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Impact of calcium propionate on cellular behavior in A549 and DMS114 lung cancer cell lines

  • Tugba Muhlise Okyay ORCID logo , Eray Metin Güler ORCID logo , Ebru Kale ORCID logo , Fatih Gultekin ORCID logo and Macit Koldas ORCID logo EMAIL logo
Published/Copyright: September 30, 2024
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 49 Issue 5

Abstract

Objectives

To investigate the cellular and apoptotic effects of food additive calcium propionate by in vitro methods.

Methods

Cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, intracellular Glutathione (GSH) by luminometric method, mitochondrial membrane potential (MMP) by fluorometry, apoptosis by dual Acridine Orange/Ethidium Bromide (AO/EB) fluorescent staining were studied in lung cells.

Results

It has been observed that calcium propionate reduced GSH and MMP levels significantly while increased cytotoxicity, apoptosis, and reactive oxygen species (ROS) levels in both A549 and DMS114 cancer cells in a dose-dependent manner.

Conclusions

Cytotoxic effects of calcium propionate were more pronounced in cancer cells compared to healthy cells, suggesting its potential suitability as a chemo-preventive agent.

Keywords: calcium propionate; cancer; food additive; cell viability; lung cancer

Introduction

Calcium propionate, a widely used food preservative, has gained considerable attention for its multifaceted roles in food preservation, safety, and quality enhancement. The Food and Drug Administration (FDA) classifies it as a Generally Recognized As Safe (GRAS) compound, underlining its established safety for consumption in various food products [1]. The assigned E code for calcium propionate is E282, and it exhibits high solubility in water, while its solubility in alcohol is comparatively lower [2]. Calcium propionate, derived from propionic acid, is a versatile additive used in a variety of food products, including bread, bakery items, dairy, and processed meats [3]. Utilizing calcium propionate in bread formulation can effectively extend its shelf life, preserving its freshness for a period of 10–12 days [4]. Functioning as an antimicrobial agent, calcium propionate is typically incorporated into the bread dough at a concentration ranging from approximately 0.1–0.3 % relative to the weight of the flour [5]. Furthermore, a synergistic effect has been observed when calcium propionate is combined with sodium chloride, further contributing to the extension of the bread’s shelf life [6].

There are studies examining research on its genotoxicity. In specific tests, including the Bacillus subtilis recombination test, Escherichia coli, and Salmonella typhimurium reversion test [7], calcium propionate did not exhibit any mutagenic effects. Conversely, in a different study, calcium propionate induced chromosomal abnormalities in Chinese hamster lung cells [8]. When assessing its effects on Allium cepa chromosomes calcium propionate significantly increased the frequency of abnormalities compared to the control group and notably reduced the mitotic index [9].

Calcium propionate treatment at concentrations of 0.5 and 1 % has been observed to induce the formation of aflatoxins, preventing Aspergillus fungus formation [10]. In investigations involving BRCA1 mutated breast cancer cells, a key genetic mutation associated with breast cancer, the HCC1937 cell line was subjected to calcium propionate at various concentrations (0.5–2.0 mg/mL). The treatment with calcium propionate led to a decrease in superoxide dismutase levels within the cells, thereby impacting oxidative stress [11].

Recently, there has been a substantial focus on investigating the connection between dietary factors and various types of cancers 12], [13], [14], [15. Food additives are being investigated for their potential role in reducing cancer risk [16]. Lung cancer ranks first in cancer deaths among others [17]. However, as of the current knowledge, there hasn’t been any specific study focusing on the use of calcium propionate for the prevention or treatment of lung cancer. The aim of this study is to investigate the cancer cell viability and apoptotic effects induced by calcium propionate by in vitro methods.

Materials and methods

Materials

Calcium propionate, fetal bovine serum (FBS), MTT, Dulbecco’s modified Eagle medium (DMEM), dimethylsulfoxide (DMSO), PBS, 2’7’-dichlorodihydrofluorescein diaceticacid (H2DCF-DA), penicillin/streptomycin (P/S), acridine orange (AO), ethidium bromide (EB), 3,3’-dihexylocarbocyanine iodide (DiOC6(3)), 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA) Sigma-Aldrich (Germany) were used. GSH-Glo Glutathione Assay (Promega) was purchased. Other chemicals and solutions used in the experiments were of analytical grade.

Cell culture

In the study, A549 (ATCC® CCL-185), DMS114 (ATCC® CRL-2066) lung cancer cell lines and Beas-2B (ATCC® CRL-9609) cell lines were used as the control group. All cells were grown in DMEM media at 37 °C containing 10 % FBS and 1 % P/S. For cell viability, intracellular ROS, GSH, and MMP measurements 15 × 103 cells per well in 96-well plates, and apoptosis measurements 50 × 103 cells per well were seeded in 6-well plates. All experiments were performed in triplicate.

MTT assay

MTT assay was used to evaluate the cytotoxic effects of calcium propionate to determine cell viability. The cells were treated with various concentrations of calcium propionate (0, 0.25, 0.5, 1, 4, 8, 16 mg/mL in distilled water). After 24 h of incubation, MTT solutions (50 µL) from the stock (5 mg/mL) was added, and cells were incubated in a CO2 incubator for 2 h at 37 °C. The medium was removed, and formazan crystals formed by the cells were dissolved using DMSO (200 µL). The absorbance was read at 570 nm using 630 nm as reference wavelength on a multi-well plate reader (Biotek Instruments, USA) 18], [19], [20. The absorbance value of cells not treated with calcium propionate was considered to represent 100 % viability, as these cells were incubated with only the cell culture medium. The viability in other cell groups was calculated as a percentage relative to this control value.

ROS detection

The cells were treated with calcium propionate at different concentrations (0, 0.25, 0.5, 1, 4, 8, 16 mg/mL) for 24 h. Plates were washed 3 times with PBS. Then, the cells were collected and incubated with DCFA-DA (100 μL, 10 μM) in the dark at 37 °C for 30 min. The fluorescence intensity (Ex/Em=488/525 nm) of DCF formed after incubation was measured using a microplate reader (Biotek Instruments, USA). Results were calculated by comparing relative to the control group cells that receive no treatment [21].

Glutathione assay

A commercially purchased GSH-Glo™ Glutathione Assay kit manufactured by Promega was used to measure intracellular GSH levels in the study. Cells seeded in 96-well plates with 15 × 103 cells per well were incubated for 24 h with calcium propionate at different concentrations (0.25–16 mg/mL). After incubation, the culture medium removed from the wells GSH (50 μL) solution was added to the wells and incubated for 30 min. Luciferin Detection Reagent (50 μL) was added to each well and luminescence measurement was taken in a microplate reader within 5 min (Biotek Instruments, USA) [22].

Mitochondrial membrane potential

After 24 h of incubation of the prepared concentrations of calcium propionate (0.25–8 mg/mL), the medium is removed and washed, then incubated with DiOC6(3) (40 nM) for 15 min at 37 °C in the dark. After washing 3 times with PBS, fluorescence intensity Ex:484 nm/Em:501 nm fluorescence was measured using the plate reader (Biotek Instruments, USA) [23]. The results were calculated by comparison to the untreated control group, and expressed as a percentage relative to the control value (100 % MMP).

Apoptosis

AO/EB stain is a double staining was used to evaluate morphological changes in cells. Cells seeded in 6-well plates were removed with trypsin-EDTA 24 h after treatment with calcium propionate at different concentrations (0.25–8 mg/mL). Cells were washed with PBS were centrifuged at 1,000 rpm for 5 min at +4 °C, and their supernatants were discarded. Cell pellet (10 μL) and AO/EB solution (10 μL, 100 μg/mL AO + 100 μg/mL EB) mixture were transferred to an empty slide and covered [24]. A minimum of 100 cells were counted and recorded under a fluorescence microscope (Zeiss, Germany).

Statistical analysis

Results are given as mean ± standard deviation. Data in all experiments were analyzed by analysis of variance (One-Vay ANOVA), and Mann-Whitney U tests using the SPSS package program (Version 25 for Windows, Chicago, USA). IC50 values ​​of calcium propionate on cell lines were calculated by nonlinear regression analysis. Numerical analyses were performed on the invasion images using the Image J (NIH, USA) program. Relationships between all parameters were analyzed with the Pearson correlation coefficient. A p-value of <0.05 was considered statistically significant.

Results

Cell viability

The effects of various concentrations of calcium propionate on A549, DMS114 and Beas-2B cell proliferation were determined using MTT assay. The A549 cell viability dramatically decreased to 40 , 28, and 16 % at concentrations of 4 mg/mL, 8 mg/mL and 16 mg/mL, respectively. For the control group cell line Beas-2B, only 8 and 16 mg/mL concentrations of calcium propionate inhibited cell viability below 80 % (Figure 1).

Figure 1: Percent cell viability of cells treated with various concentrations of calcium propionate. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.
Figure 1:

Percent cell viability of cells treated with various concentrations of calcium propionate. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.

By linear regression analysis, and IC50 values were subsequently determined for each cell line. The IC50 concentration for A549 cells was 5.44 mg/mL, whereas DMS114 cells reached its IC50 value at 12.15 mg/mL. Beas-2 B cells maintained viability above 70 % at all concentrations, preventing the calculation of its IC50 value. The highest non-toxic concentration of calcium propionate for the IC50 of the normal cells was used to calculate selectivity index of A549 (0.34 mg/mL) and DMS114 (0.75 mg/mL) cells.

Intracellular ROS

Cells were pretreated with various concentrations of calcium propionate (0.25–16 mg/mL), and after 24-h incubation period, intracellular ROS productions of calcium propionate were evaluated using the H2DCFDA assay in cells. High metabolic activity and higher ROS amount in metabolism are expected in cancer cells compared to healthy cells. The noticeable increase in ROS levels was more pronounced in A549 cells compared to both DMS114 and the control Beas-2B cells (Figure 2). ROS measurements were statistically significant for all concentrations except DMS114, and Bas-2B cells exhibited statistical significance after 0.5 mg/mL (p<0.05).

Figure 2: ROS assay on A549, DMS114 and Beas-2B cells for calcium propionate. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.
Figure 2:

ROS assay on A549, DMS114 and Beas-2B cells for calcium propionate. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.

Intracellular glutathione

After 24 h of incubation with different concentrations of calcium propionate (0.25–16 mg/mL), intracellular GSH levels in A549, DMS114 and Beas-2B cells were measured luminometrically. Calcium propionate treatment at the highest concentration (16 mg/mL) resulted in a reduction of intracellular GSH levels by 20.45 % in A549 cells and 19.45 % in DMS114 cells (Figure 3). However, calcium propionate slightly reduced intracellular GSH levels of Beas-2B. Thus, it was observed that the intracellular GSH level decreased the most in A549 cancer cells, and this trend is almost the same in DMS114 cells.

Figure 3: Changes in intracellular GSH content of A549, DMS114 and Beas-2B cell lines after exposure to calcium propionate. Data are presented as the mean ± SD of three independent experiments, p<0.05 (compared with the control).
Figure 3:

Changes in intracellular GSH content of A549, DMS114 and Beas-2B cell lines after exposure to calcium propionate. Data are presented as the mean ± SD of three independent experiments, p<0.05 (compared with the control).

In comparison to the control, GSH level showed a statistically significant increase at concentrations exceeding 0.25 mg/mL for A549 and 0.5 mg/mL for DMS114 (p<0.05). However, for Beas-2B, the changes were statistically insignificant across all concentrations (p>0.05).

Changes in MMP

Cells were treated with different concentrations of calcium propionate (0.25–8 mg/mL). Then DiOC6(3) staining was applied, and fluorescence intensities were read using a fluorescence spectrophotometer. At the highest concentration of calcium propionate (8 mg/mL), the MMP ratio in A549 cells was reduced by 30.12 %, in DMS114 cells by 28 %, and approximately 14 % in Beas-2B cells compared to the control. The cell line whose membrane potential was least affected by calcium propionate was Beas-2B (Figure 4).

Figure 4: The effect of calcium propionate on MMP in cells. All data were expressed as mean ± SD of three experiments, p<0.05.
Figure 4:

The effect of calcium propionate on MMP in cells. All data were expressed as mean ± SD of three experiments, p<0.05.

Our data demonstrated that calcium propionate treatment led to the dramatic decrease in MMP for A549 and DMS114 cells in a dose-dependent manner.

Apoptosis assessment

Acridine orange/ethidium bromide (AO/EB) staining shows apoptosis induction by calcium propionate. Increasing concentrations of calcium propionate induced apoptosis in all cells. Depending on the concentration, the rate of apoptotic cell death increased most rapidly in A549 cells. At the highest concentration of calcium propionate, apoptotic cells were 16.5 % in A549 cells, 9.7 % in DMS 114 and 6.82 % in control cell Beas-2B (Figures 5–6).

Figure 5: The effect of calcium propionate on the apoptotic activity of lung cells. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.
Figure 5:

The effect of calcium propionate on the apoptotic activity of lung cells. All values are expressed as mean ± SD (error bars) of three measurements, p<0.05.

Figure 6: Representative fluorescence images showing AO/EB stained A549 and DMS114 cells untreated and treated with different concentrations of calcium propionate.
Figure 6:

Representative fluorescence images showing AO/EB stained A549 and DMS114 cells untreated and treated with different concentrations of calcium propionate.

Discussion

Lung cancer holds the top position in cancer-related fatalities [25], and nutrition is acknowledged as one of the preventive factors in the development of cancer [26]. The wrong diet or the chemicals we ingest with food have genetic and environmental effects. A meta-analysis found a positive association between consumption of food additives and increased cancer risk, suggesting that disruption of cellular oxidative status by food additives may be a contributing factor [27]. Commonly present in various foods, particularly in bread, calcium propionate has been studied for its impact on human health [28, 29]. However, the anti-cancer activity of calcium propionate has never been reported before. The limited available information prompted our research investigation into this subject.

Calcium propionate induced superoxide dismutase activity through SKBR3 breast cancer cell line in range 0.5–2 mg/mL [11]. Based on the findings, our research marked the exploration into the molecular effects of calcium propionate on human lung cells compared to healthy cells of a wider range of calcium propionate concentrations, from 0.25 to 16 mg/mL. The findings revealed a notable dose-dependent inhibition of A549 cell growth induced by calcium propionate (Figure 1). The calculated IC50 of calcium propionate for A549 was found at 5.44 mg/mL, and for DMS114 was 12.15 mg/mL after a 24-h exposure. However, calcium propionate did not cause any toxicity reaching the IC50 value in Beas-2B cells. The resistance of A549 to calcium propionate was lower, so that A549 cells showed a different response than the Beas-2B in terms of cell viability.

Moreover, we delved into the molecular mechanisms leading the calcium propionate-induced cell death in A549 and DMS114 cells. Previous studies have indicated that chemo-preventive food additives can instigate cell death and apoptosis, partially by fostering the accumulation of ROS and perturbing redox homeostasis [30]. It is well-established that ROS buildup can lead to endoplasmic reticulum stress and DNA damage, consequently triggering changes in the expression of downstream proteins such as p53 [31]. Usually, cancer cells demonstrate elevated baseline levels of ROS compared to normal cells, stemming from an imbalance between oxidants and antioxidants [32]. Therefore, it can be expected that cell death due to apoptosis caused by the cumulative effect of ROS and basal ROS created by the pro-oxidant activity of the active substances in cancer cells is higher than in healthy cells. Elevated levels of ROS result in damage to proteins, nucleic acids, lipids, membranes, and organelles, ultimately culminating in cell death [33]. Our data showed that upon treatment with calcium propionate, a high amount of ROS was reached, and a positive correlation was found between the increased ROS level and the resulting cytotoxicity (Figure 2).

Certain food additives can influence cellular GSH and ROS levels through several mechanisms such as induction of oxidative stress, disruption of mitochondrial function and impairment of cellular signaling [34]. GSH is a critical antioxidant molecule that can neutralize ROS and reactive nitrogen species, helping to protect cells from oxidative stress [35]. It has been found that sodium benzoate, a food additive, has a toxic effect by increasing the amount of intracellular ROS [36]. Increasing the amount of ROS in the cell creates a synergistic effect with the decrease in GSH level, increasing cytotoxicity, genotoxicity and apoptosis in the cell simultaneously [37]. In our study, calcium propionate depleted GSH significantly in A549 and DMS114 more than Beas-2B (Figure 3). This observed decrease in GSH levels can be attributed to excessive oxidative stress caused in cancer cells and hence to the increased amount of ROS [38, 39].

The fundamental strategy for cancer therapy is to induce apoptosis in cancer cells, a process that can be categorized into either extrinsic or mitochondria-dependent pathways [40]. MMP is an essential parameter for mitochondrial function and also an indicator of cell health. The cellular levels of ATP and MMP are maintained within a specific range. Decreased MMP is indicative of loss of mitochondrial membrane integrity, reflecting the initiation of proapoptotic signaling. Prolonged reduction in MMP can lead to pathological events, causing damage to the membrane and triggering apoptosis in the cell [41]. Calcium propionate significantly reduced MMP levels in lung cancer cells (Figure 4). It is thought that this decrease is first activated by cellular apoptotic mechanisms. Hence, calcium propionate reduced MMP and caused apoptosis in lung cancer cells. This concludes that the generation of MMP was enhanced upon treatment with calcium propionate, which affected the apoptosis cells and reduced cell proliferation which indicates anticancer activity in vitro (Figure 5).

Conclusions

In this study, it was found that increasing concentrations of calcium propionate on A549 and DMS114 cells in vitro increased cytotoxicity, apoptosis and intracellular ROS levels, while decreasing MMP and glutathione levels remarkably. Nevertheless, it is imperative to conduct further comprehensive studies that concentrate on molecular pathways in detail. Utilizing models such as co-cultured diverse cells and in vivo models is crucial to evaluate the findings derived from our research.

Corresponding author: Macit Koldas, Department of Medical Biochemistry, Hamidiye Faculty of Medicine, University of Health Sciences, Istanbul, Türkiye, E-mail:

Acknowledgments

This study was undertaken for Tuğba Muhlise Okyay’s Doctor of Philosophy degree (PhD) thesis. We acknowledge coordinatorship of scientific research projects of the University of Health Sciences, Turkey for their financial support to the project titled as “Effects of calcium propionate on invasion, migration, proliferation, apoptosis and long non-coding RNAs (lncRNA) in lung cancer cells”.

  1. Research ethics: Non applicable.

  2. Informed consent: Non applicable.

  3. Author contributions: T.M.O. conceived, designed the experiments, analyzed the data, wrote the paper and performed the MTT experiments, E.M.G. performed the GSH, MMP and ROS experiments, F.G. edited the paper, E.K. edited the paper, M.K. supervised and coordinated the research project.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: Coordinatorship of scientific research projects of the University of Health Sciences Turkey with 2020/077 number.

  7. Data availability: Not applicable.

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Received: 2024-04-01
Accepted: 2024-07-05
Published Online: 2024-09-30

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0054
Keywords for this article
calcium propionate; cancer; food additive; cell viability; lung cancer
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Translational medicine and a new potential example: brain-derived neurotrophic factor (BDNF)
  4. Review
  5. Extraction, purification, characterization and antidiabetic mechanisms of plant polysaccharides: a critical review
  6. Research Articles
  7. Some animal protein antigens identified in cells of two plant species
  8. Determining the kinetic and optimum characteristics of glucose oxidase immobilized on polyurethane
  9. Efficacy and safety of sulforaphane-loaded emulsomes as tested on MCF7 and MCF10A cells
  10. Effect of silibinin on GAS6/sAXL and JAK/STAT pathways in human cholangiocarcinoma cell line
  11. The comparative evaluation of cell viability, inflammatory response, and antimicrobial activity of calcium hydroxide-bovine dentin grain
  12. Curcumin suppresses cell viability in breast cancer cell line by affecting the expression of miR-15a-5p
  13. Impact of calcium propionate on cellular behavior in A549 and DMS114 lung cancer cell lines
  14. The diagnostic value of serum exosomal miRNA-587 combined with hypersensitive C-reactive protein as noninvasive biomarker in early-stage non-small cell lung cancer
  15. Effects of YM087 and VPA985 on the T237M mutant receptor functionality in nephrogenic diabetes insipidus
  16. Relationship of paraoxonase-1 and paraoxonase-3 with routine laboratory tests and oxidative stress in type 2 diabetes mellitus
  17. Chromosomal breakage and sister chromatid exchange analysis in breast cancer patients with heterozygous BLM gene variants
  18. Investigation of SARS-CoV-2 release in fecal specimens of discharge COVID-19 patients
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