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ORIGINAL ARTICLE
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Vanillin extracted from proso and barnyard millets induces cell cycle inhibition and apoptotic cell death in MCF-7 cell line


 Department of Biotechnology, School of Bioengineering, College of Engineering and Technology, Faculty of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Chengalpattu, Chennai, Tamil Nadu, India

Date of Submission17-Dec-2019
Date of Acceptance12-Apr-2020
Date of Web Publication22-Jul-2021

Correspondence Address:
Nageswaran Sivalingam,
Department of Biotechnology, School of Bioengineering, College of Engineering and Technology, Faculty of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Chengalpattu District - 603 203, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1128_19

 > Abstract 


Context: Consuming whole grain food has been motivated due to numerous health benefits arising from their bioactive components.
Aims: This study aims to study whether the active compound extracted from Proso and Barnyard millets inhibits cell proliferation and induces apoptotic cell death in MCF-7 cell line.
Materials and Methods: Cell proliferative effect was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using MCF-7 cell line. Cytotoxicity was determined by release of lactate dehydrogenase (LDH) enzyme from cells. Apoptotic morphological changes in MCF-7 cells were observe under fluorescence microscope using double staining of Hoeschst 33342/propidium iodide (PI). Induction of apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/PI through flow cytometry.
Results: In this study, cell proliferative effect of the bioactive compounds from proso millet (Compound 1) and barnyard millet (Compound 2) was evaluated using MCF-7 cell line. Both the compounds significantly inhibited the proliferation of MCF-7 cells after treated with 250 μg/ml and 1000 μg/ml concentration for 48 h. Cytotoxic activity of compounds was assessed by the release of LDH showed that these extracted compounds were not toxic to the cells. Apoptosis was confirmed by Hoechst 33,342/PI dual-staining, Annexin V-FTIC/PI staining, and flow cytometry results of cell cycle analysis shows that there was a significant cell arrest in the G0/G1 phase and increased the apoptotic cells in sub-G0 phase in a dose-dependent manner.
Conclusions: This study suggests that the extracted vanillin compound from these millets have effectively induced apoptotic cell death in breast cancer cell line.

Keywords: Apoptosis, barnyard millet, breast cancer cell line, proso millet, vanillin



How to cite this URL:
Ramadoss DP, Sivalingam N. Vanillin extracted from proso and barnyard millets induces cell cycle inhibition and apoptotic cell death in MCF-7 cell line. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322158




 > Introduction Top


Worldwide annually around 2%–3% of deaths was recorded is due to different types of cancer.[1] Apoptosis is a programmed cell death; during the past several decades, it has been widely considered as an ideal technique to kill cancer cells. Natural products, plant derivatives, and chemical agents are been reported to control malignant cancer which promotes apoptosis.[2] This natural product with antitumor activity reports an exceptional pharmacotherapeutic potential.[3]

Millets are served as a major staple food[4] and considered to be the first cereal grains for populations in the world. Moreover, in several developing countries, 80% of millet species were utilized and unexploited as a food.[5] Proso millet (PM) or common millet (Panicum miliaceum) and Barnyard millet (BM) (Echinochloa frumentacea) are vital minor millet grown widely in the arid and semiarid region of India, Africa, Japan, other Asian countries, and Europe.[6] The grains of millets are nutritionally greater than rice and wheat.[7] Millets act as antinutrients, these antinutrients are well established to reduce the risk of cancer in animals mainly breast and colon cancer.[8] Particularly, whole grains fiber has been revealed to conserve against breast cancer.[9] According to Dr. Liu, whole grain have high potential against cancer.[10]

Vanillin is a characteristic natural bioactive component generally found in many plant species having multiple properties.[11] Based on the relationship among antimutagenic and anticarcinogenic activities, it was liable that vanillin presents anticarcinogenic effect, though very few literature studies accomplished to study its anticarcinogenic effects have indicated merely conflicting outcomes.[3] However, there is a lack of scientific studies that evaluate the cytotoxicity, apoptosis, and cell cycle inhibition inducing the effect of PM and BM or its active compound in human breast cancer cell line.

MCF-7 cells are appropriate for in vitro studies because cell line has sustained many ideal characteristics specifically to the mammary epithelium. MCF-7 cell line is most broadly utilized as a model of estrogen positive breast cancer cell line for the investigation of breast cancer as it narrates to the sensitivity of the cells to apoptosis.[12] Hence, the human breast cancer cell line (MCF-7) were used to study whether the bioactive component extracted from PM and BM inhibits cell proliferation and induce apoptotic cell death. We hypothesized that vanillin extracted from PM and BM has antiproliferative and proapoptotic effect in breast cancer cell line.


 > Materials and Methods Top


Plant materials

Dried seeds of PM (CO[PV] 5) and BM (CO[KV] 2) were purchased and authenticated from the Department of millet, Tamil Nadu Agriculture University, Coimbatore, Tamil Nadu, India. The seeds were thoroughly washed with tap water and rinsed with distilled water twice and dried at room temperature. Before proceeding to the extraction process, the dried seeds were ground in a blender to form a powder.

Extraction of bioactive compounds

In this study, bioactive compounds were extracted by supercritical fluid extraction system with pressure 10 MPa, temperature 40°C and dynamic extraction time 60 min using liquid carbon dioxide and methanol as co-solvent. The flow rate of co-solvent and CO2 was retained at 6 g/min and 1 ml/min (14%), respectively.[13] The total amount of solute collected and weighed. The structure of the extracted bioactive compounds from PM (C1) and BM (C2) was elucidated using spectroscopic methods (UV, IR, LC-MS, 1H-and 13C-NMR).

Cell culture

Breast cancer cell line (MCF-7) was obtained from national center for cell sciences, Pune, India. MCF-7 cells were cultured in DMEM medium containing 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate supplemented with 10% heat-inactivated fetal bovine serum and 100U/ml penicillin G, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin. The cells were maintained at 37°C in a humid of 5% CO2 incubator. The experiments were performed when the cells reach 80% confluence; all the experiments with these cells were carried out with the passage number between (12 and 23).

In vitro studies, cells were seeded in a 96 well plate for 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell viability and incubated in 5% CO2 incubator for 24 h at 37°C. The cells were treated with 250 μg/ml and 1000 μg/ml concentration of extracted PM and BM compounds (C1 and C2) and incubated for 24, 48, and 72 h and assays were performed. The DMSO (Dimethyl sulfoxide) treated cells served as controls. The extracted C1 and C2 dissolved in less than 0.01% DMSO with the medium. We have also used commercially available synthetic vanillin as a positive control group which has been shown to induce apoptosis.[14]

In vitro bioactivity assay

Antiproliferative activity

The cell proliferation was determined by assessing the reduction of MTT to formazan.[15] The resulting MTT products were determined by measuring the absorbance (OD) at 570 nm using ELISA reader (Model 680 Microplate Reader, Bio-Rad).[16]

Cell viability

Cell viability assay was performed by trypan blue dye exclusion assay to quantify the percentage of viable cells and dead cells.[17]

Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate in the presence of coenzyme NAD+. LDH released from cells during plasma membrane damage indicates non-apoptotic cell death. In MCF-7 cells, the cytotoxic effect of extraction compounds was measured by LDH assay as described previously by Donald W Moss.[18]

Morphological detection of apoptosis

Inverted and phase contrast microscope

MCF-7 cells were seeded in 6 well plates at a density of 1 × 106 cells/well and grown for 24 h. The cells were treated with extracted compounds at 250 μg/ml and 1000 μg/ml concentration and incubated for 48 h. After the treatment period, the medium was aspirated and the cells were washed with phosphate-buffered saline. Morphological changes were examined using inverted (Leica, Germany) and phase contrast microscopy (Olympus, CKX41/CKX31) and images were photographed.

Fluorescence microscopy

Hoechst 33342 and propidium iodide (HO/PI) double staining method was used to examine the apoptotic morphological changes in each cell under fluorescence microscopy.[19] MCF-7 cells were seeded at a density of 1 × 106 cells/well were grown over the coverslip and incubated for 48 h with extracted C1 and C2 at concentrations of 250 μg/ml and 1000 μg/ml in a 6 well plate.

Cell cycle analysis by flow cytometry

PI is been frequently used dye for DNA content/cell cycle analysis. MCF-7 cells were seeded onto the 60 mm petri dish at a density of 1 × 106 cells/ml and grown for 24 h. The cells were then treated with 250 and 1000 μg/ml concentration of extracted compounds for 48 h. After 48 h treatment, the cells were analyzed by FACScalibur flow cytometry (BD Bioscience) using CellQuest pro software (Becton Dickinson, San Jose, CA, USA) as previously described by Carlo and Nicoletti.[20]

Annexin V-fluorescein isothiocyanate/propidium iodide staining assay

The Annexin V fluorescein isothiocyanate (FITC)/PI double staining assay was performed to detect cellular apoptosis. MCF-7 cells (1 × 106) were cultured in 6 well plates and grown for 24 h. The cells were treated with extracted compounds at 250 and 1000 μg/ml for 48 h. After the treatment period, the cells were analyzed by FACScalibur flow cytometry (BD Bioscience) within 1 h of staining as described earlier by Chang et al.[21] Data were analyzed using CellQuest Pro software (Becton Dickinson, San Jose, CA, USA).

DNA fragmentation

DNA fragmentation assay is a definitive assay to confirm apoptotic cell death. After treating the cells with 250 μg/ml and 1000 μg/ml concentration of extracted compounds for 48 h, DNA was extracted as described by Samarghandian and Shabestari[22] and separated using 1% agarose gel and then DNA was visualized in Chemidoc (Bio-Rad Molecular Imager ChemiDoc XRS + imaging system, California, USA).

Statistical analyses

All experiments were performed in triplicates and expressed as mean ± standard deviation of the results of three independent experiments, n = 3 and statistical analyses were performed by one-way ANOVA with the Tukey's multiple comparison test and two-way ANOVA with the Bonferroni post-test using GraphPad Prism 5 software to determine the significant differences between the groups. P < 0.05 was considered to be statistically significant between the control and treated groups.


 > Results Top


The compounds extracted from PM and BM using supercritical fluid extraction method. The extracted compounds structure was elucidated using spectroscopic methods (LC-MS, IR, 1H-and 13C-NMR). Based on the spectroscopic data, it was identified that bioactive component extracted from PM (C1) and BM (C2) was found to be a phenolic aldehyde-vanillin (4-Hydroxy-3-methoxybenzaldehyde), molecular formula: C8H8O3, molecular weight: 152.0 (data not shown).[23]

Antiproliferative activity

In this study, we have evaluated the anticancer activity of extracted PM compound (C1) and BM compound (C2). 250 and 1000 μg/ml concentration of extracted C1 and C2 was used for testing the proliferative potential (MTT assay) and cell viability of breast cancer cells (MCF-7) at various time intervals (24 h, 48 h, and 72 h) [Figure 1]a. It was found that extracted compounds (1 and 2) indicate a significant reduction in the number of viable cells at the concentration of 1000 μg/ml than 250 μg/ml when compared with control at a different time interval [Figure 1]b. This result implies that the viability of MCF-7 cells decreases as the concentration of compounds increases showing a dose-dependent manner. Based on these results, 250 μg/ml and 1000 μg/ml concentration of extracted compounds with 48 h treatment period was chosen for the further assay, which shows significant results without cytotoxicity.
Figure 1: Effect of the extracted compound 1 and 2 on antiproliferative and cell viability against MCF-7 cell line. (a) Antiproliferative activity of compound 1 and compound 2 extracted from proso and barnyard millet (b) percentage viability of cells after treating with compound 1 and compound 2 extracted from proso and barnyard millet. Values are expressed as mean ± standard deviation *Indicates a significant result when compared with control (P < 0.05). Whereas, Proso millet – Compound 1, Barnyard millet-Compound 2

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Lactate dehydrogenase cytotoxicity assay

The significant results of MTT assay and cell viability assay shows that C1 and C2 possess antiproliferative activity. Furthermore, we evaluated if these compounds induce cytotoxic cell death or non-cytotoxic cell death. LDH is a stable cytosolic enzyme present in various forms of cells. Cytotoxicity assay measures the releases of the enzyme activity through damaged plasma membrane when cytotoxic cell death occurs, LDH was released from the cells into the medium is a marker to determine the cytotoxic activity was analyzed. The cytotoxicity of the extracted compounds by LDH release assay was determined on MCF-7 cells treated with 250 and 1000 μg/ml concentration of C1 and C2 for 48 h, the activity of LDH was measured in the medium. LDH release in the medium implies the loss of membrane integrity due to necrosis. Although there was a difference in LDH release between control and treated cells it was not a significant difference in LDH release at 250 μg/ml of C1 and C2 and 1000 μg/ml of C2 showed a significant difference when compared with control. This result confirms that the compounds induce non-cytotoxic cell death [Figure 2]. We have shown that the cell death induced by vanillin extracted from PM and BM was through up-regulating apoptotic proteins using western blotting analysis.[24]
Figure 2: Effect of compound 1 and compound 2 on Lactate dehydrogenase release on MCF-7 cell line. Cytotoxicity assay measures the activity of a cytosolic lactate dehydrogenase enzyme release into the medium after cells treated with extracted compounds (250 and 1000 μg/ml) for 48 h. Whereas, Proso millet-Compound 1, Barnyard millet-Compound 2. Values are expressed as mean ± standard deviation *Indicates a significant result when compared with control (P < 0.05)

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Morphological studies

Morphological changes of MCF-7 cells untreated and treated with 250 and 1000 μg/ml concentration of extracted compounds (1 and 2) for 48 h were observed under an inverted microscope and phase contrast microscope. The most apparent changes characteristics of apoptosis were observed in the treated cells. Morphological observation reveals that more than half of the cells exhibited characteristic features of apoptosis such as detachment of cells, shrinkage of cells, and a decrease in the cell number prominent with an increase in the concentration of C1 and C2 [Figure 3].
Figure 3: Effect of vanillin extracted from proso and barnyard millets on morphological changes in MCF-7 cell line. Cells were treated with C1 and C2 for 48 h induce morphological changes in MCF-7 breast cancer cells. (a) Control and treated cells observed under Inverted microscope (magnification 20x) and photographed (A) control (B) 250 μg/ml (C1) (C) 1000 μg/ml (C1) (D) 250 μg/ml (C2) (E) 1000 μg/ml (C2) (F) Vanillin. (b) Control and treated cells observed under Phase contrast microscope (magnification 20×) and photographed (G) control (H) 250 μg/ml (C1) (I) 1000 μg/ ml (C1) (J) 250 μg/ml (C2) (K) 1000 μg/ml (C2) (L) vanillin. Arrows indicate Shrinkage of cells. Whereas, C1-Compound 1, C2-Compound 2

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Fluorescence microscopy

Apoptosis is a physical pattern of cell death considered by morphological characteristics and wide DNA fragmentation.[19] Apoptosis was appraised cytologically using the dual staining of HO and PI. Morphological changes in cell nuclei were determined by fluorescence microscope. Hoechst 33342 (blue fluorescent) is a cell-permeable nucleic acid dye and the propidium iodide (red fluorescent) is cell impairment DNA-binding dye. The viable cells shown the blue nuclei, dead cells exhibited red nuclei, and apoptotic cells signified pinkish red nuclei, as shown in [Figure 4.
Figure 4: Effect of vanillin extracted from proso and barnyard millets on nuclear morphology in MCF-7 cells. Cells were detected with double staining of Hoechst 33342/ propidium iodide. MCF-7 cells were treated with extracted compounds (1 and 2) for 48 h and imaged by fluorescence microscope (magnification 10×) (a) Control (b) 250 μg/ml (C1) (c) 1000 μg/ml (C1) (d) 250 μg/ml (C2) (e) 1000 μg/ml (C2) (f) Vanillin. Arrows indicate 1-Viable cells, 2-Apoptotic cells. Whereas, C1-Compound 1, C2-Compound 2

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The morphological study in the cell nuclei of MCF-7 cells treated for 48 h with 250 and 1000 μg/ml concentration of C1 and C2 displayed major morphological alterations when compared to control and this was imaged by fluorescence microscope. The apoptotic nuclei exhibited uniform fluorescent distribution due to highly fragmented and condensed chromatin. Apoptotic cells were initiated that there is an increase in a dose-dependent manner, the number of apoptotic cells was higher in 1000 μg/ml than in 250 μg/ml. Vanillin (400 μg/ml) used as a standard was also found to increase apoptotic cells when compared with control. Thus, HO/PI staining of MCF-7 cells displayed that the cells had undergone prominent morphological changes in apoptotic bodies.

Cell cycle analysis

Two foremost mechanisms involved in anticancer drug treatment are cell cycle arrest and apoptosis, which can be measured by DNA content. Cell cycle plays an important role in cell replication, cell death and its function.[25] To determine cell growth inhibition involving cell cycle changes, the cell cycle phase distribution was examined by flow cytometry. There was a statistically significant difference in the cell cycle stages between treated cells and control in the Sub-G0 phase which is a marker for apoptosis, G0/G1, S, and M/G2 phases. Our results showed that MCF-7 cells after 48 h treatment with 250 and 1000 μg/ml of the extracted compounds 1 and 2 have induced cell cycle arrest in the G0/G1 phase significantly when compared to control. The percentage of G0/G1 phase cells in control was found to be 71.34%, whereas it was found to be 45.23%, 17.85% (C1) and 35.63%, 19.46% (C2), respectively. The percentage of the S phase cells in the control, it was 8.19%, whereas there was a slight increase of 9%, 5.06% (C1) and 9.52%, 4.33% (C2). The percentage of G2/M phase cells, in control it was found to be 12.95%, whereas it was increased to 23.91%, 4.72% (C1) and 26.52%, 3.96% (C2) for 250 μg/ml and 1000 μg/ml concentration, respectively. The percentage of sub-G0 phase (apoptotic cells) was found to be 4.99% in control, whereas it was increased to 14.54%, 56.91% (C1) and 11.50%, 54.81% (C2), respectively. Vanillin (400 μg/ml used as standard) induces cell cycle arrest and apoptosis. Our results suggested that C1 and C2 inhibited the cell cycle progression by inhibiting cells at the G0/G1 phase and increased the apoptotic cells in sub-G0 phase in a dose-dependent manner. This result confirms that these compounds induced cell cycle arrest led to growth inhibition of MCF-7 cells [Figure 5]a and [Figure 5]b.
Figure 5: E ffect of extracted compounds 1 and 2 on cell cycle arrest and apoptosis in MCF-7 cell line. MCF-7 cells were treated with 250 and 1000 μg/ml concentration of extracted compounds for 48 h and analyzed by flow cytometry after staining with propidium iodide. (a) Percentages of DNA content at sub G0, G0/G1, S, and G2/M phases of MCF-7 cells were analyzed (A) Control (B) 250 μg/ml (C1) (C) 1000 μg/ml (C1) (D) 250 μg/ml (C2) (E) 1000 μg/ml (C2) (F) Vanillin. (b) Flow cytometric cell cycle analysis and quantification of the percentage of DNA content. Whereas, A and C1–Compound 1, B and C2-Compound 2. Values are expressed as mean ± standard deviation *Indicates a significant result when compared with control (P < 0.05)

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Annexin V-fluorescein isothiocyanate/propidium iodide staining assay

Apoptosis is a programmed cell death with a typical physical method that plays a vital role in internal stability and progress of normal and cancer cells, additionally, dysregulation of apoptosis generally studied as a main property of cancer.[26] In this study, MCF-7 cells were double stained with annexin V-FITC and PI which can differentiate between necrotic cells, live cells, early apoptotic cells, and late apoptotic cells or dead cells. Annexin V specifically binds to phosphatidylserine and hence allows the use of a fluorescein labeled Annexin V to recognize the cells that are undergoing apoptosis. The cells were furthermore stained with PI to distinguish early apoptotic cells from necrotic cells.

Our results revealed that in control 95.24% of the cells were viable, 2.10% cells were in early apoptosis, 1.70% of the cells were necrotic cells, and 0.96% of the cells were in late apoptosis or dead cells. Whereas, MCF-7 cells treated with extracted compounds 1 and 2 at the concentration of 250 and 1000 μg/ml for 48 h exhibited a decreased percentage of viable cells of 25.60%, 8.13% (C1) and 35.06%, 11.17% (C2), respectively. The percentage of necrotic cells was found to be increased with 23.53%, 5.15% (C1) and 29.29%, 7.33% (C2). Late apoptotic or dead cells was found to be increased to 49.82%, 86.19% (C1) and 33.25%, 80.38% (C2) and early apoptotic cells was found to be decreased to 1.05%, 0.40% (C1) and 0.73%, 1.12% (C2), respectively. Similarly, in vanillin (Standard) 26.69% of the cells were viable, 1.10% cells were in early apoptosis, and 18.82% of cells necrotic cells and 55.24% of cells were in late apoptosis when compared with control. These results have concluded that MCF-7 cells treated with extracted compounds 1 and 2 have significantly increased the percentage of late apoptotic cells in a dose-dependent manner when compared with control [Figure 6]a and [Figure 6]b.
Figure 6: Effect of extracted compounds 1 and 2 on induction of apoptosis in MCF-7. (a) The MCF-7 cells were treated with 250 and 1000 μg/ml concentration of extracted compounds for 48 h and labelled with Annexin V-fluorescein isothiocyanate/propidium iodide. The upper left quadrant-primary necrotic cells, the upper right quadrant-late apoptotic or secondary necrotic cells, the lower left quadrant-viable or live cells and the lower right quadrant-Early apoptosis (A) Control (B) 250 μg/ml (C1) (C) 1000 μg/ml (C1) (D) 250 μg/ml (C2) (E) 1000 μg/ml (C2) (F) Vanillin. (b) Bar chart representing the percentage of apoptosis. Whereas, A and C1 – Compound 1, B and C2-Compound 2. Values are expressed as mean ± standard deviation *Indicates a significant result when compared with control (P < 0.05)

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DNA fragmentation

One of the last stages of apoptosis is the internucleosomal DNA fragmentation resultant after the initiation of endogenous DNase, which cut the internucleosomal regions into double-standard fragments of 180–200 base pairs units.[27] To illustrate whether extracted compounds initiate apoptotic cell death through the induction of DNA fragmentation, genomic DNA isolated from MCF-7 cells that were treated with 250 and 1000 μg/ml concentration of C1 and C2 for 48 h and DNA fragmentation was evaluated using agarose gel electrophoresis. The compounds treated cells shows smear DNA pattern in the lanes [Figure 7]. The smearing might be due to apoptosis that took place as a result of treatment. In contrast, the DNA from control cells did not show any fragmentation or smearing. This result implies that C1 and C2 induced an internucleosomal cleavage of DNA in the MCF-7 cells prominent to the generation of fragments which is the induction of apoptosis.
Figure 7: Effect of vanillin extracted from proso and barnyard millets on DNA fragmentation in MCF-7 cell line. MCF-7 cells were treated with extracted compounds at 250 and 1000 μg/ml concentration for 48 h and genomic DNA was extracted and separated using 1% agarose gel and visualized with ethidium bromide in Chemidoc. Lane 1-Marker (100 bp), Lane 2-Control, Lane 3-250 μg/ml (C1), Lane 4-1000 μg/ml (C1), Lane 5-250 μg/ml (C2), Lane 6-1000 μg/ml (C2), Lane 7-Vanillin

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 > Discussion Top


The health benefits of whole grains correlated with phytochemicals have important functional properties.[28] Dietary phytochemicals have the capacity to act on various mechanisms to deliver defense against cancer.[29] In the present study, MTT assay and cell viability of extracted compound vanillin were assessed using the MCF-7 cell line for 48 h. The results revealed that extracted compounds showed a significant reduction in viable cells in a dose-dependent manner. Lirdprapamongkol et al. postulated that vanillin has the potential to be developing an effective anticancer agent having low toxicity and therapeutic properties preventing angiogenesis and metastasis.[30] Deb et al. stated that synthetic vanillin showed weak toxicity against the SW480 cell line with the IC50 value of 380 μg/ml.[31] Janicke et al. have reported that dietary phenolic compound exhibited an antiproliferative effect on Caco-2 cells.[32] Zhang et al. reported that both bound and free phytochemical extracts of PM show higher antiproliferative activity with a lower EC50 value of 46.47 mg/ml in MDA human breast cancer cells in a dose-dependent manner.[33] Furthermore, food derived phenolic acids have shown antiproliferative activity against human breast cancer cells T47D.[34] Whereas, in our study, vanillin compound extracted from PM and BM inhibited the cell proliferation in a low concentration of 250 μg/ml when compared with control.

The natural analogs from plant sources offer a chance to assess the influence of structure in the compounds cytotoxicity. In previous studies, only a few investigations have been carried out on the cytotoxic mechanism of vanillin. Based on the LDH results, it was concluded that C1 and C2 from PM and BM effectually induces non-cytotoxic cell death at a low dose which suggests that the cell death might be mediated through the induction of apoptosis in MCF-7 cells. It has been further proved by analyzing the apoptotic proteins using western blotting analysis.[24]

Apoptosis (programmed cell death) with a characteristic physical process which plays an important part in the internal balance and development of normal and cancer cells. Usually, the main property of cancer is dysregulation of cancer is instigated by alternations in the DNA.[26] During mutation, a normal cell will repair the alternation or die, while cancer cells survive with the transformations and proliferate in an uncontrolled manner, resulting in tumor formation. Frequently those tumors affect the healthy tissues and extend to the different parts of the muscles.[35]

The MCF-7 cells were double stained with PI/HO revealed nuclear morphological changes such as condensed chromatin and DNA fragmentation. When cells treated with 1000 μg/ml concentration a high number of apoptotic cells were identified than treating cells with 250 μg/ml. It was found that there is an increase in the apoptotic cells in a concentration-dependent manner. These characteristic features form the foundation for the most extensively worn methods for the measurement and identification of apoptosis. Fluorescence microscopy and phase contrast microscopy are the most ideal approaches to characterize apoptosis.[36] Shi et al. have documented that HCT-116 exhibits apoptosis features after treating with foxtail millet bound polyphenol extract and stained with HO.[37] Songpol et al. have identified that PI/HO dual stained NCI-H460 lung cancer cells showed apoptotic cells at a 200 μM concentration of vanillin after 24 h treatment.[38]

Cell cycle progression is a symbol for the proliferation of cells. Cell cycle has arisen as one of the appealing targets of cancer therapy.[39] To measure the changes in the cells the MCF-7 cells were stained with PI (DNA stain) to analysis cell cycle using flow cytometry. The PI stain will diffuse into cells due to membrane permeability in apoptosis cells.[40],[41] This is an appropriate technique to measure the different phases of the cell cycle.[42] Shan et al. have reported that bran protein of foxtail millet induces G1 phase arrest in colon cancer cells.[43] The findings obtained in this study corroborated with previous studies in cancer cell lines suggested that extracted compound 1 and 2 inhibited the cell cycle progression in a concentration-dependent manner, by inhibiting cells at the G0/G1 phase and increasing the apoptotic cells in sub G0 phase This confirms that these compounds induced cell cycle arrest led to growth inhibition of MCF-7 cells.

Vanillin regulated the gene complicated in oxidative damage and DNA damage in HCT-116 colon cancer cell line.[44] Loss of mitochondrial membrane integrity and fragmentation of DNA leads to apoptosis.[45] In MCF-7 cell extracted compounds generated a cleavage of nucleosomal DNA which promoted DNA fragmentation. In the present study, it was detected that C1 and C2 from PM and BM treated MCF-7 cells evidently induced DNA fragmentation. It was documented that derivatives of synthetic vanillin induced cell death by interacting AT-rich sequences of the nucleic acid lead to DNA fragmentation in the leukemic cell line.[46]

Apoptosis is observed as a regulated energy-dependent mechanism, distinguished by appropriate biochemical and morphological features. It is one of the main therapeutic targets for cancer research. In this study, the results evidence that bioactive compound vanillin extracted from PM and BM has an anticancer property. It was concluded that C1 and C2 efficiently induce non-cytotoxic cell death which implies that it might be intermediated through the induction of apoptosis in MCF-7 cells. Our in vitro study indicated that extracted vanillin compound from PM and BM can be a natural effective anticancer agent against breast cancer cells. Hence, further broad investigations are essential to unveil the complete molecular mechanism of apoptosis induced by an extracted compound in vitro and whether proso and BM consumption will have an effect on breast cancer in an animal model.

Financial support and sponsorship

The authors would like to acknowledge SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu, India for the financial support.

Conflicts of interest

There are no conflicts of interest.



 
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