in primary tumors. Bands were detected for 27 kDa, 151 kDa and 150 kDa, respectively. The immune expression of GAPDH (36 kDa) was used to confirm equivalent amounts of total proteins loaded per lane. B) The immunoblot bands were quantified by an optical densitometer. The OD (optical density) values of the VEGF-A, VEGFR-1 and VEGFR-2 bands were normalized to the OD values of GAPDH bands. The data in the graph is presented as mean ± SEM. p < 0,05 is a statistically significant difference.

Discussion

There are many in vitro and in vivo studies which have highlighted an important oncostatic role of melatonin. These studies have in the context of the oncostatic effects of melatonin, due to different experimental conditions, such as doses, time of dosage, and length of treatment.^5,9,13,14,21 The aim of our study was to investigate the effects of melatonin either given before or after the injection of highly aggressive metastatic breast carcinoma cells in a mice model of breast cancer. Based on our results, angiogenesis and distant metastasis were significantly decreased with melatonin administration. To the best of our knowledge, anti-proliferative, anti-angiogenic and anti-metastatic time dependent efficacy of melatonin on tumor immune microenvironment have not been reported before.

It has been previously demonstrated that melatonin might have a control on tumor growth.^5,9,13,14,21 Our tumor growth measurements were consistent with previously published scientific findings. Inflammation promotes tumor development and activates tumor microenvironment for metastasis. The inflammatory microenvironment is characterized by the tumor-infiltrating lymphocytes and leukocytes and the cells responsible for cancer-associated inflammation that represents an attractive strategy for cancer therapy. ²² We performed peripheral blood analysis. The decreased numbers of neutrophil, lymphocytes and monocytes were related to anti-inflammatory effects of melatonin.

In studies using melatonin, the effect of this antioxidant agent on animal weights has never been evaluated. However, we know that in many diseases triggered by inflammatory responses and cancers, the body weight is significantly reduced. ²³ In our study, it was observed that animal weights did not change significantly during the study.

Akbarzadeh et al. investigated the effects of melatonin on cancer stem cells isolated from ovarian cancer cells, they showed that melatonin prevents invasion, which is one of the important steps of metastasis. ²⁴ Borin et al. reported that metastases metastases were significantly lower in melatonin treated groups. ¹³ In the current study, and in another ongoing study that has not been published yet, we have provided evidence that melatonin reduces metastasis in breast cancer (unpublished data).

Tumor cells need a unique microenvironment to metastasize and create a new tumor in a distant region. In sequence of inflammation, there are various immune system compounds around the tumor such as macrophages, neutrophils, mast cells, MDSCs, dendritic cells and natural killer cells. It has been known that immature myleoid cells migrate to the tumor microenvironment and forms MDSCs.^25–28 MDSCs have been associated with worse breast cancer stages and metastatic tumor microenvironment ²⁹ ; bad prognosis and survival in stage-IV breast and colorectal cancer patients. ³⁰ Toor et al. stated that tumor infiltrating MDSCs were remarkably higher in breast cancer patients. ³¹ Moreover, studies have shown that MDSCs are associated with recurrence and metastasis in breast cancer.^25,28,30,32

CD11b⁺ and GR1⁺ proteins are known as MDSC markers. ²⁶ Similarly, CD33 has been used as a MDSC marker in various studies and increase in CD33⁺ cells in the tumor microenvironment has been associated with metastasis.^33,34 Diaz-Montero et al. showed that blood MDSC levels correlated with breast cancer and metastatic tumor microenvironment. ²⁹ Yan et al. showed that in 4T1 cells injected mice, GR1⁺/CD11b⁺ cells infiltrated to the lung before metastasis of tumor cells. ³⁵ In our study, MDSCs were shown in the liver tissues, similar to previous literature. First time in the literature, our study showed that in melatonin treated groups, CD11b, GR1 and CD33 expressions in tumor microenvironment were significantly lower than in other groups. This might suggest that melatonin is decreasing the immune suppressive response by affecting MDSCs.

Angiogenesis is an important factor in tumor development. Since the tumor needs more oxygen as it grows, it tends to increase the angiogenic activity to prevent hypoxia. VEGF-A, which is a trigger for the angiogenesis, is a key molecule in tumor formation. In primary tumors with VEGF-A deletion, the amount of pericytes and vascular structures were reduced and angiogenic transition was blocked, tumorigenesis was regressed and apoptosis was increased. ³⁶ VEGF-A molecule can bind to three main VEGFR receptors, 1, 2 and 3. ³⁷ Melatonin has an indirect oncostatic activity, with its anti-angiogenic effect and regulation of oncogene expressions. ³⁸ Alvarez-Garcia et al. stated that melatonin causes antiangiogenic activity in tumoral tissue and plays a role in the regulation of VEGF. ³⁹ In another study, it has been stated that endogenous VEGF expression is suppressed by melatonin. ⁴⁰ In a study on the MDA-MB-231 triple breast cancer cell line, it has been shown that VEGF and VEGFR-2 expressions decreased with melatonin administration. ¹¹ Similar to the above, our study also showed that VEGF-A, VEGFR-1 and VEGFR-2 expressions were significantly lower in the pre- and post-melatonin administration groups. These results may indicate that melatonin has an effect on tumor growth and metastasis by suppressing angiogenesis.

One of our hypotheses in this study was that the use of melatonin prior to tumor may have effects on the tumor parameters. Nonetheless there were no differences between the M1 and M2 groups in any parameter. These findings suggested that the usage of melatonin before tumor injection did not significantly induce regression of the tumors. To the best of our knowledge there are no other studies on the effects of using melatonin prior to tumor.

Melatonin might have a therapeutic role in aggressive breast carcinoma which inhibits inflammatory cells in peripheral blood, tumor progression, angiogenesis and metastasis. Also, inhibits MDSC cells in primary tumor and distant metastatic tissue microenvironment. This study would suggest that melatonin may control tumor growth by inhibiting angiogenesis-dependent cancer proliferation. In this context this study emphasized that melatonin has an adjuvant effect of tumor growth. Further investigation needs to enlight possible underlying molecular mechanisms of this hormone in metastatic breast cancer.