In recent years, the incidence of cancer has grown exponentially, so finding the best strategy for its treatment is one of the main challenges for health scientists. Chemotherapy is one of the main approaches to cancer treatment. However, drug toxicity and the development of tumor resistance constitute serious limitations of current cancer treatments [1]. The search for new anticancer with improved activity and less toxicity than conventional therapies has drawn particular interest in natural plant compounds.

In the last years, research has focused on the utilization of phytochemicals as potential anticancer candidates because of a lot of successful reports of high efficacy, low toxicity, and minimal side effects [2]. This is the case for the phenolic compounds (PCs), which have been shown to induce apoptosis and cell cycle arrest, and produce antiproliferative effects against many human cancer cell lines [3]. Therefore, these compounds could be safe and effective in potential cancer prevention and therapy. Most of the investigations have been carried out with individual PC for a better understanding of the biological and cellular mechanisms of their anticancer efficacy. However, several studies have confirmed the improvement when they are applied in combination with other different PC, exhibiting synergistic effects and improved bioavailability [4].

Viticulture generates a significant volume of wastes, including bunch stem in the winery and canes from vineyards, which are investigated as alternative sources of a wide range of PCs with potential bioactive properties [5]. Canes are the result of a management practice on grapevine plants performed every year aimed to prune the plants to improve grape yield stability and quality of grapes. Normally, this activity annually generates an average amount of roughly 2.5 tons per ha of grape cane residues [6]. Bunch stems are the other lignocellulosic by-product that accumulates during the destemming step and constitutes 5% of the processed grapes at harvest time [7]. In previous studies, bunch stem and cane extracts have shown considerable amounts of PC-like flavonoids, phenolic acids, and stilbenes [5, 8]. Consequently, the extracts have also reported significant antioxidant activities. Apostolou et al. [9] demonstrated the protective activity of bunch stem extracts against ROS-induced DNA damage. In another work, bunch stem PC modulated H₂O₂-induced oxidative stress by maintaining the redox balance in human epidermal keratinocytes (HaCaT cells) [10]. This protective antioxidant capacity was also reported in an in vitro intestinal barrier model (differentiated Caco-2 cells) [11], so bunch stem extracts could be useful not only in the treatment of cancer but also in the prevention of its appearance. Whereas for cane extracts, Medrano-Padial et al. [12] reported a protective and reducing role against oxidative stress induced in human cell lines. Some authors also reported that bunch stem and cane extracts can inhibit the growth of cancer cell lines as lung (SK-MES-1), liver (HepG2), cervix (HeLa), stomach (AGS), bladder (J28), colon (Caco-2, HT-29, Lovo), breast (MCF-7, MDA-MB-231), kidney (786-0, Caki-1), thyroid (k1), and prostate (PC-3) [9, 12–17]. This wide range of inhibitory action in several types of cancers suggests that the extracts of both matrices have great potential for the prevention and/or treatment of the disease and could be used as nutraceutical and/or pharmaceutical agents.

The aim of this study was to chemically characterize and evaluate the in vitro cytotoxicity of the bunch stem and cane extracts of Vitis vinifera L. cv. Malbec against human HCT-116 (colorectal cancer) in comparison with non-tumoral cell lines (epithelial HBL-100).

In the last few years, several studies have been carried out on the valorization of grape canes [5, 8], although only some of these studies have assessed the cytotoxic activity of the obtained extracts [12, 15–17, 20]. Most studies show high concentrations of stilbenes trans-resveratrol and ε-viniferin. However, the quantification varies depending on the cultivar, the growing conditions, and the extraction conditions used. Sáez et al. [16] reported a 6.3% of stilbenes in the total composition of a Pinot Noir cane extract [Table 2, entry (5)]. The insolated stilbenes were mainly ε-viniferin (3.5%) and trans-resveratrol (1.7%). Medrano-Padial et al. [12] obtained an extract enriched in stilbene (45.4%, w/w) from a mixture of Merlot and Cabernet Sauvignon [Table 2, entry (2)]. ε-viniferin (16.3%) and trans-resveratrol (8.1%) were also the majority stilbene compounds. Moreira et al. [20] showed the same qualitative composition of the cane extracts obtained from different varieties by subcritical water extraction (SWE), but the levels of flavanols were higher than stilbenes [Table 2, entry (1)]. Fuchs et al. [15] prepared a PC-rich extract from cv. Riesling cane and the content was expressed on dry extract (12,000 µg/g) [Table 2, entry (3)]. Instead, Jesus et al. [17] informed 654 µg/g of the total stilbene level from freeze-dried cane extract (cv. Loureiro) [Table 2, entry (4)]. On the other hand, bunch stem has been extensively characterized for its PCs [5, 8]. Only a few studies have evaluated its in vitro cytotoxic potential [9–11, 13, 21]. In terms of phenolic content, this matrix is mainly rich in flavanols. The main components are (+)-catechin, followed by (–)-epicatechin, and procyanidins. Quercetin derivatives were the main flavonols found. Phenolic acids were reported, mainly caftaric and gallic. The literature also reported lower concentrations of stilbenes, with ε-viniferin followed by trans-resveratrol being the most common. In Table 3, flavanols have been reported with concentrations between 9.48 and 31,310 µg/g of dry extract, followed by stilbenes with a concentration range from 7.78 to 25,410 µg/g of dry extract [9, 11, 13]. Domínguez-Perles et al. [10] reported levels of stilbenes (ε-viniferin) between 2,940 and 5,820 µg/g dw from different varieties, and also other PCs (data not shown). Sei-ichi et al. [21] separated and characterized the isolated compounds from the grape stem, focusing this study on the proanthocyanidins. A number of factors explain the variability in the concentration of individual PCs, although there is an overall trend in the PCs profiles of these grapevine by-products. Some of them are reported in the literature, e.g., genetic background, environmental growth conditions, vineyards’ management, winemaking practices, pretreatment/storage of residuals, and the extraction/characterization procedures used [5, 8, 22].

This study, unlike those cited, is the first to evaluate the cytotoxic effect of an extract from pruning Vitis vinifera L. cv. Malbec on human non-tumoral breast epithelial cells (HBL-100) and colorectal adenocarcinoma (HCT-116). Therefore, a direct comparison with the rest of the studies is not possible, despite the fact that some have used colon cells but from other cell lines (Caco-2 and CCD 841 CoN) [12, 17]. However, several reports demonstrated that pruning extracts generate a cytotoxic action on various cell lines at the in vitro level (Table 2) [12, 15–17, 20]. Moreira et al. [20] found no adverse effects on dermal cell lines (HaCaT and HFF-1) treated with cane extracts from different varieties at concentrations below 100 and 1,000 μg/mL, respectively [Table 2, entry (1)]. However, for concentrations higher than 1 µg/mL from Tinta Roriz (Dão Region), the cell viability of human fibroblasts (HFF-1) was reduced to < 7.6%. In contrast, Medrano-Padial et al. [12] reported a concentration- and time-dependent decrease in the growth of two cell lines (HepG2 and Caco-2) when exposed to a cane extract rich in stilbenes (45.4%, w/w) [Table 2, entry (2)]. This extract reduced cell viability in the concentration range of 30 to 100 μg/mL after 48 h of exposure in both cell lines. Likewise, Fuchs et al. [15] also reported dose- and time-dependent cytotoxic effects on HepG2 for an extract from this matrix [Table 2, entry (3)]. When using a concentration of 500 μg/mL, cell viability was reduced by 50% after 24 h of incubation. Jesus et al. [17] reported the inhibitory action of cane extracts on the growth of four cancer cell lines (MDA-MB-231, MCF-7, HepG2, Caco2) and one non-cancer line (CCD 841 CoN) [Table 2, entry (4)]. The range of IC₅₀ values was 49.7–89.7 μg/mL for the 48 h treatment. The authors hypothesized that these results could be attributed to the abundant presence of PC such as apigenin, quercetin, ellagic acid, and hesperidin in the extracts, which have reported the ability to induce apoptosis and inhibit the progression of human cancer cell lines. Sáez et al. [16] also evaluated the antiproliferative activity, but of oligostilbenes isolated from pruning residues, in four cell lines (MRC-5, AGS, SK-MES-1, and J82) [Table 2, entry (5)]. trans-resveratrol presented the best IC₅₀ value for the MRC-5 and AGS lines, while trans-piceatannol was more effective for the SK-MES-1 and J82 lines. In addition, they evaluated the crude extract, obtaining IC₅₀ values of 57.5, 23.3, 45.0, and 9.6 μg/mL for MRC-5, AGS, SK-MES-1, and J82, respectively.

In recent years, the ability of PCs to modulate (prevent or induce) apoptosis, a mechanism of programmed cell death, has been tested by monitoring the specific ability to respond to inducing factors such as oxidative stress [23]. Using mainly in vitro experiments, PCs have been tested in cancer and normal cells. These studies provide experimental evidence for the selective apoptosis of malignant cells, while the cell viability of normal cells was not affected by these compounds [24]. This apoptotic selectivity has been demonstrated with quercetin, by inhibiting the growth of prostate cancer tumor cells, while it did not affect normal cells (PC-3 and DU-145 cell lines, respectively). Domínguez-Perles et al. [10] reported an antiapoptotic effect from a bunch stem extract on HaCaT cells [Table 3, entry (2)].

A number of studies have pointed out the anticancer potential of bunch stem extracts [23]. Apostolou et al. [9] have suggested the use of bunch stem extracts as cytotoxic agents, given their ability to inhibit the growth of liver and cervix cancer cell lines [Table 3, entry (4)]. The mean IC₅₀ values for HepG2 and HeLa cancer cells were 50 ± 12 and 32 ± 16 μg/mL, respectively. In another study, Sahpazidou et al. [13] demonstrated anticancer potential of bunch stem extracts of Greek varieties against colon, breast, kidney, and thyroid cancer [Table 3, entry (5)]. IC₅₀ values were: 121–230 (MCF-7), 121–184 (MDA-MD-23), 175–309 (HT29), 159–314 (K1), 180–225 (786-0), and 134 to > 400 (Caki-1) μg/mL. Quero et al. [11] studied the effect on cell viability of a bunch stem extract on the Caco-2, MCF-7, and MDA-MB-231 cancer lines, and the results showed that it was capable of inducing cell death by apoptosis [Table 3, entry (1)].

The success in in vitro cytotoxic results of the majority of stem-derived extracts is related to their PCs content and profile. Although the relative amounts of individual PCs in extracts derived from this matrix may vary due to various factors mentioned above, they stand out for their abundant content of flavonoids and phenolic acids. Some PCs in the extract are highly reported as active compounds against cancer cells, though the potential synergism between them and other chemically different compounds present in the extracts should not be forgotten [13, 25]. For example, the most potent bunch stem extract against all cancer cell lines tested, except 786-0, was characterized by the highest content of gallic acid and trans-resveratrol (Voidomato variety) [13]. Proanthocyanidins have shown inhibition of cancer cell proliferation, and epicatechin pentamers and hexamers were reported to inhibit cancer cell invasive activities [21]. trans-resveratrol has reported the ability to inhibit carcinogenesis at multiple stages (initiation, promotion, and progression) [25, 26]. The in vitro antiproliferative and proapoptotic effects of trans-resveratrol in tumor cell lines have been widely documented [27, 28]. In the treatment of colorectal cancer, this compound has been effective, causing a 70% inhibition of CaCo-2 cell growth. In in vivo trials, trans-resveratrol has shown efficacy in the prevention and treatment of tumors of the skin, esophagus, intestine, and colon [4]. Oral administration of high doses of trans-resveratrol in potable water and diet reduces the incidence of tumors in mice. However, its short half-life and low bioavailability have made its use difficult [28], limiting its efficacy to tumors with which it may come into direct contact (e.g., skin or gastrointestinal tract cancers). Although serum concentrations may be relatively low, local concentrations at the site of action may be sufficient to exert a pharmacological effect. On the other hand, trans-resveratrol together with other PC, such as quercetin and (+)-catechin, have exhibited synergies that could overcome their scarce bioavailability [26]. By way of an example, a freeze-dried extract of natural red wine promoted differentiation and reduced the proliferation of HCT-116 colon cancer cells [27]. trans-piceatannol, reported antiproliferative activity against hepatocellular cancer cells and suppressed metastasis of these cells in rats [25]. Meanwhile, pterostilbene inhibited in vitro the proliferation of pancreatic cancer cells. OH-tyrosol, also present in the extract, has shown a wide range of antitumor effects, inhibiting proliferation and promoting apoptosis in various human tumor cell lines through various mechanisms [26].

Data confirming the in vitro bioactivity of PC usually use the pure compounds or their mixtures in (primary) plant extracts. Therefore, their bioavailability and biotransformation after ingestion is a parameter that not considered. That is, in vitro micromolar concentrations may not be operative in vivo. Hence, it is hard to draw reliable conclusions about any “real” health-promoting properties of PCs, knowing that they may have low bioavailability and extensive metabolism. Biological activity and bioavailability, are rarely investigated at the same time, so the bioactivity of their derived metabolites, present in human blood and tissues, are almost completely unknown [29]. It is not possible to obtain meaningful insight into biological effects without counting on sufficient evidence about the uptake of specific PCs in humans when exposed to cultured cells. Except when these compounds can go into direct contact with cells without undergoing uptake or metabolism [30]. So, this supports the idea of investigating the in vivo effects of PCs and/or their extracts on colon or skin cancer cells. Synergistic and/or additive effects of PC could be increased by their encapsulation with conventional anticancer therapies or their combination with other PC. In this context, future research that advances the bioavailability of PCs will be relevant, as well as the study of its metabolites, which facilitate the extrapolation of the results to in vivo studies first in animal models. Subsequently, clinical studies will be needed to explain the in vitro/vivo findings. This will help to understand the potential benefits of these extracts and their active compounds in patients with cancer.

The current study reports the content of PCs of cane and bunch stem extracts Vitis vinifera L. cv. Malbec and their in vitro cytotoxicity in human tumor and non-tumor cell lines. Cane extract did not inhibit cell proliferation at the in vitro level for the tested lines, which is an advantage for its potential application as a functional or nutraceutical agent. On the other hand, the bunch stem extract showed cytotoxic activity against the human tumor line HCT-116, representative of colorectal adenocarcinoma, with selective and significantly lower effects on non-tumor cells. Colorectal cancer is the third leading cancer in the world. There is great interest in the development of new chemopreventive products and/or anticancer agents from natural and sustainable sources such as agro-industrial residues. This disease, due to its condition in the intestinal lumen, does not necessarily require the intestinal absorption of the active principles to exert its antitumor function. Importantly, this is the first time that the cytotoxic effect of Vitis vinifera L. cv. Malbec bunch stem and cane extracts were assessed in the HBL-100 and HCT-116 cell lines. This information reinforces the importance of further investigation of these extracts as potential preventive and/or therapeutic phytoderivatives that could lead to valuable biological effects in targeted cells without causing toxic side effects in normal cells. Likewise, a potential alternative can be opened for its combined use with traditional drugs with the aim of reducing treatment doses or preventing their toxicities, especially in detoxification organs such as the liver and kidneys. Additional studies are needed to gain a deeper knowledge of the molecular mechanisms involved, the effects and the bioavailability of the compounds (and/or their metabolites) in target tissues.