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Effect of Curcumin on Docetaxel-Induced Apoptosis in the DU145 (Prostate) Cell Line Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay
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Mohammad Shokrzadeh1    , Elahe Gharehkhani *2 , Mahboube Rahmati Kukandeh3 , Mahsa Hosseini4 |
1- Professor, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
2- Ph.D in Toxicology, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. , egh199012@gmail.com
3- Ph.D Candidate in Toxicology, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
4- Pharmacy Student, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Ramsar, Iran. |
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Keywords: Docetaxel [MeSH], Curcumin [MeSH], Apoptosis [MeSH]
Article ID: Vol27-17 |
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Type of Study: Original Articles |
Subject:
Pharmacology
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Abstract: (146 Views) |
Extended Abstract
Introduction
Curcumin is the primary active compound of the turmeric plant (Curcuma longa). This yellow-colored herbal product also contains other compounds, such as demethoxycurcumin and bis-demethoxycurcumin. Curcumin typically exerts its effects through various pharmacological processes, including antioxidant, anti-inflammatory, antithrombotic, apoptotic, and hepatoprotective actions. In addition to its antitoxigenic properties, some studies have shown that curcumin possesses antinephrotoxic and antihepatotoxic effects. The antioxidative, antiproliferative, and anti-angiogenic properties of curcumin have garnered significant attention in recent years. However, its poor solubility and rapid degradation have hindered its clinical application. Consequently, recent research has focused on designing and developing polymeric micelles (PMMCs) at the nanoscale to enhance the cellular delivery of curcumin. Studies have shown improved cellular uptake of curcumin when formulated with these polymeric compounds. Specifically, F68-Cis-Cur micelles have demonstrated greater cytotoxic effects against A2780 and SMMC 7721 cells compared to pure curcumin, culminating in a more pronounced reduction in mitochondrial membrane potential and induction of cellular apoptosis.
Curcumin is a flavonoid compound, and flavonoids are present in various plants. In in vitro studies, these compounds have been identified as antioxidants due to their unique structure, acting through the inhibition of lipid peroxidation (LPO), scavenging free radicals, and chelating metal ions. Additionally, flavonoids are recognized as antioxidant, cardioprotective, anti-inflammatory, and anticancer agents. Overall, flavonoids exert several protective effects in the brain, including positive effects against neurotoxins, suppression of neuroinflammation, and the ability to promote memory, cognitive functions, and learning.
Docetaxel (DTX), a chemotherapy drug marketed under the brand name Taxotere and others, is used to treat various cancers, including breast cancer, head and neck cancers, gastric cancer, prostate cancer, and non-small cell lung cancer (NSCLC), either as a monotherapy or in combination with other chemotherapeutic agents. DTX is a cytotoxic chemotherapeutic agent that binds to β-tubulin subunits, prevents microtubule depolymerization, and consequently leads to mitotic arrest, apoptosis, and inhibition of cell proliferation. Additionally, in vitro studies show it inhibits androgen receptor (AR) nuclear translocation and AR expression.
Apoptosis is a physiological and biological process essential for active and normal development, as well as for maintaining homeostasis. In situations where the survival of a cell jeopardizes the existence of the living organism, the cell commits suicide through programmed death. When a cell is exposed to various environmental or even internal factors, such as ionizing radiation, cytotoxic drugs (used in cancer treatment), hyperthermia, and glucocorticoid hormones, its contents, including DNA, undergo changes. If the cell were to continue living with these alterations, it could lead to severe abnormalities, including cellular carcinogenesis. Intracellular pathogenic bacteria, such as Salmonella, Shigella, Listeria, and Legionella can also contribute to guiding the cell toward this specific type of cell death during infection by altering certain intracellular metabolic and biochemical pathways.
Programmed cell death is initiated through various pathways. Some of these pathways are triggered by the binding of ligands to cell surface receptors, while others are activated by the absence of certain growth factors. Severe damage to the cell's genetic material can also initiate various pathways leading to apoptosis.
This study aimed to determine the effect of curcumin on DTX-induced apoptosis in the DU145 (prostate) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Methods
This descriptive analytical study was conducted on the DU145 (prostate) cell line, purchased from the National Genetic Resources Cell Bank, at the Cell Culture Laboratory of the Faculty of Pharmacy, Mazandaran University of Medical Sciences.
Cellular Culture: The cell line was cultured in the Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin antibiotic. Cells were maintained in an incubator at 37°C with adequate humidity and 5% carbon dioxide. For various experiments, when cell confluence reached at least 70-80%, cells were detached from the flask bottom using trypsin-ethylenediaminetetraacetic acid (EDTA), without any shaking or tapping of the flask, and then centrifuged at 2000 rpm for 5 minutes. The resulting cell pellet was resuspended in 1 mL of culture medium, and the viability of cells in the suspension was determined by mixing equal parts of trypan blue and counting with a hemocytometer under a light microscope.
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay Procedure: In the pre-treatment phase, curcumin at concentrations of 0, 10, 25, 50, 100, and 200 µM was applied to 96-well plates 24 hours prior to the addition of DTX at concentrations of 1.65, 0.87, 13.39, 3.0, and 6.25 mg/mL. This method serves as a competitive metabolic assay and evaluates mitochondrial function.
The cell viability percentage was calculated using the following equation:
Cell Viability Percentage = Mean Optical Density (OD) of Treated Cells ¸ Mean OD of Control × 100
The grouping was as follows:
Group 1: DTX at a concentration of 4.46 µM (optimal)
Group 2: Curcumin at a concentration of 2 µM combined with DTX at a concentration of 4.46 µM (optimal)
Group 3: Curcumin at a concentration of 10 µM combined with DTX at a concentration of 4.46 µM (optimal)
Group 4: Curcumin at a concentration of 10 µM alone
Cell Apoptosis Measurement: Cellular apoptosis was investigated at the cellular level using an Annexin flow cytometry kit to assess the effects of drug exposure. Flow cytometric analysis was performed on DU145 (prostate) cells stained with Annexin V-FITC/PI.
Results
The Effect of Curcumin on Docetaxel-Induced Cell Viability in DU145 (Prostate) Cell Line Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay: Treatment with DTX at a concentration of 46.4 µM (IC50) resulted in a 50% reduction in cell viability compared to the control group (P < 0.001). In groups treated with DTX combined with curcumin (2, 4, 6, 8, and 10 µM), there was a statistically significant difference compared to the DTX-alone group (P < 0.001), leading to an increase in cell viability (Figure 1). Furthermore, the highest cell viability was observed in the control group, while the lowest cell viability was in the DTX-alone group.
The Effect of Curcumin on Docetaxel-Induced Apoptosis in DU145 (Prostate) Cell Line: Cells were distributed into four quadrants (Q1-Q4). Q1 cells were defined as necrotic PI (+) cells. Q2 cells indicated late apoptosis PI (+) and Annexin (+). Q3 cells represented early apoptosis with Annexin V (+). Finally, live cells were in Q4, characterized as PI (-) or Annexin V (-). Evaluation of the drug groups was performed at optimal drug concentration alone, protective agent at minimum and maximum concentrations combined with the drug at optimal concentration, and protective agent at maximum concentration alone. Significant changes in apoptotic and necrotic cells were observed in treatment using curcumin at its highest concentration (Figure 2-C) and curcumin at its highest concentration combined with DTX (Figure 2-D). Moreover, the DTX group exhibited 18.7% viable cells. The observed antitumor effect in DU145 cells treated with curcumin in combination was greater than that of DTX alone.
Conclusion
DTX reduced the viability of DU145 (prostate) cells by approximately 50%. Additionally, groups treated with curcumin in combination with DTX showed a dose-dependent decrease in cytotoxicity, leading to an increase in DU145 (prostate) cell viability. Additionally, curcumin was able to enhance the induction of apoptosis in DU145 (prostate) cells.
Ethical Statement
The study received approval from the Research Ethics Committee at Ramsar Self-Governing Campus, Mazandaran University of Medical Sciences (IR.MAZUMS.RIB.REC.1403.006).
Funding
This article has been extracted from the doctoral dissertation of Ms. Mahsa Hosseini in Pharmacy at Ramsar Self-Governing Campus, Mazandaran University of Medical Sciences. This study was funded by the Vice-Chancellor for Research and Technology, Mazandaran University of Medical Sciences.
Authors' Contributions
Mohammad Shokrzadeh: Project administration and design and approval of the final manuscript.
Elahe Gharehkhani: Data analysis, interpretation of the results, and drafting of the initial manuscript.
Mahboube Rahmati Kukandeh: Data collection.
Mahsa Hosseini: Project execution.
Conflicts of Interest
No conflicts of interest.
Acknowledgments
The authors would like to thank the research officials of the Faculty of Pharmacy, Mazandaran University of Medical Sciences, and the thesis committee for their assistance in conducting and improving the quality of this study.
Key Message: DTX led to a decrease in cell viability, while in the groups receiving curcumin, cell viability increased. Additionally, curcumin was able to increase the rate of apoptosis in DU145 (prostate) cells. Therefore, the use of antioxidant compounds is suggested to improve the efficacy and reduce the complications of chemotherapy.
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Shokrzadeh M, Gharehkhani E, Rahmati Kukandeh M, Hosseini M. Effect of Curcumin on Docetaxel-Induced Apoptosis in the DU145 (Prostate) Cell Line Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay. J Gorgan Univ Med Sci 2025; 27 (2) :61-69 URL: http://goums.ac.ir/journal/article-1-4498-en.html
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