Research Articles
Antitumor potential of dark sweet cherry sweet (Prunus avium) phenolics in suppressing xenograft tumor growth of MDA-MB-453 breast cancer cells

https://doi.org/10.1016/j.jnutbio.2020.108437Get rights and content

Abstract

This study investigated in vivo the antitumor activity of dark sweet cherry (DSC) whole extracted phenolics (WE) and fractions enriched in anthocyanins (ACN) or proanthocyanidins (PCA) in athymic mice xenografted with MDA-MB-453 breast cancer cells. Mice were gavaged with WE, ACN or PCA extracts (150 mg/kg body weight/day) for 36 days.

Results showed that tumor growth was suppressed at similar levels by WE, ACN and PCA compared to control group (C) without signs of toxicity or significant changes in mRNA oncogenic biomarkers in tumors or mRNA invasive biomarker in distant organs. Tumor protein analyses showed that WE, ACN and PCA induced at similar levels the stress-regulated ERK1/2 phosphorylation, known to be linked to apoptosis induction. However, ACN showed enhanced antitumor activity through down-regulation of total oncogenic and stress-related Akt, STAT3, p38, JNK and NF-kB proteins. In addition, immunohistochemistry analysis of Ki-67 revealed inhibition of tumor cell proliferation with potency WE ≥ ACN ≥ PCA. Differential quantitative proteomic high-resolution nano-HPLC tandem mass spectrometry analysis of tumors from ACN and C groups revealed the identity of 66 proteins associated with poor breast cancer prognosis that were expressed only in C group (61 proteins) or differentially up-regulated (P<.05) in C group (5 proteins). These findings revealed ACN-targeted proteins associated to tumor growth and invasion and the potential of DSC ACN for breast cancer treatment. Results lead to a follow-up study with highly immunodeficient mice/invasive cell line subtype and advanced tumor development to validate the anti-invasive activity of DSC anthocyanins.

Introduction

Breast cancer, as defined [1], “is characterized by uncontrolled growth of malignant cells in the mammary epithelial tissue”. It is the most frequently diagnosed and leading cause of cancer deaths among women worldwide. According to the American Cancer Society’s “Cancer Facts and Statistics,” breast cancer mortality in 2019 is estimated to be 41,760 women and 500 men. The reason behind the high breast cancer death rate is that it can metastasize in the body even before its diagnosis. An approach that could be effective for prediction and prevention of metastatic breast cancer could be the implementation of personalized medicine to identify high-risk metastasis patients. For example, the “Flammer Syndrome” (FS) study identified common symptoms in breast cancer patients and has helped to advance the insights regarding individual predisposition to develop premetastatic niches, which are created prior the tumor onset [2]. This study was later discussed to support the role that chronic inflammation and impaired wound healing play in the development of premetastatic niches [3]. Moreover, FS symptoms indicate suboptimal health condition that promotes systemic hypoxia early in life and predispose young woman to the development of pregnancy-associated breast cancer (PABC) [4]. PABC incidence has been reported to affect from 1 in 10,000 to 1 in 3000 pregnancies [5]. PABC development is favored by the pre-existence of cancerous prelesions and the pathophysiologic conditions associated with pregnancy including a supportive hormonal profile, compromised immune tolerance, and breast tissue involution after delivery and discarded breastfeeding. These conditions constitute a fertile environment for the transformation of pre-existing lesions into aggressive and metastatic breast cancer [4].

Once developed, each breast cancer subtype has a unique etiology, with some subtypes being less preventable [6]. Based on the prognostic markers, breast cancer has been classified as luminal A, luminal B, human epidermal growth factor receptor 2 enriched (HER2+), and triple-negative (TN) subtypes [7]. Luminal A breast cancer subtype [estrogen/progesterone receptor positive (HR+), Ki-67<15% and HER2] is preventable and has shown the best survival in early stage and metastasis [8]. Luminal B breast cancer subtype is HR+ and HER2+ and has Ki-67>15% [8]. The Ki-67 nuclear protein is associated with cellular proliferation [9,10]. The HER2+ highly expresses Ki-67 and is associated with overall poor prognosis among breast cancer patients [11,12]. The TN is HR and HER2, has the worse prognosis and because of its heterogeneity has been classified into basal, claudin-low, metaplastic breast cancer and interferon-rich [13].

Alcohol consumption and physical inactivity are considered as modifiable risk factors for postmenopausal breast cancers and PABC. However, some other risk factors are specific for postmenopausal breast cancers (weight gain, breastfeeding and menopausal hormone therapy) [14] and for PABC (smoking, rotating shift and night work, abnormally low body mass index, diabetes mellitus and FS) [4]. These environmental and lifestyle risk factors are modifiable and contribute to approximately 90% of all breast cancer cases worldwide [15]. They promote epigenetic alterations, which can be reversible, and involve the dysregulation of DNA methylation, histone chemical modifications and expression of noncoding RNAs to regulate translation of RNA into protein [16]. The dysregulation of DNA methylation is characterized by hypomethylation of oncogens and hypermethylation of tumor-suppressor genes. These conditions lead to the expression of oncogene genes and the silencing of tumor-suppressor genes, respectively. Likewise, methylation of histones influences chromatin structure to activate/repress transcription. In general, high levels of methylation and acetylation in histone are associated with luminal-like breast cancer and better prognosis, whereas moderate and low levels of methylations on histone lysine and arginine amino acid residues are found in the most aggressive HER2+ and TN breast cancer subtypes associated with poorer prognosis [16].

The early screening to recognize breast cancer high-risk patients (e.g., FS phenotype, systemic hypoxia that predisposes young women to PABC development, epigenetic mechanisms) is particularly important for patient profiling, stratification and implementation of customized chemopreventive treatments. The intake of dietary phytochemicals can be adopted as preventive medicine to reverse aberrant epigenetic modifications. This approach, known as nutriepigenetics, is widely recognized for its potential in personalized breast cancer chemoprevention and management [16]. It is also supported by epidemiologic, case–control and clinical studies showing that dietary patterns rich in polyphenols (e.g., flavonols, flavones, flavan-3-ols, anthocyanins) are associated with lower breast cancer risk and recurrence [[17], [18], [19]].

For example, the HER2+ breast cancer subtype has features common to the luminal and basal subtypes and affects an estimate of one in five women diagnosed with breast cancer worldwide [11,13]. The current standard treatment for HER2+ involves the use of monoclonal antibody targeting HER2 directly combined with chemotherapy. This treatment induces significant cytotoxic effects on normal tissues, decreasing the patient's quality of life [20]. Currently, the use of dietary bioactive components is being recognized as a promising, effective, selective, more affordable and less toxic treatment approach [20]. For example, anthocyanins have shown to suppress tumor growth, the expression of angiogenesis factors and pulmonary metastasis in BALB/c nude mice bearing xenografted MDA-MB-453 tumors [[21], [22], [23], [24]]. Furthermore, we have found that DSC phenolic extracts enriched in anthocyanins and proanthocyanidins inhibit tumor growth and invasion of MDA-MB-453 breast cancer cells in vitro through regulation of markers related to cell growth, apoptosis and invasion [25]. Hence, the anticancer potential of DSC phenolics deserves to be further investigated in vivo.

The objective of this study was to investigate the tumor growth suppression and anti-invasive properties of DSC total phenolics (WE) and the isolated fractions enriched in anthocyanins (ACN) or proanthocyanidins (PCA) in vivo using MDA-MB-453 breast cancer cells xenografted into athymic nude mice.

Section snippets

Chemicals, antibodies and reagents

The Folin–Ciocalteu reagent and solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA). Antibodies against phospho-ERK1/2 and IHC-specific Ki-67 were purchased from Cell Signaling Technology (Danvers, MA, USA). Western blotting chemiluminescence luminol reagent was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Direct-zol RNA MiniPrep Plus mRNA extraction kit was purchased from Zymo Research (Irvine, CA, USA), while iScript reverse transcription supermix and

Tumor growth inhibition

Orally administered, WE, ACN and PCA (150 mg/kg body weight/day) reduced significantly and at similar extent tumor growth compared to C group (Fig. 1A). The human dose equivalent is ~ 5 glasses (200 ml) of DSC drinking juice/day [25]. This calculation was performed considering total phenolics in DSC concentrate juice used for this study and assuming a dilution factor of 6.2 to achieve a natural juice Brix level of 11 °Brix from the 68 °Brix of concentrate juice.

The WE contained a mixture of

Discussion

Tumor volume growth was significantly reduced by WE, ACN and PCA treatments; however, there was no significant effect on tumor weight. This is not uncommon as previously discussed in a study of tumor biology which demonstrated that measuring changes in xenografted tumor weights alone is not an accurate assessment of tumor growth kinetics and treatment response. This study demonstrated that there is no conclusive direct relationship between significant inhibition of cellular proliferation and

Conclusions

DSC phenolics in WE and fractions enriched in ACN and PCA delayed the growth of xenografted MDA-MB-453 tumors with similar potency and without physiological signs of toxicity. No invasion to lungs, liver, spleen, hearts and kidneys was detected when assessed through mRNA levels of hβ2G. Biomarkers associated with tumor growth/survival, apoptosis and invasion/angiogenesis in tumor tissues were, in general, similar at gene expression levels among experimental groups. Tumor protein expression

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Authors thank Andrea Urrutia from University of Zamorano, Honduras, for her technical and valuable assistance during animal treatments; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing Nara N. Lage the PDSE scholarship/process number 88881.132176/2016-01; and the Office of International Linkages, University of the Philippines, for providing Marjorie Anne A. Layosa the scholarship.

Funding

This work was supported by the Northwest Cherry Growers, Washington State Fruit Commission.

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