INTRODUCTION:

Cancer is a non- communicable diseases that are uncontrolled cell proliferation and overexcited cell division and differentiation¹. The search for effective cancer treatments is a complex undertaking requiring a thorough comprehension of the complicated cellular mechanisms that underlies the aetiology and course of the illness.

In order to increase the survival rates of those afflicted by the disease, scientists are always working to develop safer and more effective medicines for cancer therapy, this is a dynamic and active area of scientific research². Based on mRNA gene expression levels, breast cancer may be categorised into molecular subtypes that provide insights into new treatment modalities and patient stratifications that influence breast cancer patients' care. Treatment for breast cancer is complex and involves several different treatments to be carried out in different sequences, such as hormone therapy, biological therapies, radiation therapy, chemotherapy, and surgery³. In 2020,the worldwide mortality-to-incidence ratio for breast cancer was 0.30, indicating a favourable 5-year survival rate. The comparative survival rates for localised and regional breast cancer were 76.3% and 47.4%. respectively, in less developed nations like India ^4,5. The toxicity of agents towards normal cells and tissues, along with the emergence of treatment resistance, lead to side effects that hinder the efficacy of anti-cancer therapies that target molecules⁶.

Pharmacokinetics and getting enough active medication into cancer cells in vivo are the two most difficult aspects⁷. New anticancer drugs with unique mechanisms of action are constantly needed, as is the discovery of fresh potential cellular targets and effective treatment plans that target only cancer cells⁸. In this context, we predicted in our work that 7,3′- dihydroxyflavone would function as an anti-proliferative drug in breast cancertreatment with the ability to interact with Nfkb, p21, and p53. Better alignment to the allosteric area and a better fit within the binding pockets of p53, p21, and Nfkb were expected outcomes of the greater conformational flexibility provided by the amide bond, leading to increased binding affinity.

METHODOLOGY:

Cell Culture:

Cell Culture The cells were cultured in tissue flasks using a 1:1 combination of MEM (Minimum Essential media) and Ham's F12 media, enriched with foetal bovine serum and antibiotic, at a temperature of 37℃. Upon reaching a confluence density of 70 to 80%, the cells were trypsinized and seeded to a 96-well plate at a density of 1×106 cells per well. The plate was then incubated at 37℃ for 48 hours in a 5% CO2 incubator⁹.

MTT Assay for evaluation of Cell Proliferative Activity:

Dimethyl Sulfoxide (DMSO) was used to dissolve the chemical 7,3'-Dihydroxyflavone, whichwas subsequently added to the wellsat concentrations of 31.25μg/mL, 62.50μg/mL, 125μg/mL, and 500 μg/mL.Following which these samples were placed in an incubator for a duration of 24hours.The untreated (Control) and treated cells exhibited morphological changes, which were identified and photographed using a digital inverted microscope at a magnification of 40X.Thereafter Phosphate Buffer Saline (PBS) was used to wash the cells. at a pH of 7.4, and then 20 microliters of MTT solution (5 milligrammes per millilitre in PBS) were introduced into each well. The resultant mixture was subjected to incubation for a duration of 3 hours. After a 3 hour incubation period, the formazan crystals produced by metabolically active cells were allowed to air-dry in a dark place for 30 minutes. Following that,in 100 microliters of DMSO the crystals were dissolvedand the UV absorbance was quantified at a wavelength of 570nm using an UV-VIS Spectrophotometer. The authors¹⁰.

Annexin V-FITC/PI Apoptotic Assay for Measurement of Apoptosis:

Necrotic and Apoptotic cells were differentiated on the basis of Annexin VFluorescein Isothiocyanate and Propidium Iodide (Annexin V-FITC/PI) doublestaining. In order to estimate various phases of apoptosis, with the inhibitory concentration (IC50) of 7,3’-Dihydroxyflavone the MCF-7 cells were treated and cultured for 24hours. Untreated cells cultured in the presence of 1% DMSO (v/v) were taken as the negative control. Following treatment, after rinsing the cells with 1X binding buffer, they were treated for 10 to 15 minutes at room temperature in a dark environment with 10 μL of Propidium Iodide (50 μg/mL in PBS) and 5 μL of Annexin V-FITC. Finally, the cells were visualised using a fluorescent microscope.^11,12.

Gene Expression analysis of p21, p53, NFkB:

Assessment of the p21, p53, and NFkB gene expression, was done using real time PCR. Trizol Reagent (Sigma), a standardised procedure was used to separate the entire RNA. Using a PrimeScript first strand cDNA synthesis kit, 2μg of RNA were utilised for reverse transcription-based cDNA synthesis. (TakaRa, Japan). The targeted genes were amplified using specific primers. SYBR green dye and all of the necessary PCR components are included in the GoTaq® qPCR Master Mix (Promega), which was used to run the PCR reaction. Utilising a Biorad CFX96 PCR instrument, real-time PCR was carried out. The outcomes were analysed using the comparative CT technique, and Schmittgen and Livak's (2008) 2−∆∆CT method was used to calculate the fold change¹³.

Molecular Docking:

Using AutoDock Vina 4.2 software, molecular docking studies were carried out for the 7,3'-dihydroxyflavone inside the active sites of p53 (PDB: 3DCY), p21 (PDB: 2AST), and nuclear factor kappa B (PDB: 1NFK). It was used to conduct a molecular docking study of 7,3'-dihydroxyflavone on the active site of human p21, p53, and NFkB^14,15. The threedimensional crystal Nucleotide kappa B (PDB: 1NFK), p53 (PDB: 3DCY), and p21 (2AST) structures have been retrieved from http://www.rcsb.org, the Protein Data Bank. Using the Dock Prep tool of the UCSF Chimaera programme¹⁶, the structures of three proteins were generated to eliminate heteroatoms and water molecules all of the receptor structure, insertion of polar hydrogens, and repair of missing side chain atoms. Using Open Babel, the 7,3'-dihydroxyflavone SDF format was changed to the pdb format¹⁷. Using MGLTools 1.1.6, the receptor and ligands wereconverted to pdbqt format. The coordinates of the ligand in the PDB record served as the centre of the docking search space, which had values of X=20, Y=20, and Z=20. The maximum binding affinity with receptors was determined by considering the binding pose that adopts the lowest energy conformation. We were able to locate the protein binding site by molecular visualising the 7,3′- dihydroxyflavone in its lowest energy conformations. The software called Discovery Studio 2021 was used to analyze the protein-ligand interaction¹⁸.

In-Silico Drug-likeness and ADMET Study:

Using the pkCSM online server, the drug-likeness and ADMET analysis were carried out.(https://biosig.lab.uq.edu.au/pkcsm/) (19).

RESULTS:

The findings indicate that the 7,3′-dihydroxyflavone inhibited the proliferation of cancerous cells in MCF7 cell line. The outcomes obtained from the MTT assay of 7,3′-dihydroxyflavone against MCF-7cell line breast of cancer to validate the anti-cancer activity of 7,3′-dihydroxyflavone at various concentration are shown in Table 1 and Doxorubicin was used a positive control.

Table No.1: Summary of Anti-Proliferative Effect by MTT Assay

Concentrations (μg/mL)	Average of Absorbance	Cell Viability (%)	Percentage of Inhibition
Control (No drug treatment)	0.753	100	0
31.25	0.691	91.766	8.234
62.5	0.571	75.830	24.170
125	0.306	40.637	59.363
250	0.190	25.232	74.768
500	0.090	11.886	88.114

Gene Expression analysis of p21, p53, NFkB:

Effects on mRNA expression of p21, p53, NFkB on MCF7. The administration of 7,3′-dihydroxyflavone up-regulated the mRNA levels of p21, p53, whereas downregulated NFkB. Moreover, we found that there are significant differences in the gene expression levels (Figure 3). The verification of p21, p53, and NFkB mRNA expression in MCF7 cells by RT-qPCR among control groups (doxorubicin, negative control). The standard error of the mean (SEM) is shown by error bars. Gene products for NFkB, p21, and p53 have been connected to pathways leading to programmed cell death. We have examined the expression of p53, p21, and NFkB in a number of 1 1 1 4 31 32 39 human breast cancer cell lines. We discovered a negative connection between NFkB, p21, and p53 expression. According to our findings, in breast cancer cell lines, 7,3′- dihydroxyflavone downregulated NFkB and elevated the p53 and p21 genes.

(A)

(B)

(C)

Figure No.5: Gene Expression Results. (A) Effect of 7,3′-dihydroxyflavone (187.801 μg/ml) on p53 gene expression in breast cancer cell line (MCF-7). (B) Effect of 7,3′-dihydroxyflavone (187.801 μg/ml) on p21 gene expression in breast cancer cell line (MCF-7). (C) Effect of 7,3′-dihydroxyflavone (187.801 μg/ml) on p21 gene expression in breast cancer cell line (MCF-7). The results are given as a fold change from the control after the target gene expression is normalised to GAPDH mRNA expression. The mean + SEM of three separate observations is shown by each bar. ‘*’ indicates the statistical significance, at the p<0.05 between the drug treatment and control groups.

Molecular Docking:

Crystallographic structures of human p53 (PDB: 3DCY), p21 (PDB: 2AST), and nuclear factor kappa B (PDB: 1NFK) were retrieved from the Protein Data Bank in order to determine the molecular mechanism involved in the anti-proliferative action of the 7,3′-dihydroxyflavone. Molecular docking requires a certain level of binding affinity between the ligand and protein. Molecular docking was performed by computing the affinities between 7,3′-dihydroxyflavone and protein targets. We assessed the 7,3′-dihydroxyflavone's binding affinities and modalities of binding to each protein using the open-source AutoDock Vina programme, which optimises docking by utilising a scoring function Lamarckian Genetic algorithm. The conformation binding mode of the lowest Gibbs free energy of binding (ΔG) was used to evaluate the interactions between the ligand and the protein.

The amino acids in p21 involved in this interaction are ARG 2167 and MET 3058 also by hydrogen bonding and hydrophobic interactions for 7,3′- dihydroxyflavone (Figure 6B). The amino acids in Nfkb involved in this interaction are GLY 166, LEU 176, GLN 177, and ARG 228 by hydrogen bonding and hydrophobic interaction for 7,3′-dihydroxyflavone (Figure 6C). The catalytic core of protein targets, which includes amino acids p53, p21, and Nfkb, may account for the inhibitory impact of 7,3′-dihydroxyflavone.

Figure No.6: Ribbon representation of the binding mode of 7,3′-dihydroxyflavone to human p53. The protein is represented as rainbow ribbons. The ligand, 7,3′-dihydroxyflavone is was represented in stick model.

Figure No.7: Ribbon representation of the binding mode of 7,3′-dihydroxyflavone to human p21. The protein is represented as rainbow ribbons. The ligand, 7,3′-dihydroxyflavone is was represented in stick model.

Figure No.8: Ribbon representation of the binding mode of 7,3′-dihydroxyflavone to human Nfkb. The protein is represented as rainbow ribbons. The ligand, 7,3′-dihydroxyflavone is was represented in stick model.

(A) (B)

(C)

Figure No.9: 2D Interaction Plot of Docked Poses. (A) p53, (B) p21, and (C) Nfkb.The structure and interactions of 7,3′-dihydroxyflavone with Nfkb, p21, and p53 active sites. The primary source of binding energy to the 7,3′-dihydroxyflavone's binding affinity for p53, p21, and Nfkb was shown to be this substructure. For hydrogen bonds, interactions are displayed in green, for π-alkyl in pink, and for cation-π in orange.

Table No.2: Binding energies from in silico docking towards human p53, p21, and Nfkb

Receptors	Minimum Binding Energies (Kcal/mol)
p53	-7.0
p21	-7.4
Nfkb	-6.4

Drug-likeness and ADMET Study:

When it comes to potential drug like candidates, 7,3′-dihydroxyflavone conforms to Lipinski's "Rule of Five," also known as Pfizer's rule of five.(Table 3). These guidelines enable us to assess 7,3′-dihydroxyflavone's potential as a medication.The majority of oral active drugs with excellent bioavailability are said to meet all four requirements: number of hydrogen bond acceptors ≥ 10, number of hydrogen bond acceptors ≥ 500 Da, limited lipophilicity represented as logP ≥ 5, and number of hydrogen bonds.

Table No.3: Drug-likeness Parameters

Descriptor	Value
Molecular Weight	254.241
LogP	2.8712
No. of Rotatable Bonds	1
No. of Acceptors	4
No. of Hydrogen Bond Donors	2
Surface Area	107.725

When using a hit-to-lead strategy, pharmacokinetics (PK) characteristics are crucial in the hit compound selection process. Therefore, we used the pkCSM programme to examine these properties in silico. The results of this evaluation revealed that 7,3′- dihydroxyflavone has 94.22% of bioavailability, -3.605 mol/L (water solubility), skin permeability of -2.751 log Kp, binding with P-glycoprotein substrate, no interaction with P-glycoprotein I and II inhibitor, In theview of distribution, Volume of distribution (VDss) (human) (0.063 L/kg), Fraction unbound (human) (0.146 Fu), BBB permeability of -0.057 log BB, and CNS permeability of-1.927 log PS. The metabolism prediction showing inhibition towards CYP1A2 inhibitior, CYP2C19 inhibitior, CYP2C9 inhibitior, no inhibition towards CYP2D6 substrate,CYP3A4 substrate, CYP2D6 inhibitior, CYP3A4 inhibitior. The excretion process shows 0.242ml/min/kg of total clearance, and no interaction with Renal OCT2 substrate. In terms of toxicity, it shows no toxicity towards AMES, hERG I inhibitor, hERG II inhibitor, Hepatotoxicity, Skin Sensitisation, T.Pyriformis toxicity (0.654μL), Minnow toxicity (1.178mM). The Maximum tolerated dose (human) (0.175 log mg/kg/day), Oral Rat Acute Toxicity (LD50) (2.208mol/kg), and Oral Rat Chronic Toxicity (1.231 log mg/kg_bw/day) was also calculated. The drug-likeness prediction of 7,3′-dihydroxyflavone was summarized in Table 3.

DISCUSSION:

Worldwide, over a million women receive a breast cancer diagnosis each year, and more than half of them will pass away as a result of the illness. The most frequent cancer and the main reason why women die from cancer is breast cancer. The general prognosis for breast cancer has improved due to widespread screening programmes and the introduction of novel therapies. For women with late-stage or metastatic breast cancer, the average annual survival rate is still rather poor. Five years after diagnosis, just 35 percent of women with advanced breast cancer still live. When breast cancer is detected at a late stage, when there is little chance of recovery, one-third of women receive this diagnosis ³². In both industrialised and developing nations, breast cancer is regarded as one of the worst illnesses. To design and create more selective, effective, and low-toxicity therapeutic medicines for the treatment of cancer, ongoing research is necessary³⁴. The increased ability to diagnose and treat patients has resulted in a steady rise in survival rates. Many physical and psychological changes are brought about by the diagnosis and treatment of cancer³¹. The survival rates of breast cancer in India are low because the detection takes place late. The only way to change these numbers is by increasing awareness^{35,36,37,38,39,40}.

A collection of cells that proliferate uncontrollably and grow malignantly is called cancer. Currently, chemotherapy, radiation therapy, gene therapy, immunotherapy, molecularly targeted therapy, and surgery are the main forms of cancer treatment. However, some tumour characteristics have thus far hampered the therapeutic effectiveness of these treatments. Unusual signalling pathway activation contributes to tumour pathogenesis and is essential for the development, advancement, and recurrence of malignancies. Cancer patients now have better results thanks to targeted medicines that target oncogenic signalling effectors²⁵. It is believed that 50% of cancers especially breast cancer are caused by gene alteration of cell cycle regulating genes, which continue to be the most frequently associated genes in many common human cancers.Higher eukaryotes have developed many checkpoint mechanisms to detect and respond to cellular disruption.Modifications to checkpoint mechanisms compromise the integrity of the genome, promote the development of cancer, and significantly reduce the efficacy of anticancer treatments.²⁶. The tumour suppressor protein p53 activates several genes that limit development or cause apoptosis, which controls the DNA damage-induced checkpoint.The 165 amino acid protein known as p21 mediates the p53-dependent G1 growth arrest. Additionally, earlier studies have shown that the p21 protein acts as a growth suppressor of cancers by inducing cell cycle arrest in response to a variety of stimuli.

Superior modulator of many tumour suppressor pathways, facilitating anti-proliferative effects not mediated by the p53 tumour suppressor pathway²⁷. According to recent studies, p21 may sometimes promote cellular proliferation and carcinogenicity. Although p21 is typically dysregulated in human cancers, its expression suggests that it could still be functional. Depending on the cellular milieu, either as an oncogene or a tumour suppressor.The simple hypothesis that p21 serves as a tumour suppressor has been called into question by the finding that it may exhibit carcinogenic properties. Several human cancers overexpress p21, including those of the breast, prostate, cervical, and squamous cell carcinomas. The grade, invasiveness, and aggressiveness of the tumour are often positively correlated with this overexpression, which is a poor prognostic indicator²⁸. An essential signalling system involved in the aetiology and management of cancer is NFκB. Studies on the NFκB-signaling pathway for targeted cancer therapy have revealed excessive activation of this pathway in a variety of tumour tissues ²⁹.

In a related work, Mohammed et al. (2023) designed a novel medication by molecular docking with ERa and applying the Structure Based medication Design (SBDD) technique to predict the drug's ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profile. The software utilised for result analysis and prediction were BIOVIA Discovery Studio Visualizer 2020, SwissADME, and GOLD (Genetic Optimisation for Ligand Docking)³⁰. In an another study, the author assessed the cytotoxic activity of sulfonamide derivatives against a panel of cell lines representing breast cancer, lung cancer, and NCI-H23, NCI-H522, as well as one normal cell line, HEK-293T (a human embryonic kidney cell line); the results were compared with those of the standard medication, doxorubicin³³.

CONCLUSION:

In conclusion, we established the anti-cancer properties of 7,3′-dihydroxyflavone. The compound 7,3′-dihydroxyflavone were assessed in vitro using the human cancer cell line MCF7. The other experimental molecular studies were validated by the molecular docking research. 7,3′-dihydroxyflavones have the potential to be powerful anticancer candidates since they can upregulate the expression of p53 and p21 and downregulate NFkB.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 08.03.2024 Revised on 19.07.2024

Accepted on 27.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):481-488.

DOI: 10.52711/0974-360X.2025.00073

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.