IN-SILICO STUDY: MYRICETIN FROM THE STEM BARK OF SYZYGIUM CUMINI INHIBITS THE GROWTH OF NON-SMALL CELL LUNG CANCER CELLS THROUGH THE EGFR PATHWAY

IN-SILICO STUDY: MYRICETIN FROM THE STEM BARK OF SYZYGIUM CUMINI INHIBITS THE GROWTH OF NON-SMALL CELL LUNG CANCER CELLS THROUGH THE EGFR PATHWAY

M. Viji, S. Aishwariya, C. Ireen, S. Hinduja, S. Indu, P. Vijayalakshmi and M. Rajalakshmi *

DBT-BIF Centre, Holy Cross College (Autonomous), Tiruchirappalli, Tamil Nadu, India.

ABSTRACT: Around the world, cancer frequently leads to death. Lung cancer is the primary cause of cancer-related deaths globally. Through in-silico analysis, this study sought to assess the anticancer effectiveness of myricetin on human lung cancer cells.  The study's goal is to assess how well myricetin adheres to the Lipinski rule of 5. To evaluate the ADMET properties (Absorption, Distribution, Metabolism, Excretion, Toxicity) for myricetin. To predict the target-ligand interaction.  The results from these studies supported that the myricetin follows the Lipinski rule of 5 and ADMET properties. Myricetin has a better H bond interaction. The results of the in-silico analysis used in this work serve as encouraging evidence for the prediction of the mechanism by which myricetin acts to cause apoptosis via the EGFR pathway. The results from the molecular docking study suggested that myricetin inhibited critical tumorigenic effects through apoptosis. Thus myricetin could be further explored as a promising chemotherapeutic agent in lung cancer treatment.

Keywords: Epidermal growth factor receptor (EGFR), Small cell lung cancer (SCLC), Non-Small cell lung cancer (NSCLC), Protein data bank (PDB), Myricetin, Docking

INTRODUCTION: Of all malignant tumours, lung cancer has the highest rates of morbidity and mortality in the globe ¹. In 2020, lung cancer accounted for 18% of all cancer-related deaths and 11.4% of all new cancer cases. In 2025, it's anticipated that there will be a substantial increase in cases, reaching 81,219 cases among men and 30,109 cases among women. In India, there are grave concerns about lung cancer's rising prevalence and slow diagnosis ².

Lung adenocarcinoma, the most typical type of lung cancer, affects both smokers and nonsmokers as well as persons under 45 years old. Adenocarcinoma accounts for roughly 30% and 40%, respectively, of primary lung cancers in smokers who are men and women. These numbers among non-smokers are about 60% for males and 80% for women ³.

Smoking, using alcohol, and eating betel nuts are the three main lifestyle factors linked to an increased risk of cancer. Smoking has been linked to an increased risk of developing lung, hepatoma cellular carcinoma, oral cavity, neural progenitor cells, oesophageal, urinary bladder, and cervical cancer in a dose-response relationship ⁴. It has been demonstrated that lung cancer is connected with a diminished capacity to detoxify particular types of cancer-causing chemicals or tobacco carcinogens, even though it is uncommon for lung cancer to result from genetic abnormalities of oncogenes or tumour suppressor genes ⁵.  The two primary types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with non-small cell lung cancer accounting for 80–85% of diagnoses ⁶.

Every kind of cancer cell develops and disperses differently.  Based on histological classifications, NSCLC is typically divided into three categories: squamous cell carcinoma, adenocarcinoma, and large-cell carcinoma ⁷. Only 3-4% of cases of other, less frequent subtypes, such as bronchioloalveolar carcinoma (BAC), occur, while BAC features are present in 10-15% of adenocarcinomas. SCLC accounts for about 15–25% of cases of lung cancer with neuroendocrine morphological features ⁸.

Therapy for Lung Cancer: Every cancer treatment aims to kill cancerous cells while leaving healthy ones alone. The most widely used cancer therapies include chemotherapy, radiation therapy, and surgery. These therapies can be applied independently, in combination with other therapies, or both. Surgery is frequently used as the initial line of treatment for the majority of malignancies, especially solid tumours, and entails the removal of any tissue that is malignant. To confirm a biochemical diagnosis and further ascertain the size and distribution of the tumours, ultrasound and/or CD scanners are utilised as diagnostic instruments. In radiation therapy, high-energy X-rays are utilised to reduce cancer size. It is frequently used in conjunction with surgery or an alternative kind of chemotherapy as a neo-adjuvant therapy to facilitate surgery by reducing tumours. It is regarded as a local treatment because it only affects the region surrounding the cancer ⁹.

Chemotherapy Drugs and Treatment: Chemotherapy, which employs drugs or chemicals to kill cancer cells, has a wide range of effects. Anticancer drugs currently come in a variety of distinct categories based on their mechanisms of action, including the following:: a) anti-metabolites that substitute for the normal RNA and DNA building blocks b) mitotic inhibitors that prevent mitosis and cell division; c) alkylating agents that damage DNA; d) antibiotics that interfere with DNA replication enzymes; e) topoisomerase inhibitors that prevent topoisomerase I or II from unwinding DNA during replication and transcription; and f) corticosteroids that are used to treat cancer and to relieve the side effects from other drugs ¹⁰.

Lung cancer is treated with bleomycin, doxorubicin, etoposide (VP-16), cisplatin, and methotrexate, among other anticancer drugs. Given that these drugs have been demonstrated to increase the expression of the Fas ligand (FasL) on the surface of cells that express the Fas receptor, it is possible that Fas cross-linking is the mechanism by which these drugs cause apoptosis ¹¹.

Natural compounds have long been known to contain anticancer agents ¹².  Natural products and their derivatives make up around 63% of the process in the development of innovative medications ¹³. Cancer has long been treated using traditional Chinese medicine (TCM) in China and other nations ¹². Herbal medicines frequently cost minimal money, are easily accessible, and show little harmful consequences when used in therapeutic settings ¹⁴.

Myricetin, a flavonoid is frequently discovered in tea, wines, berries, fruits, and medicinal herbs ¹⁵.  A wide variety of plant-based foods and beverages contain flavonoids, which can be broadly categorised as flavonols, flavones, anthocyanins, catechins, flavanols, and isoflavones ¹⁶. Some of the biological actions of flavonoids that may help prevent cancer include free radical scavenging, antimutagenic and antiproliferative properties, modulation of cell signalling and cell cycle, and suppression of angiogenesis ¹⁷. According to certain studies, myricetin has anti-inflammatory, anti-oxidative, and anti-proliferative properties ¹⁸. Myricetin inhibits the proliferation of cells that are cancerous of the lung, breast, thyroid, liver, leukaemia and prostate, according to earlier research ¹⁹. In this study, we have analysed the ability of myricetin from the stem bark of Syzygium cumini to induce apoptosis through the EGFR pathway by an in-silico approach.

Methodology:

Ligand Preparation: The 2D structure of ligand Myricetin was generated using ACD/ Labs Chemsketch. Their pharmacokinetic properties like the LIPINSKI rule of 5 and ADMET properties were analysed using the SwissADME tool.

Determination of Lipinski Rule: Another crucial element of computer-based drug synthesis is the Lipinski rule. When a chemical interacts with the lipid bilayers in the body, the "rule of five" is used to estimate how much of it will be absorbed or how permeable it will be. When evaluating these features, Lipinski's "rule of five" states that a substance satisfies all five of the following conditions to meet the drug-like criteria. 500 g/mol or less is the minimum molecular weight. The octanol/water partition coefficient is less than 5 as determined by (log P 5). Less than five hydrogen bond donors are present. There are just 10 hydrogen bond acceptors, mostly N and O atoms.

Determination of the Data of ADMET: An extensive investigation of the ADMET projection is regarded to be necessary for the development and discovery of novel medicinal drugs. The ADMET qualities are the most useful for measuring the properties and in-silico pharmacokinetic parameters of the predicted drugs. It enables rapid and provisional assessment of ADMET properties before a complete in-vitro analysis of the compounds. This is advantageous because recently created substances could be harmful to health. To project ADMET data, the online web tool SwissADME has been used. The chemical properties and molecular structures are used to calculate and measure these ADMET values.

Protein Preparation: The three-dimensional structure of EGFR (PDB ID: 4ZAU_A), KRAS (PDB ID: 4OBE_A), RAF (PDB ID:3OMV_A), MEK (PDB ID:3W8Q_A), ERK (PDB ID:2ERK _A),  were obtained from Protein Data Bank (PDB). The receptors were prepared by using the BIOVIA Discovery Studio Visualizer 2021 Client software.

Molecular Docking: PyRx software was used to create the in-silico investigations, while the BIOVIA Discovery Studio Visualizer 2021 Client software was used to visualise the outcomes.

RESULTS AND DISCUSSION:

FIG. 1: 2D STRUCTURE OF MYRICETIN

TABLE 1: LIPINSKI RULE OF 5 -MYRICETIN WAS OBTAINED THROUGH SWISSADME

TABLE 2: ADMET PROPERTIES OF MYRICETIN WAS OBTAINED THROUGH SWISSADME

TABLE 3: DEPICTING THE BINDING AFFINITY AND H BOND INTERACTION WHEN THE PROTEINS WERE DOCKED WITH LIGANDS

FIG. 2: MOLECULAR DOCKING ANALYSIS OF MYRICETIN AGAINST EGFR, KRAS, RAF, MEK AND ERK

Epidermal Growth Factor Receptor (EGFR) is a transmembrane tyrosine kinase receptor and a member of the ErbB family of receptors. Members of the ErbB family include EGFR (ErbB1), ERBB2 (HER2/neu), ERBB3 (HER3), and ERBB4 (HER4) ²⁰. The cytoplasmic domain of EGFR, which has the tyrosine kinase region and tyrosine autophosphorylation sites, the transmembrane domain, and the extracellular ligand-binding domain all contribute to its structure. According to earlier research, EGFR overexpression was found in 60% of NSCLC and was linked to a poor prognosis ²¹.

The EGFR mutation is commonly acknowledged as the primary oncogenic-driven mutation in certain NSCLCs because of the well-studied activating mutation in the EGFR kinase domain ²².  Exons 18–24, which are proximal to the enzyme's ATP-binding pocket, encode the EGFR kinase domain. The bulk of EGFR kinase domain mutations are found in exons 18–21. These mutations boost the EGFR's kinase activity, which in turn causes signal pathways that encourage NSCLC cell growth to become overactive. Phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR, interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) are the three primary downstream signalling pathways that EGFR activates ²³.

The rat sarcoma virus (Ras) gene family includes the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene. About 30% of lung adenocarcinomas (34% in smokers and 6% in non-smokers) have KRAS mutations. They typically occur at codons 12 or 13 in lung adenocarcinoma ²⁴. Ras/Raf/MEK/ERK pathway activation increases cell growth and prevents apoptosis ²⁵ and cell senescence ²⁶.

All multicellular organisms rely on the process known as programmed cell death (PCD) or apoptosis to control cell division, keep tissues in a state of homeostasis, and get rid of harmful or undesired cells. Defects in physiological apoptosis may have a role in several human diseases, including cancer. The discovery of apoptosis mechanisms, its effector proteins, and the genes encoding apoptosis has enabled the discovery and development of novel medications that can decrease the apoptotic threshold or increase apoptosis susceptibility in cancer cells ²⁷.

Targeting the EGFR Pathway: In this study, natural compound myricetin was screened with the active site of EGFR, KRAS, RAF, MEK and ERK. Myricetin was previously recognised as an anticancer substance. The results show that Myricetin obeys Lipinski’s rule of 5 and ADMET properties.

Myricetin interacted with LEU A:718, LYS A:745, VAL A:726, ALA A:743, LEU A:844 and ASP A:855 residues of  EGFR. THR A:790 and  MET A:793 residues of EGFR H-bonded with myricetin. Myricetin interacted with LYS A:147, ALA A:146, ALA A:18, LYS A:117 and  PHE A:28 residues of KRAS. TYR A:32 residues of KRAS H-bonded with myricetin. Myricetin interacted with THR A:421, VAL A:363, LEU A:406, LYS A:375, ALA A:373, GLY A:485, PHE A:475 and TRP A:423 residues of  RAF. ASP A:486 and CYS A:424 residues of  RAF H-bonded with myricetin. Myricetin interacted with VAL A:82, LEU A:74, ALA A:95, LEU A:197, CYS A:207 and  MET A:143 residues of MEK. LYS A:97   residues of MEK H-bonded with myricetin. Myricetin interacted with PRO A:91, PRO A:354, ALA A:90, GLN A:353,  ILE A:88 and ARG A:89  residues of ERK. ASP A:86, PHE A:352 and ASP A:18 residues of ERK H-bonded with myricetin.

FIG. 3: SCHEMATIC REPRESENTATION OF MYRICETIN IN THE TREATMENT OF LUNG CANCER THROUGH EGFR PATHWAY

Myricetin, a naturally occurring substance, binds to the EGFR (tyrosine kinase receptor) and prevents it from being expressed. Myricetin binding causes phosphorylation, which activates RAS. Once Ras is turned on, the downstream RAF is attracted to the cell membrane and activated. After RAF is phosphorylated, MEK phosphorylates and overactivates the extracellular regulatory protein to cause ERK1/2 to become overactive. This causes an accumulation of intracellular oxygen free radicals that sets off the DNA damage response and cell senescence ²⁸. Finally, myricetin inhibits tumour growth, cell proliferation, survival and migration but induces the apoptosis through EGFR pathway.

CONCLUSION: In conclusion, myricetin shows better Hydrogen bond interaction with the EGFR, RAS, RAF, MEK and ERK proteins. Myricetin obeys Lipinski’s rule of five and it has a better binding affinity. It can inhibit lung cancer growth by inducing programmed cell death through the EGFR pathway. This compound can be taken for further studies.

ACKNOWLEDGEMENT: I acknowledge the DBT-BIF centre at Holy Cross College for providing infrastructure facility to carry out the in-silico analysis.

CONFLICTS OF INTEREST: The authors have no conflicts of interest.

REFERENCES:

1. Song Y, Zhou B, Du X, Wang Y, Zhang J, Ai Y, Xia Z and Zhao G: Folic acid (FA)-conjugated mesoporous silica nanoparticles combined with MRP-1 siRNA improves the suppressive effects of myricetin on non-small cell lung cancer (NSCLC): Biomed Pharmacother 2020; 125: 109561.
2. Nath A, Sathishkumar K, Das P, Sudarshan KL & Mathur P: A clinicoepidemiological profile of lung cancers in India - Results from the National Cancer Registry Programme. The Indian Journal of Medical Research 2022; 155(2): 264–272.
3. Huang CY, Ju DT, Chang CF, Muralidhar Reddy P & Velmurugan BK: A review on the effects of current chemotherapy drugs and natural agents in treating non-small cell lung cancer. BioMedicine 2017; 7(4): 23.
4. Chen CJ, Liang KY, Chang AS, Chang YC, Lu SN, Liaw YF, Chang WY, Sheen MC & Lin TM: Effects of hepatitis B virus, alcohol drinking, cigarette smoking and familial tendency on hepatocellular carcinoma. Hepatology (Baltimore, Md.) 1991; 13(3): 398–406.
5. Wei Q, Cheng L, Amos CI, Wang LE, Guo Z, Hong WK & Spitz MR: Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular epidemiologic study. Journal of the National Cancer Institute 2000; 92(21): 1764–1772.
6. Zhou H, Xu L, Shi Y, Gu S, Wu N, Liu F, Huang Y, Qian Z, Xue W, Wang X and Chen F: A Novel Myricetin Derivative with Anti-cancer Properties Induces Cell Cycle Arrest and Apoptosis in A549 Cells. Biol Pharm Bull 2023; 46(1): 42-51.
7. Brambilla E, Travis WD, Colby TV, Corrin B & Shimosato Y: The new World Health Organization classification of lung tumours. The European Respiratory Journal 2001; 18(6): 1059–1068.
8. Huang CY, Ju DT, Chang CF, Muralidhar Reddy P and Velmurugan BK: A review on the effects of current chemotherapy drugs and natural agents in treating non-small cell lung cancer. Biomedici (Taipei) 2017; 7(4): 23.
9. Shinoura N, Yamada R, Okamoto K, Nakamura O & Shitara N: Local recurrence of metastatic brain tumor after stereotactic radiosurgery or surgery plus radiation. Journal of Neuro-oncology 2002; 60(1): 71–77.
10. Hotta K, Matsuo K, Ueoka H, Kiura K, Tabata M & Tanimoto M: Meta-analysis of randomized clinical trials comparing Cisplatin to Carboplatin in patients with advanced non-small-cell lung cancer. Journal of clinical oncology. Official Journal of the American Society of Clinical Oncology 2004; 22(19): 3852–3859.
11. Müller M, Strand S, Hug H, Heinemann EM, Walczak H, Hofmann WJ, Stremmel W, Krammer PH & Galle PR: Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. The Journal of Clinical Investigation 1997; 99(3): 403–413.
12. Hsiao WL & Liu L: The role of traditional Chinese herbal medicines in cancer therapy--from TCM theory to mechanistic insights. Planta Medica 2010; 76(11): 1118–1131.
13. Chantarawong W, Chamni S, Suwanborirux K, Saito N & Chanvorachote P: 5-O-Acetyl-Renieramycin T from Blue Sponge Xestospongia sp. Induces Lung Cancer Stem Cell Apoptosis. Marine Drugs 2019; 17(2): 109.
14. Harvey AL & Cree IA: High-throughput screening of natural products for cancer therapy. Planta Medica 2010; 76(11): 1080–1086.
15. Imran M, Saeed F, Hussain G, Imran A, Mehmood Z, Gondal TA, El-Ghorab A, Ahmad I, Pezzani R, Arshad MU, Bacha U, Shariarti MA, Rauf A, Muhammad N, Shah ZA, Zengin G and Islam S: Myricetin: A comprehensive review on its biological potentials. Food Sci Nutr 2021; 9(10): 5854-5868.
16. Kopustinskiene DM, Jakstas V, Savickas A and Bernatoniene J: Flavonoids as Anticancer Agents. Nutrients 2020; 12(2): 457.
17. Qamar M, Akhtar S, Ismail T, Wahid M, Abbas MW, Mubarak MS, Yuan Y, Barnard RT, Ziora ZM and Esatbeyoglu T: Phytochemical Profile, Biological Properties, and Food Applications of the Medicinal Plant Syzygium cumini. Foods 2022; 11(3): 378.
18. Akash S, Kumer A, Rahman MM, Emran TB, Sharma R, Singla RK, Alhumaydhi FA, Khandaker MU, Park MN, Idris AM, Wilairatana P and Kim B: Development of new bioactive molecules to treat breast and lung cancer with natural myricetin and its derivatives: A computational and SAR approach. Front Cell Infect Micro 2022; 12: 952297.
19. Han SH, Lee JH, Woo JS, Jung GH, Jung SH, Han EJ, Park YS, Kim BS, Kim SK, Park BK, Choi C and Jung JY: Myricetin induces apoptosis through the MAPK pathway and regulates JNK mediated autophagy in SK BR 3 cells. Int J Mol Med 2022; 49(4): 54.
20. Hsu PC, Jablons DM, Yang CT and You L: Epidermal Growth Factor Receptor (EGFR) Pathway, Yes-Associated Protein (YAP) and the Regulation of Programmed Death-Ligand 1 (PD-L1) in Non-Small Cell Lung Cancer (NSCLC). Int J Mol Sci 2019; 20(15): 3821.
21. Nicholson RI, Gee JM & Harper ME: EGFR and cancer prognosis. European journal of cancer (Oxford, England: 1990), 37 Suppl 2001; 4: 9–15.
22. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J & Haber DA: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. The New England J of Med 2004; 350(21): 2129–2139.
23. Sharma SV, Bell DW, Settleman J & Haber DA: Epidermal growth factor receptor mutations in lung cancer. Nature reviews. Cancer 2007; 7(3): 169–181.
24. Dogan S, Shen R, Ang DC, Johnson ML, D'Angelo SP, Paik PK, Brzostowski EB, Riely GJ, Kris MG, Zakowski MF & Ladanyi M: Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-mutant cancers. Clinical Cancer Research: an Official Journal of the American Association for Cancer Research 2012; 18(22): 6169–6177.
25. Thirunavukkarasu MK, Suriya U, Rungrotmongkol T and Karuppasamy R: In-silico screening of available drugs targeting non-small cell lung cancer targets: a drug repurposing approach. Pharmaceutics 2021; 14(1): 59.
26. Wen Z, Jiang R, Huang Y, Wen Z, Rui D, Liao X and Ling Z: Inhibition of lung cancer cells and Ras/Raf/MEK/ERK signal transduction by ectonucleoside triphosphate phosphohydrolase-7 (ENTPD7). Respir Res 2019; 20(1): 194.
27. Goldar S, Khaniani MS, Derakhshan SM & Baradaran B: Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pacific Journal of cancer prevention. APJCP 2015; 16(6): 2129–2144.
28. Wang Z, Liu Y, Takahashi M, Van Hook K, Kampa-Schittenhelm KM, Sheppard BC, Sears RC, Stork PJ & Lopez CD: N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence. Proceedings of the National Academy of Sciences of the United States of America 2013; 110(1): 312–317.
    

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    How to cite this article:

    Viji M, Aishwariya S, Ireen C, Hinduja S, Indu S, Vijayalakshmi P and Rajalakshmi M: In-silico study: myricetin from the stem bark of Syzygium cumini inhibits the growth of non-small cell lung cancer cells through the EGFR pathway. Int J Pharm Sci & Res 2024; 15(5): 1384-90. doi: 10.13040/IJPSR.0975-8232.15(5).1384-90.

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