Introduction

Sulforaphane is a naturally occurring isothiocyanate (Wu et al. 2024), that can perform strong pharmacological activity, particularly with preventive and anticancer effects (Coutinho et al. 2023). It is a substance with strong anticancer activity (Ramirez and Singletary 2009). Sulforaphane inhibits the activation of proto-carcinogenic substances and promotes the decomposition of carcinogenic chemicals in the body, which leads to cancer prevention (Choi et al. 2014). Sulforaphane also has anti-inflammatory and antioxidant properties (Ibrahim 2023). Consequently, the study of sulforaphane activity against cancer cells has attracted researchers’ interest in recent years (Kang and Yu 2017). Broccoli (Brassica oleracea L. var. italica) has attracted considerable attention due to its richness in sulforaphane and glucoraphanin. However, sulforaphane is obtained from broccoli seedlings or seeds, which poses challenges associated with a high cost of production and low yield (Tusevski et al. 2017).

Biotechnological tools like genetic transformation and in vitro regeneration are very important for the propagation and metabolic engineering of medicinal plants. Hairy root cultures are considered a powerful tool for producing secondary metabolites, usually biosynthesized in the plant roots or the aerial organs of mature plants (Li and Wang 2021). Therefore, in vitro Agrobacterium rhizogenes-mediation is a key element in plant biotechnology (Niazian et al. 2023). Agrobacterium rhizogenes is a gram-negative soil bacterium responsible for the induction of hairy roots at the infection site through the transfer of TDNA or Ri plasmid (Tao and Li 2006). Increasing the content of bioactive compounds by introducing their biosynthetic or regulatory genes into the hairy roots through a transgenic system offers promising prospects. Hairy root cultures have great potential for producing high yields of secondary metabolites by manipulating the pathway of genes for synthesizing secondary metabolites with elicitors and other strategies (Li and Wang 2021). The efficiency of plant transformation can be increased by either manipulating the explant type and/or the Agrobacterium genotypes (Sharafi et al. 2014a, b). The yield of secondary metabolites in hairy root cultures can be enhanced by elicitation, which appears to be the most efficient strategy (Alcalde et al. 2022). Hairy root cultures are preferable for elicitation due to their higher growth rate, genetic stability, and pathogen-free existence (Biswas et al. 2023). Elicitors act as signals recognized by elicitor-specific receptors on the plant cell membrane that trigger defensive responses through elicitation (Halder et al. 2019). This action causes an enhancement in the production and accumulation of secondary metabolites (Humbal and Pathak 2023). The effect of elicitation depends on the elicitor type, exposure duration, concentration, and treatment schedule (Tien 2020).

Copper (Cu) is an essential micronutrient for plants, participating in many physiological processes as a co-factor of different enzymes (Chen et al. 2022). In addition, zinc (Zn) is a heavy metal that acts as a nutrient for plants (Stuiver et al. 2014) and is required in various physiological pathways such as enzyme structure, protein synthesis, auxin metabolism, energy production, gene expression, and pathogen resistance (Suganya et al. 2020). However, high levels of heavy metals are phytotoxic because they can replace essential metals in proteins, react with thiol groups of proteins and glutathione, and stimulate the formation of reactive oxygen species (ROS) that result in lipid peroxidation, protein denaturation, and DNA mutation reactions (Morelli and Scarano 2004; Yruela 2009). In Brassica species, tolerance to metal stress is acquired mainly through the synthesis of sulfur-containing compounds like the amino acids methionine, cysteine, phytochelatins, glutathione, and metallothionein (Gill and Tuteja 2011; Reich et al. 2018).

Breast cancer is the most common cancer among women, posing a global public health challenge. It comprises a group of molecularly and biologically heterogeneous diseases that start in the breast (Feng et al. 2018). About one-third of women with breast cancer develop metastases that eventually lead to death. Most breast tumors are often poorly responsive or non-responsive to treatments and develop resistance to therapies after increasing the doses of chemotherapeutic drugs. It would therefore be necessary to search for alternative anticancer drugs that can trigger the cell death process in tumor cells (Pelicano et al. 2004). Isothiocyanates elicit chemo-preventive potency through multiple mechanisms that include modulation of phase I and II detoxification pathway enzymes, control of cell growth, regulation of cell cycle arrest, induction of apoptosis, anti-angiogenic effects, antioxidant activity, and regulation of epigenetics (Esteve 2020).

A study by Ma et al. (2022) indicated that hairy roots produced by infecting broccoli’s leaf discs with A. rhizogenes are genetically stable and have large quantities of the anticancer substance sulforaphane. The present work aims to investigate the effect of ZnSO4 and CuSO4 metal ions as elicitors of sulforaphane production by over-expressing the MY gene in broccoli’s hairy root cultures. In addition, the cytotoxicity and anti-proliferative effects of different treatments of sulforaphane, extracted from hairy roots following elicitation, on the human breast cancer cell line MDA-MB-231 were studied.

Materials and methods

Plant material

Broccoli F1 hybrid Agassi RZ seeds were germinated on the MS basal medium (Murashige and Skoog 1962) containing 30 g/L sucrose and 2.5 g/L Gelrite to produce multiple seedlings. The cultures were kept at 25 ºC in the incubation room under a daily photoperiod of 16 h light and 8 h dark. The seedlings were used for hairy root induction.

Hairy root induction

Preparation of Agrobacterium culture for transformation

One week before transformation, Agrobacterium rhizogenes strain A4 from glycerol stock was plated on LB solid medium (Bertani 1951) containing 50 mg/L (w/v) kanamycin for screening and selecting the desired host bacterial colony and was later incubated for 48 h at 28 °C. One day before transformation, bacterial cultures were prepared by incubating one colony in 20 ml of LB medium at 28 °C for 12–16 h on a rotary shaker (130 rpm). Bacterial cells were harvested by centrifugation at 4,000 rpm at 4 °C for 15 min, and the pellet was resuspended in 20 ml of MS liquid medium containing 50 µM acetosyringone used for infecting broccoli leaf explants.

Transformation and establishment of hairy root cultures

For transformation, broccoli leaf explants obtained from the leaves of 21-day-old seedlings were immediately immersed in 20 ml bacterial suspension for 1 h at room temperature. The explants were blotted with sterile filter paper and cultured on MS solid plates containing 50 µM acetosyringone without antibiotics (Baskar et al. 2016) to enhance the efficiency of Agrobacterium infection and facilitate the transformation. Plates were incubated for 4 days in the dark at 25 °C. Explant tissues were transferred to MS solid medium containing 500 mg/L carbenicillin after 4 days of co-cultivation to eliminate A. rhizogenes. Cultures were then incubated at 25 °C in complete darkness. Hairy roots emerged from the wound sites on the broccoli leaf explants within seven days after inoculation. After four weeks of co-cultivation with A. rhizogenes, the hairy roots were excised from the necrotic explants and subcultured on a fresh MS solid medium containing 500 mg/L carbenicillin for four weeks. They were then transferred and grown in 50 ml of liquid MS medium supplemented with 400 mg/L carbenicillin in 250 ml Erlenmeyer flasks maintained on a rotary shaker (80–90 rpm) at 25 °C under a photoperiod (16 h light and 8 h dark). The hairy roots were subcultured every four weeks into a fresh medium.

Elicitors preparation and treatments

A stock solution of ZnSO4 (Fisher Scientific, Loughborough, UK, Catalog No: Z68-500) or CuSO4 (Acros Organics, New Jersey, USA, Catalog No: 422871000) was prepared as 1 mM of each compound dissolved in dd.H2O. Hairy roots were subcultured in 50 ml of MS liquid medium in a 250 ml Erlenmeyer flask and grown at 25 °C under agitation (80–90 rpm). After 15 days of cultivation in the basal medium, hairy roots were transferred to a medium supplemented with ZnSO4 or CuSO4 at the following final concentrations: 0, 4, 8, and 16 µM for 8 and 16 h (Mohammadi et al. 2014). Six replications were performed for every treatment. Treated hairy roots were harvested, washed several times with sterile d.H2O, and then ground to a fine powder using a pestle and mortar in liquid nitrogen and stored at -80 ºC until analysis.

Sulforaphane extraction and determination

For sulforaphane extraction, broccoli's hairy roots were lyophilized and squashed into a fine powder. Sulforaphane was then extracted from 0.5 g of treated and untreated root powder according to the protocol of Ares et al. (2014). Sulforaphane was purified with a glass column using the protocol of Azizi et al. (2011). The obtained extract was stored at -20 ºC until analysis.

The determination of sulforaphane in broccoli’s hairy roots was done as described by Amer et al. (2021).

Quantitative real-time PCR analysis of the Myrosinase gene

Total RNA was extracted from both control hairy roots and roots treated with the two elicitors, ZnSO4 or CuSO4. After isolation, the RNA samples were standardized to a concentration of 300 ng/µl for each treatment. Real-time PCR was then conducted to quantify RNA levels, as described by Amer et al. (2021). All quantitative assays were done in triplicate. Gene expression analysis focused on the MY gene as the target and β-actin as the housekeeping gene and was performed on the Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, Singapore). The specific primer sequences used for MY and β-actin genes (Yang et al. 2015) are provided in Table 1. Delta Delta Threshold cycle (ΔΔCq) expression values were calculated to determine MY gene expression, normalized to β-actin. The specificity of the reactions was validated using the standard melt curve analysis. Statistical analysis was performed using SPSS software version 16. A one-way ANOVA followed by Turkey’s Honestly Significant Difference (HSD) test was conducted to compare the significance between the mean values of MY gene expression from different treatments.

Table 1 Sequence-specific primers of Myrosinase (MY) and β-actin genes (Yang et al. 2015)
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Cell culture and viability testing

The human breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection (Manassas, VA, USA) via the Nawah Scientific Research Center (Cairo, Egypt). The cell culture protocol and their viability testing were done as described by Amer et al. (2021). Table 2 describes the treatments used for viability testing. Each experiment was repeated three times, and six replicates were tested for each concentration. These treatments included two crude sulforaphane extracts from hairy roots following exposure to 16 µM ZnSO4 or 4 µM CuSO4 at concentrations of 10, 40, 80, 100, 200, and 300 µg/ml in addition to standard sulforaphane at concentrations of 0.2, 0.5, 1, 5, and 10 µg/ml. Standard and crude sulforaphane were dissolved in DMSO. The control group was treated with 0.1% DMSO to eliminate possible toxic effects of the solvent, and a control blank of untreated cells was used to eliminate the effects of non-specific binding of the neutral red dye.

Table 2 Description of sulforaphane treatments used for viability testing of the human breast cancer cell line MDA-MB-231
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Transcription analysis of the apoptosis genes

The cells of the human breast cancer cell line MDA-MB-231 were treated using the IC50 of each treatment. Following treatment, total RNA was extracted from both treated and untreated cells. The transcription of apoptosis genes was assessed by quantitative real-time PCR as described in Amer et al. (2021). All reactions were performed in triplicate. The apoptosis genes used in transcription analysis were Caspase-3, Caspase-8, Caspase-9, Bax, Bcl-2, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene. The forward and reverse primers for these genes were obtained from Table 2 described in Amer et al. (2021).

Statistical analysis

The analysis of the results was done using SPSS software version 16. Graph Pad Prism (version 8.3) was used to determine the IC50. A one-way ANOVA followed by Turkey’s Honestly Significant Difference (HSD) test was conducted to compare the significance between the mean values of the measured data at P < 0.05.

Results

Transformation and production of hairy root cultures

A. rhizogenes strain A4 was used for transformation and hairy root production. The transformation protocol is illustrated in Fig. 1. The steps were as follows: Leaf explants were inoculated with A. rhizogenes for 1 h at room temperature and then cultured on hormone-free MS plates containing 50 µM acetosyringone to encourage the growth of A. rhizogenes at the cut ends of explants. After four days of co-cultivation, explants were transferred onto MS selection plates supplemented with carbenicillin to kill A. rhizogenes. Plates were incubated at room temperature in a clean and dark place and checked regularly for hairy root production every two days.

Fig. 1
figure 1

Procedure of the transformation and hairy roots production experiment: (a) Explants inoculated with Agrobacterium rhizogenes, (b) Co-cultivation of leaf explants for 4 days on hormone-free MS medium plates, (c) Selection of transformed roots on carbenicillin antibiotic-containing medium, (d) Hairy root formation on the selection medium, (e) Excised and sub-cultured hairy roots on solid MS medium, (f) The transferred hairy roots in liquid MS medium

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The transformation rate in broccoli leaf explants infected with A. rhizogenes is explained in Table 3. The leaf explants produced hairy roots at an average transformation rate of 45 ± 8.9% (Table 3) in leaves infected with A. rhizogenes. Hairy roots resulting from A4 infection were yellowish in color, branched, and thick, forming a callus at the root base.

Table 3 Transformation rate in the broccoli leaf explants infected with Agrobacterium rhizogenes as indicated by the hairy root production
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Effect of zinc sulfate and copper sulfate on sulforaphane production

The ZnSO4 treatments showed a dose-dependent increase in sulforaphane production. The production of sulforaphane increased when the time of ZnSO4 treatments was prolonged from 8 to 16 h (Table S1, Fig. 2). The highest amount of sulforaphane was recorded in the hairy roots exposed to 16 µM ZnSO4 after 16 h of elicitation (54.758 mg/g dry weight), representing an increased percentage of 136.5% with a significant difference compared to the control. On the other side, treatment with CuSO4 for 8 h and 16 h resulted in a dose-dependent increase in sulforaphane production (Table S1, Fig. 3). It is observed that sulforaphane showed the most significant increase in hairy root cultures exposed to 4 µM CuSO4 after 16 h of elicitation; the amount of sulforaphane was 62.754 mg/g dry weight, reflecting a 148.4% increase in sulforaphane content in the hairy root cultures treated with CuSO4 for 16 h in comparison to the corresponding control.

Fig. 2
figure 2

Sulforaphane content (mg/g dry weight) of control and ZnSO4 (Zinc sulfate) treated broccoli hairy roots after the treatment time (8 and 16 h). Bars with different letters are significantly different at p < 0.05, according to one-way ANOVA and HSD test

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Fig. 3
figure 3

Sulforaphane content (mg/g dry weight) of control and CuSO4 (Copper sulfate) treated broccoli hairy roots after treatment time (8 and 16 h). Bars with different letters are significantly different at p < 0.05, according to one-way ANOVA and HSD test

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Effect of zinc sulfate and copper sulfate on MY gene expression

The expression level of the MY gene was significantly up-regulated compared to the control following exposure to elicitation with ZnSO4 treatments for 8 and 16 h (Fig. 4). It was significantly increased by 845% in hairy roots treated with 8 µM of ZnSO4 after 8 h of elicitation. However, the highest percentage of MY gene up-regulation was recorded at 4 µM of ZnSO4 after 16 h of treatment, indicating an increase of 1446%. Additionally, a high level of MY gene expression was observed at 8 µM of ZnSO4 after 16 h of exposure, which gave a fold gene expression of 13.978, representing an increase of 1298% (Table S2). The higher concentration of 16 µM of ZnSO4 induced much lower levels of MY gene up-regulation after 8 h and 16 h of treatment (Table S2, Fig. 4).

Fig. 4
figure 4

The effect of ZnSO4 (Zinc sulfate) treatments on the Myrosinase (MY) gene expression after 8 and 16 h. Bars with different letters are significantly different at p < 0.05, according to the one-way ANOVA and the HSD test

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The expression level of the MY gene in broccoli’s hairy root cultures exposed to CuSO4 treatments was also significantly up-regulated compared to the control (Fig. 5). It resulted in the highest percentage increase in gene up-regulation (1551%) at 4 µM CuSO4 after 8 h of treatment. Moreover, a high level of MY gene up-regulation (1377.1%) was also observed at 8 µM CuSO4 after 8 h (Table S2). Thus, lower doses of 4 µM CuSO4 after 8 h induced a higher level of MY gene up-regulation in broccoli’s hairy root cultures compared to the higher dose of 16 µM ZnSO4 after 8 h and 16 h of treatment (Table S2, Fig. 4).

Fig. 5
figure 5

The effect of CuSO4 (Copper sulfate) treatments on the Myrosinase (MY) gene expression after 8 and 16 h. Bars with different letters are significantly different at p < 0.05, according to the one-way ANOVA and the HSD test

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Cytotoxicity of sulforaphane treatments on MDA-MB-231 breast cancer cells

As described in Table 2, the cytotoxic effect of three sulforaphane treatments on the viability of the MDA-MB-231 breast cancer cell line was evaluated. All sulforaphane treatments increased the percentage of MDA-MB-231 breast cancer cell death, as measured by the neutral red uptake assay, in a dose-dependent manner. Figure 6 illustrates the dose-dependent increase in cell death of the MDA-MB-231 breast cancer cells by sulforaphane crude extracts derived from hairy root cultures treated with (a) 16 µM ZnSO4, (b) 4 µM CuSO4, and (c) Sulforaphane standard concentrations. Additionally, it displays the IC50 values (µg/ml) of different sulforaphane treatments (d). After the 48 h incubation period, the IC50 for the treatment with (a) crude extract-16 µM ZnSO4 and (b) crude extract-4 µM CuSO4 were 63.032 ± 0.35 µg and 23.563 ± 0.25 µg respectively. In addition, the IC50 for the treatment with (c) sulforaphane standard was 0.532 ± 0.03 µg. Thus, crude extract-4 µM CuSO4 induced a higher inhibitory effect on the MDA-MB-231 breast cancer cell line compared to crude extract-16 µM ZnSO4 (Table 4 and Fig. 6d).

Fig. 6
figure 6

The dose-dependent increase in cell death of the MDA-MB-231 breast cancer cells by sulforaphane crude extracts derived from hairy root cultures treated with (a) 16 µM ZnSO4- (b) 4 µM CuSO4- (c) Sulforaphane standard concentrations and (d) IC50 values (µg/ml) of different sulforaphane treatments. The data are means ± SEM (n = 3). The significant difference between Crude-16 µM ZnSO4 and Crude-4 µM CuSO4 was indicated by *p < 0.05. Standard error values were multiplied by ten to be visible

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Table 4 IC50 values (µg/ml) of different sulforaphane treatments from treated hairy root cultures in the MDA-MB-231 breast cancer cell line determined by neutral red uptake assay
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Effect of sulforaphane treatments on apoptosis genes expression

The relative expression of apoptosis genes; Caspase-3, Caspase-8, Caspase-9, Bax, and Bcl-2 was determined in the MDA-MB-231 cell line after 48 h incubation with the crude extract of broccoli hairy roots sulforaphane and the sulforaphane standard using real-time PCR. The given values express gene transcription levels of the above genes normalized to the constitutive reference gene GAPDH at IC50 of different sulforaphane treatments (Fig. 7). The transcription of the Caspase-3 gene in cells treated with sulforaphane extracts obtained from treated hairy root cultures showed significant (p < 0.05) up-regulation compared to the control cells (Fig. 7). The highest percentage of Caspase-3 gene up-regulation was 156.7% in cells treated with crude sulforaphane extract following exposure to 4 µM CuSO4.

Fig. 7
figure 7

The relative expression of apoptosis genes in the MDA-MB-231 cell line after 48 h incubation with crude extract of sulforaphane from broccoli hairy roots treated with the two elicitors ZnSO4 and CuSO4 and the sulforaphane standard. Treatment 1: (crude extract at 16 µM ZnSO4), Treatment 2: (crude extract at 4 µM CuSO4), Treatment 3: (Sulforaphane standard). Bars with different letters within each group are significantly different at p < 0.05, based on the one-way ANOVA and the HSD test

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The results shown in Fig. 7 also indicate that all sulforaphane crude extracts of hairy roots up-regulated the Capsase-8 gene transcription significantly (p < 0.05) in the treated cells compared with the control cells. The cells treated with crude sulforaphane extracted from hairy roots exposed to 16 µM ZnSO4 recorded the highest percentage of Capsase-8 transcription (218.2%) compared to the control. Data presented in Fig. 7 also indicated that Caspase-9 transcription in all cells treated with sulforaphane extract derived from hairy roots was significantly up-regulated (p < 0.05) in comparison to the control cells. The highest percentage of 138% Caspase-9 gene up-regulation was detected in cells treated with crude sulforaphane extracted from hairy roots exposed to 16 µM ZnSO4 (treatment 1).

On the other hand, the proapoptotic Bax gene transcription level was significantly up-regulated (p < 0.05) in all treated cells compared to the control cells (Fig. 7). Bax was mostly up-regulated to 568.1% in cells treated with the sulforaphane standard, followed by cells treated with the sulforaphane extracted from hairy roots exposed to 16 µM ZnSO4. Furthermore, the transcription level of the antiapoptotic Bcl2 gene was significantly low (p < 0.05) in all treated cells (Fig. 7). The lowest percentage of Bcl-2 gene down-regulation was detected in cells treated with sulforaphane extracted from hairy roots exposed to 16 µM ZnSO4.

Discussion

The above results showed increased production of hairy roots derived from leaf explants of broccoli inoculated with A. rhizogenes at a transformation rate of 45 ± 8.9%. The application of synthetic phenolic compounds, including acetosyringone, enhances the efficiency of transformation through the activation of vir-genes involved in T-DNA transfer during plant transformation in A. rhizogenes (Sharma et al. 2013). By enhancing the activation of vir genes, acetosyringone increases the efficiency of T-DNA delivery and integration into the plant genome, leading to higher transformation rates. Another factor is the selection of different explants (Sharma et al. 2013) and modified medium (Sharafi et al. 2014a). In our experiment, the addition of 50 µM acetosyringone to the co-cultivation medium facilitated the transformation in broccoli. The factors affecting the A. rhizogenes-mediated transformation in broccoli were investigated by Henzi et al. (2000), who reported similar findings on the role of acetosyringone in broccoli transformation. An efficient and reliable protocol for inducing genetically transformed roots in the medicinal plant Nepeta pogonosperma was also documented by Valimehr et al. (2014). Similar results were also proclaimed by Jaberi et al. (2018), who used acetosyringone in the co-cultivation medium to facilitate transformation in the medicinal plant Cosmos bipinnatus.

The degree of metabolite production is influenced by both the timing of the treatment (Vahdati et al. 2004) as well as the type of elicitor (Patel and Krishnamurthy 2013). In the current study, the effect of CuSO4 and ZnSO4 as abiotic elicitors on sulforaphane production in the hairy root cultures of broccoli was studied at different times and concentrations. In the hairy roots treated with CuSO4 for 8 h, increasing sulforaphane content was produced by all tested concentrations (4 µM, 8 µM, and 16 µM) of CuSO4. The highest amount of sulforaphane was detected at 16 µM CuSO4, with significant differences from the control. Otherwise, sulforaphane content in hairy roots exposed to all concentrations of CuSO4 after 16 h of elicitation increased significantly in contrast to the control. The highest amount of sulforaphane was recorded in hairy root cultures exposed to 4 µM CuSO4, with significant differences compared to control, and by increasing CuSO4 concentration, the amount of sulforaphane was significantly reduced at 8 µM and 16 µM CuSO4 after 16 h of elicitation. The results could be attributed to the fact that longer elicitor contact leads to disrupted cell permeability, osmotic conditions, and changes in membrane potential (Vasconsuelo and Boland 2007).

Yaoya et al. (2004) revealed that the addition of CuSO4 (64 µg/ml) to the hairy root culture of Pharbitis nil resulted in the increased release of the coumarin derivatives umbelliferone and scopoletin into the medium. However, excessive levels of copper are phytotoxic because of their action on indolic glucosinolate gene expression and other enzymes for their biosynthesis in Chinese cabbage (Aghajanzadeh et al. 2020a). It was reported that Cu may replace essential metals in proteins, react with thiol groups of proteins and glutathione, and induce the formation of reactive oxygen species (ROS), which lead to lipid peroxidation, protein denaturation, and DNA mutation reactions (Morelli and Scarano 2004; Yruela 2009).

In the hairy roots treated with ZnSO4, the results indicated a dose-dependent increase of sulforaphane following all tested ZnSO4 concentrations (4 µM, 8 µM, and 16 µM) after 8 h and 16 h. The highest amount of sulforaphane was recorded in hairy roots exposed to 16 µM ZnSO4 after 8 h and 16 h of elicitation, with a significant difference compared to the control. Aghajanzadeh et al. (2020b) reported that exposure of Chinese cabbage to 5 μM and 10 μM ZnCl2 led to enhancements of organic sulfur and total sulfur concentrations. This was attributed to the differential partitioning of thiols and glucosinolates between root and shoot after exposure to an excess amount of zinc.

Various genes and transcription factors have been identified in the secondary metabolism of plants under exogenous elicitors (Broun et al. 2006). qRT-PCR is a valuable technique for analyzing gene expression (Goharrizi et al. 2016). The gene expression level is generally affected by the treatment time and the type of elicitor employed in the elicitation process (Chong et al. 2005). In the current study, the level of MY gene expression under CuSO4 and ZnSO4 elicitation at different time courses was investigated. The qRT-PCR results indicated that the MY gene was significantly up-regulated in broccoli’s hairy roots treated with all concentrations of CuSO4 for 8 h and 16 h compared to the control. The gene up-regulation recorded the highest percentage at 4 µM CuSO4 after 8 and 16 h of elicitation. Amer et al. (2021) disclosed that over-expression of the MY gene leads to increased sulforaphane production. The increase in indolic glucosinolates in Chinese cabbage was accompanied by enhanced transcript levels of two genes, CYP79B2 and CYP83B1, involved in the biosynthesis of indolic glucosinolates, along with enhanced MYB51, a transcription factor involved in the regulation of the indolic glucosinolate biosynthesis pathway, at elevated copper concentrations (Aghajanzadeh et al. 2020a). Furthermore, exposure to all treatments of ZnSO4 induced a higher percentage of gene up-regulation. The highest percentage of gene up-regulation after 8 h of elicitation was detected at 8 µM of ZnSO4. After 16 h, the highest percentage was determined at 4 µM of ZnSO4. Similarly, elevated glucosinolate content in Chinese cabbage was also attributed to exposure to a high level of zinc, which coincided with an increase in gene expression of the key genes responsible for the biosynthetic enzymes and regulators CYP83B1, CYP79B3, and MYB34 (Aghajanzadeh et al. 2020b).

The most appropriate method for targeting anticancer therapy is apoptosis (Chimento et al. 2023). Apoptosis is one of the main pathways by which chemotherapeutic and chemo-preventive agents inhibit cancer cell growth (Ming et al. 2012). It was observed that the anticancer activity of sulforaphane extracted from hairy roots exposed to 4 µM CuSO4 has a higher inhibitory effect than sulforaphane extracted from hairy roots treated with 16 µM ZnSO4. The results revealed that increasing sulforaphane concentrations led to an increased rate of cell apoptosis. Our results agree with the results of Chung et al. (2016), who reported the anticancer activity of a hairy root extract of turnip (Brassica rapa ssp. rapa) by inhibiting the division of human breast and colon cancer cell lines. Various in vivo and in vitro studies have documented that indoles and isothiocyanates elicit chemo-preventive potential through various mechanisms, including phase I and phase II detoxification pathway enzyme modulation, regulation of cell cycle arrest, and cell growth control, apoptosis induction, anti-angiogenic effects, antioxidant activity, and regulation of epigenetics (Esteve 2020).

Apoptosis often occurs through the caspase pathway (Fiandalo and Kyprianou 2012). Caspases are defined as a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Apoptosis is regulated by several caspases, including Caspase-3, Caspase-8, and Caspase-9 (Dehkordi et al. 2022). The family members of the B-cell lymphoma 2 (Bcl-2) protein are key regulators with proapoptotic activity like Bax and antiapoptotic activity like Bcl-2, which regulate apoptosis, tumorigenesis, and cellular responses to anticancer therapy (Qian et al. 2022). In our results, the transcription levels of Caspase-3, Caspase-8, and Caspase-9 genes in treated cells showed significant (p ≤ 0.05) up-regulation compared to the control cells. Apoptosis is induced in cancer cells through two major pathways: the extrinsic and intrinsic pathways (Mortezaee et al. 2019). Caspase-8 is a key initiator of caspase-inducing apoptosis via the death receptor pathway (extrinsic pathway), while Caspase-9 has a vital role in initiating apoptosis via the mitochondrial pathway (intrinsic pathway), which plays a key role in apoptosis (Yang et al. 2007). The activation of Caspase-8 can trigger intrinsic Caspase-9-mediated apoptosis through truncation of the BH3 interacting domain (Inao et al. 2018). MDA-MB‑231 cells that exhibit lower levels of Caspase-8 were distinguished by their high metastatic capacity (De Blasio et al. 2016). Therefore, promoting Caspase-8 gene transcription might seem good for cancer treatment by inducing apoptosis.

The transcription level of the Bax gene (proapoptotic) was significantly (p ≤ 0.05) high in all MDA-MB-231-treated cells. The Bax up-regulation increases the cellular sensitivity to apoptosis and is considered therapeutically relevant, whereas its down-regulation confers resistance of tumor cells to apoptosis (Ghasemi et al. 2018). On the other hand, the Bcl-2 gene (antiapoptotic) transcription level was significantly (p ≤ 0.05) down-regulated in all treated cells compared to the control cells. The members of the Bcl2 family play critical roles in mitochondria-mediated apoptosis in many tissues as well as in the breast (Williams and Cook 2015). The ratio of proapoptotic to antiapoptotic molecules determines the sensitivity of cells to a death signal. The up-regulation of gene expression of Bcl-2 is one of the factors that leads to breast malignancy (Kordezangeneh et al. 2015). On the other side, down-regulation of Bcl-2 using Bcl-2 inhibitors enhances the effects of treatments against both hematological malignancies and different types of solid tumors, such as human breast cancer cells (Inao et al. 2018). Sulforaphane from broccoli hairy root cultures caused enhanced Bax expression while also triggering the down-regulation of Bcl-2, leading to the subsequent promotion of apoptotic activity in MDA-MB-231 breast cancer cells.

Conclusions

The results of the current investigation revealed that CuSO4 and ZnSO4 treatments increased sulforaphane production in broccoli’s hairy root cultures. The data showed that CuSO4 elicitation was more effective in sulforaphane production. The analysis of apoptosis gene transcription revealed that all sulforaphane treatments up-regulated the proapoptotic genes Bax, Caspase-3, Caspase-8, and Caspase-9 while down-regulating the transcription of the antiapoptotic gene Bcl-2. The increased production of sulforaphane extract may enhance its antiapoptotic activity in MDA-MB‑231 breast cancer cells. These findings may provide therapeutic insight for the treatment of cancer using sulforaphane derived from the hairy root cultures of broccoli.