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Relative effect of Malayer Shahani and Asgari grapes seed extract on inducing apoptosis in human leukemia cells


1 Department of Biology, Faculty of Science, Research Institute for Grape and Raisins, Malayer University, Malayer, Iran
2 Department of Chemistry, Faculty of Science, Research Institute for Grape and Raisins, Malayer University, Malayer, Iran
3 Department of Biology, Malayer University, Malayer, Iran

Date of Submission18-Sep-2019
Date of Decision19-Nov-2019
Date of Acceptance07-Jan-2020
Date of Web Publication22-Oct-2020

Correspondence Address:
Maryam Rahimi,
Department of Biology, Faculty of Sciences, Research Institute for Grape and Raisins, Malayer University, P. O. Box: 65719-95863, Malayer
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_766_19

 > Abstract 


Background: Previous studies have suggested that consuming fruit and vegetable can lower the risk of several cancers, including breast, colorectal, and lung cancers.
Aims: The present study aims to investigate the in vitro anticancer effects of Shahani and Asgari grape seed extract (GSE) grown in Malayer City of Iran on HL-60 cancer. However, to the best of the author's knowledge, it is the first time in this study that the antiproliferative effect of Shahani and Asgari GSE is compared.
Materials and Methods: Shahani and Asgari GSE Was extraction white method of Liquid/liquid extraction with ethyl acetate. Then assessing cytotoxic activities of Shahani and Asgari GSE on the HL-60 cells was tested using MTT assay.
Results: The results show that compared with the control group, seed extract of both Shahani and Asgari at the various concentrations (25, 50, 100, and 200 μg/ml) had a significantly inhibitory effect on HL-60 cell proliferation that was dose dependent. However, Shahani GSE at different concentrations (50, 100, and 200 μg/ml) indicated a significantly higher inhibitory effect compared to Asgari GSE. In addition, GSE can induce cell cycle arrest at G0/G1 cells. Furthermore, GSE of Asgari and Shahani remarkably increased the induction of HL-60 cell apoptosis depending on its dose. However, at the concentration of 200 μg/ml, GSE induced cell necrosis rather than apoptosis.
Conclusion: Seed extract of both Shahani and Asgari at the various concentrations had a significantly inhibitory effect on HL-60 cell proliferation that was dose dependent.

Keywords: Apoptosis, G0/G1, grape seed extract, leukemia



How to cite this URL:
Rahimi M, Pakravan N, Babaei A, Mohammadi M, Atafar E. Relative effect of Malayer Shahani and Asgari grapes seed extract on inducing apoptosis in human leukemia cells. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 2]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=298867




 > Introduction Top


Acute myeloid leukemia (AML) is the most common myeloid leukemia with common mutations and chromosomal abnormalities, which occurs due to the transformation of an immature hematopoietic stem.[1],[2],[3] The survival rates of patients with AML have remarkably improved over the past decades because of the current attempts, such as chemotherapy, irradiation, and operating room, to induce cell cycle arrest and apoptosis to limit uncontrolled cell proliferation and survival of cells in malignant neoplastic diseases. However, the side effects of cytotoxic agents in chemotherapy are notorious.[4] Since cytotoxic agents decrease the quality of life in cancer patients, improvement in the quality of their life depends on cytotoxic chemopreventive drugs with high efficacy and low toxicity.[5] Grape seed extract (GSE) offers promising new options for the development of more effective chemotherapeutic strategies for the treatment of different types of cancers such as breast, colorectal, leukemia, and lung cancer.[6],[7],[8],[9] GSE is a mixture of several polyphenolic components forming dimers, trimers, tetramers, and oligomers/polymers of monomeric catechins and/or epicatechins, which result in its antioxidant, free radical scavenging, and anticancer properties.[10],[11],[12] Malayer city in Hamadan province, called the city of “The Golden Bunches of Grapes,” is known for its high-quality grape juice and raisin. The present study aims to compare anticancer activity of GSE of two varieties of Malayer grapes, i.e., Askari and Shahani, on human promyelocytic leukemia HL-60 cells.


 > Materials and Methods Top


Cell culture

Human myelocytic leukemia cells (HL-60 cells) were purchased from the Pasteur Institute, Tehran, Iran, maintained in RPMI-1640 medium (Gibco, UK) and supplemented with 10% fetal bovine serum (Gibco), 100 U/ml of penicillin, and 100 μg/ml of (Gibco) streptomycin in a humidified incubator with 5% CO2 at 37°C. The medium was replaced every 48 h.

Liquid/liquid extraction with ethyl acetate

Liquid/liquid extraction is the main method mostly used to separate proanthocyanidins from grape seeds. Among the polar solvent, ethyl acetate combined with water is the most generally applied liquid/liquid solvent system of this procedure.[13] A typical procedure using this solvent mixture contains a 24 h wait, decantation of the extract, drying, and mixing with a precipitating agent, such as n-hexane, which permits the solid to be filtered out late on.[14] Although other extraction methods applying methanol, ethyl alcohol, and acetone (all with water) are effective, ethyl acetate and water play a more important role in extracting proanthocyanidins.

Assessing cytotoxic activities

To determine the cytotoxic effects of Askari and Shahani GPS on the HL-60 cells, cell viability was tested using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were first seeded in 96-well cell culture plates (Nunc, Denmark) at a density of 3 × 104 cells/ml/well and subsequently incubated in a humidified 5% CO2 environment for 1 h. Then, they were treated with 10, 25, 50, and 100 μg/ml GPS of Askari and Shahani, respectively, for 24 h. Untreated cells were also used as controls. In brief, after each treatment, 100 μl of 5 mg/ml MTT (Sigma, Aldrich, MO, USA) was added to each well in 96-well plates. The crystals of viable cells were solubilized by acid-isopropanol (100 ml of 0.04 N HCl in isopropanol) after incubation for 4 h at 37°C. Then, the optical density (OD) of each well was measured at a wavelength of 570 mm using a spectrophotometer after overnight incubation in darkness. The OD values of the experimental groups were divided by those of control group, and the results were presented as the percentage of cell viability.

Cell cycle analysis

HL-60 cells were treated with different concentrations of GSE. After 48 h, cells were harvested and fixed in 70% ethanol at 4°C overnight. Fixed cells were stained with 5 μL of propidium iodide (PI) for 20 min on ice in the dark. Finally, the fluorescence emitted by PI-DNA complex was tested at 488 nm. The portions of mobile phones in several stages of the cell cycle, namely G0, G1, S, and G2/M, were measured using flow cytometry (Becton Dickinson, Japan) and analyzed by Cell Quest software.

Acridine orange/ethidium bromide staining

Acridine orange/ethidium bromide (AO/EB) staining of HL-60 cells was performed to detect the apoptotic and necrotic patterns as reported before.[15] Briefly, HL-60 cells (2 × 105 cells/ml) were treated by GPS (10, 25, 50, and 100 μg/mL) for 24 h and then washed three times with phosphate-buffered saline. The cells were stained with 100 μg/ml AO/EB for 5 min. At least 200 cells were observed under a fluorescence microscope. The cells were sorted as viable, apoptotic, or necrotic. Then, the percentage of apoptotic cells was estimated as follows:

Percentage of apoptotic cells (%) = (amount of apoptotic cell/total cell examined) × 100%.

Statistical analysis

Student's t-test and one-way analysis of the variation were used to define the statistical meaning of differences between values for various experimental and control groups. P < 0.05 was considered statistically significant.


 > Results Top


Grape seed extract inhibits the proliferation of HL-60 cells

MTT assay showed that, compared with the control group, seed extract of both Shahani and Asgari grapes at various concentrations (25, 50, 100, and 200 μg/ml) significantly inhibited HL-60 cell proliferation depending on its dose. However, Shahani GSE at various concentrations (50, 100, and 200 μg/ml) has a significantly higher inhibitory effect compared with Asgari GSE (P < 0.05) [Figure 1].
Figure 1: Effect of GPS concentration of Shahani and Asgari on HL-60 cell proliferation in 24 h: HL-60 leukemia cells were treated with 10, 15, 25, 50, 100, and 200 of grape seed extract of Shahani and Asgari and percentage of viable cells/control was determined by 3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The experiment was repeated more than three times with similar effects. *,†Statistically significant cells that treated with grape seed extract of Shahani and Asgary alone difference (P < 0.001) compared to control cells and cells that treated with Shahani compared to Asgari (P < 0. 05), respectively

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Grape seed extract induces cell cycle arrest of HL-60 cells

To determine whether GSE inhibits the proliferation of HL-60 cells through regulating cell cycle progression and apoptosis, flow cytometry was first carried out using PI staining. A significant increase from 15.2% and 16.4% was observed in G0/G1 cells in the control group of Shahani and Asgari GSE, respectively, to 51.65% in the GSE-treated group Shahani (50 μg/mL) and 50.31% in the GSE-treated group Asgari (100 μg/mL). However, the seed extract of both Shahani and Asgari grapes at 200 μg/mL and Shahani GSE at 100 μg/mL could not further increase the cell ratio in G0/G1 phase [Figure 2], implying that GSE can lead to cell cycle arrest at G0/G1.
Figure 2: HL-60 cells were treated by GPS of Shahani and Asgari at the indicated concentration for 24 h, and the ratio of cells at G0/G1 was calculated based on flow cytometry. *P < 0.001 versus control

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Grape seed extract induces apoptosis in HL-60 cells

Based on AO/EB staining, uniformly green live cells with normal morphology were seen in control HL-60 cells, while green early apoptotic cells with nuclear margination and chromatin condensation occurred in HL-60 cells treated by 25 and 50 μg/ml of GSE, and orange later apoptotic cells with fragmented chromatin and apoptotic bodies were observed in HL-60 cells treated by 100 μg/ml of it. The percentage of apoptotic cells increased gradually with an increase in GSE concentration from 25, 50, to 100 μg/ml, compared with the control group [Figure 3]. Yet, at the concentration of 200 μg/ml, GSE induced cell necrosis rather than apoptosis, suggesting that GSE induce HL-60 cell apoptosis.
Figure 3: HL-60 cells were treated by GPS of Shahani and Asgari at the indicated concentration since 24, and the percentages of apoptotic cells were estimated based on acridine orange/ethidium bromide staining. *P < 0.001 versus control

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 > Discussion Top


GSE that is industrially derived from grape seeds is regarded as one of the natural food additives recommended for use as an antioxidant. The component of interest is polyphenol, mainly proanthocyanidins, which are condensed tannins.[12] Based on previous studies, GSE has anticancer effects on different tumors,[6],[7],[8],[9] which is mainly ascribed to its catechin and procyanidin. The present study indicates that Shahani and Asgari GSE can inhibit proliferation and stimulate the apoptosis of HL-60 cells in vitro. Antitumor drugs determine cell cycle, inhibit cell proliferation, and induce apoptosis in tumor cells.[16] Another study reported that GSE can induce apoptosis and G1 phase cell cycle arrest of human pancreatic cancer cell line. Here, GSE induced the apoptosis of HL-60 cells in a dose-dependent way within the range of 25–100 μg/ml. However, higher doses of GSE at 200 μg/ml induced cell necrosis, suggesting the toxic effect of GSE high dosage. In addition, crossing at the dose of 25–100 μg/ml induced G0/G1 phase arrest of HL-60 cells depending on the dosage. Thus, the results suggested that GSE inhibited HL-60 cell proliferation by inducing G0/G1 arrest and apoptosis of HL-60 cells. Furthermore, this study demonstrated that Shahani GSE at various concentrations (50, 100, and 200 μg/ml) had a significantly higher inhibitory effect compared with Asgari GSE. Medicinal herbs exert antitumor effects by inducing programmed cell destruction in malignant neoplastic disease cells such as leukemia.[17],[18],[19] Gao et al. showed that the induction of human leukemia cell death activates JNK, which plays a vital role in modulating the cell death response.[20] Based on other studies, GSE exhibits cytotoxicity against some cancer cells in skin,[21] breast,[22] colon,[23] lung,[24] gastric,[18] and prostate cancers[25] and enhances the growth and viability of normal cells.[26] In addition, GSE causes programmed death in human leukemia cells through activation of caspases such as caspase-3.[20]


 > Conclusions Top


In vitro studies demonstrate that seed extract of Shahani and Asgari grapes inhibits the proliferation of HL-60 cells, which may be mediated by the trigger of programmed cell death and cell cycle arrest The findings suggest that, compared with Asgari GSE, Shahani GSE at various concentrations has higher potential to be developed as a novel drug with high efficacy and low toxicity for the treatment of cancer of the ancestry.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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