Notes
Comparative analysis of the effect of resveratrol derivatives on microRNAs
2024 Volume 30 Issue 6 Pages 697-704
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2024 Volume 30 Issue 6 Pages 697-704
Dietary factors are associated with an increased risk of the development of several diseases. Some phytochemicals regulate animal microRNAs (miRNAs) that have been implicated in disease development. Understanding the beneficial changes in miRNA expression mediated by food ingredients or food-derived natural products is important for maintaining good health and finding novel dietary functions. Previous studies reported that resveratrol controls breast cancer cell proliferation by inducing the expression of tumor-suppressive miRNAs. Although several stilbene derivatives in foods exist, their functional activity in regulating miRNAs has not yet been elucidated. Here, we conducted a preliminary study to investigate whether resveratrol derivatives (gnetin C, ε-viniferin, and piceid) regulate similar tumor-suppressive miRNAs as resveratrol in breast cancer. We first reported that slight differences in the structure could influence the effect of each compound on cell proliferation and miRNA expression.
Cell Culture MDA-MB-231-luc-D3H2LN (Xenogen) cells were cultured in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and 1 % antibiotic-antimycotic (Thermo Fisher Scientific) at 37 °C in 5 % CO2. Cyanidin (FUJIFILM Wako, ≥ 98 %), delphinidin chloride (FUJIFILM Wako, ≥ 95 %), nobiletin (FUJIFILM Wako, ≥ 95 %), gnetin C (FUJIFILM Wako, ≥ 97 %), ε-viniferin (FUJIFILM Wako, ≥ 98 %), piceid (FUJIFILM Wako, ≥ 99 %), and trans-resveratrol (Cayman Chemical, ≥ 98 %) was dissolved in dimethyl sulfoxide and used at concentrations ranging from 5–50 μM.
Cell proliferation assay MDA-MB-231-luc-D3H2LN cells were seeded into 96-well plates. The following day, the cells were treated with trans-resveratrol, piceid, ε-viniferin, gnetin C, cyanidin, delphinidin, or nobiletin and cultured for 72 h. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories). The plates were incubated for 2 h at 37 °C in 5 % CO2 after adding the Cell Counting Kit-8 solution. The absorbance at 450 nm was measured with a Synergy H4 microplate reader (Biotek). The experiments were repeated independently at least three times. Statistical analyses of data containing more than two groups were performed using one-way analysis of variance followed by Dunnett’s test.
Quantitative real-time PCR analysis MDA-MB-231-luc-D3H2LN cells were cultured with or without each phytochemical treatment for 2 days. Total RNA and microRNA (miRNA) were extracted from the cells using the QIAzol and miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. cDNA was generated using the PrimeScript RT reagent Kit (TaKaRa) and the TaqMan® MicroRNA Reverse Transcript Kit (Applied Biosystems). TaqMan® probes (hsa-miR-34a, hsa-miR-141, and hsa-miR-200c; TaqMan® Gene Expression Assays and TaqMan® MicroRNA Assays) were purchased from Applied Biosystems. qRT-PCR reactions were performed with the TaqMan probes using the Premix Ex Taq™ (Probe qPCR) (TaKaRa) and TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG (Thermo Fisher Scientific) on the StepOne® Real-Time PCR system (Applied Biosystems). Expression levels of interest genes were normalized to the expression of RNU6B. Experiments were performed in four replications. Statistical analyses of data containing more than two groups were performed using one-way analysis of variance followed by Dunnett’s test.
Fruits and vegetables provide vital nutrients, including vitamins, minerals, dietary fiber, and phytochemicals, which can prevent the occurrence of chronic diseases and reduce mortality, including cancer, cardiovascular diseases, and mental illnesses (Bellavia et al., 2013; Key, 2011; Liu et al., 2016; Slavin and Lloyd, 2012; Wang et al., 2014). Epidemiological studies have shown that the intake of a phytochemically rich diet is inversely related to the incidence of breast, colorectal, lung, and pancreatic cancers (Feskanich et al., 2000; Gandini et al., 2000; Kunzmann et al., 2016; Li et al., 2015; Liu and Lv, 2013; Voorrips et al., 2000). Phytochemicals in fruits and vegetables are necessary to maintain healthy conditions. For instance, the polyphenolic phytoalexin, resveratrol, is known for the “French paradox” that describes the relationship between the consumption of resveratrol in red wine and the relatively low incidence of coronary heart disease in the French population despite a high dietary fat intake (Catalgol et al., 2012; Lippi et al., 2010). Resveratrol has garnered attention owing to its effects on sirtuin genes and longevity (Howitz et al., 2003; Michan and Sinclair, 2007). It exerts antitumorigenic effects, such as inhibiting tumor initiation and progression, by inducing cell cycle arrest and apoptosis (Scott et al., 2012). However, the potential mechanisms underlying the prevention and treatment of these diseases remain unclear.
Several recent studies have shown that food-derived phytochemicals positively control tumor-suppressive and oncogenic miRNAs (Otsuka and Ochiya, 2021). miRNAs are single-stranded non-coding RNA molecules of approximately 22 nucleotides, which regulate the translation of multiple target genes by binding specific sequences of the 3′-untranslated regions of the targets (Esteller, 2011). The expression of specific miRNAs is often altered through genetic and epigenetic factors during carcinogenesis, and their dysregulation in cancer is a potential diagnostic biomarker. Multiple studies have suggested that the early detection of cancers can be achieved by analyzing serum miRNAs and utilizing miRNA expression patterns as specific and sensitive markers, with miRNA profiles in body fluids serving as plausible indicators of health conditions (Matsuzaki and Ochiya, 2017; Otsuka and Ochiya, 2021; Schwarzenbach et al., 2014). The anticancer effects of resveratrol have been demonstrated in mouse models; consequently, food-derived natural products, including resveratrol, have been reported to increase tumor-suppressive miRNAs and decrease oncogenic miRNAs (Hagiwara et al., 2012; Han et al., 2012; Jang et al., 1997; Li et al., 2013; Liu et al., 2013; Otsuka, Yamamoto and Ochiya, 2018; Ren et al., 2015; Tili et al., 2010; Yu et al., 2013). Understanding the beneficial changes in miRNA expression mediated by food ingredients or food-derived natural products is important for maintaining optimal health and uncovering novel dietary functions.
Notably, each compound regulates specific miRNAs in a specific cancer cell line. However, the differences in the effects of phytochemicals on miRNA expression have not yet been elucidated. Apart from resveratrol, there are several derivatives of stilbene, such as resveratrol dimer, gnetin C, and ε-viniferin, and its glucoside trans-polydatin piceid. The functional activity of stilbenes against miRNAs has not been examined despite the possibility that they might have more activity than resveratrol. Here, we conducted a preliminary study to confirm whether resveratrol derivatives, namely gnetin C, ε-viniferin, and piceid, regulate similar tumor-suppressive miRNAs similar to resveratrol and non-stilbene polyphenols in breast cancer.
We first examined the anticancer effects of phytochemicals on the proliferation of aggressive breast cancer cell line (MDA-MB-231-luc-D3H2LN) before analyzing their effects on miRNA expression because cell proliferation is associated with the expression of tumor-suppressive and oncogenic miRNAs (Peng and Croce, 2016). We selected resveratrol, resveratrol dimers, gnetin C and ε-viniferin (viniferin), and polydatin piceid other than resveratrol (Fig. 1). We analyzed the non-stilbene polyphenols cyanidin, delphinidin and nobiletin, which have been reported to possess antitumor activities (Katsube et al., 2003; Kawaii et al., 1999) (Fig. 1). The non-stilbene polyphenols, delphinidin and nobiletin, exerted antitumor effects on the breast cancer cells; however, the effect of cyanidin was less pronounced than that of the other compounds (Fig. 2). Resveratrol and its derivatives inhibited cell proliferation; in particular gnetin C and viniferin showed strong antitumor effects (Fig. 2, Fig. S1). We decided to exclude cyanidin from further analysis owing to its ineffectiveness in controlling breast cancer cell proliferation.
Cell viability at breast cancer cells (MDA-MB-231-luc-D3H2LN cells) treated with non-stilbene polyphenols (cyanidin, delphinidin, and nobiletin) and stilbene derivatives (piceid, resveratrol, ε-viniferin, and gnetin C) (mean ± SEM, *p < 0.05). Number of data points per sample was eight.
Subsequently, we analyzed the miRNA expression in the cancer cells treated with the abovementioned polyphenols. We selected three major tumor-suppressive miRNAs (miR-200c, miR-141, and miR-34a) that are upregulated upon resveratrol treatment in breast cancer cells (Hagiwara et al., 2012; Otsuka, Yamamoto and Ochiya 2018). The concentrations of each compound were set based on the relative cell viability following treatment with 25 μM of resveratrol. Based on the previous research (Hagiwara et al., 2012), the concentrations were set, including the tumor-suppressive effect of each compound on the relative cell viability at 60–80 % during the analysis of the miRNA expression. In miR-200c, unlike resveratrol, delphinidin, nobiletin, and gnetin C did not upregulate miR-200c expression (Fig. 3). The expression of miR-200c was significantly increased following treatment with piceid at all concentrations tested, and upon exposure to 30 μM of viniferin (Fig. 3). In miR-141, the expression of miR-141 was significantly upregulated by resveratrol but not by delphinidin, nobiletin, piceid, or gnetin C (Fig. 3). Viniferin significantly enhanced miR-141 expression to the same level as resveratrol (Fig. 3). In miR-34a, the expression of miR-34a was significantly upregulated following treatment with 25 μM of delphinidin, 25 μM and 30 μM of nobiletin, 25 μM and 50 μM of piceid, and 30 μM of viniferin (Fig. 3). In contrast, miR-34a expression did not increase following gnetin C treatment.
miRNAs have become increasingly important in understanding diseases, particularly cancer since their discovery. Recent studies have reported that miRNAs are potential biomarkers for several diseases, suggesting that miRNA profiles in body fluids can act as indicators of numerous health conditions (Otsuka and Ochiya, 2021). Notably, food-derived natural products or phytochemicals, such as resveratrol, curcumin, quercetin, and genistein, can control tumor-suppressive and oncogenic miRNAs. Resveratrol has garnered attention for its health benefits, including anti-obesity, antitumor, and cardioprotective effects (Zhang et al., 2021). However, the effects of resveratrol derivatives on modulating miRNA expression have not been elucidated. Our study suggested that piceid and viniferin resembled resveratrol in their action, as evidenced by the expressions of these three miRNAs. Resveratrol consists of two phenolic rings connected by a double bond. Delphinidin and nobiletin differ from resveratrol in structure; they are composed of a three-ring structure with two benzene rings and a heterocyclic pyran ring, which may affect the miRNA species. Resveratrol and piceid have the same basic structure: two phenolic rings connected by a double bond. They also have hydroxyl groups at the same positions, contributing to their similar antioxidant properties. Piceid has an additional glucose molecule attached to one of its hydroxyl groups, which would influence its biological activities. Considering that piceid is the most structurally similar to resveratrol, it would exhibit similar effects on gene expression owing to their similar structural formula. Gnetin C and viniferin are dimers of resveratrol, sharing the core phenolic structure. Owing to their dimeric nature, they have more hydroxyl groups than resveratrol. In addition, the resveratrol units are linked in a specific manner that is unique to gnetin C and viniferin, providing a larger molecular framework than resveratrol. Based on the effects on cell proliferation and miRNA expression, the stilbene derivative, gnetin C, differed from resveratrol, piceid, and viniferin, suggesting that the structural formula alone is not associated with the activity. Gnetin C has the resveratrol units linked in a specific manner, distinguishing it from viniferin. The structural differences, such as different specific types of bonds, a relatively larger molecular framework, and hydroxyl groups, may affect accessibility and binding to specific molecules and proteins. In this research, we investigated three miRNAs initially; however, a more comprehensive analysis of over 2,000 miRNAs is required. Regarding the similarity of structural formulae, comparing various naturally occurring compounds can help elucidate their points of action and novel mechanisms in regard to gene regulation.
Bioavailability is an important factor in health promotion and preventive strategies. Several preclinical and clinical studies on resveratrol have been conducted, and other clinical trials are underway (Brown et al., 2024). Resveratrol exerts protective and curative effects against diseases, such as cancer, diabetes, obesity, metabolic syndrome, hypertension, Alzheimer’s disease, and cardiovascular diseases; however, its efficacy in humans remains under investigation (Zhang et al., 2021; Brown et al. 2024). Disease prevention strategies using phytochemicals, including resveratrol, are complex because of their poor bioavailability and rapid metabolism. Piceid and viniferin are promising food-derived compounds that positively regulate miRNAs. Piceid and gnetin C have more optimized bioavailability than resveratrol (Beaumont et al., 2021; Karami et al. 2022; Nakagami et al. 2019; Ota et al. 2013; Tani et al. 2014). Following oral ingestion of 25 mg of resveratrol by six healthy volunteers, only trace amounts of unchanged resveratrol (< 5 ng/mL) could be detected (Patel et al., 2011). After supplementation with Gnetin C (150 mg/day), the plasma Gnetin-C concentration was 109 ± 51 ng/mL (range, 57-203 ng/mL) on day 7 (Nakagami et al., 2019). In rats, piceid is absorbed and metabolized into resveratrol, suggesting high bioavailability compared to resveratrol administration (Wang et al., 2015). In addition, the serum molar concentration of piceid was 2.56–4.45 times as much as that of resveratrol after oral administration of piceid (Wang et al., 2015). As described above, the mechanism of action of gnetin C differs from that of resveratrol and its other derivatives. Providing phytochemicals according to our health status and miRNA profiles would be advantageous in improving health.
However, its molecular mechanisms warrant further research. Recent preclinical studies have reported that structural modifications of a compound can improve its bioavailability and efficacy. For instance, a modified resveratrol has been developed and tested in humans (Berman et al., 2017). Various approaches can improve the stability and bioavailability of naturally occurring compounds for numerous health-related applications. The pleiotropic activities of phytochemicals are beneficial in preventing the modulation of multiple pathways. It is necessary to conduct a comprehensive investigation of the effects of dietary compounds on signaling pathways, including miRNAs, to understand their health benefits.
Author contribution KO and TO directed and supervised the study. KO designed and performed the experiments, analyzed data, and wrote the manuscript. All authors critically reviewed the manuscript and approved the final version of the manuscript.
Acknowledgements We thank Norimitsu Yamagata and Masaaki Kunou for providing comments on this research. This work was supported by the Grant-in-Aid for the Japan Agency for Medical Research and Development and the New Energy and Industrial Technology Development Organization.
Conflict of interest There are no conflicts of interest to declare.
Cell viability at breast cancer cells (MDA-MB-231-luc-D3H2LN cells) treated with ε-viniferin and gnetin C (mean ± SEM, *P < 0.05). Number of data points per sample was eight.
