Anticancer Effects in HT-29 Cells and Anti-Inflammatory Effects on Mouse Splenocytes of Broccoli Cultivated with Deep Sea Water Minerals in Organic Farming

Abstract

In this study, broccoli grown with deep sea water minerals (DSWMs) confirmed anticancer effects in HT-29 colorectal cancer cells and anti-inflammatory effects in C57BL/6 mouse splenocytes. Natural dream cultured broccoli (NB) grown with DSWMs had elevated sodium (Na) and calcium (Ca) levels and enhanced the expression of p53 and p21, both of which are associated with cell cycle arrest in HT-29 colorectal cancer cells. It also decreased the expression of Bax, Bad, Bim, Bak, caspase-9, and caspase-3. In lipopolysaccharide (LPS)-treated C57BL/6 mouse splenocytes, NB produced little nitric oxide (NO). ELISA research indicated that NB decreased IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 expression while increasing NK cell activity. As a result, broccoli cultivated with deep water minerals has better anticancer and anti-inflammatory properties than conventional and organic farming.

1. Introduction

Broccoli (Brassica oleracea L. spp. italica) is a Brassicaceae family vegetable widely consumed worldwide. It has been discovered that eating cruciferous vegetables reduces overall cancer risk, notably breast, colon, and lung cancer [1]. Broccoli contains several vitamins, including vitamins C and E, and other possibly helpful components, such as fiber, flavonoids, quercetin, and kaempferol glycosides [2]. Phytochemicals, such as glucosinolates and isothiocyanates, are also found in broccoli. Dietary isothiocyanates have been extensively examined for their effects on human health and are assumed to be responsible for the specific anticancer activity of cruciferous vegetables, such as broccoli, cabbage, and cauliflower. The major glucosinolate found in broccoli is hydrolyzed to sulforaphane [4-(methylsulfonyl) butyl isothiocyanate], which has been recognized as a potent anticancer agent in humans and animals and has antitumor action that also inhibits cell proliferation and promotes death in cancer cells [3,4].

In today’s world, agricultural management globally tends to favor organic farming systems. The main principles of organic farming involve abstaining from the use of chemical inputs, reducing soil contamination, and avoiding the use of machinery that compacts the soil [5]. The use of organic and biological inputs has been shown to increase health safety while reducing the use of chemical fertilizers and associated environmental risks [6,7]. Organic farming also contributes to health benefits such as improved farming conditions and increased content of antioxidants and vitamins [8]. However, the productivity, labor efficiency, and yield stability of organic farming are considerably lower than those of conventional farming systems. It was confirmed that when crops were grown using mineral fertilizers to improve this, productivity was increased compared with organic farming, and environmental risks associated with pesticide use were prevented [9]. Additionally, mineral fertilizers can affect soil microbial communities and consequently soil quality, which in turn affects plant growth, so it is important to better understand the effects of mineral fertilizers [10].

Deep sea water (DSW), defined as sea water deeper than 200 m, is generally low in temperature, high in cleanliness, rich in nutrients, and rich in minerals, such as calcium, magnesium, potassium, sodium, and zinc [11]. As a result, DSW is often used in aquaculture, agriculture, food processing, and cosmetics [12]. Due to over-cultivation in agriculture, the soil and crops have been depleted of nutrients in recent years, and DSW, which has a high mineral content and is free of artificial pollution, can be used as a water source to compensate [13]. Furthermore, DSW inhibited the metastasis of HT-29 colon and breast cancer cells. The anticancer effect of DSW is believed to be attributed to the ion action of several minerals found in DSW, such as calcium, magnesium, and potassium [14].

Colorectal cancer is the fourth most prevalent cancer killer, particularly among people aged 40–50 years [15]. The most prevalent signs of colorectal cancer are chronic inflammation and etiologies, such as chronic and increasing constipation, bloody stools, and gastrointestinal tract [16]. Most chemotherapeutic medicines used as adjuvant therapy for colorectal cancer treatment are nonspecific and can harm healthy tissues [17]. As a result, research into the interaction between new natural products that can replace existing chemotherapeutic medicines and plant secondary active metabolites in the suppression of human colorectal cancer is required [15]. As a result, in this study, HT-29 colorectal cancer cells, a human colon adenocarcinoma cell line, were used to evaluate several cell signaling pathways and techniques, such as cell cycle arrest and apoptosis [18].

Data from genetic, pharmacological, and epidemiological studies have revealed a relationship between inflammation and cancer. Inflammatory bowel illness is a major risk factor for colorectal cancer development, and inflammation is linked to different types of sporadic and hereditary colorectal cancer [19]. Inflammation is a biological response that protects the body through complicated systems that involve immune cells and inflammatory mediators generated by cells that act against infections and damaged cells. The inflammatory process eliminates the source of cell injury, eliminates necrotic cells, and restores tissues [20]. Several inflammatory mediators, including nitric oxide, prostaglandin E2 (PGE2), and pro-inflammatory cytokines, induce discomfort, fever, and redness throughout this process [21]. LPS, a key inflammatory component of gram-negative bacteria, induces a distinctive pattern of cytokine release that regulates inflammation and can activate NF-κB through several signal pathways to induce the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 [22]. LPS-mediated splenocyte activation produces reactive oxygen species (ROS), spleen-derived macrophages increase (NO) production, TNF-α, and NF-κB in response to LPS activation [23]. This study aims to investigate how broccoli cultivated with deep sea water minerals affected mRNA expression of apoptosis and cell cycle arrest genes in the HT-29 human colorectal cancer cell line and inflammatory cytokine expressions in C57BL/6 mouse splenocytes.

2. Materials and Methods

2.1. Broccoli Preparation

iCOOP Natural Dream Co. (Goesan, Chungcheongbuk-do, Korea) offered fresh broccoli. Organic broccoli (OB) and conventional broccoli (CB) were grown using conventional and organic farming methods, respectively, and natural dream broccoli (NB) was supplemented with two times foliar fertilization and five times soil drench by diluting 1000 times DSW minerals (DSWMs; Gurye, Jeollanamdo, Korea). Broccoli was cultivated in the same environment, specifically a 200-square-meter greenhouse. CB was cultivated using commonly used pesticides and chemical fertilizers. OB and NB were grown without artificial fertilizers or pesticides and were certified by the Republic of Korea’s Ministry of Agriculture, Food and Rural Affairs. The Korea Quality Testing Institute (Suwon, Gyeonggi-do, Korea) did a mineral content examination of DSWM, and the mineral concentrations were Cu at 0.87 mg/L, Na at 9565.36 mg/L, Mg at 60,000 mg/L, and Mn at 0.01 mg/L, Mo at 0.51 mg/L, Se at 0.01 mg/L, Zn at 0.19 mg/L, Ca at 49.5 mg/L, K at 11,588 mg/L, NO₃ at 27 mg/L, Si at 0.61 mg/L, Cl at 147,000 mg/L, SO₄ at 65,000 mg/L, and B at 179 mg/L. Prior to the mineral analysis of the broccoli sample, it underwent pretreatment using the microwave method, a micronutrient test method approved by the Ministry of Food and Drug Safety. The following elements were analyzed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES): sodium, magnesium, zinc, iron, potassium, and calcium. Additionally, the contents of copper and manganese were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Broccoli was cleaned, dried, and frozen at −20 °C before being ground into a powder with a freeze dryer. For 24 h, 20 g of broccoli powder was agitated twice with 400 mL methanol. The broccoli extracted was then powdered using a rotary evaporator and dissolved in dimethyl sulfoxide (DMSO) at 250 mg/mL before being used in the experiment.

2.2. Assessment of 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Inhibition Rate of Broccoli

Broccoli samples extracted with methanol were dissolved in DMSO and kept at 4 °C until needed. The 96-well plate was filled with 100 μL of CB, OB, NB, methanol, and 150 μM DPPH solution, and the reaction was run in the dark for 30 min. To confirm the DPPH inhibition rate, the absorbance at 517 nm was measured and computed using the following formula [24]:

$$\mathrm{DPPH}\mathrm{inhibition}\mathrm{rate}(\%)=[1-\frac{\left(\mathrm{S}\mathrm{D}-\mathrm{S}\mathrm{M}\right)}{\left(\mathrm{M}\mathrm{D}-\mathrm{M}\mathrm{M}\right)}]\times 100$$

SD: sample + DPPH; SM: sample + methanol; MD: methanol + DPPH; MM: methanol + methanol

2.3. Assessment of Total Phenol (TP) Content

The Folin–Denis method determined the total phenolic (TP) component content. We combined 15 µL of Folin–Ciocalteu reagent (Sigma-Aldrich Co., St. Louis, MO, USA) with 5 µL of a specific quantity of broccoli methanol extract and reacted at room temperature for 5 min. The light was then blocked for 40 min at room temperature after adding 40 µL of 7.5% Na₂CO₃ solution and 140 µL of distilled water (DW). A Wallac Victor3 1420 Multilabel Counter (Perkin-Elmer, Wellesley, MA, USA) was used to detect absorbance at 765 nm. The standard curve was created with gallic acid as the reference (standard concentration was 0.03125–1 mg/mL), and it was used to quantify the TP content of broccoli [25].

2.4. Assessment of Total Flavonoid (TF) Content

Add 200 μL of diethylene glycol to 20 μL of sample extract and set aside for 5 min at room temperature. Then, dispense 20 μL of 1 N NaOH with vortexing and heat the block for 1 h at 37 °C. The absorbance at 420 nm was measured using a Wallac Victor3 1420 Multilabel Counter. The flavonoid content was determined using the standard calibration curve generated by drawing the standard curve with Quercetin (Sigma-Aldrich Co., St. Louis, MO, USA) as the reference (standard concentration is 0–1280 μg/mL) [25].

2.5. HT-29 Cell Culture

The Cell Line Bank in Seoul, Korea, provided the HT-29 human colon cancer cells. The cells were cultured in RPMI 1640 (Welgene Inc., Daegu, Republic of Korea) with 1% penicillin–streptomycin solution (GIBCO BRL., Rockville, MD, USA) and 10% inactivated fetal bovine serum (Welgene Inc., Daegu, Republic of Korea) at 37 °C in a 5% CO₂ incubator. The cancer cells were refed two to three times per week, and after washing with PBS, they were detached and centrifuged with 0.05% trypsin–0.02% EDTA. Following centrifugation, the accumulated cancer cells and media were thoroughly mixed with a pipette, and 10 mL of each was injected into a 75T cell culture flask, subcultured every 2–3 days, and used for the experiment.

2.6. HT-29 Cell MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay

The MTT test is extensively used to determine cell growth and death rates. HT-29 cells were planted at a 2.0 × 10⁴ cells/mL density in a 96-well plate. After 24 h of incubation, broccoli extract samples were administered to cells at 15.63, 31.25, 62.5, and 125 μg/mL. MTT solution at a concentration of 5 mg/mL was diluted tenfold with the culture medium, resulting in a final concentration of 500 μg/mL, and 100 μL was added to each well after 48 h of incubation. The cells were then incubated for 4 h under the same culture conditions. The absorbance at 540 nm was measured using a Wallac Victor3 1420 Multilabel Counter after dissolving the produced formazan crystals in DMSO [26].

2.7. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

A cell counter (Luna automated cell counter; Logos Biosystems) was used to count the cultured HT-29 human colorectal cancer cells, and cells at 5.0 × 10⁴ cells/mL per well were distributed into a 6-well plate and cultivated for 24 h. Following that, HT-29 cancer cells were treated for 48 h with media containing 62.5 µg/mL broccoli. After removing the media, RNA was extracted from the cells and dissolved in 0.1% diethyl-pyrocarbonate (DEPC) using Trizol (Invitrogen, Carlsbad, CA, USA). Total dissolved RNA was quantified with a NanoDrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA), and the quantified RNA was synthesized into cDNA with Invitrogen’s Superscript II reverse transcriptase. The cDNA was tested for gene expression using a BioRad CFX-96 real-time thermal cycler (Bio-Rad, Hercules, CA, USA). p53, p21, Bcl-2, Bcl-xL, Bim, Bad, Bax, Bak, caspase-9, caspase-3, and GAPDH were used as genes, and the primer sequences are presented in

Table 1. Primer sequences of RT-qPCR assay.

Gene Name	Primer Sequence
p53	F: 5’-ATGGAGGAGCCGCAGTCAGA-3’
	R: 5’-TGCAGGGGCCGCCGGTGTAG-3’
p21	F: 5’-ATGTCAGAACCGGCTGGGG-3’
	R: 5’-GCCGGGGCCCCGTGGGA-3’
Bcl-2	F: 5’-AAGATTGATGGGATCGTTGC-3’
	R: 5’-GCGGAACACTTGATTCTGGT-3’
Bcl-xL	F: 5’-GCTGGGACACTTTTGTGGAT-3’
	R: 5’-TGTCTGGTCACTTCCGACTG-3’
Bim	F: 5’-AGATCCCCGCTTTTCATCTT-3’
	R: 5’-TCTTGGGCGATCCATATCTC-3’
Bad	F: 5’-CAATGACCCCTTCATTGACC-3’
	R: 5’-GACAAGCTTCCCGTTCTCAG-3’
Bax	F: 5’-TGCTTCAGGGTTTCATCCAG-3’
	R: 5’-GGCGGCAATCATCCTCTG-3’
Bak	F: 5’-TCTGGCCCTACACGTCTACC-3’
	R: 5’-AGTGATGCAGCATGAAGTCG-3’
Caspase-9	F: 5’-CTAGTTTGCCCACACCCAGT-3’
	R: 5’-CTGCTCAAAGATGTCGTCCA-3’
Caspase-3	F: 5’-TTTTTCAGAGGGGATCGTTG-3’
	R: 5’-CGGCCTCCACTGGTATTTTA-3’
GAPDH	F: 5’-AGGTCGGTGTGAACGGATTTG-3’
	R: 5’-GGGGTCGTTGATGGCAACA-3’

.

2.8. Western Blot

The HT-29 cells were seeded into 6-well plates for 24 h at 5.0 × 10⁴ cells/mL. Cell proteins were reacted with 1 mL radioimmunoprecipitation assay (RIPA, Invitrogen, Carlsbad, CA, USA) buffer, and the proteins were separated by centrifugation at 13,000 rpm for 15 min at 4 °C. The separated protein was quantified by the Bradford assay method. The extracted proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred into polyvinylidene fluoride (PVDF, Bio-Rad, Hercules, CA, USA) membrane, and nonspecific proteins were blocked with 5% skimmed milk containing phosphate-buffered saline with Tween 20 (PBST). After blocking, the PVDF membrane was washed three times with PBST and once with PBS, and the primary antibody was reacted overnight at 4 °C. The PVDF membrane was then washed three times with PBST and once with PBS, and the secondary antibody was reacted for 2 h at room temperature. For caspases 3, caspase 9, and α-tubulin, Santa Cruz (Dallas, TX, USA) primary antibodies were used, and the bands of the proteins were detected using an Amersham Imager 680 (GE Healthcare Life Sciences, Chicago, IL, USA) [25].

2.9. Raw 264.7 Cell Culture

The RAW 264.7 (a murine monocyte/macrophage cell line) cells used in the research were obtained from the Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). The RAW 264.7 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Media, GIBCO) with 1% penicillin–streptomycin solution (GIBCO BRL., Rockville, MD, USA) and 10% inactivated fetal bovine serum (FBS; Welgene Inc., Daegu, Republic of Korea) at 37 °C in a 5% CO₂ incubator. RAW 264.7 cells were cultivated two to three times per week.

2.10. Splenocyte Culture

The following animal experiments were carried out with the agreement of CHA University’s Animal Experimentation Ethics Committee (IACUC220126). Ten 6-week-old C57BL/6 mice were acquired and acclimated for one week (Orient Bio, Seongnam, Republic of Korea). The mice’s spleens were excised in a pathogen-free (SPF) environment, and the cells in the spleen were separated using a cell strainer. The cells were cultured in a medium supplemented with Roswell Park Memorial Institute (RPMI) 1640 (Welgene Inc., Daegu, Republic of Korea), 10% FBS (Sigma-Aldrich Co., St. Louis, MO, USA), and 100 units/mL penicillin–streptomycin (Welgene Inc., Daegu, Republic of Korea).

2.11. RAW 264.7 Cells MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay

The MTT test is widely used to evaluate cell growth and death rates. RAW 264.7 cells were plated in a 96-well plate at a density of 1.0 × 10⁵ cells/mL. Following a 24 h incubation period, broccoli extract samples were used to treat the cells at doses of 1.5, 2.0, 2.5, and 3.0 mg/mL. After 48 h of incubation, the MTT solution (500 μg/mL) was mixed in the medium, and 100 μL was added to each well, followed by 4 h of incubation under the same culture conditions. The absorbance at 540 nm was measured using a Wallac Victor3 1420 Multilabel Counter after dissolving the produced formazan crystals in DMSO.

2.12. NO Production

NO generation was determined by measuring nitrite concentration in the medium with a Griess reagent (Sigma-Aldrich, St. Louis, MO, USA). RAW 264.7 cells and splenocytes were plated in a 6-well plate at 1 × 10⁶ cells/mL each and incubated for 24 h. Following incubation, the cells were treated for 48 h with broccoli extracts (2 mg/mL) and LPS (1 μg/mL), and the supernatant was isolated. The supernatant was treated with Griess reagent and its absorbance at 550 nm was measured using a Wallac Victor3 1420 Multilabel Counter (PerkinElmer, Wellesley, MA, USA) [27,28].

2.13. Measurement of Inflammatory Cytokines Using Enzyme-Linked Immunosorbent Assay (ELISA)

After dispensing splenocytes at a concentration of 1 × 10⁶ cells/mL per well in a 6-well plate, the supernatant was incubated for 24 h in a 37 °C, 5% CO₂ incubator. Following incubation, the cells were treated for 48 h with broccoli extracts (2 mg/mL) and LPS (1 μg/mL), and the supernatant was isolated. The supernatant was then tested for IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 concentrations using enzyme-linked immunosorbent assay kits (ELISA kits, BioLegend, San Diego, CA, USA). The detailed experimental technique was conducted in accordance with the manufacturer’s instructions [27].

2.14. Measurement of Natural Killer Cell Activity

Splenocytes were employed as effector cells and YAC-1 cells (NK-sensitive cell line) as target cells, with the ratio of effector cells to target cells adjusted to 5:1 in each well. They were also given broccoli extract samples simultaneously at a concentration of 2 mg/mL. After 4 h of incubation, the NK cell activity was evaluated by measuring the lactate dehydrogenase (LDH) produced by the cells at 450 nm using an EZ-LDH cytotoxicity assay kit (Dogenbio, Seoul, Republic of Korea) to determine cytotoxicity [29].

2.15. Statistical Analysis

Graph Pad Prism 9.4.1 (GraphPad, San Diego, CA, USA) was used to analyze the data, and the experiments’ results were presented as mean ± standard deviation. Duncan’s multiple range test was used to confirm the significance between each group using two-way ANOVA and one-way ANOVA. p < 0.05 was used to determine significance, and all experimental data were analyzed with the SPSS v18 statistical software package (SPSS Inc., Westlands, Hong Kong).

3. Results

3.1. Mineral Analysis of Broccoli Samples

The analysis of the mineral content of cultivated broccoli was conducted at the V&B Center (Goesan, Chungcheongbuk-do, Korea) (

Table 2. Mineral contents of broccoli. CB: Conventional cultivation broccoli; OB: Organic cultivation broccoli; NB: natural dream cultivation broccoli. Means with different letters (a–c) above the bars are significantly different (p < 0.05) by Duncan’s multiple range test.

mg/kg	Na	Mg	Ca	K	Zn	Fe	Mn	Cu
CB	260.91 ± 4.56 b	2390.55 ± 25.43 a	2628.37 ± 36.54 c	33158.42 ± 584.82 a	50.35 ± 0.74 b	60.68 ± 1.46 b	50.86 ± 2.37 a	4.68 ± 0.15 b
OB	196.53 ± 1.27 c	1807.60 ± 10.74 c	2856.75 ± 37.34 b	30753.30 ± 458.85 b	44.94 ± 0.35 c	70.21 ± 0.46 a	22.70 ± 1.02 c	5.30 ± 0.13 a
NB	855.29 ± 0.81 a	2194.33 ± 14.24 b	4134.48 ± 18.06 a	31289.17 ± 179.25 b	56.25 ± 0.20 a	61.35 ± 0.19 b	32.97 ± 1.16 b	4.98 ± 0.04 b

). As a result of the analysis, it was confirmed that the contents of sodium and calcium greatly increased in NB. Sodium (Na) increases 3.28 times more than CB and 4.35 times more than OB. Calcium (Ca) content increases 1.57 times and 1.45 times compared with CB and OB. These trace elements seem to be the influence of cultivation with DSWM.

3.2. Antioxidant Capacities of Broccoli Samples

The antioxidant capacity of broccoli samples was determined using DPPH free radical scavenging activity and the total phenol and flavonoid levels (Figure 1). At all concentrations, OB and NB had higher oxygen scavenging activity. The total phenol content of broccoli samples increases as the treatment dose increases, and at 4 mg/mL, NB increases more than CB (p < 0.0001) and OB (p < 0.001). The overall flavonoid content of the samples does not differ up to 0.25 mg/mL, but at 1 mg/mL, the flavonoid content of NB increases compared with CB and OB, and at 4 mg/mL, NB has the greatest flavonoid content (p < 0.0001).

3.3. HT-29 Cell Inhibition Rate by Broccoli Extract

The HT-29 cancer cell growth inhibition rate also increases as the broccoli extract concentration increases (Figure 2). The HT-29 cell growth inhibition rate of NB is higher than that of CB and OB at 62.5 μg/mL concentration, and the apoptosis rate of NB is the highest at 125 μg/mL (p < 0.0001). The concentration of 62.5 μg/mL was selected for the follow-up experiment, and CB demonstrated inhibition rates of 34.78% ± 0.29%, OB 35.14% ± 1.34%, and NB 40.94% ± 2.08% at this concentration.

3.4. mRNA Expressions of Cell Cycle Arrest Genes in HT-29 Cells

NB significantly increases mRNA expression levels compared with other groups after validating the expression of p53 and p21 genes associated with cell cycle arrest (Figure 3). OB is increased by 1.26 times and NB by 1.79 times in p53 compared with the CON group. Additionally, OB is increased by 1.17 times, and NB is increased by 1.40 times in p21. These results show that organic broccoli grown with deep sea water minerals increases gene expression in cell cycle arrest.

3.5. mRNA Expressions of Apoptosis Genes in HT-29 Cells

Figure 4 shows the mRNA expression levels of apoptosis-related genes Bax, Bad, Bim, Bak, caspase-9, caspase-3, Bcl-2, and Bcl-xL. NB increases the expression levels of pro-apoptotic genes Bax and Bad by 1.74 and 1.86 times, respectively, compared with the CON group, and increases the expression levels of Bim and Bak by 2.10 and 1.45 times, respectively. Furthermore, caspase-9 and caspase-3 mRNA expression levels in NB were higher than in other groups. Anti-apoptosis-related genes Bcl-2 and Bcl-xL decrease by 0.61 and 0.52 times, respectively, when NB is compared with the control group. These results indicate that NB causes apoptosis in HT-29 colorectal cancer cells by regulating the mRNA expression levels of apoptosis-related factors.

3.6. Expression Levels of Proteins Associated with Apoptosis

Western blot analysis was conducted to further validate the apoptosis mechanism of HT-29 cells using broccoli extract (Figure 5). Among these extracts, the expression of Caspase 3 and Caspase 9 significantly increased in OB and NB, with NB exhibiting the highest levels of caspase 3 and 9 expression. The Western blot results are in close agreement with the mRNA analysis, suggesting that NB grown with deep sea water minerals inhibits the growth of HT-29 cancer cells through an apoptotic pathway.

3.7. Viability of RAW 264.7 Cells in Broccoli Extract

As a preliminary experiment, the viability of RAW 264.7 cells treated with broccoli samples was established to confirm the sample treatment concentration in RAW 264.7 cells and splenocytes. When broccoli samples were treated at 2.0 mg/mL, a 90% or higher survival rate was observed, and a significant decrease in cells was validated at concentrations of 2.5 mg/mL or higher (Figure 6). Accordingly, a concentration of 2.0 mg/mL was used for subsequent experiments. The viability of RAW 264.7 cells was 90.65% ± 3.73% for CB, 93.14% ± 0.80% for OB, and 95.23% ± 0.32% for NB.

3.8. Nitric Oxide Production in RAW 264.7 Cells and C57BL/6 Mouse Splenocytes

The results of evaluating NO production ability in LPS-treated RAW 264.7 cells and splenocytes (Figure 7) revealed that the cells’ NO production after LPS treatment was significantly increased relative to the CON group and significantly decreased in the broccoli-treated groups. The NO concentration in RAW 264.7 cells is 1.21 ± 0.05µM in the CON group and increases to 47.74 ± 0.83 µM in the LPS group. The concentrations of NO in the CB, OB, and NB groups are 15.51 ± 0.24, 19.08 ± 0.26, and 12.11 ± 0.21µM, respectively, with NB producing the least. Splenocytes produce the least NO in the CON group at 1.87 ± 0.12 µM and the highest in the LPS group at 22.89 ± 0.06µM. As in the RAW 264.7 cells, the broccoli treatment reduces NO production in splenocytes, and the NB has the lowest NO production among the three broccoli treatment groups at 5.59 ± 0.11µM (p < 0.05).

3.9. Production of Inflammatory Cytokines in the Splenocytes

In the supernatant of splenocytes treated with LPS, the expression of pro-inflammatory cytokines, IL-1β, IL-6, TNF-α, IFN-γ, and IL-12, is highest in the LPS group and gradually decreases in the broccoli treatment groups (Figure 8). In particular, the expression of these cytokines in the NB group is the lowest among the broccoli groups. IL-1β shows a high expression level of 428.73 ± 2.76 pg/mL in the LPS group, decreasing to 12.69 ± 0.44 pg/mL in the NB group. Furthermore, the NB group has the lowest expression levels of IL-6 and TNF-α among the broccoli groups. IL-12 expression is high in the LPS group (89.03 ± 0.62 pg/mL) but decreases to 6.87 ± 0.39 pg/mL in the NB group, which is similar to the 5.95 ± 0.12 pg/mL found in the CON group (p < 0.05). These results suggest that NB has a high anti-inflammatory effect in splenocytes.

3.10. Activity of NK Cells in Splenocytes

As a result of measuring NK cell activity by reacting splenocytes and YAC-1 cells at a ratio of 5:1 (Figure 9), a 5:1 ratio revealed that CB had an activity of 18.84 ± 2.25%, OB, 18.36 ± 0.83%, and NB, 23.69 ± 3.39%, respectively. The activity was highest in the NB group and shows a significant difference from that of the OB group (p < 0.05).

4. Discussion

DSWM showed higher levels of Na, Mg, and K compared with mineral fertilizers, and additional mineral components such as Cl, N, B, and SO₄ were also detected. These mineral components can significantly impact plant growth and are essential for the synthesis of crucial organic compounds. Moreover, they play diverse roles as ions or components of inorganic compounds, indicating that MB exhibits higher mineral content than CB and OB, which is likely attributed to their various roles in plant physiology [30].

An imbalance between the generation of ROS and the detoxification of reactive intermediates by biological systems causes oxidative stress [31]. Because of oxidative stress, sympathetic nerve stimulation increases respiratory rate, generating more available oxygen and disrupting the balance between ROS production and the antioxidant system [32]. Broccoli is an antioxidant-rich vegetable that protects the human body from oxidative stress, a major cause of some cancers and heart diseases, by removing free radicals [33]. Moreover, DSW protects against cardiovascular diseases by lowering TC, TG, atherogenic index, and MDA levels while increasing serum trolox equivalent antioxidant capacity (TEAC) and offering intestinal protection through antioxidant and anti-apoptotic mechanisms [34]. The antioxidant capacity of a substance is shown by measurements of DPPH radical scavenging activity, total phenol, and total flavonoid concentrations [35]. Unlike conventional farming practices that use chemical fertilizers and pesticides, organic farming relies solely on natural materials and is considered a cultivation method that improves soil health and enhances societal well-being [36]. Recent meta-analyses have shown that organic production methods tend to significantly increase antioxidants, phenols, and Zn compared to conventional farming [37]. Additionally, when roses were cultivated through organic farming, they exhibited superior antioxidant effects compared to those grown conventionally [38]. In this study, organically grown OB and NB also showed higher DPPH scavenging ability than CB, and in particular, the antioxidant properties of NB were superior to those of CB and OB with increasing concentration. Therefore, it is hypothesized that due to its high antioxidant activity, NB could reduce reactive oxygen species and have anticancer and anti-inflammatory properties.

Cancer cells exhibit uncontrolled cell proliferation and abnormal growth, and tumor cells are often the result of genetic damage to genes that regulate cell cycle arrest [39]. The expression of transcription factors associated with cell cycle arrest and cell apoptosis has been considered a crucial factor in cancer treatment [40]. Cell cycle arrest is mainly caused by activating one or both p53/p21 and p16/pRB tumor suppression pathways [41]. p53 may promote PUMA, BID, Bax, TRAIL2, and FASL transcription in the nucleus, as well as translocate to the mitochondrial membrane, where it can respond to cell death signals by forming an inhibitory complex with Bcl-xl and Bcl-2, triggering cell apoptosis [42]. p21, which is significantly regulated by p53, plays a vital role in cell cycle arrest by binding to cyclin E/cyclin-dependent kinases (Cdk)2 and cyclin D/Cdk4 complexes, resulting in G1 cell cycle arrest [43]. Broccoli contains various phenolic compounds, which are known to exhibit not only anti-proliferative and cytotoxic effects but also pro-apoptotic activity in animal tumor models and several cancer cell lines. Among the phenolic compounds found in broccoli, sulforaphane has been identified as a key component responsible for its anticancer effects [44]. Furthermore, it has been observed that the incorporation of CaSO₄ into the NaCl solution used for cultivation leads to an increase in sulforaphane content in broccoli sprouts [45]. Sulforaphane induces growth inhibition in breast cancer cells such as MCF-7 in a time and concentration-dependent manner, along with alterations in cell cycle regulatory molecules such as an increase in p21 and p27 and a decrease in cyclin A, cyclin B1, and CDC2 proteins [46]. In this regard, this study confirmed the influence of broccoli on p53 and p21 expression levels and found that NB, broccoli cultivated with DSWM, significantly increased p53 and p21 mRNA expression levels in HT-29 cells.

Apoptosis is a type of programmed cell death observed in multicellular organisms. Apoptosis is characterized by morphological and biochemical changes, such as nuclear membrane blebbing, mitochondrial membrane permeability, and caspase cascade activation [47]. Cell apoptosis is primarily mediated by the extrinsic apoptotic pathway involving death receptors and the intrinsic mitochondrial pathway involving multiple caspase family members [48]. A primary mechanism of cell apoptosis is the mitochondrial pathway, which is triggered by the Bcl-2 family and promotes mitochondrial outer membrane permeabilization (MOMP). Pro-apoptotic factors that influence cytochrome C release from mitochondria and anti-apoptotic factors that inhibit cytochrome C release combine to induce apoptosis [49]. Members of the caspase family play essential roles in the initiation and execution of cell death, and these caspases, which are activated in response to signals, are classified into initiator caspases and executioner caspases, influencing the stages of cell apoptosis [50]. Sulforaphane, a major compound in broccoli, was found to induce mitochondria-related cell death and increase the Bax/Bcl-2 ratio in human colorectal cancer cells, SW480. It also activated caspase-3 and caspase-9. Moreover, sulforaphane exhibited growth-inhibitory effects on HT-29 and HCT-116 cells. It was revealed that sulforaphane induced cell cycle arrest at the G2/M phase and apoptosis by upregulating phosphorylation of CDK1 and CDC25B, along with activation of the p38 and JNK pathways [46]. In a study by Bae and Lee [51], it was found that using DSW to improve the quality of green tea significantly inhibited N-nitrosation, which can cause mutagenesis and cellular damage responses. Another study by Kim et al. [14] revealed that the ability of breast cancer cell migration could be inhibited. In this study, treatment with NB, organically cultivated broccoli with DSWM, significantly increased the mRNA expression of cell death factors Bax, Bad, Bim, and Bak in HT-29 cells, as well as the mRNA and protein expression of caspase-9 and caspase-3. Furthermore, the anti-apoptotic factors Bcl-2 and Bcl-xL were less expressed.

In this study, the effect of broccoli samples on the production of the inflammatory mediator NO was investigated to evaluate the anti-inflammatory effects. NO plays a vital role in normal physiological processes, such as neurotransmission, vasodilation, and immune defense, but excessive NO production caused by increased iNOS expression promotes inflammatory responses and increases oxidative stress and tissue damage [52]. The exact mechanism by which simple NO production causes disease pathogenesis is unknown, although NO has the chemical potential to cause DNA damage via nitration, nitrosation, and oxidation [53]. In this study, CB, OB, and NB inhibited LPS-induced NO production in RAW 264.7 cells and spleen cells, and the expression levels in NB were most similar to those in the CON group.

Macrophages are activated during an inflammatory response to enhance immune defenses, producing several cytokine types. TNF-α, IL-6, IL-1β, and IL-12 are examples of pro-inflammatory cytokines produced [54]. These components are required to initiate and improve the inflammatory response, and their expression is increased in LPS-stimulated macrophages [55]. LPS activates M1 macrophages and can also accelerate the inflammatory response by activating or increasing the expression of other pro-inflammatory cytokines and mediators [56]. TNF-α is a pro-inflammatory cytokine that regulates immune cells and cell death by producing IL-1β and IL-6, activating the inflammatory cascade [57]. IL-6 promotes B and T cell development, regulates immunity and inflammation, and is regulated by IL-1β and LPS [58]. IFN-γ is a glycoprotein secreted by T lymphocytes and NK cells. When produced in excess, it increases the production of hydrogen peroxide in macrophages, promotes the release of TNF-α and cytokines, and induces local inflammation and tissue destruction [59]. IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits that enhance the link between innate and adaptive immune responses [60]. IL-12 is also required to fight against infectious diseases and tumors and promote cell-mediated immunity by stimulating Th1 cells [61].

The production of pro-inflammatory cytokines in LPS-induced splenocytes was highest when treated with LPS and gradually decreased when treated with CB, OB, and NB. In particular, the expression of the cytokines was lowest in the NB group and was found to be similar to that of the CON group. Additionally, after culturing the splenocytes, it was confirmed that the activity of the NK cells in the splenocytes was also significantly increased by NB. This suggests that broccoli cultivated with DSWMs effectively regulates the inflammatory response of splenocytes induced by LPS by regulating inflammatory responses and increasing NK cell activity in splenocytes. It was confirmed that NB, which also used DSWM in organic cultivation, had high Na and Ca contents. It also exhibited anticancer activity in HT-29 colorectal cancer cells and anti-inflammatory effect in LPS-treated C57BL/6 mouse splenocytes. These results suggest that DSWMs significantly affect plant growth and functioning.

5. Conclusions

These results show that organically cultivated broccoli enriched with DSWM has higher sodium and calcium content than conventional and organic broccoli and has higher antioxidant activity. Additionally, NB significantly suppressed the growth of HT-29 colon cancer cells and upregulated the expression of cell cycle arrest-related genes p53 and p21 and cell death genes Bax, Bad, Bim, Bak, caspase-9, and caspase-3 while downregulating the expression of Bcl-2 and Bcl-xl. NB significantly reduced NO production and showed a potential to decrease the expression of inflammatory factors IL-1β, IL-6, TNF-α, IFN-γ, and IL-12 in LPS-treated C57BL/6 mouse splenocytes. Moreover, it increased NK cell activity in response to YAC-1 cells. In conclusion, the use of DSWM in broccoli cultivation demonstrates the potential to increase the mineral content and enhance the anticancer properties, as well as elevate the anti-inflammatory functionality of plants. Moreover, the application of DSWM in the cultivation of other crops suggests the possibility of enhancing similar effects in their growth and functional properties.