Preliminary Assessment of the Protective and Antitumor Effects of Several Phytoene-Containing Bacterial and Microalgal Extracts in Colorectal Cancer

Citation: Perazzoli, G.; Luque, C.; León-Vaz, A.; Gómez-Villegas, P.; Rengel, R.; Molina-Márquez, A.; Morón-Ortiz, Á.; Mapelli-Brahm, P.; Prados, J.; Melguizo, C.; et al. Preliminary Assessment of the Protective and Antitumor Effects of Several Phytoene-Containing Bacterial and Microalgal Extracts in Colorectal Cancer. Molecules 2024,29, 5003. https://doi.org/10.3390/ molecules29215003 Academic Editor: Wing-Leung Wong Received: 11 September 2024 Revised: 16 October 2024 Accepted: 18 October 2024 Published: 22 October 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). molecules Article Preliminary Assessment of the Protective and Antitumor Effects of Several Phytoene-Containing Bacterial and Microalgal Extracts in Colorectal Cancer Gloria Perazzoli 1,2,3, Cristina Luque 1, Antonio León-Vaz 4, Patricia Gómez-Villegas 4, Rocío Rengel 4, Ana Molina-Márquez 4,Ángeles Morón-Ortiz 5, Paula Mapelli-Brahm 5, JoséPrados 1,2,3 , Consolación Melguizo 1,2,3 , Antonio Meléndez-Martínez 5and Rosa León4,* 1Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research Center (CIBM), 18100 Granada, Spain; [email protected] (G.P.); [email protected] (C.L.); [email protected] (J.P.); [email protected] (C.M.) 2Instituto de Investigación Biosanitaria de Granada, Ibs. GRANADA, 18012 Granada, Spain 3Department of Anatomy and Embryology, University of Granada, 18016 Granada, Spain 4 Laboratory of Biochemistry, Center for Natural Resources, Health and Environment (RENSMA), University of Huelva, Avda. de las Fuerzas Armadas s/n, 21071 Huelva, Spain; [email protected] (A.L.-V.); [email protected] (P.G.-V.); anamariamolinamar[email protected] (A.M.-M.) 5Food Color and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (Á.M.-O.); [email protected] (P.M.-B.); [email protected] (A.M.-M.) *Correspondence: [email protected] Abstract: The identification of new functional food constituents is a priority to improve the prognosis and prevention of colorectal cancer (CRC). In this study, several bacterial and algal phytoeneenriched extracts were obtained, and their potential activity against oxidative damage and their ability to inhibit proliferation and cell migration in several human colon-adenocarcinoma-derived cell lines were assessed. The main conclusions indicate that total extracts of Sphingomonas echinoides and Chlorella sorokiniana exhibited the highest protective effect against oxidative damage. All extracts enhanced the activity of detoxifying enzymes, particularly importantly the increase of NAD(P)H:quinone oxidoreductase activity, which reached a value 40% higher than that of untreated control cells upon exposure to Escherichia coli extracts. Staphylococcus haemolyticus and transgenic E. coli extracts significantly arrested the migration capacity of both cell lines, while S. haemolyticus and C. sorokiniana extracts inhibited cell proliferation by 15 to 20% compared to untreated cells. These results point to these extracts as potential antioxidant complements able to protect cells against oxidative damage and with a moderate ability to inhibit the proliferation and migration of CRC tumor cells, paving the way to design functional foods or probiotic formulations with preventive properties against oxidative stress-related diseases, such as cancer, or as starting point for purifying anticancer compounds. Keywords: antitumor extracts; colorless carotenoids; colorectal carcinoma; healthy diets; microalgae; phytoene 1. Introduction The current treatment of CRC (colorectal cancer), the third most common type of cancer and second leading cause of cancer-related mortality, is mainly based on surgery or systemic chemotherapy and/or radiotherapy treatments. However, chemotherapeutic agents (i.e., 5-fluorouracil, capecitabine, oxaliplatin, irinotecan, etc.) show serious limitations, including a large number of adverse effects that exert a substantial impact on the general well-being of the patient, which may require the interruption of treatment [ 1 ]. Taking into account the high frequency of this type of cancer, its high mortality, and the foreseeable increase in its incidence over the years [ 2 ], the development of new therapeutic strategies and the Molecules 2024,29, 5003. https://doi.org/10.3390/molecules29215003 https://www.mdpi.com/journal/molecules Molecules 2024,29, 5003 2 of 17 identification of new functional nutrient components is essential for prevention and for improving the prognosis of patients with CRC. Fruits and vegetables contain bioactive compounds with antioxidant, anti-inflammatory, and anti-aging properties which have been demonstrated to enhance the effect of antitumor classical drugs [ 3 , 4 ]. Among these bioactive phytochemicals, carotenoids, versatile isoprenoid compounds biosynthesized by all higher plants and algae, as well as by some bacteria, archaea, fungi, and arthropods, have received considerable attention. The importance of carotenoids in food science and nutrition has been based for many years in their role as natural colorants and their ability to combat vitamin A deficiency, as some of them are precursors of this essential nutrient. However, a growing body of evidence indicates that carotenoids can benefit human health in several ways [ 5 ]. Their dietary intake has been related to a reduced incidence of several chronic diseases, such as obesity, diabetes, cardiovascular disease, neurodegenerative disorders, macular degeneration, and some types of cancer. Mechanistically, the primary benefits of carotenoids can be explained by their antioxidant potential, either interacting directly with reactive oxygen species or through the induction of the expression of antioxidant enzymes. However, carotenoids may also provide health benefits through their conversion into vitamin A, absorption of light, modulation of gene expression, modulation of communication between cells, or interaction with the microbiota, among others [6,7]. Carotenoids are also very important in feeds for aquatic and terrestrial animals which, like human beings, are not able to synthesize carotenoids de novo but need to acquire them from the diet [ 8 ]. There are around one thousand cataloged carotenoids, of which only very few are colorless. The two main dietary colorless carotenoids are phytoene and phytofluene, which are the precursors of virtually all carotenoids. Although much attention has been paid to colored carotenoids, uncolored ones have been largely neglected in studies related to food science and technology, nutrition, or health. However, it is now clear that they are present in commonly consumed foods, are readily bioavailable, and can be involved in health-promoting biological actions and even provide aesthetic benefits [ 9 ]. Phytoene and phytofluene are present in food staples, such as tomatoes, carrots, apricots, or citrus, among others, and contribute significantly to total carotenoid intake, as demonstrated in two countries with different dietary patterns: Luxembourg and Spain. Particularly, phytoene was the second most consumed carotenoid in both countries [10,11]. In addition to traditional plant foods, microorganisms of different origins can be alternative sources of colorless carotenoids and other secondary metabolites. Along with their higher growth rate and productivity compared to plants, their easier metabolic modulation, their ability to grow in harsh conditions, and their contribution to CO 2 sequestration and wastewater bioremediation [ 12 ], some are part of the human and animal microbiota and can synthesize essential compounds with health-promoting effects [13]. Despite their already commented on importance in the diet, there are still few studies addressing the potential health benefits of colorless carotenoids. This is especially surprising as one of the earliest works reporting the possible antitumor activity of carotenoids featured phytoene, which was reported to retard the appearance of UV-B-induced skin tumors and reduce their number in a rodent model [ 14 ]. Possible anti-prostate cancer activities of phytoene have also been reported in rodents [ 15 ]. Regarding absorbing UV light, there are several studies suggesting that the colorless carotenoids phytoene and phytofluene, on their own or from tomato products, can protect against light-induced damage, as shown in guinea pigs [ 16 ] and humans [ 17 , 18 ]. More recently, there has been evidence that phytoene, phytofluene, and lycopene can protect against nicotine-induced oxidative stress-mediated pancreatic islet dysfunction in rats. The nicotine-induced changes in fasting blood glucose, oral glucose tolerance, and glycated hemoglobin levels were reduced by the intake of the individual carotenoids and a combination of them [ 19 ]. On the other hand, phytoene and phytofluene have been shown to account for ~25% of the carotenoids in human adipose tissue, and their content in such tissue is inversely correlated with diastolic Molecules 2024,29, 5003 3 of 17 blood pressure [ 20 ]. All these precedents suggest that new uncommon colorless carotenoid sources can provide new therapeutic adjuvant therapies and new ingredients of sustainable healthy diets for CRC prevention. Although phytoene is usually present in small amounts as a biosynthetic intermediate, its accumulation has been reported to be induced in higher plants [ 21 ] and microalgae [12,22] by treatment with bleaching herbicides, such as norflurazon, which causes inhibition of the enzyme phytoene desaturase by competition with its cofactors [ 23 ]. On the other hand, although high phytoene-producing bacteria are mostly unknown, the bacterial species Sphingomonas echinoides and Staphylococcus haemolyticus have been described as able to accumulate phytoene. S. echinoides (strain B-3127) forms colorless colonies and is a rarity within species of the Sphingomonas genus, which are typically pigmented by colored carotenoids [ 24 ]. Staphylococcus haemolyticus is a coagulase-negative Staphylococcus species (CoNS), found for the first time on the skin of healthy individuals. Coagulase is considered a virulence factor, hence CoNS are usually regarded as part of the normal human mucous membrane microbiota and are mostly harmless [ 25 ]. In addition, the enterobacterium E. coli, which does not naturally produce carotenoids, can be genetically modified to express genes from carotenogenic bacteria or plants, generating recombinant strains specifically designed to accumulate the desired carotenoids, including phytoene [26,27]. In this study, methanolic extracts from phytoene-accumulating bacteria and from the microalga Chlorella sorokiniana, specially treated to induce the accumulation of phytoene, were characterized. Their potential activity against oxidative damage, their ability to inhibit proliferation and cell migration in several human colonic adenocarcinoma-derived cell lines, and their potential ability to induce the activity of detoxifying antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase and the glutathione S-transferase, were assessed as part of a preliminary study on their protective and antitumor effects. 2. Results 2.1. Obtaining Functional Extracts from Phytoene-Enriched Algal and Bacterial Biomass In this study, four functional extracts were obtained from different phytoene-enriched bacterial and microalgal cells. Bacterial extracts were obtained from recombinant E. coli bacteria harboring the plasmid pAC-PHYTipi and from the Gram-negative soil bacterium Sphingomonas echinoides and the Gram-positive commensal bacterium Staphylococcus haemolyticus, pinpointed as good bacterial sources of phytoene by previous screening. The algal extract was obtained from the chlorophyte microalga Chlorella sorokiniana, which was chemically induced to accumulate phytoene. The microalga C. sorokiniana, which in normal conditions does not accumulate phytoene, can be treated with the bleaching herbicide norflurazon to block the carotenoid biosynthetic pathway at the level of phytoene desaturase and induce the accumulation of uncolored carotenoids. To determine the optimal non-lethal norflurazon-inducing doses, well-grown cultures (about 1 µ g DW biomass mL −1 ) were harvested by centrifugation, suspended in fresh TAP culture medium with growing concentrations of the herbicide norflurazon, between 0.5 and 10 µ g L −1 , and incubated for 48 h. After this incubation, the cultures were harvested by centrifugation at 4400 rpm, and the carotenoids were extracted and analyzed as indicated in Section 4.2 (Figures S1 and S2). It was concluded that treatment with 1 µ g mL −1 of the herbicide for 48 h produced an algal biomass with 6.56 ±0.13 mg g−1 DW of 15Z-phytoene, which represented about 61% of the total carotenoids accumulated by the microalgae. Although the specific growth rate decreased to about 70% of the value observed for the control culture without norflurazon, the microalga retained its growth capacity (Table 1). For higher concentrations of norflurazon, the intracellular concentration of phytoene was lower, as was the specific growth rate and the productivity of total carotenoids, due to the toxic effect of the herbicide. Molecules 2024,29, 5003 4 of 17 Table 1. Assessment of the optimal non-lethal concentration of norflurazon to induce the accumulation of phytoene in C. sorokiniana after 48 h of incubation. Norflurazon (µg mL−1) 15Z-Phytoene (mg g−1DW) Total Carotenoids (mg g−1DW) Specific Growth Rate µ (h−1) 0 0 14.92 ±0.04 0.055 ±0.006 0.5 6.45 ±0.46 10.98 ±0.77 0.042 ±0.002 1 6.56 ±0.13 10.67 ±1.06 0.038 ±0.005 1.5 4.97 ±0.03 10.47 ±0.21 0.026 ±0.013 2 0.92 ±0.49 6.74 ±0.43 0.023 ±0.008 5 0.32 ±0.02 5.56 ±0.03 0.019 ±0.003 10 0.1 2 ±0.02 5.59 ±0.14 0.015 ±0.001 Specific growth rate, content of total carotenoids, and phytoene in C. sorokiniana cultures treated with increasing concentrations of the herbicide norflurazon. Values are the mean of three independent determinations ± standard deviation. DW: dry weight. Regarding the bacteria, E. coli harboring the plasmid pAC-PHYTipi, S. echinoides, and S. haemolyticus, cultured as indicated in Materials and Methods, contained 80.2 ±12 , 306.02 ±8.63 , and 11.03 ± 0.93 µ g g −1 of 15Z-phytoene, at the end of the exponential phase of growth, respectively (Table 2). The three bacterial strains accumulated phytoene as the only carotenoid: the all-trans phytoene level was undetectable in E. coli and S. haemolyticus, while it represented around 10% of total phytoene in the case of S. echinoides (Figures S3–S5). Table 2. Intracellular concentration of carotenoids in the bacterial species studied. Bacterial Species Culture Conditions Phytoene (µg g−1DW) (15Z) (All-E) E. coli with pAC-PHYTipi plasmid LB, Chl (30 µg mL−1), 28 ◦C80.2 ±12 n.d. Sphingomonas echinoides TGY, 26 ◦C 11.03 ±0.93 n.d. Staphylococcus haemolyticus NB, 37 ◦C 306.02 ±8.63 30.17 ±1.19 Content of phytoene in three different bacterial strains, cultured at the indicated conditions. Values are the mean of three independent determinations ± standard deviation. DW: dry weight. Chl: chloramphenicol. n.d.: not detected. Functional extracts were obtained from the indicated algal and bacterial strains, grown (as detailed in Section 4.2) at the optimal conditions for phytoene accumulation, and the content of phytoene and total carotenoids was newly determined in these extracts. The concentration of phytoene in the obtained extracts was 2- and 2.7-fold higher than that in the microbial biomass in the case of S. echinoides and S. haemolyticus, respectively, and 2.5-fold higher in the case of E. coli and the induced microalga Chlorella sorokiniana (Table 3). Table 3. Concentration of carotenoids in the functional extracts obtained. Species 15Z-Phytoene (µg g−1) All-E-Phytoene (µg g−1) Total Carotenoids (µg g−1) Phytoene-enriched E. coli 193 ±28 n.d. 193 ±28 Sphingomonas echinoides 22.06 n.d. 22.06 Staphylococcus haemolyticus 765.05 75.43 840.48 Phytoene-enriched Chlorella 10,335 ±90 n.d. 16,670 ±180 Content of phytoene isomers and total carotenoids (TCCs) in four functional extracts obtained from the indicated species. Values are the mean of three independent determinations ± standard deviation. n.d.: not detected; DW: dry weight. Molecules 2024,29, 5003 5 of 17 2.2. Protective Effect of the Extracts Against Oxidative Damage To evaluate the protective ability of the obtained extracts against oxidative damage in T84 CRC cell lines, the cells were subject to pre-treatment for 24 h with two non-toxic doses of each extract and subsequently exposed to oxidative damage by incubation with two doses (1.2 mM and 1.5 mM) of H2O2. These experiments revealed that extracts from E. coli (pAC-PHYTipi), S. echinoides, and C. sorokiniana exhibited a statistically significant protection ability against the oxidative damage caused by H 2 O 2 in the T84 CCRC cell line (Figure 1) (p< 0.05). Specifically, E. coli (pAC-PHYTipi) extract at 0.01 µ g mL −1 resulted in 6.6% cellular protection from the oxidative damage caused by 1.5 mM H 2 O 2 (p< 0.05), while S. echinoides extract exhibited between 9.3% and 11.9% cell protection across all conditions tested (p< 0.01). Furthermore, C. sorokiniana at 0.01 µ g mL −1 resulted in 10% and 7% cellular protection in treatments with 1.2 mM and 1.5 mM H 2 O 2 , respectively (p< 0.05). No significant protective activity of the S. haemolyticus extract was noted. It was also observed that increasing the extract concentration from 0.01 to 0.1 µg mL−1did not improve protection against oxidative damage. Molecules 2024, 29, x FOR PEER REVIEW 5 of 17 Content of phytoene isomers and total carotenoids (TCCs) in four functional extracts obtained from the indicated species. Values are the mean of three independent determinations ± standard deviation. n.d.: not detected; DW: dry weight. 2.2. Protective Effect of the Extracts Against Oxidative Damage To evaluate the protective ability of the obtained extracts against oxidative damage in T84 CRC cell lines, the cells were subject to pre-treatment for 24 h with two non-toxic doses of each extract and subsequently exposed to oxidative damage by incubation with two doses (1.2 mM and 1.5 mM) of H2O2. These experiments revealed that extracts from E. coli (pAC-PHYTipi), S. echinoides, and C. sorokiniana exhibited a statistically significant protection ability against the oxidative damage caused by H2O2 in the T84 CCRC cell line (Figure 1) (p < 0.05). Specifically, E. coli (pAC-PHYTipi) extract at 0.01 µg mL−1 resulted in 6.6% cellular protection from the oxidative damage caused by 1.5 mM H2O2 (p < 0.05), while S. echinoides extract exhibited between 9.3% and 11.9% cell protection across all conditions tested (p < 0.01). Furthermore, C. sorokiniana at 0.01 µg mL−1 resulted in 10% and 7% cellular protection in treatments with 1.2 mM and 1.5 mM H2O2, respectively (p < 0.05). No significant protective activity of the S. haemolyticus extract was noted. It was also observed that increasing the extract concentration from 0.01 to 0.1 µg mL−1 did not improve protection against oxidative damage. Figure 1. Analysis of the protective potential of methanolic extracts obtained from E. coli (EC), S. echinoides (SE), C. sorokininana (CS), and S. haemolyticus (SH). An in vitro antioxidant activity was performed on the T84 cell line pretreated with non-toxic doses (0.01 and 0.1 µg mL−1) of each extract Figure 1. Analysis of the protective potential of methanolic extracts obtained from E. coli (EC), S. echinoides (SE), C. sorokininana (CS), and S. haemolyticus (SH). An in vitro antioxidant activity was performed on the T84 cell line pretreated with non-toxic doses (0.01 and 0.1 µ g mL −1 ) of each extract and then subjected to oxidative damage by exposure to H 2 O 2 (1.2 and 1.5 mM). Data are shown as the mean ± SD of eight independent replicates. * p< 0.05, ** p< 0.01, and *** p< 0.001 compared to cells treated with H2O2at the doses shown. Molecules 2024,29, 5003 6 of 17 2.3. Effect of the Extracts on the Activity of Detoxifying Enzymes The possible effect of the different extracts on the activity of two detoxifying enzymes, NAD(P)H:quinone oxidoreductase (QR) and glutathione S-transferase (GST), was tested by exposing HT29 human colonic adenocarcinoma cells to non-toxic doses (0.01 µ g mL −1 ) of the extracts for 48 h of treatment (Figure 2). Our results show that exposure to E. coli (pAC-PHYTipi), S. haemolyticus, and C. sorokiniana extracts resulted in a significant increase in QR enzyme activity compared to untreated control cells, increasing this activity by 1.42, 1.3, and 1.2, respectively (p< 0.05). For the enzyme GST, interesting results were also obtained, as E. coli (pAC-PHYTipi), S. haemolyticus, and S. echinoides extracts significantly induced the activity of this enzyme. S. echinoides extracts showed the highest induction ability, causing an increase in GST activity by 1.34 times compared to the control (p< 0.001). Molecules 2024, 29, x FOR PEER REVIEW 6 of 17 and then subjected to oxidative damage by exposure to H2O2 (1.2 and 1.5 mM). Data are shown as the mean ± SD of eight independent replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to cells treated with H2O2 at the doses shown. 2.3. Effect of the Extracts on the Activity of Detoxifying Enzymes The possible effect of the different extracts on the activity of two detoxifying enzymes, NAD(P)H:quinone oxidoreductase (QR) and glutathione S-transferase (GST), was tested by exposing HT29 human colonic adenocarcinoma cells to non-toxic doses (0.01 µg mL−1) of the extracts for 48 h of treatment (Figure 2). Our results show that exposure to E. coli (pAC-PHYTipi), S. haemolyticus, and C. sorokiniana extracts resulted in a significant increase in QR enzyme activity compared to untreated control cells, increasing this activity by 1.42, 1.3, and 1.2, respectively (p < 0.05). For the enzyme GST, interesting results were also obtained, as E. coli (pAC-PHYTipi), S. haemolyticus, and S. echinoides extracts significantly induced the activity of this enzyme. S. echinoides extracts showed the highest induction ability, causing an increase in GST activity by 1.34 times compared to the control (p < 0.001). Figure 2. Effect of the extracts on the activity of the detoxifying enzymes QR and GST. After 48 h of treatment with non-toxic doses of each extract (EC: E. coli (pAC-PHYTipi); SE: Sphingomonas echinoides; SH: Staphylococcus haemolyticus; CS: Chlorella sorokiniana), the activity of the QR and GST enzymes was determined. DL-sulforaphane (SFN) was established as a positive control. Results are shown as the mean ± standard deviation of three independent experiments. ** p < 0.01, and *** p < 0.001 compared to untreated cells. 2.4. Effect of Extracts on the Migration of T84 and HCT-15 CRC Cells Cell migration is a key process at the early stages of the complex tumor metastasis process; thus, we studied the effect of E. coli (pAC-PHYTipi), S. echinoides, S. haemolyticus, and C. sorokiniana extracts on cell migration (Figure 3). Figure 2. Effect of the extracts on the activity of the detoxifying enzymes QR and GST. After 48 h of treatment with non-toxic doses of each extract (EC: E. coli (pAC-PHYTipi); SE: Sphingomonas echinoides; SH: Staphylococcus haemolyticus; CS: Chlorella sorokiniana), the activity of the QR and GST enzymes was determined. DL-sulforaphane (SFN) was established as a positive control. Results are shown as the mean ± standard deviation of three independent experiments. ** p< 0.01, and *** p< 0.001 compared to untreated cells. 2.4. Effect of Extracts on the Migration of T84 and HCT-15 CRC Cells Cell migration is a key process at the early stages of the complex tumor metastasis process; thus, we studied the effect of E. coli (pAC-PHYTipi), S. echinoides,S. haemolyticus, and C. sorokiniana extracts on cell migration (Figure 3). Cell migratory capacity was measured in the two colon tumor cell lines (T84 and HCT-15) and the control non-tumor L929 murine fibroblast cell line by monitoring wound healing once per day during the assay for a period of 72 h. As shown in Figure 3, the extracts obtained from S. haemolyticus and S. echinoides triggered a significant decrease in HCT-15 tumor cell migration, with values of 9.3% at 24 h and 9.1% at 72 h, respectively. T84 cancer cell line migration was significantly reduced by E. coli (pAC-PHYTipi) extract, by up to 12.4% (p< 0.05) and C. sorokiniana extract, between 18.7% at 24 h and 8.9% at 72 h of treatment (p< 0.01). In contrast, migration of fibroblast L929 control cells was promoted by S. haemolyticus and S. echinoides by 28.6% and 27.2% at 72 h, respectively (p< 0.001). Molecules 2024,29, 5003 7 of 17 Molecules 2024, 29, x FOR PEER REVIEW 7 of 17 Figure 3. Wound-healing assay carried out to determine the influence of several bioactive extracts (EC: E. coli (pAC-PHYTipi); SE: Sphingomonas echinoides; SH: Staphylococcus haemolyticus; CS: Chlorella sorokiniana) on cell migration of two colon cancer cell lines: T84 (A) and HCT-15 (B), and the nontumor murine fibroblast cell line L929 (C) every 24 h over 72 h of treatment with non-toxic doses of each extract (0.1 µg mL−1 ). Results are expressed as the mean ± standard deviation of at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to control cells. Cell migratory capacity was measured in the two colon tumor cell lines (T84 and HCT-15) and the control non-tumor L929 murine fibroblast cell line by monitoring wound healing once per day during the assay for a period of 72 h. As shown in Figure 3, the extracts obtained from S. haemolyticus and S. echinoides triggered a significant decrease in HCT-15 tumor cell migration, with values of 9.3% at 24 h and 9.1% at 72 h, respectively. T84 cancer cell line migration was significantly reduced by E. coli (pAC-PHYTipi) extract, by up to 12.4% (p < 0.05) and C. sorokiniana extract, between 18.7% at 24 h and 8.9% at 72 Figure 3. Wound-healing assay carried out to determine the influence of several bioactive extracts (EC: E. coli (pAC-PHYTipi); SE: Sphingomonas echinoides; SH: Staphylococcus haemolyticus; CS: Chlorella sorokiniana) on cell migration of two colon cancer cell lines: T84 (A) and HCT-15 (B), and the nontumor murine fibroblast cell line L929 (C) every 24 h over 72 h of treatment with non-toxic doses of each extract (0.1 µ g mL −1 ). Results are expressed as the mean ± standard deviation of at least three independent experiments. * p< 0.05, ** p< 0.01, and *** p< 0.001 compared to control cells. 2.5. Effects of Extracts on Proliferation of T84 and HCT-15 Colon Cancer Cell Lines To evaluate if the four phytoene-rich functional extracts obtained exhibited antiproliferative activity, the T84 and HCT-15 colon cancer cell lines were exposed to increasing concentrations, from 5 to 100 µ g mL −1 , of the indicated extract (Figure 4). The effect of DMSO, used to dissolve the lyophilized extracts, was also analyzed and subtracted from the toxic effect of the corresponding extract. Our results indicate that neither the extracts obtained from E. coli nor those obtained from S. echinoides showed a significant antitumor Molecules 2024,29, 5003 8 of 17 effect on the T84 and HCT-15 cell lines below the dose of 100 µ g mL −1 . Only extracts obtained from C. sorokiniana and S. haemolyticus at a concentration of 100 µ g mL −1 exhibited antiproliferative activity in these two cell lines. Extracts from S. haemolyticus caused an inhibition of 15% and 20% on the relative proliferation of the T84 and HCT15 cell lines, respectively. Treatment with the extract obtained from the microalga C. sorokiniana inhibited the proliferation of T84 and HCT15 cells by 10% and 15%, respectively. Molecules 2024, 29, x FOR PEER REVIEW 8 of 17 h of treatment (p < 0.01). In contrast, migration of fibroblast L929 control cells was promoted by S. haemolyticus and S. echinoides by 28.6% and 27.2% at 72 h, respectively (p < 0.001). 2.5. Effects of Extracts on Proliferation of T84 and HCT-15 Colon Cancer Cell Lines To evaluate if the four phytoene-rich functional extracts obtained exhibited antiproliferative activity, the T84 and HCT-15 colon cancer cell lines were exposed to increasing concentrations, from 5 to 100 µg mL−1, of the indicated extract (Figure 4). The effect of DMSO, used to dissolve the lyophilized extracts, was also analyzed and subtracted from the toxic effect of the corresponding extract. Our results indicate that neither the extracts obtained from E. coli nor those obtained from S. echinoides showed a significant antitumor effect on the T84 and HCT-15 cell lines below the dose of 100 µg mL −1. Only extracts obtained from C. sorokiniana and S. haemolyticus at a concentration of 100 µg mL −1 exhibited antiproliferative activity in these two cell lines. Extracts from S. haemolyticus caused an inhibition of 15% and 20% on the relative proliferation of the T84 and HCT15 cell lines, respectively. Treatment with the extract obtained from the microalga C. sorokiniana inhibited the proliferation of T84 and HCT15 cells by 10% and 15%, respectively. Figure 4. Effect of functional phytoene-rich extracts obtained from the bacteria E. coli (pAC- PHYTipi), S. haemolyticus, and S echinoides and the chlorophyte microalga C. sorokiniana on relative proliferation of the colon cancer cell lines T84 and HCT-15 during 72 h of treatment. Data are shown as the mean ± standard deviation of at least three independent experiments. * p < 0.05 compared to control cells. 3. Discussion Colorectal cancer is a multifactorial malignant disease with increasing impact in developed countries, whose prevalence has been associated with lifestyle and dietary habits by different epidemiological studies [28]. Although some studies suggest that the Figure 4. Effect of functional phytoene-rich extracts obtained from the bacteria E. coli (pAC-PHYTipi), S. haemolyticus, and S echinoides and the chlorophyte microalga C. sorokiniana on relative proliferation of the colon cancer cell lines T84 and HCT-15 during 72 h of treatment. Data are shown as the mean ±standard deviation of at least three independent experiments. * p< 0.05 compared to control cells. 3. Discussion Colorectal cancer is a multifactorial malignant disease with increasing impact in developed countries, whose prevalence has been associated with lifestyle and dietary habits by different epidemiological studies [ 28 ]. Although some studies suggest that the intake of vegetables and fruits could be inversely related to colorectal cancer risk, there are contradictory conclusions, and it is still uncertain whether this positive association is true for men and women of all ages and ethnic backgrounds [ 28 , 29 ], or if it can be applied to all kinds of vegetables and fruits [ 30 ]. In addition, bacteria in the human microflora can produce bioactive compounds involved in the promotion of health and protection against different pathologies, including cancer. Several studies have demonstrated that alterations in the gut microbiota have a link with tumorigenesis, affecting the immune response or the effectiveness of antitumor agents [31]. Carotenoids are bioactive compounds existing in practically all natural fruits and vegetables, frequently together with other bioactive molecules such as flavonoids, S- compounds, and other bioactive components with well-known antioxidant and anti- Molecules 2024,29, 5003 9 of 17 inflammatory activities, which in many cases interact with each other or have synergic activities, usually making it a great challenge to investigate the activity of individual compounds [ 32 ]. Although the anti-cancer activity of carotenoids, either as isolated compounds or as food ingredients, has been widely studied, the potential activity of phytoene and other colorless carotenoids has been left behind [ 9 ]. We obtained extracts of different microalgal and bacterial cells that can accumulate phytoene to investigate their antitumor activity. These studies were performed using whole extracts, due to the importance of studying natural isomeric forms and the importance of the synergic interaction of different molecules present in natural extracts. Moreover, the present tendency in the fight against cancer is not only based on drugs, but also on healthy nutrients. Nutraceutical supplementation may contribute, together with classical chemotherapy, to successful anticancer therapy and can be essential in the response and compliance of patients [33]. In accordance with previous research, we found that treatment of the microalga Chlorella sorokiniana with the herbicide norflurazon induced the accumulation of phytoene [ 22 ]. After optimizing the herbicide doses, microalgal biomass with high concentrations of phytoene was obtained (Table 1). On the other hand, by the selective expression of relevant genes from carotenoid-producing bacteria in E. coli, a transgenic bacterial strain able to accumulate phytoene was obtained (Table 2). This strategy has been successfully used to obtain a large variety of recombinant bacteria able to accumulate different carotenoids [ 26 , 27 ] and has been widely used to carry out functional complementation assays and uncover the function of many genes of the carotenoid biosynthetic pathway [ 34 ]. Methanolic extracts of these manipulated microalgal and bacterial strains, in addition to the extracts of two natural bacteria Sphingomonas echinoides and Staphylococcus haemolyticus, were obtained, and their phytoene content was characterized (Table 3). The antioxidant activity of these extracts and their ability to inhibit proliferation and cell migration in two human colon adenocarcinoma-derived cell lines, line T84 and the drug-resistant line HCT-15, were studied. Oxidative stress is considered the primary cause of many human health disorders, including cancer [ 35 ]. The ability of the methanolic extracts obtained in this study to offer protection against the oxidative damage induced by H 2 O 2 in the T84 CRC cell line (Figure 1) is in agreement with previous data reported for extracts obtained from other microalgae, such as Chlorobotrys gloeothece,Chlorobotrys regularis, and Characiopsis aquilonaris, which showed high antioxidant activity, analyzed through DPPH assays, correlating in turn in C. regularis with a greater amount of β -carotene in breast cancer cell lines [ 36 ]. In general, the presence of significant amounts of pigments such as carotenes and chlorophylls, and vitamins, such as vitamin C, have been related to high antioxidant capacity, as demonstrated in Chlorella vulgaris [ 37 ]. The antioxidant and cell signaling role of carotenoids and their effects in the prevention of cancer have been widely explored and reviewed by several previous reports [38]. In addition, there is increasing evidence that one of the main mechanisms by which many nutritional bioactive components, including carotenoids, contribute to reducing the risk of cancer development is the induction of the activity of detoxifying enzymes, such as glutathione S-transferase (GST) and NAD(P)H:quinone reductase (QR), which play an essential role in the initial detoxification stages of numerous potentially harmful xenobiotics. High levels of these enzymes have been positively correlated with chemoprevention and cancer inhibition. Numerous plant and bacterial extracts containing natural products, such as triterpenoids, vitamins, or carotenoids, have been shown to induce the activity of these enzymes [ 39 , 40 ]. However, the effect of phytoene or phytoene-rich extracts on the activity of GST and QR detoxifying enzymes has not been studied so far. Our results showed that bioactive extracts with a high concentration of phytoene obtained from E. coli harboring the pACCRT-EB plasmid, Staphylococcus haemolyticus, and Chlorella sorokiniana-induced cells resulted in a significant increase in QR enzyme activity compared to untreated control cells. Similarly, E. coli (pAC-PHYTipi), Staphylococcus haemolyticus, and Staphylococcus haemolyticus extracts significantly induced the activity of the GST enzyme (Figure 2). Molecules 2024,29, 5003 16 of 17 25. Rossi, C.C.; Ahmad, F.; Giambiagi-deMarval, M. Staphylococcus haemolyticus: An updated review on nosocomial infections, antimicrobial resistance, virulence, genetic traits, and strategies for combating this emerging opportunistic pathogen. Microbiol. Res. 2024,282, 127652. [CrossRef] 26. Misawa, N.; Shimada, H. 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