Muscadine grape (Vitis rotundifolia
Michx.) is one of the important native fruit crops in southeastern United States of America (USA), and was recently introduced in China [1
]. The berries are used commercially to manufacture wine, juice, and preserves. To date, more than 100 different cultivars are being grown and they produce berries differing in size, color, and flavor [2
]. The unique characteristic of muscadine grape berry is its thick skin [3
The berry has been extensively studied for its phytochemical composition. The presence of polyphenolics, anthocyanins, stilbenes, ellagic acid, ellagitannins, organic acids, amino acids, and sugars has been reported for berry extracts [4
]. Antioxidant, anti-microbial [4
], anti-cancer [6
], anti-diabetic [7
], anti-inflammatory [8
], immunomodulatory [9
], cardioprotective [10
], anti-obesity [11
], and inhibition of lipid accumulation [12
] properties of muscadine berries have been studied using in vitro (cell-free and cell lines), in vivo, and human trials. Berry extracts have been shown to be effective in controlling the proliferation of blood [13
], colon [14
], prostate [15
], and breast [6
] cancers. Whole berry, skin, or seed extracts rich in phenolics, including ellagic acid, have been linked to their anti-cancer effect [6
]. However, studies have not tried to identify and map the exact phytochemicals responsible for the biological activity.
Stilbenes are a major class of bioactive phenolic compounds present in grapes. The presence of common stilbenes like t-piceid, t-resveratrol, ε-viniferin, and t-pterostilbene in muscadine grape berries has been reported [16
]. Resveratrol and other stilbenes have been extensively studied for their biological activities, including anti-cancer, anti-inflammatory, antioxidant activity, etc. [17
]. Generally, the stilbenes are isolated in groups, but publications have failed to address and understand their synergistic action.
The aim of the current study was to do a comparative evaluation of the stilbene-rich muscadine berry extracts of selected cultivars and pure resveratrol for their anti-cancer activity in pulmonary, breast, and liver cancer cell lines.
2. Experimental Section
Analytical grade methanol, acetonitrile, ethyl acetate, trans-resveratrol, trans-piceid, trans-pterostilbene, and ε-viniferin were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Berry Samples
Berries from two different cultivars of muscadine grapes (Pineapple and Southern Home) were collected from the vineyard at the Center for Viticulture and Small Fruit Research, Florida Agriculture and Mechanical University, Tallahassee, FL, USA (30.47 latitude and 84.17 longitude) during vintage of 2018 (August and September). Ripe berries (EL-38 stage) [18
] were collected from different vines.
2.3. Extraction of Stilbenoids
The stilbenoids were extracted according to the described protocol [19
], with slight modifications. Ten grams (10 g) of berry was ground with liquid nitrogen and the ground powder was mixed with 7 mL of methanol, and then homogenized for 10 min on a vortex and further extracted for 24 h under at darkness. The suspension was centrifuged at 14,000 rpm for 15 min. The supernatant was removed carefully, and the resulting residue was extracted a second time with 3 mL of methanol and ethyl acetate (1:1, v
), as described above. The pooled organic solvent extracts were vacuum-dried in a concentrator (SpeedVac concentrator, Savant Instruments, Inc. Holbrook, NY, USA) at room temperature in the dark. The dried samples were then re-dissolved in methanol (1 mL) for HPLC studies and DMSO for bioactivity studies.
HPLC Analysis of Stilbenoid Content
The vacuum concentrated extracts of the berries were dissolved in 1 mL of HPLC grade methanol and filtered through a 0.22 µm nylon membrane filter (Sartorius Stedim Biotech GmbH 37070 Gottingen Germany, Goettingen, Germany). HPLC analysis of stilbenoids was carried out with a system equipped with a 2487 dual UV detector and 1525 gradient pump (Waters Corporation, Milford, MA, USA). The HPLC pumps, autosampler, and detectors were controlled via Waters Empower software (Empower 3 service pack 2) supplied by Waters Corporation (Milford, MA, USA). The analytical column Luna RP C18 (4.6 × 250 mm; particle size, 5 μm) and guard cartridge (C18 4 × 3.0 mm) were obtained from Phenomenex (Torrance, CA, USA). The column temperature was maintained at 25 °C. The gradient elution profile was as follows: 90% solvent water (B), 10% solvent acetonitrile (A) (0–18 min); 85% A, 15% B (18–23 min); 85% A 15% B (23–30 min); 10% A, 90% B (30–35 min). The flow rate was set at 0.4 mL/min. A volume of 2 µL of each sample was injected to resolve and measure individual stilbenoids. Three injections were performed in sequence for each biological replicate. UV absorbance detection was recorded using dual wavelengths at 285 and 305 nm. A mixture of the standards was prepared using 1, 2, 3, 4, 5, and 6 ng of each of the four stilbenoids and used for calibration and quantification of stilbenoids. The standards, t-piceid, t-resveratrol, ε-viniferin, and t-pterostilbene (Sigma Aldrich, St. Louis, MO, USA) were prepared and used as described above. Samples and calibration standards were run in triplicates. Chromatograms were acquired with different retention times for each of the stilbenes and the area under the curve (AUC) was calculated using Empower III software (Waters Corporation, Milford, MA, USA). The linearity ranges of the calibration curves were R2 = 0.9906. Quantification of stilbenoids from the two muscadine cultivars was based on the calibration curves obtained from the respective standards.
2.4. Cell Culture and Reagents
The A549 (human lung carcinoma), HCC1806 (human triple negative breast cancer) and HepG2 (human hepatocellular carcinoma) cell lines were directly obtained from ATCC (Manassas, VA, USA) for this study. The A549, HCC1806 cell lines, and HepG2 cells were cultured in RPMI1640 basal medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. All of the cells were grown at 37 °C in a humidified incubator containing 5% CO2. Cell growth and viability were determined using a Bio-Rad TC-20 automated cell counter (Hercules, CA, USA).
2.4.1. Cytotoxicity Assay
The cytotoxicity assay was performed according to the method by Mosmann [20
] with modifications. Confluent cells were seeded into three 96-well plates at 1 × 105
cells/mL and incubated for 24 h in a humidified atmosphere of 5% CO2
at 37 °C. Cells were treated with serum-free medium containing varying concentrations of extracts dissolved in DMSO. The volume of DMSO was maintained not to exceed 2% v
per well, and a DMSO treatment group was also maintained as control. Cells were then incubated in a humidified atmosphere of 5% CO2
at 37 °C. Cell viability was assessed using the Bio-Rad TC-20 automated cell counter at 24, 48, and 72 h. Results are expressed as percentage cell viability in comparison to the untreated control.
2.4.2. Total RNA Extraction and cDNA Synthesis
The TRI Reagent-based method was used for RNA extraction [21
]. In brief, 24 h extract (1 µg/mL) and resveratrol (50 µg/mL)-treated HepG2 cells were released from the substratum by scraping and by repeated pipetting. The cell suspension was centrifuged at 12,000 rpm for 10 min at 4 °C. Cells from different replicates were pooled to obtain about 3–4 × 106
cells. To the pellet, 1 mL of TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) was added by repeated pipetting. After 5 min at room temperature, 0.2 mL of chloroform was added and vigorously shaken for 15 s. The mixture was allowed to stand at room temperature for 15 min and centrifuged at 12,000 rpm for 10 min at 4 °C. The upper aqueous phase was transferred to a fresh tube and 0.5 mL of ice-cold iso-propanol was added and allowed to stay at room temperature for 10 min. RNA was pelleted by centrifugation at 12,000 rpm for 10 min at 4 °C. The pellet was washed with 1 mL of 75% ethanol, dried, and dissolved in DEPC (Diethyl pyrocarbonate; 0.1%)-treated water. RNA was stored at −80 °C until use. The quantity and quality of RNA was checked using a nano-quantity spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), using oligo(dt) according to the manufacturer′s instruction, using 5 µg of the total RNA.
2.4.3. Semi-Quantitative PCR
Semi-quantitative analysis of gene expression was assessed following the protocol described by Marone et al. [22
]. Briefly, the cDNA (2 µL) was amplified with gene specific primers (0.3 µM) in a PCR mix composed of 50 mM KCl, 10 mM Tris-HCl pH 9.0, 1.5 mM MgCl2
, 200 µM each dNTP and 2 Taq DNA polymerase, in a 10 μL final volume reaction. The reaction conditions were as follows: Initial denaturation at 95 °C for 5 min, followed by 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 5 min in a thermal cycler (Eppendorf, Master Cycler Gradient, Hamburg, Germany). The amplicons were resolved alongside standard 100 bp DNA marker in a 2% agarose gel prepared with tris-acetic acid EDTA (TAE) buffer and documented using gel documentation system (Bio-Rad, Hercules, CA, USA). The bands were analyzed using the quantity and density analysis tools of Quantity One software (version 4.6.3). β-actin
were used as standards to normalize the expression of the genes of interest.
2.5. Statistical Analysis
Data are presented as means ± standard deviation. Comparison of means was performed by Student′s t-test. A 95% level of confidence was considered; thus, p < 0.05 referred to statistical significance.
Fruits are a major source of bioactive phytochemicals, which are consumed without much processing. Anti-cancer effects of several muscadine grape cultivars have been reported earlier [6
]. The most commonly studied genotypes are noble, carlos, and dixie red. While the majority of studies have used pomace, skin, and seed extracts, some have used juice and wine. The magnitude of cancer cell cytotoxicity responses varies with the muscadine varieties, parts used, and method of preparation. This can be attributed to the phytochemical content of the extract. In the current study, we used two of the commonly found varieties that produce black (Southern Home) and bronze (Pineapple) berries. The extraction method followed in this study extracted the major stilbenes present in muscadine grape berries. The varieties also showed variation in the composition of stilbenes tested. This study is the first report on the anti-cancer property of Southern Home and Pineapple cultivars of muscadine grapes. The secondary metabolite content of plants can greatly vary based on various factors, but certain cultivars of muscadine grapes, including the cultivars selected for this study, were found to consistently produce stilbenes [16
Stilbenes are a family of plant secondary metabolites derived from the phenylpropanoid pathway and the acetate–malonate pathway [16
]. Over the last two decades, stilbenes have gained popularity due to the health benefits associated with their consumption. However, resveratrol is the most studied and has been reported to be effective against various cancers, including breast, lung, colon, skin, prostate, ovarian, liver, oral cavities, thyroid, and leukemia [28
]. Though plants contain multiple stilbenes, the health benefits of other stilbenes like piceid, viniferin, pterostilbene, and their combinations have not been focused on much.
In vitro studies have shown effective cytotoxic potential of resveratrol at concentrations up to 200 µM, while the physiological concentrations of resveratrol do not go beyond 50 nM [29
]. This is due to the poor bioavailability of resveratrol and leads to differential anti-tumor effects in in vitro and in vivo. Higher doses of resveratrol were found to impair cell division and induce pro-apoptotic factors in normal cells [30
]. The current study shows that the combination of stilbenes could be more effective than the individual resveratrol. The cytotoxic effect found with 50 µg/mL of resveratrol was comparable with 1 µg/mL of the stilbene-rich extract of the muscadine grape cultivars. Further, modulation of tumor suppressor and promotor gene expression was more predominant with the extract than the resveratrol alone.
Pterostilbene has been tested for its cytotoxicity against lung, breast, and hepatic cancer cells. The pterostilbene showed anti-proliferative activity (IC50
) in MCF7 (breast cancer) and NCI H-460 (lung cancer) cell lines at 30.0 and 47.2 µM [31
]. However, a combination of resveratrol (15 μM) and pterostilbene (5 μM) inhibited proliferation of HCC1806 cell lines [32
]. Piceid was found to have anti-proliferative effects in intestinal epithelial Caco-2 cells in a concentration-dependent manner from 1 to 50 μM [33
]. Studies have shown that the piceid is metabolized by cells to form resveratrol, which induces biological effects. However, Storniolo et al. [33
] did not observe deglycosylation of piceid to resveratrol by Caco-2 cells. So, the cytotoxic effect was attributed to piceid in the medium. ε-viniferin has been reported to have a cytotoxic effect against various human cancers. Viniferin tested against melanoma cells (HT-144 and SKMEL-28) and healthy dermal fibroblast (HDF) cells showed an IC50
value at concentrations around 90 μM, while that of resveratrol was at >100 μM [34
All of the major stilbenes tested in this study showed cytotoxic effect against various cancer cells. In fact, stilbenes like piceid performed better than resveratrol in in vitro studies. Nevertheless, the effective concentrations reported depends on various factors, but it is evident that the inhibitory concentrations of stilbenes differ with cancer cell type. Diseases like cancer are systemic, involving multifactorial etiology; in such conditions, treating them with one molecule and one target paradigm cannot be successful. Multicomponent drugs consist of several compounds that interact with multiple targets [35
]. System biology understanding of multicomponent drugs indicates that they have superior efficacy and reduced toxicity. The optimal combinations of these stilbenes might have a better outcome in inducing cancer cell death.
Cancer cell lines have been widely used in research for understanding the biological mechanisms involved in cancer. The observations of the research in cancer cell lines can usually be extrapolated to in vivo human tumors and largely used in primary screening in cancer drug discovery. To date, several thousands of molecules have been tested positive for their anti-cancer effect against various types of cancer. However, the majority of them fail to reproduce the effect in vivo. So, further studies with the muscadine grape extracts will be required before using them for therapeutic purposes.
The mode of action of stilbenes is mainly through arresting mitosis, inhibiting telomerase activity, arresting DNA damage repair processes, inducing oxidative stress, activating mitophagy, and leading to apoptosis [36
]. However, other additional effects, such as anti-inflammatory activity, and reduction in growth factors like VEGF
are also observed [28
]. In the current study, stilbene-rich muscadine grape extracts of Southern Home and Pineapple cultivars were also found to induce apoptosis-related gene expression (Bcl2
). Though there was a difference in the magnitude of different genes expressions, the trend was found to be the same with both of the cultivars. The overall molecular process by which muscadine grape extracts tested induce cell death is summarized in Figure 8