Vol 8 No 1 2023 – 92

Cytotoxic potential activity of quercetin derivatives on MCF-7 breast cancer cell line

Husam Salman Jasim 1, *, Zainab A. Al-kubaisi 2and Hanady S. Al-Shmgani 3
1    Chemistry Department, Ibn Al-Haitham, University of Baghdad, Baghdad 61023, Iraq 1; husam_alaythawy78@yahoo.com
2    Biology Department, Ibn Al-Haitham, University of Baghdad, Baghdad 61023, Iraq 2
3  Biology Department, Ibn Al-Haitham, University of Baghdad, Baghdad 61023, Iraq 2; hanadysalim@yahoo.com
*   Correspondence: husam_alaythawy78@yahoo.com; Tel.: (9647711250525l).
Available from: http://dx.doi.org/10.21931/RB/2023.08.01.92
Many previous investigations have found quercetin to be a powerful antioxidant and antitumor flavonoid, but its poor bioavailability has limited its use. This current study investigated the effects of two newly synthesized Quercetin Schiff bases containing 2-amino thiadiazole-5-thiol (Q1), and its benzyl derivatives (Q2) on MCF-7 human breast cancer cells. Cell viability and apoptosis were assessed to determine the toxic effects of Q1 and Q2. Cytotoxicity valuation showed that both compounds inhibited MCF-7 cell growth, and lactate dehydrogenase (LDH) activity increased in a dose-dependent aspect compared to the control group. Comet assay results observed that Q1 and Q2 induce more serious DNA damage than the control (untreated cell); however, in all curried experiments, Q2 showed higher effects than Q1. Hence two synthesized quercetin Schiff bases can take action as a promising anticancer agent.
Keywords: quercetin derivatives, Schiff base, breast cancer, MCF-7 Cytotoxic.
Cancer considerable one of the most significant and dangerous diseases. Breast cancer, one of the most widespread types of cancer in women, causes high mortality and morbidity. Modern studies emphasized that quercetin can prevents breast cancer by avoiding signal transduction, the exhortation of cancer cell apoptosis, and repressing proliferation, inroad, and metastases of tumor cells1. Generally, in the human diet, many biomolecules, such as flavonoids, can inhibit the growth of cancer cells and act as «chemo-preventers.» The cancer-prophylactic effects of these biomolecules can be assigned to diverse mechanisms, containing the investment of cell-cycle arrest and/or apoptosis besides the antioxidant ability. The antioxidant properties of chemo-preventers have lately had a broad interest, ultimately the oxidative stress involved in the inception and progression of diverse pathological conditions, which includes cancer. The antioxidants are known for their capability to prohibit oxidative damage and the extensive utilization of natural food-derived antioxidants dad major consciousness as potential anti-carcinogens. Quercetin, one of these flavonoids rated as an excellent free-radical scavenger.  Despite this, this activity mightily counts on the intracellular availability of reduced glutathione. Besides the antioxidant properties of quercetin as well performs a direct, proapoptotic effect in cancer cells, and prohibits the growth of diverse human cancer cell lines at diversified phases of the cell cycle. These impacts are reported in a broad assortment of cellular models and animal patterns. The altitude toxicity strives by quercetin on tumor cells skillfully corresponds with the roughly total non-attendance of any deterioration for normal, non-transformed cells2.
Quercetin, as a significant flavonoid existing in many vegetables, fruits, and beverages3, has another property like anti-hyperlipidemia, anti-hyperglycemia, antiviral, anticancer4, anti-microbial5, and neuroprotective6, antioxidant7, anti-inflammatory8 and anti-apoptotic9 effects that might help reduce swelling, kill cancer cells10, arthritis11, control blood sugar12, quercetin derivatives are most commonly used for conditions of the blood vessels and help prevent heart disease13. Furthermore, the impacts of quercetin on the amelioration of the signs of Metabolic syndrome (MetS), counting sublime of glucose level, obesity, hyperlipidemia, as well blood pressure. Quercetin also participates in the management of metabolic disorders by assorted mechanisms that resemble rising adiponectin, diminishing leptin, antioxidant ability, lowering of insulin resistance, rising insulin level, and preventing calcium channel 14. Furthermore, studies of Quercetin on cellular models display nearly comprehensive demonstration of the mechanisms which link Quercetin with oxidative cell balance also to the control of cell-cycle phases. Many studies have been raised impacts of quercetin on abnormal and normal cells. The remarkable toxicity of Quercetin for cancer cells, accompanied by the distinctive extended antiproliferative and proapoptotic influences on normal cells only at high concentrations, are decisive aspects in research of the anticancer field, Which is considered a serious objective is the recognition of drugs that selectively kill cancer cells in the absence of causes any damaging normal cells15. Quercetin Schiff bases also gained significant interest due to their variety of biological activities 16-18. The thiadiazol ring also demonstrated a large spectrum of physical activities such as antioxidant19, antibacterial, anti-influenza agents20, anti-amnesic effect in mice21, antiproliferative and antiangiogenic activity22, anti-inflammatory23 besides a large number of its derivatives obtained anticancer activity24-26.
In this research, we demonstrated two new Quercetin derivatives as Schiff base of 1,3,4-thiadiazole 5-thiol and thio benzyl derivative and studied their toxicity and anticancer activity against the MCF-7 breast cancer cell line. The research focused its attention on understanding and fighting this disease and finding an appropriate treatment with low side effects and less toxicity.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone, ≥ 95%) was purchased from Aldirch-Sigma (St Louis, MO, USA). All cell culture medium (RPMI-1640) and other consumable reagents were at analytical grade and obtained from Gibco (Invitrogen, MD, USA). LDH kit was purchased from Jiancheng BioEngineering (Nanjing, China). The Schiff bases of quercetin with 2-amino-1,3,4-thiadiazole-5-thiol (compound 1) was synthesized utilizing procedures described by HS Al-Shmgani et.al.27 and the thio etherification with bromobenzyl bromide (compound 2) was done according to procedure described by SA Saoud et al. 28. These compounds were identified from their FT-IR ,1H-NMR,13C NMR and mass spectroscopy.
Cell culture
MCF-7 human breast cancer cell lines were obtained from the Center of Biotechnology/ Al-Nahrain University. Cells were grown in RPMI-1640 medium completed with 1% penicillin-streptomycin and 10% fetal bovine serum and maintained under sterile culture conditions.
Determination of cell viability assay
3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium colorimetric assay (MTT) was assessed to evaluated cell variability according to Al-Kubaisi et al.29. Cells were cultivated at a concentration of 106 cell/ml in 96 cell well plates, and 100 µl of Q1and Q2 were add at different concentrations (25, 50, 100, 200 µg/ml) to each well. MTT was added after 24 h of incubation and the outcoming formazan was perfectly dissolved in 100 μL of 10% sodium dodecyl sulfate.  Absorbance was read at 620 nm using microplat reader (Versa Max TM, Molecular Devices, Sunnyvale, CA). The inhibition percentage was calculated as the following:
Cell viability% = (Ab of control – Ab of treatment) / Ab of control × 100
Where Ab = optical density
Determination of lactate dehydrogenase
lactate dehydrogenase (LDH) activity was measured to assess the cytotoxicity of Q1 and Q2 derivatives on cells following manufacture instructions. Cells were exposed to different (25,50,100, and 200 µg/ml) concentrations for 48 h.  The absorbance was then read at 450 nm using (Versa Max TM, Molecular Devices, Sunnyvale, CA)  microplate reader.
Determination of DNA damage
Comet assay (single-cell gel electrophoresis) was used to assess cell DNA strand damage. Briefly, cells were cultured at 106 concentration, treated with 100 µg/ml of Q1 and Q2 and incubated at  37 ºC (5% CO2) for 24 h. Low melting point agarose (in phosphate-buffered saline) was mixed with 106 cells then spread on pre-coated slides with 1% normal-melting agarose. Slides were then kept in lysis buffer for 1h at 4ºC, then placed in an electrophoresis buffer. Electrophoresis has conducted at a field of 24V and 300 mA for 30 min. After neutralization slides were stained with ethidium-bromide (2 mg/ml) and kept in the dark until observed using Leica DMR fluorescence microscopy fitted with 510 nm excitation wavelength and 590 nm emission wavelength.  Images were analyzed using Komet image analysis software (version 5) from Andor Technology (Belfast, UK).
Statistical analysis
The data offered as mean ± standard error. One-way analysis of variance (ANOVA) was outright using SPSS software version 16.0. Statistical difference was accepted at P≤0.05.
The chemical structure for compounds 1 and compound 2 was depicted in (Figure 1).
Cell viability
The cell viability of MCF-7 breast cancer was measured by MTT assay. Cells treated with different concentrations (25, 50, 100, 200 µg/ml) of quercetin derivative Q1 and Q2. Results on MCF-7 cells after 24 showed a significant different at the highest concentrations (100 and 200 µg/ml) compared to untreated cell (control cell). Also a considerable variance reveled between the 200 and 100 µg/ml with 25 µg/ml concentration. Effect caused dose-dependent increase manner (Figure 2).  However, there was no difference between the two synthesized derivatives, indicating that the quercetin Schiff bases and the thiadiazole ring have the major effect, not the 5-thiol group.
Assessment of cytotoxicity
LDH-released enzyme widely used as a good indicator of cell membrane integrity. Results of LDH released into MCF-7 cells media after treatment with different concentrations of the two synthetic quercetin derivative for 48 h revealed significant dose increase response. Both derivatives showed a significant elevation compered to control group where the highest impact was at 200 µg/ml (Figure 3).
DNA damage assessed by Comet assay
The results from the Alkaline Single-Cell Gel Electrophoresis (SCGE) or comet assay revealed the significant damage to MCF-7 cell induced by treatment with quercetin derivative at 100 μg/ml concentration, as shown in (Figure 4A). Both compounds caused an increase in the tail DNA percentages compared to control (figure 4B). A marginally significant increase in DNA strand breaks was observed in Q2 compared to Q1.
Figure 1. The molecular structure of compounds 1 and 2.
Figure 2. MCF-7 human breast cells growth inhibition in control (untreated cells) and groups treated with quercetin (Q1 and Q2) derivative compounds. * indicated a significant decrease compared to the control group. # indicated a significant decrease in 100 and 200 µg/ml compared to 25 µg/ml group. Data are checked in as mean ± SD from three independent experiments.
Figure 3. lactate dehydrogenase (LDH) activity on MCF-7 human breast cells in control (untreated cells) and treated groups with quercetin (Q1 and Q2) derivative compounds. * indicated a significant decrease compared to the control group. # indicated a significant decrease in 200 µg/ml compared to other treated groups. The data are checked in as mean ± SD from three independent experiments.
Figure 4. (a) Comet assay results in MCF-7 human breast cancer cell after treatment with quercetin derivative (Q1 and Q2) at 100 µg/ml; (b) the results showed that both compounds had significant difference increase in the median DNA percentage compared to control (untreated cell). Data represent mean ± SD from three independent experiments.
The anticancer activity of quercetin has been previously studied30, but this study was carried out to determine the specific functional group and the structure responsible for that effect. Both compounds induced cell growth inhibition considering the semi-similarity in the chemical system. Inhibition of cancer cell growth index could be through suppression of cell cycle and proliferative pathways or induced apoptotic pathways. Possible mechanisms explaining its anticancer activity are either by stimulating apoptotic pathways or suppressing cell survival pathways. Their chemical structure can regulate the tendency of flavonoids to scavenge free radicals; therefore, the position and number of substitutions can impact their free radicals’ inhibition activity. It is well known that compounds having an electron donating group and the lipophilic group have less cell cytotoxicity due to increasing its ability of membrane penetration and lipophilicity activity 31.
Cell death can also determine by LDH release into the medium. In this study, the phenomenon that derivative compounds structurally related to parent quercetin influenced solid inhibitory effects in a dose-dependent manner on MCF-7 human cancer cell line. A previous study has shown that reactive oxygen species  (ROS) played a vital role in apoptosis, and increased ROS levels are associated with elevated LDH level32. Also, ROS production led to cell death through mitochondria pathway 33. some previous reports indicated that quercetin and its derivatives possess strong antioxidant activity in MCF-7 cell line. Another mechanism involved in the anticancer activity of quercetin and its products is due to cell circle arrest at G2/M and G1/S34.
One of the suggested mechanisms by which DNA damage in cells occurs is the increased production of free radicles and especially reactive oxygen species (ROS), causing oxidative stress. Oxidants induce both biochemical and morphological alterations via the oxidation of cellular components. ROS can induce DNA damage either by base modifications or strand breaks; this strand breaks due to the activation of nucleases or the formation of H2O2 and OH•. H2O2 is required for lipid peroxidation and probably the DNA damage detected by the current results. Studies have reported the preventive role of quercetin as a potent antioxidant agent through reducing ROS levels, increasing antioxidant enzyme activity and directly affecting glutathione level35. This study is in contrast with36, who suggested an anti-carcinogenic activity of quercetin based on their funding that quercetin is able to protect against H2O2-caused DNA damage in human lymphocytes. Moreover, quercetin has been demonstrated to restrain tumor growth in human ovarian cancer by provoked apoptosis via increasing Bax and p21 expression and decreased Bcl-2 expression37.
Evolution of newly synthesized quercetin derivative cytotoxicity on MCF-7 cell line by MTT, LDH and Comet assay indicated significant cytotoxic to the cell in a dose-dependent aspect. These findings highlight the potential role in breast cancer treatment, so it can be used as possible alternative therapy or supplement. Therefore, it is essential to identify the effective functional structure responsible for cytotoxicity and further to study their in vitro and in vivo effects.
Acknowledgments: We would like to thank the Center of Biotechnology/ Al-Nahrain University staff.
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Received:  January 15, 2023 / Accepted: February 25, 2023 / Published:15 March 2023
Citation.  Jasim H S, Al-kubaisi Z A, Al-Shmgani H S. Cytotoxic potential activity of quercetin derivatives on MCF-7 breast cancer cell line. Revis Bionatura 2023;8 (1) 92 http://dx.doi.org/10.21931/RB/2023.08.01.92

Vol 9 No 2 2024