ES2956393A1 - CEREAL POLYSACCHARIDE EXTRACTS, METHOD FOR OBTAINING THEM, PHARMACEUTICAL COMPOSITION THAT CONTAINS IT AND ITS USE AS AN ANTITUMORAL AND/OR ANTIFIBROTIC AGENT (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Extracts of cereal polysaccharides, method for obtaining them, pharmaceutical composition that contains them and their use as an antitumor and/or antifibrotic agent. The present invention describes a procedure for obtaining extracts of polysaccharides of plant origin to be used as antitumor and/or antifibrotic agents, from cereals, specifically rigo and more specifically Triticum aestivum, as well as the use of these extracts as a medicine and especially for the treatment of cancer and fibrosis. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

CEREAL POLYSACCHARIDE EXTRACTS, METHOD FOR

OBTAIN THEM, PHARMACEUTICAL COMPOSITION THAT CONTAINS IT AND ITS USE

AS AN ANTITUMORAL AND/OR ANTIFIBROTIC AGENT

TECHNIQUE SECTOR

The present invention is within the field of biology, botany, nutrition and biomedicine. Specifically, it refers to obtaining extracts of polysaccharides of plant origin to be used as antitumor and/or antifibrotic agents, from cereals, specifically Wheat and more specifically Triticum aestivum.

BACKGROUND OF THE INVENTION

Despite recent developments in prevention and treatment, cancer causes millions of deaths annually and poses an enormous economic burden worldwide [1]. In 2020, cancer caused 10.0 million deaths worldwide, including 915,000 due to colorectal cancer (CRC) [2]. Globally, between 1 and 2 million new cases of CRC are diagnosed each year, making it the third most common type of cancer, and the fourth leading cause of cancer-related death [3,4,5].

Among the risk factors for the development of CRC are i) age, since it is diagnosed more frequently in patients over 50 years of age (90% of cases) without other pathologies or predisposing diseases; ii) dietary factors, which play an essential role and are under continuous research; In fact, excessive alcohol consumption, overweight and obesity, and certain types of foods (processed meat) have been linked to this pathology; iii) predisposing diseases, especially the presence of intestinal polyps or inflammatory bowel disease, which mean that these patients should be considered at high risk for the development of CRC; iv) the presence of a previous CRC; v) the so-called genetic or family factors, given that in 25% of cases there is a family history and in 10% a hereditary component and vi) finally, the style of life, in which physical inactivity should be highlighted, have also been related to CRC.

Although recent advances in cancer treatment are promising, new components that can combat cancer with fewer side effects on non-cancerous cells still need to be discovered. The development of new therapeutic and prevention strategies covers a large number of research fields that extend from nanotechnology, gene therapy, immunotherapy using strategies that activate the immune system or the use of new compounds that can be active in the immune system. CRC treatment or adjuvants to current therapies to improve the response to treatment. In this sense, in recent decades compounds extracted from natural sources have been of special interest [6,7], so it is urgent to explore them in search of beneficial active compounds.

Polysaccharides of natural origin

In recent years there has been growing interest in the exploration of new compounds derived from renewable natural sources to safely and effectively treat and prevent cancer. Polysaccharides (PSs) are related to the class of natural substances that have been obtained from microorganisms, fungi, algae and plants. They have been shown both in vitro and in vivo to have immunomodulatory antioxidant properties and antitumor effects [7,8,9]. Furthermore, in preclinical studies it has been observed that the biological activity of natural PSs is capable of reducing tumor growth and prolonging survival through immune stimulation, apoptosis, and cell cycle arrest [10].

In the last 20 years there has been a tendency to search for new PSs with antitumor properties and that cause minimal damage to the host cell. In this regard, PSs isolated from plants have often been considered preferable to other sources because they are less toxic and cause fewer side effects [11]. Furthermore, PSs from plants are complex macromolecules with wide structural and functional variability [12,13], which makes them a powerful molecule with a multitude of biological effects. PSs derived from various plants (apple, ginseng, aloe and many others) have already been shown to have antitumor, immunomodulatory and antioxidant properties [14,15].

The antioxidant activity of PSs has been shown to have a protective effect against damage due to cellular oxidation, minimizing the risk caused by reactive oxygen species. In turn, it is known that many chronic diseases (including cancer, diabetes, cardiovascular diseases, etc.) are associated with oxidative stress and PSs act as free radical scavengers [16,17]. This enormous magnitude of beneficial effects of PSs makes them highly valuable for preventing and treating cancer and many other chronic disorders.

Among other PSs of plant origin, medicinal herbs have been most frequently investigated and used in adjuvant cancer therapies. Some of these PSs from fungi and medicinal plants have been used to develop pharmaceutical products such as lentinan, schizophyllan, Krestin, GanoPoly, astragalan, GCS-100, and PectaSol, with low toxicity as well as potent antitumor activity, and currently exist in the market as nutritional supplements or medications. In Japan, China and Korea, bioactive PSs have already been introduced as adjuvants, complementing standard radio- and chemotherapy treatments [10]. However, although all of them have shown a direct anticancer effect in vitro and/or in vivo, they are preferably used as immunomodulators and adjuvants during these treatments. This fact is probably due to the fact that these polysaccharides stimulate the immune system at low doses (0.5-100 ^g/ml), while this direct anticancer effect requires much higher doses (40-10000 ^g/ml) (Table 1). For example, for CRC the necessary dose was 5000 ^g/ml. The use of PSs at high concentrations for antitumor therapy is compromised by the inhibition of the growth of normal cells along with cancer cells.

Table 1. Comparison of natural polysaccharides on the market

The chemical structure and functional characteristics of PSs from cereals have also been investigated, after being suggested by their use in food, cosmetics and the drug industry [18,19]. There is evidence of antitumor and immunomodulatory activity in rice bran PSs [20], and recently the antitumor effect of wheat bran PSs has also been revealed [21]. However, there are no studies of the biological effect of PSs obtained from cereal cell culture.

It has been shown that plant callus tissues can be a source of valuable PSs [22,23]. Cultivating plant cells is a biotechnological approach that allows different chemical compounds to be obtained with numerous advantages: no control of organisms, absence of changes in climatic conditions, homogeneity, possibility of optimization, standardization and greater performance [24].

The cultivation of plant tissue in vitro makes it possible to focus on the production of those PSs that have special properties [25]. Furthermore, the cultivation of plant cells allows the production and regulation of the output of phytochemicals with biotransformation, which contributes to the obtaining of natural substances with novel activity [26].

Calluses with friable morphology

Specifically, embryogenic plant calli with friable morphology show distinctive characteristics compared to other types: single embryogenic competent cells and embryogenic cell complexes, cells with signs of programmed cell death, and a dense network of polysaccharides in the extracellular space. The use of the culture of embryogenic callus of friable morphology as a source of obtaining a suspension culture for the isolation of polysaccharides with anticancer and/or antifibrotic activity has not been described in other patents or articles. This type of callus makes it possible to regulate the production and chemical composition of exopolysaccharides by changing the balance of phytohormones in the culture medium, which in turn correlates with growth, morphogenesis and cell differentiation in this type of tissue. .

Molecular mechanisms of cancer

There are numerous processes that favor the appearance and progression of cancer, including the deregulation of apoptosis, increased cell proliferation, inability to differentiate, or the presence and accumulation of free radicals. Focusing on solving these alterations has until now been the approach to finding new solutions and treatments that will stop and reverse this disease until it is eliminated.

It is widely known that apoptosis, type I programmed cell death, is an essential mechanism to maintain the balance between cell death and proliferation. Its deregulation usually causes the progression of cancer cells. Notably, many cancer cells appear to resist apoptosis signaling mechanisms, suggesting the need to search for compounds that induce apoptosis in these cells [27,28]. Cells that undergo this process are characterized because their chromatin condenses and defragments [29], the mitochondria become enlarged and damaged and their internal membranes become disorganized, a large number of autophagic vacuoles appear with particles inside, the Golgi apparatus appears. It swells, the nuclear membrane disappears, and blisters and multilamellar bodies appear.

There are two routes that trigger apoptosis: the intrinsic or mitochondrial route, and the extrinsic route through death receptors. Both pathways lead to the activation of caspases, and cause intracellular changes such as chromatin condensation, DNA fragmentation, membrane blebbing, and cell shrinkage [30,31]. There are a multitude of signaling pathways through which apoptosis can take place, involving p53, Bcl-2, NFkB, MAPK and other related proteins [27]. It has been reported that the cytotoxic effect of PSs from plants is achieved through one or more mechanisms: MAPK, NF kB, JNK, p38, p53, PARP, etc. [32,33,34].

Specifically, proteins from the Bcl-2 family [35] participate in the intrinsic activation pathway, including Bax, which leads to the release of cytochrome C from the mitochondria to the cytoplasm [36], followed by the activation of caspase 9 and finally the caspase that executes apoptosis, caspase 3. On the other hand, the extrinsic pathway, which occurs with the activation of death receptors (Fas, TNFR-I, TRAIL, etc.), continues with the activation of caspase 8, which triggers apoptosis through caspase 3 [37].

Regarding the inhibition of cell growth, it is usually associated with a decrease in beta-catenin levels [38,39], which would cause the blockage of cyclin D1 and the APC (adenomatous polyposis coli) gene pathway; and to the decrease of the c-Myc protein [40]. The latter causes a change in the tumor cells to become more differentiated, thereby growing at a lower rate [41,42]. Differentiation processes have also been related to decreased CD44 levels. In general, cell cycle arrest contributes to differentiation, but without being an ultimately necessary element for it, but accompanied by the downregulation of cyclins or the activation of inhibitors of cyclin-dependent kinases (CDKs). ) [43]. In addition to the intrinsic pathway of apoptosis, cytochrome C has also been related to cell differentiation, usually accompanied by caspase cleavage, but without causing cell death [44].

The CD44 receptor has also been detected to be related to the progression of colorectal cancer cells and appears to be a promising indicator for the prognosis of this disease [45]. CD44 expression may correlate with the Wnt pathway and epithelial-mesenchymal transition. Furthermore, CD44 is a biomarker of cancer stem cells (CSCs) in some tumors and is considered to contribute to cancer growth and metastasis [46]. It has been shown that the differentiation of CSCs in the breast can be induced by knocking out CD44, causing the loss of the CSC phenotype along with the reduction of the expression of Bcl-2, Muc-1 proteins and the consequent reduction of metastasis and tumorigenesis [47].

There are also proteins frequently associated with cancer progression, such as NFkB. This protein is widely used by cells as a regulator of genes that control cell proliferation and survival. An overactivation of the associated gene leads to the protection and induction of proliferation of cells, which normally should die by apoptosis, while its inhibition increases the susceptibility of cells to apoptosis. There are two types of NFkB pathways, which are activated differently and induce the activation of different genes: classical route, of NFkB 1 through the p65/p50 proteins; and alternative pathway, of NFkB 2 through the p100/p52 proteins [48]. Both mechanisms are important in tumorigenesis, although the alternative pathway has not been thoroughly investigated in the case of colorectal cancer cells. Furthermore, there are antitumor drugs that have been tested to inhibit the classical NFkB pathway, and not so many for the alternative route [49], which leaves open the possibility of research and applications of new drugs [50].

Molecular mechanisms of fibrosis

Fibroblasts produce the structural proteins of the extracellular matrix (ECM), such as fibrillar collagen and elastin, adhesive proteins, such as laminin and fibronectin, and the ground substance, which comprises glycosaminoglycans such as hyaluronic acid, and glycoproteins. However, fibroblasts also play several additional roles beyond ECM production. For example, they participate in ECM maintenance and resorption, wound healing, inflammation, angiogenesis, cancer progression, and in both physiological and pathological tissue fibrosis. Ancillary, fibroblasts produce and respond to a wide range of paracrine and autocrine signals, such as cytokines and growth factors. Targeting these auxiliary signaling events is the main strategy we It underlies multiple lines of research that seek a new generation of treatments for disorders related to fibroblasts.

Fibroblasts are mesenchymal cells derived from embryonic mesoderm tissue, and are not fully differentiated. They can be activated by a variety of chemical signals that promote cell proliferation and differentiation to result in myofibroblasts, with an increased rate of matrix production. Activation of fibroblasts plays a vital role in wound healing. However, in some cases and due to reasons that have not yet been fully clarified, its activation becomes uncontrolled, producing a pathological fibrotic response that promotes multiple diseases and affects a variety of organs. In fact, fibrosis plays a very important contributing role in most cases of organ failure. Examples are abundant, from systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, renal fibrosis, and cardiac fibrosis seen in cardiac hypertrophy that leads to heart failure. The major role of fibroblasts in pulmonary fibrosis was validated by lineage-specific deletion of the TGFp type II receptor [51].

Since fibroblasts are a central effector in fibrosis, they become potential therapeutic targets. One of the routes of fibroblast activity is the Wnt pathway, related to the proliferation, migration and invasion of these cells.

On the other hand, it is known that galectin-3 (gal-3) is a sugar-binding protein with pleiotropic effects with a key role in tumor progression, and may be a therapeutic agent [52]. Gal-3 contributes to the overexpression of c-Myc, beta-catenin, related to the Wnt pathway, as well as other pathways involved in tumorigenesis and, in addition, pathways that contribute to keeping cancer cells undifferentiated [53]. Inhibition of gal-3 has been shown to trigger cell accumulation in the G2/M phase [54], suppress tumor cell adhesion to the endothelium, and thereby prevent metastasis and intravascular deposits [55].

However, gal-3 inhibitors can be used for other purposes, in particular to prevent fibrosis. This is possible since gal-3 activates a multitude of profibrotic factors, promotes the proliferation and transformation of fibroblasts, and modulates collagen production. The importance of gal-3 has been demonstrated in fibrogenesis of different organs, including the liver, kidney, lung, and myocardium [56]. Specifically, it promotes fibroblast activity, including the production, secretion and maturation of collagen [57,58], and also stimulates the secretion of the proinflammatory cytokines IL-1 and IL-6, as well as fibronectin [59].

In previous research, the inventors were able to identify bioactive PSs in cereal cell suspension cultures [60]. They explored the chemical composition and physical properties of PSs from wheat, which are represented by arabinogalactans, and by xyloglucans, arabinoxylans and glucans, according to their monosaccharide composition [61,62]. Furthermore, they demonstrated that the monosaccharide composition changed significantly depending on the type and concentration of phytohormones in the culture medium, as well as the culture time in the different stages [63]. From the literature it is known that these types of PSs are considered suitable for application in medicine, including cancer treatment [10]. Therefore, the results suggest that arabinogalactans [64], beta-glucans [65], and arabinoxylans [66] have the ability to suppress the proliferation of cancer cells in vitro [66]. On the other hand, previous experiments with modified citrus pectins, which inhibit gal-3, prevent cardiac fibrosis, inflammation, and other functional alterations [67].

The closest state of the art known to the applicant is reflected in CN101705268 [68], which describes a method of extraction of wheat bran polysaccharides with antitumor and immunomodulatory activity. The composition of the extracted monosaccharides consists only of arabinoxylans, and the extraction consists of a multi-step treatment: soaking in HCl, starch removal, enzymolysis with xylanases (steps that also modulate the monosaccharide composition), double extraction with ethanol, and inactivation of enzymolysis products.

In the present invention, a method of extracting PSs from wheat cell cultures and using them for the treatment, alleviation and prevention of colorectal cancer and/or fibrosis is proposed. In this method, modifications are made to the composition of phytohormones and cultivation times to obtain extracts with different compositions. The extracts obtained have low toxicity, which would solve the existing problems with current treatments, whose activity also harms cells. healthy, causing numerous side effects. Therefore, the use of PS extracts from wheat cell cultures represents an effective and safe alternative to current treatments.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on the obtaining and use of polysaccharide extracts from a plant species of the genus Triticum, more specifically, obtained from cell cultures of embryogenic callus tissue of the species Triticum aestivum, in its soft spring wheat variety, for the treatment and prevention of colorectal cancer and/or fibrosis.

In contrast to the pharmaceutical products obtained from fungi and plants mentioned in the “Background of the Invention” (lentinan, schizophyllan, Krestin, GanoPoly, astragalan, GCS-100 and PectaSol), in the present invention extracts of PSs have been obtained whose activity against cancer cells it is effective at low concentrations between 10 and 1600 ^g/ml (colorectal cancer cells, Table 1), in addition to having low toxicity for healthy cells. Consequently, the therapeutic index of the most effective extracts would be between 9 and 70.

The closest state of the art known to the applicant is reflected in CN101705268, as already mentioned in the “Background of the invention”. When comparing them with the present invention, the following differences were found:

• Source of obtaining: in patent CN101705268, PSs have been isolated from wheat bran, which consists of the hard outer layer (aleurone and pericarp) of the mature grain of the cereal. However, in the present technology the polysaccharides are obtained from a suspension culture of wheat cells capable of maintaining their biosynthetic potential in the long term (several years) by performing in vitro subcultures.

• Monosaccharide composition: while patent CN101705268 determines that a wheat polysaccharide extract with high antitumor activity and low toxicity for the body consists only of arabinoxylans (xylose - 62.25%, arabinose - 33.77%, glucose - 4.03%), in the present invention, two of the polysaccharide extracts with high activity antitumor and low toxicity consist mainly of glucans (glucose -86-90%, arabinose - 1.5%, xylose - 2-5%, galactose - 1-2%, mannose - 1-2%, glucuronic acid - 1%, galacturonic acid - 1%), as previously published [63].

• Extraction method: the method for obtaining PSs in CN101705268 consists of a multi-step treatment: soaking in HCl, starch removal, enzymolysis with xylanases, double extraction with ethanol, and inactivation of the enzymolysis products; while the method described here requires only one step, and another additional step optionally, since PSs are secreted naturally into the extracellular space.

• Modulation of monosaccharide composition. In patent CN101705268, special chemical methods are used to obtain the desired composition: soaking in HCl, starch removal, enzymolysis for adequate bioactivity and molecular structure. On the other hand, in the present invention the composition is modulated using an optimal balance of phytohormones and different cultivation times.

• The antitumor activity of the polysaccharides obtained from wheat bran in patent CN101705268 is difficult to compare with that obtained with the use of the extracts described here, given that in vivo studies were carried out in which 50% of cancer cells were inhibited with concentrations of 400 mg/kg and 10 mg/kg, and this technology was tested in vitro, where the IC50 range was between 10 ^g/ml and 1600 ^g/ml.

The present invention is based on:

1) The development of different polysaccharide extracts from soft spring wheat Triticum aestivum, grown for different periods of time, and in the presence of different phytohormones, which gives rise to extracts with different compositions and different properties. The extracts are obtained from immature embryos cultured on solid agar for 28 or 70 days in the presence of 2,4-D 5.0 mg/mL, to originate young or mature embryogenic calli, respectively, whose cells will be cultured in suspension for one, three or six weeks in medium with different phytohormones: 2,4-D 5.0 mg/mL, or ABA 1.0 mg/mL. Polysaccharides are obtained by ethanolic extraction of the extracellular medium.

2) The antitumor activity of Triticum aestivum extracts when tested in human colon cancer cell cultures (HCT-116 cells), using the human colon fibroblast line CDD-18CO as a control. Likewise, the mechanisms of action by which the extracts act on tumor cells have been studied to elucidate the molecular pathways that activate cell death, inhibit cell growth and induce cell differentiation.

3) The antifibrotic activity of Triticum aestivum extracts when tested in CDD-18CO human colon fibroblast cell cultures. Likewise, the mechanisms of action by which the extracts act on fibroblasts have been studied to elucidate the molecular pathways that inhibit cell growth and activate cell death.

In short, the technology presented here shows the following technical advantages compared to the state of the art:

• All polysaccharides on the market with anti-cancer properties are obtained from fungi and medicinal plants that need to grow in the soil. Therefore, large-scale production requires large areas, irrigation or, at a minimum, greenhouses. In many cases, there is a strong dependence on climate and seasons. At the same time, the literature shows that PSs can also be obtained from fungal cell culture. The advantage of this technology is that it allows PSs to be obtained throughout the year, regardless of the climate and season of the year, through cell culture in suspension, under controlled conditions (temperature, light intensity, photoperiod, etc.), without requiring large fields, irrigated or greenhouses.

• The desired composition of PSs on the market is achieved through a multi-step chemical modification, followed by a multi-step purification, which represents a large investment of time and effort. On the other hand, the present invention is much simpler. The desired PSs composition is achieved simply by changing the phytohormone balance of the culture medium and the culture times, without needing to make chemical modifications, and the purification is a single step thanks to that PSs are secreted into the extracellular space.

According to the technology for obtaining PSs existing on the market, it is necessary to modify the isolation and purification protocol for each different species that acts as a source organism. The technology described here would work for any cereal crop (barley, corn, oats, rice, rye, sorghum, proso millet) using wheat cell culture as a universal model system for cereals and grasses.

Furthermore, this technology would improve profitability, in addition to simplifying the production method, optimizing, standardizing and increasing its performance, as well as the possibility of directing production towards the desired PSs.

The existing PSs on the market are effective through the activation of the immune system, and are mostly used as adjuvants in chemo- and radiotherapy treatments, and not as direct treatment, due to their negative effect on healthy cells. In the present invention it has been shown that the PSs obtained from the culture of wheat cells, preferably those cultured in suspension with abscisic acid (ABA), act as direct inhibitors of cancer cells at much lower concentrations than the PSs on the market and their toxicity in healthy cells is much lower. Thanks to this, the most effective extracts have obtained therapeutic indices that meet the requirements of antitumor drugs and can be used at higher concentrations.

Finally, when investigating the mechanism of action of PS extracts from wheat cell cultures, it has been seen that not only apoptosis is induced, but cell proliferation and differentiation is also inhibited. When analyzing the markers, a reduction in the levels of beta-catenin and c-Myc has been detected, which means that the Wnt/beta-catenin pathway would be inhibited. These observations, together with the fact that PSs contain galactose, which is capable of binding to the terminal sugar-binding end of the gal-3 protein, indicate that PS extracts from cell cultures, preferably those grown in suspension with 2 ,4-dichlorophenoxyacetic acid (2,4-D), act as gal-3 inhibitors, which in addition to being anticancer agents, are also effective for the prevention of fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description, where, with an illustrative and non-limiting nature, the following has been represented. following:

Figure 1. Inhibition of proliferation (in %) of the colon cancer cell line HCT-116 under the influence of different concentrations of the extracts T-010, B-010 and UB-010. T010 - complete extract, B-010 - acidic/bound extract, UB-010 - alkaline/unbound extract.

Figure 2. Inhibitory activity of PSs extracts in the healthy CCD-18 CO cell line compared to inhibition in HCT-116 colon cancer cells. Extracts T-010, Tb, Tf, Ab and Af.

Figure 3. Cell cycle and apoptosis of HCT-116 colon cancer cells treated with PSs extracts from wheat cell cultures . A - untreated cells, B - Ab extract, C - T-010 extract, D - UB-010 extract, E - Tb extract (2xIC50), F - Af extract, G - Tf extract, H - cell cycle, I - apoptosis, J - cellular debris.

Figure 4. Transmission electron microscope micrographs of HCT-116 cells treated with PSs extracts from wheat cell cultures at IC50 concentrations . (Scale: a, b, e, j, k, l, m, n, o, p, q, r - 400 nm, c, d, f - 600 nm, g, h, I - 200 nm) , a - nuclei of untreated cells, b, c - nuclei with condensed chromatin and nuclear pores, nuclear bubbles of cells treated with T-010 extract, d - nuclei of cells treated with Tb extract, condensed chromatin, nuclear pores (2xIC50) , e - nuclei of cells treated with the Ab extract, f - bubbles in the intercellular space, between the cells treated with the UB-01 extract, g - mitochondria of untreated cells, h - mitochondria treated with the T-010 extract , i - mitochondria treated with Tb extract, j - Golgi apparatus of untreated cells, k - swollen Golgi apparatus in cells treated with T-010 extract, l - swollen Golgi apparatus in cells treated with Ab extract, m - swollen Golgi apparatus in cells treated with the Tb extract, n - lysosomes and autophagic vacuole of untreated cells, or - accumulation of multiple autophagic vacuoles with double membrane and simple in cells treated with the T-010 extract, p - accumulation of multiple autophagic vacuoles with double and triple membrane, q - vesicles in cells treated with the Tb extract, r - large autophagic vesicle with double membrane in cell treated with the UB extract -010, A - diameter of a cross section of mitochondria, B - number of damaged mitochondria per image. N-nucleus, Np - nuclear pore, M-mitochondria, G- Golgi apparatus, L-lysosome, av - autophagic vesicle; ALV-large autophagic vesicle.

Figure 5. Scanning electron microscope micrographs of HCT-116 cells treated with PSs extracts . A: Magnification 3000x, 50^m: a - untreated cells, b - T-010 extract, c - Ab extract, d - UB-010 extract, e - Tb extract at IC50, f - Tb extract at IC50x2; Magnification 9975x, 10^m: g - untreated cells, h - T-010 extract, i - Ab extract, j - UB-010 extract, k - Tb extract at IC50, l - Tb extract at IC50x2. B - number of microvilli/10^m2.

Figure 6. Immunoblotting of different proteins related to cancer proliferation after treatment with different PSs extracts. Quantification was normalized with the p-actin signal. Data were obtained from independent experiments performed in duplicate and are expressed as the mean ± standard deviation of three independent experiments performed in triplicate (****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 vs. control).

Figure 7. Scheme of obtaining PSs extracts.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "extract" in the context of this document, when used in relation to the object of the invention, refers to a composition obtained from a plant species, and its content essentially comprises polysaccharides.

The term "treatment" or "treat" in the context of this document refers to the use of polysaccharides according to the invention in the form of an extract or composition to cure or alleviate a disease, pathological condition or one or more symptoms associated with said disease or condition. "Treatment" also encompasses the prevention, improvement or elimination of the physiological sequelae of the disease. Specifically, the concept "treat" can be interpreted as:

Yo. Inhibit or prevent the disease or pathological condition, that is, stop its development;

ii. Alleviate symptoms or alleviate the disease or pathological condition, that is, causes the regression of the disease or pathological condition; iii. Stabilize the disease or pathological condition.

Thus, in a first aspect, the invention refers to a polysaccharide extraction procedure, hereinafter "process of the invention", which comprises the ethanolic extraction of extracellular polysaccharides from a cell culture of embryogenic cereal callus tissue.

In a preferred embodiment, the cereal used is wheat, and more preferably Triticum aestivum, and even more preferably the Kazakhstanskaya-10 variety.

In a particular embodiment, the procedure of the invention refers to a procedure for obtaining polysaccharide extracts from cereal cell cultures, which comprises the steps of:

a) Plant an immature embryo on solid agar medium to obtain an embryogenic callus

b) Cultivate tissue from said embryogenic callus on solid agar medium c) Cultivate cells from step b) in suspension with the addition of at least one phytohormone

d) Extraction of the extracellular polysaccharides from step c) by ethanolic extraction

In another particular embodiment, the procedure has an additional fractionation step of the polysaccharide extract, to obtain the alkaline extract.

In another preferred embodiment, the embryogenic callus of step a) has a friable morphology, more preferably a friable morphology that is maintained in the long term.

In preferred embodiments, the process of the invention is carried out under the following conditions:

• The embryogenic callus is cultured for between 10 days and 24 months, preferably between 10 and 365 days, and more preferably for 28 or 70 days, and/or

• The embryogenic callus is cultured in Murashige and Skoog medium in the presence of 2,4-dichlorophenoxyacetic acid at a concentration between 0.1 and 20 mg/mL, preferably 5.0 mg/mL.

• The cells are cultured in Murashige and Skoog suspension agar medium in the presence of at least one of these phytohormones, preferably one of these phytohormones:

^o 2,4-dichlorophenoxyacetic acid at a concentration of between 0.1 and 20 mg/mL, preferably at 5.0 mg/mL.

^o Abcisic acid at a concentration of between 0.1 and 10 mg/mL, preferably at 1.0 mg/mL.

• The culture to obtain the embryogenic callus tissue cells is carried out for a period of time between 5 days and 6 months, more preferably for 7, 21 or 42 days, and/or

• The culture to obtain embryogenic callus tissue cells is carried out with a photoperiod of 16 hours.

In a second aspect, the invention relates to the extracts of extracellular polysaccharides obtained or obtainable by the process of the present invention.

A third aspect refers to the use of polysaccharide extracts in medicine, polysaccharide extracts for use as a medicine or for the manufacture of a medicine.

In a particular embodiment, the extracts are used for the improvement, relief, prevention and/or treatment of cancer. Preferably, the type of cancer is colorectal cancer.

In another particular embodiment, the extracts are used for the improvement, relief, prevention and/or treatment of fibrosis.

In a fourth aspect, the invention refers to a pharmaceutical composition that comprises in its formulation at least one extract of extracellular polysaccharides of the present invention. The composition may comprise at least one additional antitumor agent. Alternatively, the composition may comprise at least one additional antifibrotic agent.

In a particular embodiment, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or vehicles.

A final aspect of the invention refers to the use of a pharmaceutical composition according to the previous aspect, for use as a medicine or for the manufacture of a medicine.

In a particular embodiment, the composition is used for the improvement, alleviation, prevention and/or treatment of cancer. Preferably, the type of cancer is colorectal cancer.

In another particular embodiment, the composition is used for the improvement, relief, prevention and/or treatment of fibrosis.

As in the case of the process of the invention, and in the other aspects of the invention described below, embodiments corresponding to extracts obtained from wheat, preferably Triticum aestivum, are preferred.

Throughout the description and claims the term "comprises", which may also be interpreted as "consists of", and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention.

IMPLEMENTATION MODES

Materials and methods

Plant cell culture, extraction of polysaccharides and monosaccharide content

Embryogenic cells from soft spring wheat ( Triticum aestivum ) callus were cultured on solid agar (Murashige and Skoog) in the presence of 5.0 mg/L 2,4-D for four weeks to obtain young friable calli, or ten weeks to obtain mature friable calluses. The callus tissue was then placed in a liquid culture medium (Murashige and Skoog) [69] in the presence of a phytohormone [63], either 2,4-D 5.0 mg/L or ABA 1.0 mg/L. L, following a ratio of 200 to 300 mg of tissue in 30 to 40 ml of medium. The culture was carried out in a shaker at 140 rpm, maintaining the callus at a temperature of 26 ± 2 °C. To obtain the different extracts, cultures were made for 7 or 21 days for cells from young callus, and 42 days for those from mature callus, all with a photoperiod of 16h. The liquid medium was filtered to remove cells and their debris, and the extracellular liquid was concentrated in an IKA WERKE evaporator (Germany) for the isolation of PSs. These were extracted using 70% v/v ethanol and subsequently subjected to precipitation by centrifugation at 10,000 rpm for 10 minutes at 8°C.

Additionally, an optional fractionation step was carried out by ion exchange chromatography using a Tris HCL buffer on a DEAE-Sepharose column (Sigma, USA) [70], to separate the extracts into their acidic (bound extract) and alkaline components. (unjoined extract). The PSs were extracted using 70% v/v ethanol and subsequently subjected to precipitation by centrifugation at 10,000 rpm for 10 minutes at 8°C.

The total amount of polysaccharides of all extracts was determined by the Dubois method [71].

The extracts, to facilitate their identification, were named T if during cell culture in suspension they were in the presence of 2,4-D (Figure 1, middle 1); and A if it was in the presence of ABA (Figure 1, middle 2). According to the cultivation times, they were the following:

- T Extracts:

^or T-010. Short-term culture: young callus cells cultured in suspension for 7 days.

^or Tb. Medium-term culture: young callus cells cultured in suspension for 21 days.

^or Tf. Long-term culture: mature callus cells cultured in suspension for 42 days

- Extracts A:

^or Ab. Short-term culture: young callus cells cultured in suspension for 7 days.

^or Af. Long-term culture: mature callus cells cultured in suspension for 42 days.

Furthermore, the T-010 extract was subjected to the additional step mentioned above, fractionation on a DEAE-Sepharose column, giving rise to two extracts:

- Extract B-010. Acid, bound to the column ( bound).

- Extract UB-010. Alkaline, not bound to the column ( unbound).

Characterization of polysaccharide extracts

In total, seven extracts of PSs were used to study the effect on CRC cells and their mechanism of action. The extracts differed from each other in terms of nutritional composition of the liquid culture medium (addition of 2,4-D or ABA), as well as culture times (Figure 1), and 5 complete extracts were obtained (T-010, T-b, T-f, A-b, A-f), and 2 extracts by separation by ion exchange chromatography of one of the complete extracts (UB-010, B-010). The precipitate of each extract was dissolved in 0.5 ml of ultrapure water and lyophilized. For each experiment, extracts were diluted in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) to obtain the desired concentrations.

cell lines

HCT-116 colorectal cancer cell lines and healthy CDD-18CO cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% v/v fetal bovine serum. The authenticity of all cell lines was analyzed by STR (Short-Tandem Repeat) identification and passages were performed in less than 6 months. Routine mycoplasma contamination tests were performed.

In vitro cytotoxicity assays

The effect of PSs extracts on cell viability was measured by an MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide; Sigma, USA). 2.5x10 ³ cells/well were seeded in 96-well plates. They were incubated for 24 hours and then treated with different concentrations of PSs. Three days later, the wells were aspirated and treated with MTT for 3 hours, with subsequent dissolution with dimethyl sulfoxide (DMSO). Cells were analyzed using a Titertek Multiskan apparatus (Flow, Irvine, California) at 570 nm.

We assessed the linearity of the assays by calculating the number of cells in each well before each cell growth experiment. The 50% inhibitory concentration (IC ⁵⁰ ) was calculated from semi-logarithmic dose-response curves using linear interpolation [72]. All experiments were plated in triplicate wells and performed at least three times.

Apoptosis and in vitro cell cycle assays

Cell cycle phases (G0/G1, S or G2/M) are characterized by differences in DNA content. Since the fluorescent dye propidium iodide (Sigma, USA) binds strongly to DNA at a 1:1 ratio, the intensity of fluorescence by propidium iodide varies between different phases of the cell cycle.

HCT-116 cells were seeded in 6-well plates, at a concentration of 200x10 ³ cells/well, and incubated for 48 hours. Subsequently, they were treated for 48 hours with the PSs extracts at different concentrations, determined according to the IC ⁵⁰ values. Cells cultured in monolayer were harvested, washed twice with PBS, and fixed in 70% ethanol at 4°C. The cell pellet was washed twice in PBS and resuspended in a DNA extraction solution (pH = 7.8) of 0.1 M acetic acid, and 0.2 M anhydrous dibasic sodium phosphate in PBS, and incubated for 15 minutes at 37°C. The cells pelleted and were washed once more with PBS, after which they were resuspended in a PBS solution with 100 mg/ml propidium iodide and 40 mg/ml RNase in the dark for 30 minutes at 37°C [73]. The percentage of cells in the subG1, G0/G1, S and G2/M phases was determined with a FACS Calibur flow cytometer (BD, Biosciences, USA).

Electron microscopy

The HCT-116 cell line was seeded in 6-well plates, at a concentration of 200x103 cells/well, and incubated for 48 hours. Subsequently, they were treated for 48 hours with the PSs extracts at different concentrations, determined according to the double IC ⁵⁰ values. Cells were washed with cold PBS, fixed, and prepared for transmission electron microscopy (TEM) according to the standard protocol [74]. For scanning electron microscopy (SEM), the HCT-116 cell line was seeded in 24-well plates, with 100x103 cells/well, and after 48 hours they were treated with the same concentrations of PSs for 48h. Cells were fixed and samples were prepared for SEM as described in [75].

Immunoblotting

HCT-116 cells (150,000/well) were seeded in 6-well plates in DMEM medium. After 48 hours of treatment, the medium was removed from the cells, and the cells were centrifuged at 1500 rpm, washed twice with PBS, and then lysed with Laemmli buffer. Immunoblotting of whole cell lysates was performed following routine protocols [76]. Primary antibodies were used to detect the following proteins:

• caspase-3, 1:1000 dilution (Cell Signaling, Beverly, MA, USA);

• caspase-8, 1:500 dilution (caspase-8 (8CSP03): sc-56070, Santa Cruz Biotechnology);

• bax, 1:500 dilution (bax (B-9): sc-7480, Santa Cruz Biotechnology);

• cytochrome c, 1:500 dilution (cytochrome c (7H8): sc-13560, Santa Cruz Biotechnology);

• p-actin, dilution 1:15000 (A2228, Sigma);

• c-Myc, 1:100 dilution (c-Myc (9E10): sc-40, Santa Cruz Biotechnology);

• beta-catenin, 1:100 dilution (p-beta-catenin (1B11): sc-57533, Santa Cruz Biotechnology);

• NF-kB2 p100, dilution 1:1000, (NF-kB2 p100/p52, 4882, Cell signaling technology, USA);

• CD44, 1:200 dilution, (HCAM (DF1485): sc-7297, Santa Cruz Biotechnology)

The secondary antibodies used included anti-rabbit IgG conjugated with peroxidase (Sigma, A0545) and peroxidase-conjugated anti-mouse IgG (Sigma, A9044) - Protein-antibody complexes were made visible using enhanced chemiluminescence (ECL, Bonus, Amersham, Little Chalfont, UK) with the IMAGE READER LAS program. 4000 on a LAS-4000 system. The interpretation of the signal intensity was carried out using the Image J program. The values of each band were normalized by dividing them by the value of their p-actin, and they were relativized with respect to those of the control sample, which were assigned the value 1.

Statistic analysis

Values were expressed as mean ± standard deviation (SD). A student t-test was used to determine the significance of the differences between the two groups, with the p-value being considered significant when the p-value was less than or equal to 0.05. The GraphPrism 8 program was used to prepare the figures.

Results

Antiproliferative and selective inhibitory effect of polysaccharides from wheat cell cultures in colon cancer lines

Cell viability analyzes revealed that PSs inhibit HCT-116 colon cancer cells at different doses. MTT assays showed that T-010 extract inhibited a maximum of 47% of cells at concentrations of 1600 |jg/ml (Figure 2). The alkaline fractionated extract obtained from said T-010 extract had an antiproliferative effect, inhibiting 48% of the cells at concentrations of 1600 jg/m l. These values were considered IC50 since any increase in the concentration of the extracts did not produce greater inhibition (Figure 3A, 3B). On the other hand, the acid extract induced proliferation in vitro by 40% and was excluded from subsequent assays.

The Tb extract inhibits 50% of HCT-116 cells at concentrations between 50 μg /ml and 100 μg /ml, so 75 μg /ml has been considered as IC50 (Figure 3C). As for the Ab extract, it inhibits 50% of the cells at 160 μg /ml (Figure 3D). The Tf extract caused the inhibition of 47% of the cells at 800 jg /ml, and the death of 51% of the cells at 1600 jg /ml. Since the effect at double concentration is almost the same, the concentration of 1600 jg/ml was considered the IC50 (Figure 3E). The Af extract inhibited 50% of cancer cells at concentrations between 10 and 80 jg/ml, and if these values were increased, a slight decrease in the inhibition (40-45% of inhibition) (Figure 3F).

The IC50 value of each PSs extract for CCD-18CO healthy colon fibroblast cells was also determined. It was demonstrated for all extracts cultured in suspension with 2,4-D that the toxic effect of PSs on healthy cells was greater at low concentrations, and decreased when the concentration of PSs was higher. For example, the T-010 extract inhibited 35% of CCD-18CO cells at 50 μg/ml, however, an increase to 100-400 μg/ml caused a slight decrease in inhibition, to 25-27%. , and at high concentrations 20-22% was maintained (Figure 3A). On the other hand, the T-b extract produced an inhibition of between 30-40% at low concentrations 3.125 -12.5 jg /m l, however, an increase in concentration (25 - 100 jg /m l) inhibited 20-25 % of healthy cells, and at 200 jg /m l only 15% of these cells were inhibited (Figure 3C). The incubation of healthy cells with the T-f extract showed the same trend: at 50 jg /m l a 36% inhibition was observed, while in the concentration range between 100-800 jg /m l the inhibition decreased to 20-25%. and a higher concentration (1600-2000 jg /m l) slightly decreased the inhibition, up to 12-18% (Figure 3E). Due to this reversed effect, the IC50 could not be calculated for these cells.

The UB-010 extract caused a 30% - 47% inhibition of CCD-18CO cells, which increased according to concentrations from 50 jg /m l to 1600 jg /m l, and a subsequent decrease at 2000 jg /m l. The IC50 value of this extract is 800 jg /m l (Figure 3B).

Extracts grown in suspension in the presence of ABA showed lower toxicity at low concentrations compared to those grown in the presence of 2,4-D. Therefore, the calculation of the IC50 for these extracts was done using a GraphPrism program. Therefore, the IC50 for the inhibition of CCD-18CO cells is achieved at a concentration of 1480 μg /ml for the Ab extract, and at 700 μg /ml for the Af extract (Figure 3D, 3F). The selective antiproliferative effect of PSs extracts was estimated by calculating the therapeutic index (Table 2).

*Not measurable: cells proliferated

NC = not calculable

The therapeutic index was the highest for extract A-f, followed by extract A-b (70 and 9.25, respectively). These extracts were effective on cancer cells at lower concentrations, and had a lower toxic effect on healthy cells than extracts T-010, T-b, T-f, and UB-010. Extracts cultured in suspension with 2,4-D had a high IC50 in cancer cells, and in healthy cells it could not be calculated. Consequently, the therapeutic index of these extracts was not determined. However, all of them showed that at high concentrations, their toxicity towards healthy cells decreases (Figure 3). For example, T-b extract inhibits 40-50% of colon cancer cells at concentrations between 3 and 75 ^g/ml, and 86% of cells at 100 ^g/ml. In turn, this extract inhibits 40% of healthy cells at low concentrations, but if we increase the concentration to 100 ^g/ml, it inhibits 30%, and at 200 ^g/ml it inhibits 20%. Therefore, for these extracts, instead of calculating the therapeutic index, the results were shown as a ratio of the percentage of cancer cells inhibited at IC50 concentration, divided by the percentage of healthy cells inhibited at the same concentration. Consequently, it could be calculated that at IC50 levels, extracts T-010 (1600 ^g/ml) and T-f (800 ^g/ml) are, respectively, 2.35 and 2.5 times more toxic to cells. cancer cells than for healthy cells, and the T-b extract at 100 ^g/ml is 2.86 times more toxic for cancer cells than for healthy cells (Figure 3, Table 3).

Table 3. Proportion of the percentage of HCT-116 cells inhibited at IC50 and the percentage of CCD-18CO cells inhibited at the same concentration for the extracts cultured in suspension with 2,4-D and not fractionated.

These results confirm the antitumor effect of PSs extracted from wheat cell cultures and their selective antitumor activity when the optimal extracts and doses for this purpose are used.

There are extracts that at certain doses show even greater proliferation inhibitory activity in healthy CCD-18CO fibroblast cells, especially when T-010, UB-010 or T-f extracts are used at low doses (up to 100 or 400 jg/ m l depending on the extract) (Figure 3A, 3B, 3E). It would be ideal to dedicate a few lines to include inhibition percentages of these extracts and their concentrations. In this way, the first evidence of the antifibrotic potential of these polysaccharide extracts was obtained.

Induction of apoptosis and cell cycle arrest

Extract B-010 was excluded because it caused the proliferation of HCT-116 cancer cells (Figure 2). Tests were carried out with the rest of the extracts to study their ability to induce apoptosis and cell cycle arrest because they had inhibited the growth of cancer cells while being less toxic to healthy cells. Flow cytometry results revealed that treatment of HCT-116 cells for 48 h with the extracts increased the proportion of cells in the G0/G1 phase, and decreased the percentage of cells in the S phase (Figure 4).

In fact, the proportion of untreated cells in the G0/G1 phase was around 60%, while in the treated ones it was between 68% and 74%. The influence of the Tb extract increases it to 74%, the Tf extract to 69%, the Ab extract to 70%, the Af extract to 71% and the T-010 extract to 67.5%. There was no significant change in the percentage of cells treated with UB-010 extract compared to the control.

The percentage of cells in S phase was around 30% in control cells, while in treated cells it decreased to between 11% and 21% (Figure 4H). All extracts significantly decreased the number of cells in the S phase. Extract T-b up to 17%, extract T-f to 21%, extract A-b to 13%, extract A-f to 18%, extract UB-010 at 20%, and the T-010 extract at 11%.

The present investigation revealed that some extracts induced apoptosis (Figure 4I). Flow cytometry analyzes showed that the SubG1 apoptotic phase was increased when cells were treated with T-b, A-b, UB-010, and T-010 extracts. Particularly, the highest apoptosis levels (48%) were obtained by treating HCT-116 cells with T-010 extract, compared to the control (4.6%). Extract A-b showed a fairly strong effect since it caused programmed cell death in 40.35% of cancer cells. The T-b and UB-010 extracts showed a moderate increase in cells in the SubG1 phase, 20% and 12.5%, respectively. The percentage of cell fragments was also high in these extracts, between 20% and 60% depending on the extract (Figure 4J). However, no significant change in the SubG1 phase or the number of cell fragments was observed after treatment with extracts A-f or T-f compared to the control (Figure 4I, 4J).

Electron microscopy of HCT-116 cells treated with polysaccharides from wheat cell cultures

Transmission electron microscopy images revealed changes in the nucleus, cytoplasm, and intercellular space of HCT-116 colon cancer cells when under the influence of polysaccharide extracts from wheat cell cultures (Figure 5).

Core and blisters. Treating colon cancer cells with T-010 extract led to increased chromatin condensation (Figure 5b) compared to untreated cells (Figure 5a); as well as the loss of the nuclear membrane and the presence of blebs next to the cell (Figure 5c). When incubated with the Tb and Ab extracts, it was observed that the chromatin had fragmented and was very close to the nuclear membrane, and the nuclear pores were very visible (Figure 5d, 5e). Treatment with UB-010 extract caused similar changes in the nucleus, although the release of blebs into the intercellular space was more intense (Figure 5f).

Mitochondria. Some polysaccharide extracts from wheat cell cultures showed significant effects on mitochondria, such as damage and/or enlargement. Compared to control cells (Figure 5g), the effect of the T-010 extract caused the mitochondria to enlarge and swell, in addition to presenting their internal membranes disorganized, with partial damage, almost empty white vesicles and thin and barely visible mitochondrial cristae. (Figure 5h). On the other hand, treatment with the Tb extract also caused alterations (Figure 5i): the size of the mitochondria doubled compared to the control (Figure 5a) when viewing cross sections (1000 nm versus 500 nm), and the number of damaged and broken mitochondria (Figure 5b). There were no noticeable changes in size or damage when the Ab extract was used. With the UB-010 extract, a moderate increase in the number of damaged mitochondria was observed, but there were no changes in their size.

Golgi apparatus. Compared with untreated cells (Figure 5j), T-010 extract induced swelling of the Golgi apparatus (Figure 5k). The Ab extract caused a moderate increase in the size of this organelle (Figure 5l). Incubation of HCT-116 cells with Tb extract caused an enlarged and degrading Golgi apparatus (Figure 5m). There were no visible changes when the UB-010 extract was used.

Other changes in the cytoplasm. Incubating the cells with the T-010 extract, unlike control cells (Figure 5n), caused the appearance of multiple autophagic vacuoles or apoptotic bodies (400 x 400 nm and less) along with inclusions of different shapes (Figure 5o). The Ab extract caused the appearance of multilamellar bodies and autophagic vacuoles with a triple membrane with inclusions (600 × 400 nm and less) (Figure 5p). The effect of Tb extract differed in that the cell had empty white vesicles in the cytoplasm in addition to vesicles with inclusions (700 × 500 nm) or autophagosomes/autolysosomes (Figure 5q). The UB-010 extract contributed to the formation of large double-membrane autophagic vesicles (2000 × 800 nm), which contained multiple organelles or vesicles (Figure 5r).

In general, it was determined that the extracts T-010, Tb, Ab and UB-010 caused significant changes in cancer cells: damaging the mitochondria, condensing chromatin, removing the nuclear membrane, producing vesicles inside and outside (blebs) of the cell membrane, and multilamellar bodies. Interestingly, a variety of autophagic vacuoles of different sizes presented granule-like inclusions and parts of degraded organelles.

Remarkable changes in the morphology of HCT-116 colon cancer cells incubated with T-010, A-b, T-b or UB-010 extracts were detected when analyzed by SEM. It was observed that the effect of the T-010 extract, compared to control cells (Figure 6A, a and b), decreased the number of cancer cells, caused the cells to flatten, reduced the frequency and length of microvilli (Figure 6B), and stimulated the appearance of apoptotic bodies (or ball-shaped swelling of cells with loss of microvilli) (Figure 6A, c and d). Incubating cells with extract A-b also modified the morphology of cancer cells similarly to extract T-010, but with ball-shaped swelling of the cells without loss of microvilli. (Figure 6A, e and f). In addition to similar changes, the UB-010 extract contributed to the formation of two types of apoptotic bodies: with remaining microvilli, and in the absence of them (Figure 6A, g and h). Treatment of colon cancer cells with the T-b extract at IC50 concentrations caused the appearance of apoptotic bodies (Figure 6A, i and j), but at high concentrations, twice the IC50, caused the cells to dry out and only their membrane remained ( Figure 6A, k and l).

Molecular mechanisms of polysaccharide extracts from wheat cell cultures

Immunoblotting assays revealed variations in proteins involved in apoptosis, cell cycle, and differentiation pathways (Figure 7), which allowed predicting the mechanism of action of the extracts.

c-Myc

The expression of c-Myc protein (Figure 7A) was analyzed with respect to the relative value of the control (1). The Tf, Af and UB-010 extracts showed a significant decrease in the expression of this protein, with values of 0.6, 0.7 and 0.7 respectively. Other extracts did not cause any significant change in c-Myc protein expression.

Beta-catenin

Two of the extracts (Tf and Af) presented a lower expression of beta-catenin (Figure 7B) compared to the control value (1). The Tf extract was reduced to 0.5, and the Af extract to 0.7. The rest of the extracts did not show significant changes.

NFkB p100

Extracts T-f, A-b, A-f and UB-010 reduced the expression of the NFkB p100 protein (Figure 7C), the early form of the NFkB p52 protein. Extract T-f decreased the expression of this protein up to 0.65, and extracts A-b, A-f and UB-010 decreased it up to 0.8. Extracts T-010 and T-b had no significant effect on NFkB p100 expression.

HCAM ( CD44)

All extracts except T-010 caused a significant decrease in HCAM (CD44) protein expression (Figure 7D). The T-b and UB-010 extracts decreased it to an intensity value of 0.5 compared to the control (1). Extracts T-f and A-b decreased it to 0.2, and extract A-f to 0.7.

Bax

Activation of the bax protein was achieved with all PSs extracts (Figure 7E). The extracts T-b, T-f, A-b and A-f increased it to double (value of 2) the intensity of the control (1). Extracts UB-010 and T-010 caused a greater increase, of 3.5 and 5.2, respectively.

Cytochrome C

Immunoblot assays allowed us to detect the extent to which cytochrome C is released into the cytosol (Figure 7F). The release was triggered by four extracts: T-b, T-f, A-b and T-010. Extract T-b caused double the release, extract T-f increased it to 1.6, extract A-b to 1.7, and extract T-010 to 1.6. Extracts A-f and UB-010 did not cause a significant change.

Caspase3

The increase in normalized pro-caspase 3 expression values (Figure 7G) were greater compared to the control (1) after treatment with each of the extracts. The values obtained for cells treated with Tb, Tf, Ab, Af, UB 010, T-010 were, respectively, 1.9; 2.0; 1.5; 1.7; 1.15 and 1.25.

Caspase 8

In four of the extracts, namely T-b, T-f, A-b and T-010, the expression values of caspase 8 increased up to 1.5; 1.4; 1.57; and 1.3, respectively. Extracts UB-010 and A-f did not cause increased release of caspase 8 (Figure 7H).

Therefore, all extracts showed an influence on the increase in the expression of bax proteins and caspase 3. It was also established that extracts T-b, T-f, A-b, T-010 caused a significant increase in cytochrome C levels and caspase 8. Extracts T-f, UB-010 and A-f induced the reduction of c-Myc expression. Beta-catenin expression was inhibited by T-f and A-f extracts. Treating the cells with extracts A-f, T-f, UB-010 and A-b caused a reduction of the NFkB p100 protein. There were also considerable differences in the expression of NFkB p52 when treated with extracts B-010 and T-010 (not shown in the figures). Finally, all extracts decreased CD44 expression.

Discussion

It is widely known that natural PSs are often characterized based on their molecular weight, monosaccharide composition, functional groups, and chemical bonds [77]. All these characteristics help to predict the biological activity of these polysaccharides, although the isolation, purification, and fractionation processes can affect them [78]. However, given the difficulty of accurately identifying the structure of natural PSs due to their complexity and variability, structural analysis can be skipped to focus on determining their bioactivity in vitro and in vivo [7]. Therefore, in the present invention the central focus has been to determine the importance of the growth conditions of cell cultures for obtaining PSs with different biological effects, specifically on the processes of apoptosis, differentiation and cell proliferation.

In this sense, different polysaccharide extracts from suspension cultures were obtained by modifying the culture times and media using Triticum aestivum, whose physicochemical properties, chemical composition, molecular weight, and differences in composition of the extracts have previously been studied [61,62,63]. Of the rest of the extracts, obtained after a longer cultivation time, no monosaccharide composition analysis has been carried out, the priority being to know their biological activity. In previous studies it has been discussed that the time of plant cell culture could alter the resulting chemical compounds in the medium [79]. In fact, when rice was cultured in suspension for 3 weeks, a considerable antitumor effect was achieved, which the authors explained by the appearance of secondary metabolites during prolonged cultures [80]. Therefore, it was assumed that there would be a difference in the activity of PSs obtained from wheat cells when they were cultured for longer periods.

Because the different extracts presented different levels of activity, different doses of them were needed to achieve the inhibitory effect on the cells. In general, A extracts were less toxic to healthy fibroblast cells in a dose-dependent manner, while T extracts showed an inverted dose-response effect, where lower concentrations inhibit healthy cells to a greater extent, and vice versa.

In this study, differences were identified in the mechanisms of action and in the morphological changes at the cellular level caused by the different PS extracts. For most of the extracts, the IC50 could be determined, and it could be achieved even at very low concentration values, even 10 ^g/ml for extract A-f, 160 ^g/ml for extract A-b, and between 50 and 100 ^g/ml for extract T-b. Extracts T-010, UB-010 and T-f showed a much higher IC50 (1600 ^g/ml).

Overall, A extracts were found to have considerable therapeutic indices and were less toxic to healthy cells, and T extracts affected cancer cells 2.35 to 2.86 times more than healthy cells at IC50 concentrations.

Furthermore, during flow cytometry analyses, it was revealed that treatment of colon cancer cells with all polysaccharide extracts caused cell cycle arrest in the G0/G1 phase. Furthermore, Af and Tf extracts decreased the percentage of cells in the G2/M phase, while Ab, UB-010 and T-010 extracts increased it.

Regarding the molecular mechanisms, apoptosis assays were carried out that showed that extracts A-f and T-f did not cause changes in either the level of apoptosis or cell fragmentation compared to the control. Meanwhile, some extracts did induce apoptosis: after 48h of exposure, the T-010 extract at 1600 ^g/ml induced apoptosis in 48% of the cells; 160 ^g/ml of extract A-b at 40%; 1600 ^g/ml of 13.5% UB-010 extract; and 100 ^g/ml of the 16.7% T-b extract; while the controls only had 4.5% apoptosis. The percentage of cellular remains varied between 20 and 60%, higher than in the controls (10%).

MET analyzes also confirmed apoptosis in extracts T-010, A-b, UB-010 and T-b. Numerous signs of apoptosis were found: chromatin fragmentation and condensation [29], large and damaged mitochondria with disorganized membranes, degraded mitochondria, autophagic vacuoles with particles inside, swollen Golgi apparatus, absence of nuclear membrane, blisters and multilamellar bodies. . According to TEM analyses, the accumulation of autophagic vacuoles with a single, double or triple membrane could mean the blockage of autophagic flux, implying that the cell is dying [81,82]. Notably, the size of mitochondria was significantly changed by extract T-b, and the number of damaged mitochondria with missing or damaged cristae increased with three extracts (T-010, UB-010 and T-b). Using SEM, apoptotic bodies and a decrease in the number of villi were detected under the effect of PSs.

Regarding the molecular mechanisms, in this study the increase of several proteins in HCT-116 cells was detected: Bax and released cytochrome C, related to the intrinsic pathway; caspase 8, related to the extrinsic pathway; and the final executor of apoptosis of both pathways, caspase 3.

Four extracts, T-010, A-b, T-b and T-f, increased the level of caspase 8, indicating that they activate the extrinsic pathway of apoptosis, with the exception of extract T-f which, according to the results of the cell cycle assays, did not causes apoptosis.

It was also revealed that Tf and Af extracts inhibited the expression of beta-catenin protein, suggesting a blockade of cyclin D1, the APC gene pathway, limiting cell proliferation [38]. These same extracts, together with the UB-010 extract, decreased the levels of c-Myc and thus confirming the growth inhibition cellular in HCT-116 colorectal cancer cells. Since the Tf and Af extracts did not show signs of apoptosis or other types of cell death, they would have an active role in the differentiation processes given the decrease in c-Myc and beta-catenin. These same extracts also showed low levels of CD44, confirming this fact. In addition, they also block the cell cycle at all its points: G0/G1, S and G2/M. The Tf extract, for its part, caused a high release of cytochrome C without causing any type of cell death, a protein that can also participate in differentiation processes [44].

Regarding the CD44 protein, it was found that all extracts except T-010 attenuated its expression, highlighting its ability to induce the differentiation of cancer cells, and also contributing to reducing progression and metastasis.

Finally, it was shown that extracts T-f, A-f, A-b and UB-010 reduced the levels of NFkB 2 p100, and T-010 and UB-010 those of the p52 protein, inhibiting the alternative NFkB pathway and thereby also inducing the differentiation of cancer cells, along with apoptosis in the case of A-b and UB-010.

Regarding the antifibrotic potential, the results obtained by the inventors showed that PS extracts from wheat cell culture, specifically extracts T-010, T-b, A-b and UB-010, induced the accumulation of cells in the G2/ phase. M of the cell cycle. Furthermore, they confirmed apoptosis by detecting the cleavage of caspases 3 and 8 in these extracts. They also showed that beta-catenin and c-Myc levels were reduced. Finally, especially T-010, UB-010 and T-f extracts have been shown to inhibit the growth of CCD-18CO fibroblast cells. These observations, together with the fact that PSs contain galactose, which is capable of binding to the terminal sugar-binding end of the gal-3 protein, indicate that PSs extracts from cell cultures act as inhibitors of gal-3.

This allows us to conclude that the different extracts, which differ in the culture medium (being extracts A or T) and in the culture time of the embryogenic calli and subsequently of the cells in suspension, present different levels of cytotoxicity and activate different processes and routes. A extracts tend to be more effective in killing cancer cells at lower concentrations in addition to having less cytotoxicity to healthy cells. T extracts require higher doses to combat cancer cells, but their effect and that of the alkaline extract UB-010 on healthy fibroblasts show promising potential for the treatment of fibrosis.

The polysaccharides in the extracts derived from short- and medium-term cultures induce apoptosis through the intrinsic and extrinsic pathways (with the exception of UB-010), and would also contribute to the cellular differentiation of cancer cells. The polysaccharide extracts obtained after long-term cultivation inhibit APC and NFkB pathways and reduce CD44 levels, suggesting their role as differentiating agents.

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Claims (27)

1. Polysaccharide extraction procedure, hereinafter “process of the invention”, which comprises the ethanolic extraction of extracellular polysaccharides from a cell culture of cereal embryogenic callus tissue. 2. Procedure for obtaining polysaccharide extracts according to the previous claim, which includes the steps of: a) Plant an immature embryo on solid agar medium to obtain an embryogenic callus. b) Cultivate tissue from said embryogenic callus on solid agar medium. c) Cultivate cells from step b) in suspension with the addition of at least one phytohormone. d) Extraction of the extracellular polysaccharides from step c) by ethanolic extraction. and) 3. Procedure according to any of the previous claims, where the cereal used is wheat 4. Procedure according to any of the previous claims, where the cereal used is Triticum aestivum. 5. Procedure according to any of claims 2 to 4, which comprises an additional step of fractionating the polysaccharide extract from step d), to obtain the alkaline extract. 6. Method according to any of claims 2 to 5, wherein the embryogenic callus selected/obtained in step a) has a friable morphology, preferably a long-term friable morphology. 7. Procedure according to any of claims 2 to 6 wherein the callus culture from step a) is carried out for between 10 days and 24 months, preferably between 10 and 365 days, and more preferably for 28 or 70 days. 8. Method according to any of claims 2 to 7, wherein the means of Solid agar from step a) is Murashige and Skoog in the presence of 2,4-didorophenoxyacetic acid at a concentration between 0.1 and 20 mg/mL. 9. Procedure according to any of claims 2 to 8 where the cells from step c) are cultured for between 5 days and 6 months. 10. Procedure according to any of claims 2 to 9 where the cells from step c) are cultured with a photoperiod of 16 hours. 11. Procedure according to any of claims 2 to 10, wherein the suspension agar medium in step c) is Murashige and Skoog in the presence of 2,4-dichlorophenoxyacetic acid at a concentration of between 0.1 and 20 mg/mL, preferably at concentration 5.0 mg/mL. 12. Procedure according to any of claims 2 to 11, wherein the suspension agar medium in step c) is Murashige and Skoog in the presence of abscisic acid at a concentration of between 0.1 and 10 mg/mL, preferably at concentration 1, 0 mg/mL. 13. Extracts of extracellular polysaccharides obtained by the procedure according to any of claims 1 to 12. 14. Extracts of extracellular polysaccharides according to the previous claim, for use as a medicine. 15. Extracts of extracellular polysaccharides according to claims 13 or 14, for use in the improvement, relief, prevention and/or treatment of cancer. 16. Extracts of extracellular polysaccharides according to the previous claim, where the type of cancer is colorectal cancer. 17. Pharmaceutical composition that comprises in its formulation at least one extract of extracellular polysaccharides according to any of claims 13 to 16. 18. Pharmaceutical composition according to the preceding claim, comprising minus one additional antitumor agent. 19. Pharmaceutical composition according to any of claims 17 or 18, which additionally comprises one or more pharmaceutically acceptable excipients or vehicles. 20. Pharmaceutical composition according to any of claims 17 to 19, for use as a medicine. 21. Pharmaceutical composition according to the preceding claim, for use in the improvement, relief, prevention and/or treatment of cancer. 22. Pharmaceutical composition according to the previous claim, wherein the type of cancer is colorectal cancer. 23. Extracellular polysaccharide extracts according to claims 13 to 16, for use in the improvement, relief, prevention and/or treatment of fibrosis. 24. Pharmaceutical composition according to claim 17, comprising at least one additional antifibrotic agent. 25. Pharmaceutical composition according to any of claims 17 or 24, which additionally comprises one or more pharmaceutically acceptable excipients or vehicles. 26. Pharmaceutical composition according to any of claims 17, 24 or 25, for use in medicine. 27. Pharmaceutical composition according to the preceding claim, for use in the improvement, relief, prevention and/or treatment of fibrosis.