Anticancer Mechanism of and its bioactive compounds : A short Review along with computational molecular docking study

Mita Shikder1, Tasnin Al Hasib1, Md. Lutful Kabir1 1Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj- 8100, Bangladesh

Abstract: Withania somnifera, known as Aswogondha in Bangladesh and some part of India, is a shrub of Solanceae family. Parts of this plant is used as alternative medicine in this region to cure diseases from bronchitis to insomnia. Although such use of the plant is not supported by clinical research, recent studies have found anticancer activity of some proteins derived from w. somnifera. The purpose of this study is to summarize the anticancer activity of medicinal plant Withania somnifera and its bioactive compounds as well as to predict the interaction between phytochemicals (, Withaferin-A) and macromolecules that are responsible for cell proliferation. Studies suggested that Withanolide and Withaferin-A from W. somnifera can be used as a cancer chemotherapeutic agent for cancerous cell lines in mice models through modulating various signaling pathway including inhibition, autophagy, , radiopreventive pathway and pathway. Molecular docking of Withanolide and Withaferin-A against 9 types of vital protein mediators concluded that 3A8X (Protein kinase C iota type) and 1A9U (MAP KINASE P38) are the most active receptor for binding and interacting with Withanolide and Withaferin-A for the prevention and treatment of cancer. On the basis of this review and docking study, it can be concluded that Withania somnifera as well as its derivatives Withanolide and Withaferin- A may be considered as a promising anticancer agent.

Keywords: Withania somnifera; Withanolide; Withaferin-A; chemotherapeutic agent; anti-cancer agent;

Introduction

Cancer is one of the major cause of mortality all over the world. Although anti-cancer drugs are being developed day by day, the worldwide mortality and morbidity due to various types of are still on the rise. Thus, further research and drug development are always needed for the treatment and the prevention of cancer. More than 60% of medicines for cancer disease are available of plant origin [1]. A large number of phytochemicals play a vital role as a core molecule for the synthesis of novel therapeutic agents [2]. As anti-oxidant properties are present in fruits and vegetables, people who take fresh fruits and vegetables in diet have lower chance of tumor formation and carcinoma [3]. In-vitro and in-vivo studies conclude that, natural plant Withania somnifera and its phytochemicals show promise of anticancer activity in different cancer cell lines in experimental models [4][5].

Withania somnifera is known as a medicinal plant that is used in traditional medicine of Indian Ayurveda. It’s roots, leaves, flowers, barks and branches are also used by local healers as well as in many households as an alternative medicine [6]. This plant is found mostly in arid regions throughout India, Baluchistan, Afghanistan, Sri Lanka, Congo, South Africa, Egypt, Morocco, and Jordan [7]. Evidence showed the anti-inflammatory, and immunomodulatory properties of the plant [8]. The bioactive components of W. somnifera also exert pharmacological properties to treat various diseases including cancer, neurodegenerative diseases, cardiovascular diseases, stress related disorders and so on [9][10][11][12] . The herbal medicine, W. somnifera is effectively used in treatment of infertility and increases the function of reproductive system [14]. W. somnifera extract is also found to enhance the number of white blood cell, hemoglobin level and bone marrow cell [15]. Two major bioactive component, Withanolide and Withaferin-A that are derived from W. somnifera shows positive effect in suppressing carcinoma [16][17][18][19][20][21].

Withanolides are a group of steroidal on C28 skeleton [22]. They are present in leaves and roots of Withania somnifera [22]. exhibit chemopreventive effects, anti-stress effect, anti-cancer activity, anti-inflammatory effect [23][24]. Withanolides are found to reduce tumor growth of human cells, decrease viability of cancer cells, reduce cell proliferation and increase apoptosis [25][26][27]. Withaferin-A is a kind of steroidal lactones and is a highly oxygenated compound [28]. Withaferin-A functions as a cancer chemotherapeutic agent, anti-proliferative agent, anti-oxidant agent, cardioprotective agent and neuroprotective agent [29][30][31][32][33][34]. Although Withanolide and Withaferin-A may be considered as a better inhibitor of cancer disease in some studies, there is no detail interaction related docking study with macromolecules to analyze binding affinity of Withanolide and Withaferin-A against associated proteins.

In this study, the review predicts anti-cancer mechanism of Withania somnifera on the basis of many experimental literature as well as molecular docking studies that predicts binding interaction of Withanolide and Withaferin-A with targeted proteins associated with cancer.

Search Strategy

Articles were collected from various prestigious databases and journals e.g. Science Direct, Scopus, PubMed, NCBI, The American Chemical Society, Web of Science, Nature through google scholar search engine with keyword “Withania somnifera”, “Phytochemicals” pairing with “Anti-cancer activity”, “Withaferin-A”, “Anti-tumor effect of Withanolide” pairing with “experimental study”, “cell lines”, and “Human cancer diseases”. Among literature, some were review articles and some were laboratory-based experimental study that demonstrated test system, route of administration, dose concentration, mechanism of action, results, discussion and conclusion.

Anticancer Mechanism of Withania somnifera

Withania somnifera (WS) and its bioactive component have significant effect in prevention and treatment of different types of carcinoma [35][36][37][38]. W. somnifera is found to inhibit lung adenoma in Swiss albino mice [39]. The root extract of W. somnifera prevented form ROS (reactive oxygen species) induced injure after the treatment with combination of and WS in mouse models [40]. Withanolide was found to suppress Transforming growth factor-b1 (TGF-b1) and TNF-a induced Epithelial-Mesenchymal Transition (EMT) in Non-Small cell (NSCLC) A549 and H1299 cell lines [41]. Some of the anti-cancer effects of w. somnifera is tabulated in Table 1.

Table 1: Anticancer effects of Withania somnifera and its bioactive compounds.

W. somnifera/ its Cell Line/Experiment Effects References phytochemicals model W. somnifera(L.) Dunal male Swiss albino mice induced lung cancer [39] extract Withaferin-A MDA-MB-231 cell lines reduces the proliferation and cell [42] viability of breast cancer cells, inhibition of TASK-3 channels W. somnifera root extract A549 lung cancer cell down regulate the expression of PI3K [43] lines gene, reduce or prevent , treated for non-small cell lung cancer Root extract of w. Swiss albino mice decreased in the cancer cell number and [55] somnifera tumor weight Ethanol extract of w. LNCaP, 22Rv1 cell lines inhibit fatty acid synthesis, [45] somnifera root downregulate expression of fatty acid metabolism proteins [ACLY, ACC1, FASN , CPT1A] Withaferin-A Cervical (SKGII, SKGIIIb, downregulation of mortalin and PARP1; [46] ME180 and HeLa) and upregulation of tumor suppressor ovarian (SKOV3 and and DNA damage signaling; growth OKV-18) cancer cell lines arrest/apoptosis in cancer cells combination of Dox with A2780, A2780/CP70 and inhibition of cell proliferation and [47] WFA CaOV3 (epithelial ovarian induction of cell death; escalation of cancer cell lines) ROS production results in sever DNA damage, increase in elevate the level of autophagy marker LC3B Withanolides human breast cancer cell stimulate apoptotic death in MCF-7 [48] line MCF-7 cells, decrease the levels of -client proteins (Akt and Raf-1) besides stimulate Hsp70 overexpression in MCF-7 cells, decrease the expression of ER in MCF-7 cells

Withaferin-A reduced the proliferation and cell viability of breast cancer cells by inhibiting of TASK-3 channels [42]. W. somnifera root extract downregulated the expression of PI3K gene and reduced or prevented metastasis [43]. The extract of WS induced expression of Nrf-2 and Reduced filopodia formation in BV-2 cell lines, indicating immunomodulatory activity of W. somnifera [44]. Ethanol extract of Withania somnifera downregulated the expression of fatty acid metabolism proteins [ACLY, ACC1, FASN, CPT1A] and inhibited fatty acid synthesis in LNCaP, 22Rv1 cell lines, suggesting proliferative activity of WS [45]. A recent study suggested that Withaferin-A reduced the regulation of mortalin and PARP1 and induced the regulation of tumor suppressor p53 and DNA damage signaling in cancer cells [46]. Combined treatment of Doxorubicin with Withaferin-A was found to increase the level of ROS production resulting in increasing the expression of autophagy marker LC3B; inhibit cell proliferation and induce cell death [47]. Withanolides limits the levels of Hsp90-client proteins (Akt and Raf-1) and stimulates Hsp70 overexpression in MCF-7 cells resulting apoptotic death in MCF-7 breast cancer cell lines [48].

Table 2: Dose/concentration dependent anti-cancer effect of W. somnifera and its bioactive compounds

W. somnifera/ its Conc./Dose (route of Action and Effects Reference phytochemicals administration)/test system Root extract of Withania at the maximal tolerated induced skin cancer, [49] somnifera dose of 400 mg/kg about decrease in incidence and three times per week were average number of skin administered in Swiss lesions in mice, Prevents albino mice DMBA-Induced squamous cell skin carcinoma Withanolides, 0.24 F 0.01 to 11.6 F 1.9 Medication of tumors and [50] Withaferin-A Ag/mL of Withaferin-A; , prevented 0.32 F 0.05 to 0.47 F 0.15 or decreased the growth of Ag/mL of Viscosalactone tumors in human B for MCF-7 (Breast), NCI-H460 (Lung), HCT- 116 (Colon), SF-268 (Central Nervous System) cancer cell line Withaferin-A 0, 7, 14 and 28 µM blocked the MAPK/ RAS/ [51] concentrations for RAF signalling pathway in cisplatin-resistant SCC-4 the SCC-4 cells; increase human oral cancer cells in the ROS levels of SCC4 cells; Withaferin-A 0.75, 5.8, 12.4 and 22.66% Induction of apoptosis by [51] at 0,7,14 and 28 µM Withaferin-A in SCC-4 concentrations of cells Withaferin-A in cisplatin- resistant SCC-4 human oral cancer cells. Withaferin-A at a dose of 6 mg/kg in reduce tumor growth of [52] Panc-1, MiaPaCa2 and human pancreatic cells, BxPc3( induced apoptosis cell lines) significantly in Panc-1 cells, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, Withaferin-A 4 mg/kg body weight were reduce tumor growth of [53] administered in female human breast cancer cells, nude mice for MCF-7 decrease viability of (estrogen-responsive) and human breast cancer cells, MDA-MB-231 (estrogen- reduce cellular independent) human breast proliferation and stimulate cancer cells apoptosis Withanolides Withaferin-A (5 µM), 4β- induced cycle arrest and [54] hydroxywithanolide (20 apoptosis in human breast µM), anomanolide A (20 cancer cells, inhibition of µM) for 48 h in MDA- heat shock protein 90 MB-231 and MCF-7( (Hsp90), reduced human breast cancer cells) constitutive NF-κB activation

Withania somnifera

Withanolide Withaferin-A

1. Downregulation of

1. Inhibition of MCF-7 (Breast), PI3K gene expression; 1. Inhibition of the MAPK/

NCI-H460 (Lung), 2. Inhibition of TASK-3 channels; RAS/ RAF signaling

HCT-116 (Colon), 3. Prevention of DMBA-Induced pathway in the SCC-4 cells;

SF-268 (CNS) cancer cell lines; Squamous Cell Carcinoma of Skin. 2. Increased ROS production;

2. Inhibition of heat shock 4. Serves as an anti-inflammatory, 3. Reduced ER expression

protein 90 (Hsp90). antioxidant, immunomodulatory agent in MCF-7 cells.

Upregulation of DNA damage

Growth arrest

Apoptosis in cancer cells

Figure 1: Anticancer activity of Withania somnifera

At the maximal tolerated dose (400 mg/kg) of Withania somnifera root extract were administered in swiss albino mice that induced skin cancer; decreased average number of skin lesions in mice and Prevented DMBA-Induced squamous cell skin carcinoma [49]. In a potential experimental literature, it was suggested that 0.24 F 0.01 to 11.6 F 1.9 Ag/mL of Withaferin-A and 0.32 F 0.05 to 0.47 F 0.15 Ag/mL of Viscosalactone B were needed for the treatment of tumors and inflammation in MCF-7 (Breast), NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System) cancer cell lines [50]. A concentration of 0, 7, 14 and 28 µM of Withaferin-A blocked the MAPK/ RAS/ RAF signalling pathway in the SCC-4 cells [51]. At a dose (6 mg/kg) of Withaferin-A reduced tumor growth of human pancreatic cells and induced apoptosis in Panc-1 cells significantly [52]. About 4 mg/kg of Withaferin-A were administered in vivo in female nude mice that reduced tumor growth of human breast cancer cells; cease cellular proliferation and stimulate apoptosis [53]. Combined treatment of Withaferin-A (5 µM), 4β-hydroxywithanolide (20 µM), anomanolide A (20 µM) induced cycle arrest and apoptosis in human breast cancer cells; inhibited heat shock protein 90 (Hsp90); reduced constitutive NF-κB activation [54]. Root extract of Withania somnifera decreased in the cancer cell number and tumor size in Swiss albino mice [55].

Molecular docking study

In silico, molecular docking is an excellent tool used for predicting the drug candidate’s pharmacodynamic profile by scoring and orienting them to the receptor binding sites [56]. Docking was performed with the modified and original drugs against the macromolecules (protein) for examining the preferred orientations of minimum free binding energy using AutoDock vina in PyRx platform [57]. The interactions involve many types of non-covalent interactions such as hydrogen bonds, hydrophobic bonds, alkyl bonds, pi-bonds are visualized in Discovery studio visualizer for respective protein and ligand [58]. Drug discovery studio data and images were displayed for the best selected drug and proteins having best binding free energy. In this study, molecular docking was conducted with phytochemicals (Withanolide and Withaferin-A) against 9 significant macromolecules in order to predict anti-cancer activity of Withanolide and Withaferin-A.

Preparation of Ligands and proteins

Two dimensional structure of Withanolide [59] and Withaferin-A [60] were retrieved from PubChem database and saved as “sdf” format for docking analysis (showed in Figure 1)

Withanolide Withaferin-A Figure 1: Two dimensional structure of Withanolide and Withaferin-A. The crystal structure of macromolecules named 1SVC (NUCLEAR FACTOR KAPPA-B (NF-KB)) [61], 6SMB (Tyrosine-protein kinase JAK1) [62], 2YUU (Protein kinase C delta type) [63], 6MXY (TP53- binding protein 1) [64], 1ILQ (Interleukin-8 receptor A) [65], 3S7S (Cytochrome P450 19A1) [66], 1A9U (MAP KINASE P38) [67], 6XIH (Mitogen-activated protein kinase) [68], 3A8X (Protein kinase C iota type) [69] were collected from protein data bank online database. Unwanted ions, ligands and water molecules were removed from protein structure using PYMOL software [70]. Then energy minimization of protein structure was done by using Swiss PDB viewer [71] and saved as “pdb” format.

Docking Result and Non-bond Interaction Analysis and Visualization Docking result determine the measure of ligand interaction to the active site of the targeted protein [72]. The lowest energy of binding of the docked structure was considered as the best drug-receptor complex [56]. The binding energy values were presented in Table 3. Withanolide and Withaferin-A show the best binding affinity -9.7 and -9.3 respectively against 3A8X (Protein kinase C iota type) among the receptor proteins. The binding affinity of Withanolide for 1SVC (NUCLEAR FACTOR KAPPA-B (NF-KB)), 6SMB (Tyrosine-protein kinase JAK1), 2YUU (Protein kinase C delta type), 6MXY (TP53-binding protein 1). 1ILQ (Interleukin-8 receptor A), 3S7S (Cytochrome P450 19A1), 1A9U (MAP KINASE P38). 6XIH (Mitogen-activated protein kinase) proteins respectively -8.3, -8.6, -8.4, -9.0, -7.5, -9.1, -9.2, -8.3 kcal/mol were calculated using PyRx software. Withaferin-A exhibit promising binding affinity -8.9, -8.7, -8.4 kcal/mol against protein code 3S7S, 6MXY, 1A9U respectively. In this study, the binding energy of all the drugs is negative, meaning the binding of the drug to the target is likely to happen spontaneously without requiring any energy [73].

Table 3: Binding energy (kcal/mol) of drug-receptor complex were obtained from AutoDock vina in PyRx platform. Proteins Binding Energy with Binding Energy with withanolide (kcal/mol) Withaferin-A (kcal/mol) 1SVC (NUCLEAR FACTOR -8.3 -7.7 KAPPA-B (NF-KB)) 6SMB (Tyrosine-protein kinase -8.6 -7.7 JAK1) 2YUU (Protein kinase C delta -8.4 -8.0 type) 6MXY (TP53-binding protein 1) -9.0 -8.7 1ILQ (Interleukin-8 receptor -7.5 -7.7 A) 3S7S (Cytochrome P450 19A1) -9.1 -8.9 1A9U (MAP KINASE P38) -9.2 -8.4 6XIH (Mitogen-activated protein -8.3 -8.2 kinase) 3A8X (Protein kinase C iota type) -9.7 -9.3

Docking interactions were visualized using Discovery studio visualizer. Some potential non-bond interactions were presented in Table 4. The most efficient binding interaction of Withanolide and Withaferin-A against 3A8X receptor protein were illustrated in Figure 2.

Table 4: Some potential non-bond interaction of Withanolide and Withaferin-A against efficient receptor proteins Compounds Binding Hydrogen bonds Hydrophobic Bonds Energy (Amino acid …. Ligands) (Amino acid ….. Ligands) (kcal/mol) Distance(Å) Distance(Å) Withanolide-3A8X -9.7 ASP330 (2.33727) VAL259 (5.2382) interaction MET332 (4.48957) PHE333 (4.88484) PHE333 (5.24238) PHE547 (5.05612) PHE547 (4.68813) Withaferin A-3A8X -9.3 VAL259 (5.42141) interaction PHE333 (4.73367) PHE547 (5.45209) PHE547 (4.84909) Withanolide-1A9U -9.2 THR241 (2.1984) VAL290 (4.1536) interaction VAL239 (5.18633) VAL290 (5.08016) LEU291 (5.19464) LYS287 (3.98813) ILE297 (4.38699) LYS287 (4.1871) VAL290 (4.17586) LYS295 (5.31421) Withanolide-3S7S -9.1 LYS440 (2.54227) MET444 (4.80244) interaction LEU157 (4.88593) ILE350 (4.77827) LEU157 (4.16948) PHE430 (5.16758) TYR441 (5.4699) Withanolide-6MXY -9.0 SER1548 (3.52449) LEU1547 (3.78577) interaction LEU1547 (4.48485) TRP1495 (4.62534) TRP1495 (4.52216) TYR1502 (4.26601) TYR1502 (4.04159) PHE1519 (5.11949) PHE1519 (3.61835) Withaferin A-3S7S -8.9 ARG115 (2.979) ILE133 (5.04146) interaction ARG115 (2.79541) CYS437 (4.17532) ALA438 (3.83645) ILE133 (3.38531) VAL370 (4.20242) ILE442 (5.38685) ALA443 (3.37577) MET446 (5.10747) ALA306 (4.47701)

Withanolide-3A8X interaction Withaferin-A-3A8X interaction Figure 2: Binding interaction of Withanolide and Withaferin-A against 3A8X receptor. Hydrogen and hydrophobic bonds were found in those interactions. In biology, hydrogen bond is essential for DNA structure. It is suggested that hydrogen bond <2.3 Å increases the binding affinity by several magnitude [74]. When both the donor and acceptor have either significantly stronger or significantly weaker H-bonding capabilities than the hydrogen and oxygen atoms in water H-bonds boost receptor- ligand interaction. On the contrary, the presence of both the strong and weak H-bond pairings decrease ligand binding affinity due to interference with bulk water [75]. A strong hydrogen bond with THR241 (2.1984) and ASP330 (2.33727) amino acid residues observed in Withanolide-1A9U and Withanolide- 3A8X complex respectively. Withaferin-A 3S7S complex also predicts hydrogen bond with ARG115 (2.979) and ARG115 (2.79541) amino acid residues. Jacob Israelachvili & Richard Pashley described that hydrophobic interactions decay exponentially with distance and best at 0-100 Å range [76]. Thus all the ligands exhibit promising hydrophobic interaction. Hydrophobic nature of ligands indicate all of them are absorbed and execrated easily.

Conclusion

The bioactive components of W. somnifera can induce intrinsic and extrinsic apoptotic cell death on cancer cells. The antiproliferative effect of w. somnifera in cancer cells is also evident. Immunomodulatory, antioxidant, anti-inflammatory and anti-genotoxic effects in normal cells might be the key mechanisms of W. somnifera involved in anticancer activity in different test systems. Molecular docking study also provides evidence that Withanolide and Withaferin-A strongly bind with the macromolecules in order to inhibit cancer cell growth, as a possible anticancer agent.

Acknowledgment We are grateful to department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Science and Technology University for providing us the computational platform to complete the project.

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