Effects of Sulforaphane in Ferroptosis, Apoptosis, and Senescence induced by Cigarette Smoke in Human Bronchial Epithelial Cells: a Mechanistic Study

doi:10.21203/rs.3.rs-4887220/v1

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Effects of Sulforaphane in Ferroptosis, Apoptosis, and Senescence induced by Cigarette Smoke in Human Bronchial Epithelial Cells: a Mechanistic Study

https://doi.org/10.21203/rs.3.rs-4887220/v1

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Background

Cigarette smoke (CS) is a leading risk factor for pulmonary diseases. It has been implicated that ferroptosis and apoptosis are involved in CS-induced lung senescence. Sulforaphane (SFN) is a phytochemical with anti-oxidative and anti-inflammatory effects. However, we investigated the effects of CS on oxidative damage, apoptosis, ferroptosis, and senescence in the human bronchial epithelial cell line (BEAS-2B) and the preventive role of SFN.

Materials and Methods

BEAS-2B cells were exposed to CS extract (CSE) and varying concentrations of SFN (5, 10, and 20 µg/ml). Cytotoxicity and senescence were evaluated by MTT assay, clonogenic assay, Annexin V/PI flow cytometry, and SA-β-galactosidase staining method. Also, the involvement of the ferroptosis pathway and oxidative stress were measured via reactive oxygen species (ROS), glutathione (GSH), lipid peroxidation (LPO) levels, and intracellular iron assessment.

Results

Our results showed that CSE resulted in a concentration-dependent decline in the viability and clonogenic potential of BEAS-2B cells and induced senescence through intracellular ROS generation, LPO, and GSH oxidation, which led to ferroptosis and apoptosis. However, SFN protects against CSE cytotoxicity as measured by MTT and colony formation assay. Furthermore, SFN reduced CSE-induced oxidative stress and inhibited CSE-induced ferroptosis, as shown by lowering iron and MDA. Subsequently, SFN alleviated CSE-induced apoptotic and senescence in BEAS-2B cells.

Conclusion

This study strongly supports the idea that SFN could ameliorate CSE-induced lung toxicity via inhibition of oxidative redox, ferroptosis, and the apoptotic pathway, which results in a decrease in senescence and enhanced clonogenic potential in BEAS-2B cells.

Cigarette smoke

BEAS-2B

Oxidative stress

sulforaphane

ferroptosis

senescence

Cigarette smoke (CS) contains over 4000 chemicals, many of which have been shown to have diverse toxic effects on cells. Lung cancer and chronic obstructive pulmonary disease are well-recognized outcomes of cigarette smoking [1]. The World Health Organization estimates that around 6 million individuals die annually due to the direct effects of tobacco smoking [2]. With every puff, CS contains approximately 10 ^15–17 oxidative free radicals and 4,700 distinct chemical compounds [3]. Oxidative stress is defined by increased levels of reactive oxygen species (ROS) within cells, which causes harm to DNA, proteins, and lipids [4]. Studies have shown that CS can prompt lung tissue to generate abundant ROS. This, in turn, triggers the production and release of damage-associated molecular patterns and inflammatory factors, ultimately leading to lung inflammation [5, 6].

Cellular senescence, characterized by irreversible cessation of the cell cycle, is a distinctive feature of aging [7]. Cellular senescence can be triggered by external or internal factors, such as telomere shortening (replicative senescence), irreversible DNA damage, mitochondrial issues, and excessive oxidative stress [8]. Chronic exposure to CS leads to the senescence of pulmonary vascular cells, such as smooth muscle cells and endothelial cells [9]. Regulated cell death (RCD) is an irreversible biological process that plays a critical role in homeostasis within the body. Ferroptosis, a recently discovered mode of RCD, is linked to disruptions in iron metabolism and lipid peroxidation (LPO). Studies indicate that ferroptosis is characterized by disturbances in iron metabolism, the buildup of ROS, decreased levels of glutathione (GSH), and the deactivation of glutathione peroxidase 4 [10–12]. Previous studies indicated that exposure to cigarette smoke led to ferroptosis in BEAS-2B [13].

For centuries, phytochemicals have been utilized in traditional medicine, but they are now garnering attention for their pharmacological benefits with minimal side effects [14, 15]. Sulforaphane (4-methylsulfinybutyl isothiocyanate) is an isothiocyanate compound that is derived from glucoraphanin, commonly present in cruciferous vegetables [16]. SFN does not exist in whole vegetables but is instead produced from its precursor glucosinolate, glucoraphanin, through the activity of myrosinase, a thioglucosidase enzyme, when vegetable tissue is crushed or chewed [17]. Numerous studies have shown that SFN, with its antioxidant properties, can stimulate phase 2 enzymes, which are responsible for converting harmful substances into inactive metabolites [18, 19]. Moreover, studies have shown that it exhibits other pharmacological effects, including anticancer and inflammation-modulating properties [20].

Given the antioxidant and anti-inflammatory effects of SFN and the established link between smoking and chronic lung disease through mechanisms such as increased oxidative stress, inflammation, cellular senescence, and programmed cell death, we hypothesize that SFN could play a protective role by suppressing these processes. To test our hypothesis, we investigated the effect of sulforaphane on cigarette smoke-induced toxicity, oxidative stress, ferroptosis, apoptosis, and senescence in BEAS-2B.

Reagents

SFN was acquired from Sigma-Aldrich (St Louis, MO, USA) and was diluted in 10% dimethyl sulfoxide (DMSO). The maximum final concentration of DMSO in both control and treated cells never exceeded 0.1%. Fetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) were procured from Gibco Co Ltd (USA). CSE containing 40 mg/mL concentration, solubilized in DMSO, was acquired from the Kentucky Tobacco Research and Development Center at the University of Kentucky in Lexington, KY. It was derived from the Kentucky standard cigarette (1R6F) at the University of Kentucky in Lexington, KY. The iron chelator deferoxamine (DFO) was obtained from Danapharma (IRAN), and Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was derived from KeyGEN BioTECH Company (China).

BEAS-2B cell culture

The BEAS-2B cell line was cultured in DMEM-F12 medium enriched with 10% FBS, 5 mM L-glutamine, 100 g/ml penicillin, and 100 U/ml streptomycin. The cells were maintained at 37°C in a moist environment with 7.5% CO2. The groups were organized as follows: control group, where BEAS-2B cells were cultured using the conventional method; experimental group, where cultured BEAS-2B cells were treated with the IC50 (59.5 µg/ml) of CSE for 24 hours; low-dose group, where cultured BEAS-2B cells were treated with the IC50 of CSE and 5 µg/ml SFN for 24 hours; medium-dose group, where cultured BEAS-2B cells were treated with the IC50 (59.5 µg/ml) of CSE and 10 µg/ml of SFN for 24 hours; high-dose group, where cultured BEAS-2B cells were treated with the IC50 (59.5 µg/ml) of CSE and 10 µg/ml of SFN for 24 hours.

CSE preparation

The CSE was transported alongside dry ice, divided into small portions in sterilized microtubes, and stored at − 80°C until needed. Before the experiments, working solutions (0.01, 0.1, 1, 10, 20, 50, and 100 µg/mL) were prepared by dilution with DMEM. Throughout the process, the CSE was kept in darkness and sterilized before use.

Cell viability through MTT and Clonogenic Assays

The MTT test, as described previously [21], was used to assess the cytotoxicity of CSE and SFN. Cells were plated at a density of 4×10⁴ cells/well in 48-well plates with a flat bottom. BEAS-2B cells were exposed to increasing concentrations (0.01, 0.1, 1, 10, 20, 50, and 100 µg/mL) of CSE and increasing concentrations (0.01, 0.1, 1, 5, 10, 20, and 50 µg/mL) of SFN. Subsequently, the cells were cultured for 24 hours at 37˚C in a humidified environment with 7.5% CO2. After treatment, 50 µL of MTT solution (0.5 mg/ml) was added to each well and incubated for 3 hours at 37 ºC. Afterward, 170 µL of DMSO was added to each well. The absorbance was measured at 570 nm using a Chromate plate ELISA Reader (Awarness Technology, Inc.) to calculate the relative cell viability ratio.

The BEAS-2B cell lines were cultured and trypsinized for the clonogenic assay. After cell counting, 250 cells were seeded into 6-well plates with low-melting point agarose as described [22]. The cells were then incubated in a CO2 incubator at 37°C for a few hours to allow adherence to the plate. Subsequently, the cells were treated based on the predetermined experimental groupings and incubated for 2 weeks until colonies with a minimum of 50 cells per colony formed in the plates. Following this, the media was removed, and the cells were fixed with 2-3mL of a 6% glutaraldehyde fixation solution at room temperature for 10 minutes. The cells were then stained with 0.5% crystal violet staining solution for 30 minutes at room temperature, and the plates were rinsed with tap water to remove excess crystal violet. The number of stained colonies was determined using the formula: (colony number/seeded cell number) × 100%.

Measurement of intracellular ROS

Briefly, cells were plated in a 6-well culture plate and treated based on predefined experimental groupings. The cells were washed with PBS twice after removing the culture medium and trypsinizing. Then, 1 mL of 10 µmol/L DCFH-DA was added to each well, and the plates were incubated for 30 minutes in a 37°C incubator. The cells were homogenized and centrifuged at 12000 RPM for 5 minutes. Subsequently, 200 µL of the supernatant was collected and measured at 485 nm (excitation) and 535 nm (emission) using FP-6200 Spectrofluorometer (Jasco, Inc) [23].

Measurement of GSH and MDA

Briefly, cells were plated in a 6-well culture plate and treated based on predefined experimental groupings. For measurement of GSH, the cells were incubated with Triton-X-100 lysis buffer for 20 minutes and then centrifuged ((1000G 4 °C) for 10 minutes. Finally, the supernatant (50 µL) was combined with 200 µL of DTNB (200 µM) for 30 minutes, and the absorbance was assessed using a Chromate plate ELISA Reader (Awarness Technology, Inc.) at 405 nm.

The level of LPO was determined using the MDA assay. Following the incubation, the cells were trypsinized and then lysed through homogenization. 200 µL of each cell supernatant was combined with 200 µL of phosphoric acid (0.2 M). The mixture was incubated at 100°C for 30 minutes, followed by cooling on ice; 500 µL of n-Butanol was placed into the mixture and centrifugation at 10,000 × g for 10 minutes at 4°C. Subsequently, 200 µL of the resulting supernatant was put into a 96-well plate. The absorbance was measured at 532 nm.

Measurement of β-Galactosidase activity

The SA-β-gal activity was assessed using the o-Nitrophenyl-β-D-galactopyranoside (ONPG) colorimetric method [24]. ONPG is extensively utilized as a substrate in assays for β-galactosidase in bacterial and eukaryotic cell lysates. It is initially colorless, but upon hydrolysis, it produces o-nitrophenol, which turns yellow in an alkaline solution (λmax = 420 nm at pH 10.2).

Detection of Apoptosis

The levels of apoptosis in BEAS-2B cells were assessed using the Annexin V-FITC/PI apoptosis detection kit (K-01, Yasgene, Espahan, Iran). Following digestion with trypsin minus EDTA, the treated cells were gathered and reconstituted with 100 µl 1 × Binding Buffer. Subsequently, one µl Annexin V-FITC and 1 µl PI Solution were introduced to label apoptotic cells. After a 10-minute incubation in the absence of light, 400 µl 1 × Binding Buffer was added, and the proportion of apoptotic cells (Annexin V+/PI+) was determined using the flow cytometry BD FACSCalibur (BD Biosciences).

Measuring Intercellular Iron Levels

The intercellular iron levels in BEAS-2B cells were assessed using Agilent 4210 Microwave Plasma Atomic Emission Spectroscopy (MP-AES). The cells were subjected to a 14-hour incubation in 0.1% nitric acid and 0.1% Triton X-100 within a 60°C water bath [25]. Subsequently, the cell suspension was preserved in 0.2 M nitric acid for analysis at a later date.

Statistical analysis

The data were assessed using GraphPad Prism 8.4.2 software and are presented as the normalized mean with standard deviation (SD). Statistical variances were determined by using a one-way analysis of variance followed by Tukey’s multiple comparisons test. The statistical significance was established for p-values < 0.05.

Effect of CSE, SFN, and their combination on BEAS-2B cells

MTT assays were performed to evaluate the potential effect of CSE, SFN, and their combination on cell viability. The cells were exposed to rising concentrations of CSE and SFN for 24 hours. CSE demonstrated cytotoxic effects at concentrations of 20, 50, and 100 µg/mL, with a half maximal inhibitory concentration (IC₅₀) concentration of 59 µg/mL (Fig. 1A). SFN showed cytotoxic effects at 50 µg/mL after 24 hours. Still, not at concentrations less than 100 µg/mL (Fig. 1B). For combination Using the data, the treatment dose for the upcoming experiments was determined by employing three different values for SFN (5, 10, and 20) and the IC₅₀ value for CSE (Fig. 2).

SFN inhibits Clonogenic cell death due to CSE

Figure 3 illustrates the clonogenic cell death resulting from CSE treatment. BEAS-2B cells in the presence of CSE exhibited a decreased clonogenic survival fraction. Following 14 days of exposure to 59 µg/mL CSE, there was a 34.6 ± 4.5% reduction in colony formation compared to the control group. The data demonstrates that a concentration of 10 µg/mL for SFN significantly enhanced the reduction of colonies caused by CSE. In contrast, at concentrations of 5 µg/mL and 20 µg/mL, the colony count did not show a significant variance from the CSE group.

SFN prevents oxidative stress in BEAS-2B cells.

We set out to study how SFN affects oxidative stress caused by CSE. Our results showed that supplementation with SFN successfully prevented oxidative stress in BEAS-2B cells. We examined the alterations in ROS and GSH levels in BEAS-2B cells induced by CSE. We found that CSE exposure increased ROS levels and decreased GSH levels in the cells. Nevertheless, these effects were reversed by SFN supplementation(Fig. 4).

SFN inhibited ferroptosis in CSE-treated BEAS-2B cells

To assess SFN's impact on ferroptosis, we initially investigated intracellular LPO as the end product of ferroptosis and the level of intracellular iron as the initiator of this process in BEAS-2B cells (Fig. 5). Our experiments consistently revealed higher MDA levels and iron contents in the CSE group, indicating a significant ferroptosis effect of CSE on the cells. However, these effects were countered by SFN supplementation.

The impact of SFN on apoptosis induced by CSE

Apoptosis of lung epithelial cells is a critical element of developing pulmonary disease. Figure 6 demonstrates a notable increase in apoptosis rate in CSE-induced Beas-2B cells (P < 0.0001), and the administration of 10 ug/ml of SFN significantly mitigated the apoptosis rate (P < 0.0001). The findings indicate that SFN at 5 and 20 µg/mL does not offer protection against CSE-induced apoptosis.

The BEAS-2B cells treated with SFN exhibited a decrease in SA-β-GAL activity

Chromate plate ELISA Reader assessed the SA-β-GAL activity. In the CSE group, the activity of SA-β-GAL increased to 0.196 ± 0.017 compared to the control group (0.075 ± 0.01, p < 0.0001). Following cotreatment with SFN, the SA-β-GAL activity decreased to 0.103 ± 0.015 (p < 0.05) in the SFN 10 ug/ml group compared to the CSE group (Fig. 7).

Beas-2B cells are a human bronchial epithelial cell line widely used as a cell model in vitro to test or screen different chemicals and biological agents that could cause pulmonary toxicity [26]. Therefore, we utilized this cell line as a model to induce toxicity and observe its therapeutic response in this study. Research has shown that CS can cause oxidative stress, senescence, DNA damage, and various programmed cell death (PCD) forms, including ferroptosis and apoptosis in cell models [9, 13, 27]. This study demonstrated that CSE exhibited short- and long-term cytotoxic effects. In the short-term toxicity evaluation using the MTT test, a decrease in cell viability was observed from 20 µg/ml of CSE. SFN, a potent protective phytochemical, has been shown to counteract the toxicity of various harmful compounds in both in vitro and animal studies [28]. Co-treatment with SFN was most successful in facilitating smoking toxicity at 10 µg/ml, and this finding aligned with the outcomes of numerous other studies [29, 30]. It seems that sulforaphane synergizes with CSE in reducing cell viability at concentrations exceeding 10 µg/ml. In our clonogenic test, we observed that smoking has long-term toxic effects. Nevertheless, their viability improved when the cells were exposed to sulforaphane, with the most significant impact at 10 µg/ml. Hui Xie et al. demonstrated that SFN had a substantial inhibitory effect on growth, proliferation, and clone formation at 20 µg/ml, and their findings align with our study's [31].

We observed an intriguing result that both CSE and, in high concentration, SFN caused oxidative stress in BEAS-2B cells (Fig. 4). While CSE increased ROS and decreased GSH, SFN demonstrated a protective effect against this harmful impact at a concentration of 10µg/ml. Studies indicate that SFN exhibits a biphasic or hormetic-like dose response in cell culture. This means that at low concentrations (1–10 µM), SFN stimulates cell growth, while at high concentrations (> 10 µM), it inhibits cell growth [32–34]. The contrasting impact of sulforaphane at 5, 10, and 20 µg/ml utilized in this study may be attributed to the biphasic nature of sulforaphane.

Evidence regarding the stimulation of apoptosis in airway epithelial cell lines after exposure to tobacco smoke is contradictory. However, in cases where apoptosis is detected, antioxidants mitigate any observed apoptosis, supporting the theory that smoke-derived free radicals are accountable for the observed apoptosis [35, 36]. Hence, SFN, with its antioxidant characteristics, effectively counteracted the apoptosis caused by CSE at 10 µg/ml. However, at a concentration of 20 µg/ml, the induction of apoptosis was heightened, suggesting the intrinsic nature of SFN in the induction of apoptosis at high concentrations and its synergistic interaction with CSE. Our in-vitro model using DFO has provided clear evidence of the role of ferroptosis in CSE-induced cell death in the BEAS-2B cell line. Our findings indicated that despite a significant rise in intracellular iron levels in the CSE group, LPO was not fully mitigated in the DFO group. This suggests that LPO resulting from smoking exposure is not solely attributed to the ferroptosis pathway. When investigating the ferroptosis pathway in this study, we noticed that while SFN has a protective effect against ferroptosis at low concentrations, it can trigger ferroptosis at exceeding 10 µg/ml. These findings support the notion that phytochemicals should be used cautiously as they can exhibit contrasting effects at different doses [37–39].

Our present study observed a notable reduction in the anti-senescence activity of SFN in BEAS-2B cell lines, as evidenced by a decrease in SA-β-GAL enzyme levels (see Fig. 4). Cellular senescence is a significant factor in driving pulmonary diseases, often characterized by the overexpression of SA-β-GAL [40]Some studies have suggested the involvement of increased ROS in cigarette smoking-induced lung senescence. Elevated ROS levels lead to irreversible damage to DNA, proteins, and lipids. By induced oxidative stress, we showed that CSE exposure induces senescence in BEAS-2B cells. However, SFN demonstrated a protective effect against this harmful impact at a concentration of 10µg/ml.

Despite the positive data collected, conducting additional mechanistic tests would enhance the depth and scope of the results discussed. Additional pulmonary cell lines and in vivo models could be utilized to understand better how SFN reduces toxicity associated with CSE.

In conclusion, our research conducted a screening and found SFN to be a beneficial phytochemical for counteracting the toxic effects of CSE. Our study illustrated that CSE contributes to apoptosis, ferroptosis, oxidative stress, and cellular senescence in BEAS-2B cells. Furthermore, we observed a significant reduction in CSE-induced colony formation of BEAS-2B cells in clonogenic assays. SFN displayed remarkable characteristics, such as anti-inflammatory, anti-apoptotic, anti-ferroptotic, and anti-senescent properties. Even though the data gathered from this study looks promising, additional research could delve deeper into the cellular mechanisms and also include in vivo studies.

BEAS-2B: human bronchial epithelial cell line

CS: cigarette smoke

CSE: cigarette smoke extract

DMSO: dimethyl sulfoxide

DFO: deferoxamine

DCFH-DA: Dichloro-dihydro-fluorescein diacetate

DMEM: Dulbecco's Modified Eagle Medium

GSH: glutathione

LPO: lipid peroxidation

MDA: malondialdehyde

ROS: reactive oxygen species

RCD: regulated cell death

ONPG: o-Nitrophenyl-β-D-galactopyranoside

SFN: sulforaphane

Declarations of interest

The authors state that they have no financial conflicts of interest or personal relationships that could have influenced the impartiality of the findings presented in this paper.

Funding

This article is derived from a Ph.D. thesis funded by Mazandaran University of Medical Sciences in Sari, Iran, under grant code 18270 and ethical code IR.MAZUMS.AEC.1403-18270.

Data availability

No datasets were generated or analyzed during the current study.

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Effects of Sulforaphane in Ferroptosis, Apoptosis, and Senescence induced by Cigarette Smoke in Human Bronchial Epithelial Cells: a Mechanistic Study

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Version 1

Abstract

Figures

Introduction

Materials and methods

Reagents

BEAS-2B cell culture

CSE preparation

Cell viability through MTT and Clonogenic Assays

Measurement of intracellular ROS

Measurement of GSH and MDA

Measurement of β-Galactosidase activity

Detection of Apoptosis

Measuring Intercellular Iron Levels

Statistical analysis

Result

Effect of CSE, SFN, and their combination on BEAS-2B cells

SFN inhibits Clonogenic cell death due to CSE

SFN inhibited ferroptosis in CSE-treated BEAS-2B cells

The BEAS-2B cells treated with SFN exhibited a decrease in SA-β-GAL activity

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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