Xanthohumol from Hop: Hope for cancer prevention and treatment

Abstract

Cancer is a major public health concern due to high mortality and poor quality of life of patients. Despite the availability of advanced therapeutic interventions, most treatment modalities are not efficacious, very expensive, and cause several adverse side effects. The factors such as drug resistance, lack of specificity, and low efficacy of the cancer drugs necessitate developing alternative strategies for the prevention and treatment of this disease. Xanthohumol (XN), a prenylated chalcone present in Hop (Humulus lupulus), has been found to possess prominent activities against aging, diabetes, inflammation, microbial infection, and cancer. Thus, this manuscript thoroughly reviews the literature on the anti-cancer properties of XN and its various molecular targets. XN was found to exert its inhibitory effect on the growth and proliferation of cancer cells via modulation of multiple signaling pathways such as Akt, AMPK, ERK, IGFBP2, NF-κB, and STAT3, and also modulates various proteins such as Notch1, caspases, MMPs, Bcl-2, cyclin D1, oxidative stress markers, tumor-suppressor proteins, and miRNAs. Thus, these reports suggest that XN possesses enormous therapeutic potential against various cancers and could be potentially used as a multi-targeted anti-cancer agent with minimal adverse effects.

Abbreviations

6-GN

6-geranylnaringenin

6-PN

6-prenylnaringenin

8-PN

8-prenylnaringenin

ALL

acute lymphocytic leukemia

AMPK

5′ AMP-activated protein kinase

ASCL1

achaete-scute family BHLH transcription factor 1

B(a)P

benzo(a)pyrene

Bax

Bcl-2-associated X protein

Bcl-2

B-cell lymphoma

CCA

cholangiocarcinoma

CLL

chronic lymphocytic leukemia

COX

cyclooxygenase

CXC

cysteine X cysteine

CXCR4

C-X-C motif chemokine, receptor 4

CYP1A

cytochrome P450 1A

CYP3A

cytochrome P450 3A

DMX

desmethylxanthohumol

DNA

deoxyribonucleic acid

ERK1/2

extracellular signal–regulated kinase 1/2

FAK

focal adhesion kinase

Hes-1

hairy and enhancer of split-1

Hk-2

hexokinase-2

HPV

human papillomavirus

IL-8

interleukin-8

iNOS

inducible nitric oxide synthase

IXN

isoxanthohumol

MAPK or MEK

mitogen-activated protein kinases

MMP-2

matrix metalloproteinase-2

MMP-9

matrix metalloproteinase-9

mTORC1

mammalian target of rapamycin complex 1

NDMA

nitrosodimethylamine

NF-κB

nuclear factor-kappa B

OXPHOS

oxidative phosphorylation

RSK

ribosomal S6 kinase

STAT3

signal transducer and activator of transcription 3

Stim1

stromal interaction molecule 1

TRAIL

tumor necrosis factor related apoptosis inducing ligand

UPR

unfolded protein response

VEGF

vascular endothelial growth factor

XIAP

X-linked inhibitor of apoptosis

XN

xanthohumol

1 INTRODUCTION

Cancer is one of the major life-threatening diseases globally which is associated with high incidence and mortality.^1-5 It affects people of all ages and is a major health concern worldwide.^{6, 7} As per the estimates of GLOBOCAN 2020, 19.3 million new cases and 10 million mortality reported due to this disease.⁵ During the past several decades, extensive research has developed different treatment modalities for this disease, such as surgery, radiation, chemotherapy, and personalized therapy.^{8, 9} However, despite the advancement in the treatment modalities, satisfactory outcomes are not guaranteed due to severe adverse side-effects, chemoresistance, and high costs of the treatments.^10-21 Therefore, new alternative strategies are required for the prevention and treatment of this disease.

Cancer development involves changes in molecular pathways and biological processes.^22-25 These modifications of pathways lead to cell proliferation, cell death resistance, and angiogenesis and transform healthy cells into the malignant form.^26-31 Besides, chronic inflammation is also one of the hallmarks that lead to the development of cancer.^32-35 Hence, the agents that can suppress inflammation and modulate these pathways have remarkable potential in the prevention and treatment of this disease.

The research over the past several decades proved that a large number of phytochemicals from vegetables, herbs, fruits, and spices have the potential to exert numerous therapeutic effects against various human ailments, including cancer.^{28, 36-46} They are the roots of many traditional medicines due to their effective, multi-targeted, and safety profiles.^{39, 47-57} Thus, medicinal plants and their active components are the treasures in the development of novel therapeutics for the prevention and treatment of cancer.

Xanthohumol (XN) is one such agent that has high potential in the prevention and treatment of several diseases, including cancer. XN is a prenylated flavonoid extracted from the Hop plant Humulus lupulus L., (Figure 1a) which form an essential ingredient of beer.^63-65 XN exists ubiquitously in the Hop plant and is a component of the secreted resin from female inflorescences and through the glandular trichromes present on the cone bracts of hop plants.^{63, 66} The young leaves of the Hop plant also form as the source for XN.⁶⁶ The Hop (Figure 1) is a perennial dioecious plant that belongs to Cannabaceae family, which is widely found through the Northern Hemisphere, Europe, Asia, and North America.^67-69 The Hop plant, also contains other constituents in the resinous secretion of glandular lupulins which includes isoxanthohumol (IXN), desmethylxanthohumol (DMX), 6-prenylnaringenin (6-PN), 8-prenylnaringenin (8-PN), and 6-geranylnaringenin (6-GN).⁷⁰ XN is also derived from Sophora flavescens (shrubby sophora) plant.⁷¹ There are several methods to extract XN from the hop plant. One of these methods include the use of supercritical carbon dioxide (CO₂).⁷² This method highly depends on the pressure and temperature of solvent where the higher pressure improves the solubility of the compound.⁷³ The yield of XN obtained using this method was 115 mg/kg which is comparable to the yield produced by 1% carbon extraction from the Hop cones.⁷² In another study, using the same method, 10–30% of XN was isolated from 50 L volume of the spent Hop extracts.⁷³ Furthermore, in another study, the amount of XN was isolated from the Hop pellets, supercritical CO₂ extract, and Hop ethanolic extract, and it was observed that the higher content of XN was obtained from hop ethanolic extracts (3.75 ± 0.05 g per 100 g) as compared to other materials.⁷⁴ Additionally, the high-speed counter-current chromatography (HSCCC) technique was used for the extraction of high amount of XN. This method reported a yield of 93.60% XN (95% pure) from 607 mg crude extract of H. lupulus L. using the solvent n-hexane–ethyl acetate–methanol–water (5:5:4:3 ratio).⁷⁰ Furthermore, a green and efficient extraction method was used to isolate XN from the spent Hops where different choline chloride-based deep eutectic solvents (DES) were used, and the precipitates formed were extracted with methanol. This study reported a higher yield of XN (2.30 mg/g of spent hops) obtained through DES containing propylene glycol.⁷⁵

FIGURE 1

Source, structure, and analogues of Xanthohumol. (a) Humulus lupulus plant (Photograph by: Craig Chandler, University Communication) (https://cropwatch.unl.edu), (b) Xanthohumol, (c) Analogue 13n⁵⁸; (d) Compound 11⁵⁹; (e) Compound 12⁵⁹; (f) Naringenin⁶⁰; (g) Chalconaringenin⁶⁰; (h) 4-hydroxy-4′-methoxychalcone⁶⁰; (i) Compound number 13 (with para-methoxy and fluorine groups on B ring)⁶¹; (j) Double biotinylated XN⁶²; (k) 4-O-biotinyl XN⁶²

The compound, XN, has shown several remarkable therapeutic potential in the prevention and treatment of several diseases. For instance, it inhibited cell proliferation and metastasis in melanoma and hepatocellular carcinoma.⁶⁴ Furthermore, XN inhibited the proliferation, differentiation, and overproduction of cardiac fibroblast induced by TGF-β1 through modulating phosphatase and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway.⁷⁶ XN was also reported to inhibit extracellular signal–regulated kinase 1/2 (ERK1/2) pathway and FOS-related antigen 1 (Fra1) that lead to inhibition of activator protein-1 (AP-1) transcription and also decreased cyclin D1 in non-small cell lung cancer (NSCLC) cells.⁷⁷ Additionally, XN inhibits cyclin B1 and Ras/methyl ethyl ketone (MEK)/ERK signaling pathway, and induces G2/M cell cycle arrest by activating caspase-3 and 9 and increasing B-cell lymphoma protein 2 (Bcl-2) associated X protein (Bax)/Bcl-2 ratio in colon cancer cells.⁷⁸ In addition, uterine grafts with endometrial lesions in mice exhibited decreased tumor size when treated with XN.⁷⁹ Therefore, XN is a multi-targeted and potential compound for the prevention and treatment of different cancers. Thus, this review includes the updated information on the anticancer studies of XN in various cancers. It also includes scientometric analysis to depict the importance, occurrence, trend, and evolution of the studies on this compound against cancers. Furthermore, this review discusses the pharmacokinetics and pharmacodynamics, toxicity, and formulation studies and also highlights different analogues of this compound.

2 SCIENTOMETRIC ANALYSIS ON XN

To capture the essence of the research work that has been carried out over the years pertaining to the effect of XN on cancer, a comprehensive literature analysis was performed to generate intellectual structures, highlighting the important and relevant information. The dataset consisting of 291 documents was retrieved from the SCOPUS database. The keywords for the electronic research query were “xanthohumol” and “cancer.” VOSviewer version 1.6.16⁸⁰ and RStudio version 1.4.1103⁸¹ were used to perform scientometric analysis. The annual growth rate of the studies pertaining to the effect of XN on cancer is 3.2% (Figure 2a). Furthermore, it was observed that the journals that published highest number of articles in this area are “Molecular Nutrition and Food Research” with eight documents followed by the “Current Medicinal Chemistry,” “International Journal of Molecular Sciences” and “Nutrients” with six documents each (Figure 2b). Moreover, the journals with the highest source impact along with the h-index were visualized in (Figure 3a). Interestingly, the countries contributing to the highest scientific production relevant to the area were China, United States, and Italy (Figure 3b). The average article citations per year (2–17 per year) has been statistically represented in Figure 3c. The essential information related to the dataset, that is, the top 20 cited documents on XN, was compiled in Table 1. United States, Germany, Italy, China, and India were among the top-cited countries on this topic (Figure 3d). Initially, a co-occurrence network of author keywords was generated using VOSviewer version 1.6.16 (Figure 3e). It was observed that the keywords with the highest link strength were “xanthohumol” and “apoptosis.” Another co-occurrence network was generated to selectively analyze the processes that are involved with the effect of XN on cancer (Figure 3f). The nodes represented with the brighter shades of yellow signifies processes that are of recent interest. Henceforth, “ubiquitination,” “mitogen-activated protein kinases (MAPK) signaling,” and “cytokine production” were the focus of many recent studies. Another co-occurrence network was generated to visualize the various molecular targets associated with the effect of XN on cancer (Figure 3g). Furthermore, to analyze the evolution of various research trends of the author's keywords, over the years, a thematic evolution plot was generated (Figure 4a). It was observed that the evolution of “xanthohumol” over the period of 1998–2014 to 2015–2021 was divided into five subdomains including, “8-prenylnaringenin,” “breast cancer,” “xanthohumol,” “prenylated chalcones,” and “anti-proliferative activity.” “Xanthohumol,” “Hops,” “cancer,” and “breast cancer” present in the lower right quadrant comprise of the important themes that form the base of the current study. Two graphs were generated to analyze the growth in the annual occurrences of keywords and sources after the implication of loess smoothening one for each, keywords and sources (Figure 5a,b). In the word growth graph, a significant rise in the occurrence of the keyword “xanthohumol” was observed, signifying its rising importance in recent studies. To summarize, this analysis helped to extract the relevant information from the existing literature, which further holds immense importance in determining the course for future studies on XN and cancer.

FIGURE 2

Comprehensive analysis of literature pertaining to xanthohumol's effect on cancer. (a) Annual scientific production. (b) Graphical representation of journals publishing the highest literature relevant to this study

FIGURE 3

Generation of conceptual structures using Rstudio version 1.4.1103. (a) Source impact of highest relevant journals, with h index as impact measure. (b) Countries with the highest scientific production generated. Darker shades of blue signify more contribution, whereas grey signifies no contribution. (c) Average article citations per year. (d) The most cited countries. (e) Co-occurrence network of author's keywords signifying association strength among them. (f) Co-occurrence network of all keywords highlighting multiple processes regulated upon xanthohumol treatment. (g) Co-occurrence network of all keywords highlighting various molecular targets

TABLE 1. Top 20 cited documents relevant to this study

Paper	DOI	Total citations	TC per year	Normalized TC
Aichinger G, 2018, Front Nutr	https://doi.org/10.3389/fnut.2018.00085	11	2.7500	0.5957
Akazawa H, 2012, Chem Biodiversity	https://doi.org/10.1002/cbdv.201100233	14	1.4000	0.3725
Albini A, 2006, Faseb J	https://doi.org/10.1096/fj.05-5128fje	158	9.8750	0.8893
Albini A, 2007, Nat Rev Cancer	https://doi.org/10.1038/nrc2067	594	39.6000	5.5219
Albini A, 2010, Clin Exp Metastasis	https://doi.org/10.1007/s10585-010-9312-5	13	1.0833	0.1492
Albini A, 2019, Curr Med Chem	https://doi.org/10.2174/0929867324666170920144130	10	3.3333	1.4685
Ambro M, 2019, Nutrients	https://doi.org/10.3390/nu11040879	6	2.0000	0.8811
Amslinger S, 2010, ChemMedChem	https://doi.org/10.1002/cmdc.200900499	114	9.5000	1.3080
Andrae-Marobela K, 2013, Curr Drug Metab	https://doi.org/10.2174/13892002113149990095	61	6.7778	2.1150
Arajo JR, 2011, Nutr Res	https://doi.org/10.1016/j.nutres.2011.01.006	229	20.8182	3.0795
Azevedo C, 2015, Nutr Cancer	https://doi.org/10.1080/01635581.2015.1002625	41	5.8571	1.4046
Babu BV, 2013, Anti-Cancer Agents Med Chem	https://doi.org/10.2174/1871520611313020017	20	2.2222	0.6934
Bailly C, 2020, Chem-Biol Interact	https://doi.org/10.1016/j.cbi.2020.109124	4	2.0000	1.7455
Baio D, 2017, Nutrients	https://doi.org/10.3390/nu9091044	15	3.0000	0.6105
Bak MJ, 2016, Semin Cancer Biol	https://doi.org/10.1016/j.semcancer.2016.03.001	32	5.3333	0.9275
Balunas MJ, 2008, Anti-Cancer Agents Med Chem	https://doi.org/10.2174/187152008785133092	75	5.3571	0.8169
Bartmaska A, 2018, Molecules	https://doi.org/10.3390/molecules23112922	21	5.2500	1.1373
Batovska DI, 2010, Curr Clin Pharmacol	https://doi.org/10.2174/157488410790410579	206	17.1667	2.3635
Benelli R, 2012, Biochem Pharmacol	https://doi.org/10.1016/j.bcp.2012.03.006	40	4.0000	1.0643
Blanquer-Rossell MM, 2013, J Cell Biochem	https://doi.org/10.1002/jcb.24627	27	3.0000	0.9361

FIGURE 4

(a): Thematic evolution trends observed over the period of 1998–2014 to 2015–2021, in the literature pertaining to xanthumol's effect on cancer. (b) Thematic map of author's keywords

FIGURE 5

Analysis of growth trends. (a) Annual occurrences of words after applying loess smoothening. (b) Annual occurrences of journals (loess smoothening) publishing relevant studies

3 TRADITIONAL USE OF H. LUPULUS

The application of Hop for preservation and flavoring alcoholic beverages has been popular since 200 AD, whereas its medicinal applications initiated as early as ninth century.⁸² This plant served for various purposes, as for example, when identified in 79 AD, Hop was popular as a vegetable but later, it was used as a dying and food flavoring agent. It was also employed to manufacture coarse cloths and paper.⁸³ Traditionally, the Hop plants were also used as a sedative to treat insomnia and restlessness and to improve ear pain, toothache, and loss of appetite.⁸⁴ Furthermore, this plant is an essential ingredient in the brewing industry to add taste and bitterness to the beer.⁸⁵ The preparations from Hop plants are also consumed as supplements in diet due to their hypnotic and anxiolytic properties.⁸⁵ It was also used as an alternative strategy in the management of post-menopausal symptoms in women.⁸⁶ Furthermore, it is an essential ingredient in the perfumes, creams, and lotions.⁸³

4 BIOLOGICAL ACTIVITY OF H. LUPULUS

The components of Hop plant such as bitter resins, essential oil (EO), tannins, and terpenes were reported for various biological activities such as anti-microbial, anti-oxidant, and anti-cancer.⁸⁷ The extracts from H. lupulus were also reported to induce anti-microbial activity against Staphylococcus epidermidis and Cutibacterium acnes that are prevalent in skin disease.⁸⁸ The bitter extracts from Hop are also effective against inflammation and metabolic disorders. They are also a potential candidate for the treatment of diabetes mellitus and cardiovascular diseases.⁸⁹ The Hop is also an effective estrogenic agent and is used to treat sleep disorders.^{90, 91} The flavonoids from the Hop were also found to exhibit anti-cancer property in various cancers such as breast, colon, and ovarian cancer cells.⁹² In a study, different extracts of H. lupulus cons were evaluated against pests and compared with XN. This study revealed that XN induced better insecticidal activity against different pests as compared to the extracts. However, among the extracts, highest insecticidal activity was exhibited by ethylacetate extract and least activity was shown by methanolic extract.⁹³

5 CHEMISTRY OF XN

XN is characterized as phenolic chalcone, open chain flavonoids family. It predominates as principal compound in H. lupulus and S. flavescens.⁷¹ The molecular structure of XN (Figure 1b) was first discovered by Verzele in 1957. Chemically XN is designated as 1-(2,4-Dihydroxy-6-methoxy-3-[3-methylbut-2-en-1-yl] phenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one.⁷⁰ The molecular formula of XN is C₂₁H₂₂O₅ and molecular weight is 354.402 g/mol with a monoisotopic mass of 354.147 g/mol. It can be obtained as solid yellow crystals with a high melting point of 172°C (Pubchem CID 639665). The structure of XN consists of flavonoids chain with trans-A and B aromatic rings that are connected by three carbon unit and unsaturated carbonyl component. The α, β-unsaturated carbonyl component of XN are replaced at 4, 2′, and 4′ by hydroxyl groups, 3′ by a prenyl group and 6′ is substituted by a methoxy group.⁹⁴ The presence of this α, β-unsaturated carbonyl group contribute to the biological action of the XN. The lipophilic nature of this compound is improvised with the substitution of -A ring by a prenyl component and methoxy group leading to better affinity toward biological systems.^{94, 95}

6 BIOLOGICAL ACTIVITIES OF XN

XN possesses a wide range of pharmacological activities against diabetes, inflammation, viral infection, cardiovascular diseases and cancer.^{63, 96} XN and its related flavone, IXN, inhibit adipogenesis by preventing preadipocytes differentiation, alleviating lipogenic proteins, and allowing the mature adipocytes to undergo mitochondrial apoptotic pathway.^97-99 The inhibition of lipogenesis also leads to the activation of the adenosine monophosphate-activated protein kinase (AMPK) pathway. It improves insulin resistance in type II diabetic mouse model.¹⁰⁰ Furthermore, the treatment of XN in KK-A^y mice ameliorated diabetes by decreasing the plasma glucose and hepatic triglycerides.¹⁰¹ XN also acts against skin ageing, pigmentation, and promotes photoprotection.¹⁰² In accordance with this, XN inhibited skin inflammation by suppressing interleukin (IL)-12 production in an experimental model for psoriasis.¹⁰³ Additionally, XN is a potential multitargeted agent in the prevention and treatment of cancer due to its chemopreventive, anti-angiogenic, proapoptotic, autophagic, and anti-invasive properties.⁶³ In line with these, the treatment of SKOV3 and OVCAR3 ovarian cancer cells with XN downregulated Notch homolog 1 (Notch1) pathway and induced p21 and phosphorylated Cdc2. This induced S and G2/M cell cycle arrest and apoptosis and inhibited cell proliferation.¹⁰⁴ Furthermore, the treatment of pancreatic cancer cells, BxPC-3 with XN, inhibited angiogenesis through the suppression of nuclear factor-κB (NF-κB), vascular endothelial growth factor (VEGF), and IL-8.¹⁰⁵ In addition, the treatment of XN induced apoptosis in cervical cancer cells through the downregulation of Bcl-2, cleavage of poly (ADP ribose) polymerase (PARP), and activation of caspases.¹⁰⁶ Thus, XN targets multiple proteins and pathways that are involved in the development and progression of cancer and has remarkable potential in the prevention and treatment of various cancers.

7 MOLECULAR TARGETS AND MECHANISM OF ACTION OF XN

Aforementioned, cancer is associated with alterations in various molecular signaling pathways and proteins. Interestingly, XN has been reported to modulate these numerous molecular pathways that lead to the inhibition of cancer hallmarks such as survival, proliferation, invasion, and migration in various cancers (Figure 6).^107-111 In line with this, XN induced apoptosis and suppressed proliferation and invasion in MDA-MB-231 triple-negative breast cancer cells via downregulation of Bcl-2 and matrix metalloproteinases (MMP-2 and MMP-9).¹¹² XN also inhibits proliferation in A549 and H1563 lung cancer cells by suppressing ERK1/2 and p90RSK kinases and cAMP response element-binding protein (CREBP).¹¹³ In addition, XN reduced the proliferation of neuroblastoma cells through the suppression of PI3K/Akt pathway and increased the expression of apoptotic proteins, caspases, and induced PARP cleavage.¹¹⁴ Furthermore, XN suppresses angiogenesis by inhibiting the formation of tubular-like networks, growth of endothelial cells, cell invasion, and migration by downregulating Akt and NF-κB pathways.^{107, 110, 115} In addition, XN suppresses angiogenesis in endothelial cells that was associated with the induction of AMPK initiated through calcium/calmodulin-dependent protein kinase kinase-beta (CAMMKβ). This lead to decreased nitric oxide (NO) and phosphorylation of nitric oxide synthase (NOS).¹¹⁵ Furthermore, XN suppressed angiogenic marker, VEGF in other cancers such as leukemia, myeloma, pancreatic, sarcoma, and skin cancer.^{105, 116-118} Moreover, it was also observed that XN prevented the attachment of MCF-7 cells to lymph endothelial cells (LECs) by altering the expression of intercellular adhesion molecule 1 (ICAM-1).¹¹⁹ Additionally, XN inhibited the formation of circular chemorepellent-induced defects (CCIDs) in LECs by suppressing cytochrome P450 (CYP), selectin E (SELE), and NF-κB. It further blocked epithelial-to-mesenchymal transition (EMT), which is an important event for cancer cell migration, via suppression of paxillin, myosin light chain 2 (MCL2), and S100 calcium-binding protein A4 (S100A4) in MCF-7 cells.¹¹⁹ Furthermore, XN targets cysteine-x-cysteine (CXC) chemokine receptor 4 (CXCR4) and its ligand CXCL12 that leads to the inhibition of metastasis in breast cancer cells.¹²⁰ Moreover, XN induced cell cycle arrest (S phase and G2/M phase) and apoptosis by suppressing Notch1 and its target Hes1 and upregulating Hes6 and p21 in ovarian cancer cell lines.¹⁰⁴ Additionally, the induction of apoptotic pathway and cell cycle arrest due to the treatment of XN were also observed in lung cancer cells via activation of caspase-3, upregulation of p53 and p21, and inhibition of cyclin D1.¹¹³ The pathways like Akt and NF-κB contribute to the survival of cancer cells.^{11, 12, 121-124} However, XN suppressed tumor growth by inhibiting Akt/NF-κB signaling pathway that eventually decreases the expression of signal transducer and activator of transcription 3 (STAT3), cyclooxygenase (COX), and inducible nitric oxide synthase (iNOS) in cholangiocarcinoma.¹²⁵ XN also inhibited NF-κB pathway in breast cancer cells that resulted in the suppression of inflammation and angiogenesis leading to apoptosis.¹²⁶ Furthermore, the apoptosis caused by the treatment of XN in breast cancer cells induced PARP cleavage and increased E-cadherin/catenin complex.¹²⁷ Moreover, in colon cancer, XN induced apoptosis through the release of cytochrome c (cyt c) and inhibition of epidermal growth factor receptor (EGFR)/Akt signaling pathway.¹²⁸ Furthermore, this compound inhibited cell proliferation and induced apoptosis in U87 MG glioblastoma cells through the cleavage of procaspase-3/8 and PARP degradation, inhibition of Bcl-2, mitochondrial dysfunction, and reactive oxygen species (ROS).¹²⁹ Additionally, in another study, the generation of ROS due to the treatment of XN was associated with MAPK-mediated cell death in glioblastoma cells.¹³⁰ XN also induced oxidative stress and endoplasmic reticulum (ER) stress-mediated apoptosis in glioblastoma and leukemia cells.^{130, 131} Furthermore, this compound inhibited other pathways like STAT-3, focal adhesion kinase (FAK) and Ras/MEK/ERK in cholangiocarcinoma, leukemia, and lung cancer cells.^{108, 113, 125} Moreover, the inhibition of phosphorylated ERK1/2 was also illustrated with the treatment of XN in larynx and thyroid cancer cells.^{132, 133} Furthermore, the treatment of oral cancer cells with XN inhibited the Akt-Wee1- cyclin-dependent kinase (CDK1) pathway that lead to the induction of mitochondrial apoptotic pathway.¹³⁴ In addition, in B16 melanoma cells, XN inhibited microphthalmia-associated transcription factor (MITF) and cAMP-dependent pathway that resulted in suppression of melanogenesis, which regulates tumor behavior.¹³⁵

FIGURE 6

Modulation of various molecular pathways by xanthohumol

It is now well established that miRNAs also play an essential role in regulating the processes of cancer.^136-138 In line with this, miR-204-3p was found to have a tumor-suppressive role in glioblastoma. The treatment of U87 MG cells with XN upregulated miR-204-3p which leads to induction of apoptosis and inhibition of insulin-like growth factor-binding protein 2 (IGFBP2)/Akt/Bcl-2 pathways.¹²⁹ Furthermore, the treatment of glioblastoma cells with XN induced miR-4725-3p, which leads to the inhibition of stromal interacting molecule 1 (Stim1) and cell invasion.¹³⁹ Therefore, XN is a multi-targeted compound that diversely modulates several signaling pathways and molecules involved in the progression of various cancers. However, most of the studies have reported the inhibition of Akt/NF-κB pathway by XN in different cancers. As this pathway directly regulates the expression of the genes/proteins involved in cancer cell survival, proliferation, invasion, angiogenesis, metastases, chemoresistance, and radioresistance, suppression of this pathway by this compound may be the major molecular mechanism involved in the regulation of cancer.

8 ROLE OF XN AGAINST DIFFERENT CANCER TYPES

Several studies have reported the multi-targeted role of XN in combatting different cancers. The anti-cancer potential of XN and its related compounds was first evaluated by Miranda et al.¹⁴⁰ Subsequently, XN and its related flavone, IXN had been extensively studied in different cancers (Table 2) that are briefly summarized in the following.

TABLE 2. Effect of xanthohumol in various cancer

Disease	IC50/EC50/LD50	Model	Mechanism of action/outcomes	References
Brain tumor	-	T98G^a	↑ROS, ↑caspase-3,-9, ↑PARP cleavage	130
	-	U87 & SNB19^a	↑p53, ↑Bax/Bcl-2	141
	-	U87 MG^a	↑Pro-caspase-3/8 cleavage, ↑PARP degradation, ↑Apoptosis	129
	-	U87-MG & Hs-683^a	↓Stim1	139
Breast cancer	23.0 μM	MCF-7 and T47-D^a	↑PARP cleavage	127
	-	MCF-7 Xenograft^b	↑Necrosis	142
	7.1 μM	Sk-Br-3^a	↓Aromatase activity	142
	10 μM	MCF-7^a	↓ALP activity	143
	-	MCF-7^a	↓NF-κB, ↓Angiogenesis	126
	-	MCF-7^a	↓NF-κB, ↓anti-apoptotic genes	111
	-	-	↓CXCR4	120
	6.7 μM	MDA-MB-231^a	↓Bcl-2	112
	5 μM	MCF-7^a	↓CYP, ↓SELE, ↓NF-κB	119
	-	MCF-7 MCF-7/ADR^a	↓STAT3, ↑DR-4, ↑DR-5	144
	-	MCF-7^a	↓ROS, ↑OXPHOS	145
	7.3 μM, 8.2 μM	MCF-7 & SK-BR-3^a	↓E2-signaling pathway, ↓ERα	146
	-	MDA-MB-231^a	↑Bax, Caspase-3, Caspase-9	147
	1.22 μM	MCF-7, MCF-7/ADR^a	↓Bcl-2, ↑Bax, ↑cleaved-PARP, ↑γ-H2AX	148
	-	MCF-7 & MDA-MB-231^a	↓Notch, Bcl-2, ↑G0/G1 cell arrest	149
	-	4 T1 tumor mouse^b	↓Tumor growth	149
	-	BALB/c-4 T1 mice^b	↓Tumor growth	150
	12.25 μM	MCF-7^a	↓Cell growth	110
Cervical cancer	7.9 μM	HeLa^a	↓MMP; cleavage of PARP, ↑Caspase 3	151
	20.08 μM	Ca Ski^a	↑Caspases-3, -8 & -9; ↑PARP, ↑p53, ↑AIF; ↓Bcl-2, ↓XIAP	106
Cholangiocarcinoma	-	M214 & M139^a	↓STAT3; ↓Akt/NF-κB	125
	-	KKU-M214 injected mice^b	↓Tumor growth	125
	-	CCLP1, SG-231 & CC-SW-1^a	↓Notch1, ↑Akt, ↑cleaved PARP, ↑caspase-3	152
Colon cancer	-	HCT 116-derived 40–16^a	↑PARP cleavage; ↑caspases-3, -7, -8, & -9, ↓Bcl-2	153
	3.6 μg/ml	HCT-15^a	↓Topo I	154
	-	FHC, CCD841 CoN, HT29, SW480, LOVO, HCT116, SW620^a	↑cyt c, ↑Apoptosis, ↓EGFR/Akt, ↓Hk 2	128
	-	CRC Xenograft^b	↓Glycolysis, ↓cell proliferation, ↓Hk 2	128
	10 μM	HT-29^a	↑Apoptosis, ↑caspase-3 & -9, ↑Bax/Bcl-2, ↑G2/M arrest, ↓cyclin B1, ↓MEK/ERK	78
	-	SW620^a	↑ROS; ↓OXPHOS, ↓sirtuin 1	92
	-	AOM-induced rats^b	↓iNOS, ↓Bcl-2, ↓COX-2; ↑Bax, ↑caspase 3; ↑Apoptosis	155
Esophageal squamous cell carcinoma	-	KYSE70, 450 & 510 ESCC^a	↓Cell proliferation; ↑apoptosis, ↑G1 arrest, ↑cyt c, ↑caspase-3, -7, ↑PARP, ↑Bax, ↑Bim,	156
Kidney cancer	-	A293^a	↓NF-κB, ↓anti-apoptotic genes	111
Larynx cancer	-	RK33 & RK45^a	↑Caspase-9, -8, & -3, ↑p53; ↓cyclin D1, ↓Bcl-2	133
Liver cancer	22.8 μM^c	HepG2^a	↓DNA strand breaks, ↓Notch	157
	-	HA22T/VGH, Hep 3B^a	↑Apoptotic nuclei	132
	-	HepG2, Huh7^a	↓NF-κB, IL-8	109
	-	HepG2, SK-Hep-1, Hep3B^a	↓Notch, ↓Hes-1	158
	-	HepG2^a	↑Caspase-3, ↑p53; ↓NF-κB, ↓Bcl-2/Bax, ↓XIAP	159
Leukemia	24.4 μM^d	K562^a	↑PARP cleavage	160
	10 μM/L(48 hr), 5.4 μM/L(72 hr)	K562^a	↑p21, ↑p53, ↓survivin, ↓Bcr-Abl	161
	-	KBM-5, U937, HL-60, Jurkat, U266^a	↓NF-κB, ↓anti-apoptotic genes	111
	-	CLL cells^a	↓Mcl-1, ↓Bcl-2, ↑PARP cleavage	131
	-	ALL-PO & RS4, 11^a	↓FAK, ↓Akt, ↓NF-κB	108
	-	ALL Xenograft^b	↑Life span	108
	-	MM6, U937^a	↓VEGF, ↓MMP	116
Lung cancer	-	A549^a	↓topo I	154
	-	H1299^a	↓NF-κB, ↓anti-apoptotic genes	111
	-	A549^a	↑Caspase-3, -8, & -9	162
	8.6 μM	A549^a	↓MMP; cleavage of PARP, Caspase 3	151
	20.9 μM (48 hr), 7.2 μM (96 hr)	A549, H1563^a	↓ERK1/2, ↓p90RSK, ↓Cyclin D1; ↑p53, ↑p21, ↑caspase-3	113
Ovarian cancer	-	SK-OV-3^a	↓topo I	154
	-	SKOV3, OVCAR3^a	↓Notch1, ↑p21, ↑S & G2/M arrest, ↑cleaved PARP, ↑caspase-3	104
Neuroblastoma	12 μM	NGP, SH-SY-5Y, SK-N-AS^a	↑PARP, cleaved caspase-3/-7, ↑Bax, ↓Bcl-2, ↓PI3K/Akt pathway	114
Pancreatic cancer	-	PANC-1, BxPC-3^a	↓STAT3, ↓CyclinD1, ↓survivin, ↓Bcl-xL	163
	-	AsPC-1, PANC-1, L3.6pl, MiaPaCa-2, 512, 651^a	↓Notch1, ↓HES-1, ↓survivin	164
	-	MIA PaCa-2 & AsPC-1	↓NF-κB, ↓VEGF, ↓IL-8	105
	-	BxPC-3 Xenograft^b	↓Tumor volume	105
Prostate cancer	-	BPH-1, PC3^a	↓NF-κB	165
	-	PC-3, DU145^a	↑Caspase-3 independent cell death	166
	-	LNCaP^a	↑TRAIL-induced apoptosis	167
	-	LNCaP, PC-3, DU145^a	↑Procaspases -3, -8, and -9; ↓Akt, ↓NF-κB, ↓mTOR,↓Bcl-2, ↓survivin	168
	6.9 μM/L, 9.8 μM/L	DU145, PC3^a	↓FAK, ↓Akt	169
	-	LNCaP^a	↑Caspases-3, -8, -9, ↑Bid, ↑Bax; ↓Bcl-xL, ↓MMP	170
Sarcoma	-	HT-1080^a	↓triglyceride synthesis	171
	-	HT-1080^a	↓MMP-2, ↓MMP-9, ↓VEGF	117
Skin cancer	-	B16^a	↓MITF	135
	-	SK-MEL-2^a	↓topo I	154
	21.8 μM	RPMI8226, U266^a	↓cyclin D1, ↓Bcl-2, ↓sIL-6R, ↓VEGF, ↑p21,↑p53, ↑Bax, ↑caspase-3	118
	50.1 μM

• ^a In vitro.
• ^b In vivo.
• ^c EC₅₀.
• ^d LD₅₀.

8.1 Brain tumor

Brain tumor is defined by an abnormal cell growth that arises from the brain and its adjacent tissues and constitute nearly 2% of the total cancer cases.^{26, 172, 173} The glioblastoma multiforme represents the most aggressive and destructive form of this tumor.¹⁷⁴ Several studies have investigated the role of XN on various brain tumor cell lines. For example, XN treatment was shown to induce apoptosis through the activation of caspases -3, -9, PARP cleavage, mitochondrial depolarization, release of cyt c, and suppression of Bcl-2 in T98G cells.¹³⁰ The treatment of XN also resulted in MAPK-mediated cell death through ROS production in these cells.¹³⁰ Furthermore, XN induced caspase-dependent apoptosis in T98G cells by increasing the level of Annexin A1.¹⁷⁵ The treatment of XN also induced apoptosis and inhibited invasion in U87 and SNB19 cells by elevating the expression of p53 and Bax/Bcl-2 ratio.¹⁴¹ Furthermore, XN inhibited proliferation and induced apoptosis in U87 MG cells through the cleavage of procaspase-3/8 and PARP degradation.¹²⁹ Additionally, the treatment of U87-MG and Hs-683 cells with XN upregulated miR-4725-3p, which in turn suppressed Stim1 expression (a calcium sensor present in an ER and invasion.¹³⁹ In addition, the treatment of U87 MG cells with XN inhibited IGFBP2/Akt/Bcl2 pathways mediated through the activation of miR-204-3p levels that led to apoptosis.¹²⁹

8.2 Breast cancer

Breast cancer is the most diagnosed cancer worldwide.^{5, 9, 176, 177} Studies have shown the association of estrogen with the progression of estrogen-dependent breast cancer.¹⁷⁸ Therefore, suppressing the function or synthesis of estrogen could inhibit the progression of estrogen-dependent breast cancer.¹⁷⁷ Interestingly, studies have shown that XN could interfere with the aromatase activity and lower estrogen levels in blood that prevents the development of breast cancer.¹⁴² This inhibition of aromatase activity by XN suppressed cytochrome P450 enzymes, CYP1A and CYP3A by acting against the substrate androstenedione.¹⁷⁸ XN also inhibited the estrogen (E2)/estrogen receptor-α (ERα) signaling by preventing the interaction of brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) and protein prohibitin 2 (PHB2), which resulted in binding of PHB2 to nuclear- and cytoplasmic ERα.¹⁴⁶ Furthermore, the treatment of MCF7 and SKBR3 cells with this compound inhibited cell growth and proliferation.^{110, 126, 140, 142, 143} In addition, the treatment of MCF-7 cells with XN inhibited the proliferation by decreasing sirtuin 1, oxidative phosphorylation (OXPHOS) and increasing ROS.¹⁴⁵ Furthermore, XN treatment also induced cell cycle arrest and apoptosis and inhibited proliferation in MCF-7/ADR cells by increasing Bax, cleaved-PARP, gamma H2A histone family member X (γH2AX) and suppressing Bcl-2.¹⁴⁸ Additionally, the treatment of MDA-MB-231 cells with XN induced apoptosis by increasing the expression of Bax, caspase-3, and -9 and decreasing Bcl-2.^{112, 147} The NF-κB regulates genes involved in survival, inflammation, and angiogenesis.^179-182 However, the treatment of the breast cancer cells with XN enhanced necrosis and apoptosis and suppressed inflammation and angiogenesis prominently by interfering with the NF-κB signaling pathway.¹²⁶ The property of cancer cells to develop multi-drug resistance (MDR) contributes to its survival. Nonetheless, the treatment of XN and its related prenylflavonoids from Hops decreased MDR in breast cancer.¹⁸³ Furthermore, XN induced the secretion of perforin, granzyme B, enhanced the CD8+/CD25+ ratio, and upregulated the expression of different cytokines in breast cancer mouse model which lead to decreased tumor growth.¹⁵⁰ Furthermore, the treatment of 4T1 mouse model with XN also suppressed tumor growth by upregulating caspase-3 and by decreasing the expression of Ki-67, Notch1, and survivin.¹⁴⁹ In addition, XN induced apoptosis and inhibited proliferation and invasion in MCF-7 and T47-D cells through PARP cleavage and upregulation of E-cadherin/catenin complex.¹²⁷ Furthermore, XN suppressed invasion in breast cancer cells by suppressing CXCR4 and its ligand CXCL12.¹²⁰

8.3 Bone cancer

Bone cancer is defined as the malignancy of the bone tissue or skeletal system.^{184, 185} The effect of XN was also evaluated in bone cancer cells. The treatment of XN in HT-1080 fibrosarcoma cells was found to suppress the motility and proliferation of the cells and also reduced the synthesis of lipids in oxygen-deprived condition.¹⁷¹ Additionally, it was found that the effect was mediated through the suppression of MMP-2 and -9 and VEGF.¹¹⁷

8.4 Cervical cancer

Cervical cancer is one of the most diagnosed malignancies in women globally.^{2, 5, 186-188} The infection caused by viruses, mainly human papillomavirus (HPV)-16 and HPV-18, are the prime basis of its occurrence in most of the patients.¹⁸⁹ Studies have reported the potential of XN against this cancer. It was found that, the treatment of Ca Ski cells with XN induced S-phase cell cycle arrest and increased the expression of caspases leading to cell death. Altered mitochondrial membrane potential and fragmentation of DNA were also observed in Ca Ski cells with XN treatment.¹⁰⁶ Thus, XN is an efficient apoptotic mediator inducing both intrinsic and extrinsic apoptotic pathways.¹⁰⁶ Furthermore, the treatment of HeLa cells with XN resulted in apoptosis through the suppression of MMP, initiation of PARP cleavage and activation of caspase-3.¹⁵¹

8.5 Cholangiocarcinoma

Cholangiocarcinoma is one of the most common malignancies in the world and is categorized into intrahepatic (iCCA) and hilar cholangiocarcinoma (hCCA).¹⁹⁰ The investigation on the effect of XN in M214 and M139 cells showed that the administration of 20 and 50 μM XN suppressed STAT3 and Akt/NF-κB pathways. This suppression resulted in apoptosis and inhibition of cell growth by decreasing Bcl-2 and increasing Bax expression.¹²⁵ Furthermore, the oral administration of 50 μM XN in nude mice suppressed cholangiocarcinoma tumor growth.¹²⁵ In addition, XN treatment induced apoptosis and suppressed cell proliferation by initiating PARP cleavage and activating caspase-3 and inhibiting Notch and Akt signaling in CCLP1, SG-231 and CC-SW-1 cells.¹⁵²

8.6 Colon cancer

Colorectal cancer (CRC) ranks third most persistent cancer in both male and female in the United States.^{191, 192} The investigation of the anti-cancer effect of XN in colon cancer cells was shown that this compound induced G2/M cell cycle arrest and apoptosis by increasing caspases, Bax/Bcl-2 ratio and PARP cleavage.^{78, 106, 153, 155} In addition, the induction of cell cycle arrest in HT-29 cells with XN treatment resulted in the suppression of cyclin B1 and Ras/MEK/ERK pathway.⁷⁸ The enzyme hexokinase-2 (Hk-2) is often overexpressed in colon cancer and the suppression of this enzyme in these cells may inhibit survival and proliferation. A recent study has shown that, the treatment of FHC, CCD841 CoN, HT29, SW480, LOVO, HCT116, and SW620 cells with XN inhibited glycolysis and cell proliferation through the reduction of Hk-2.¹²⁸ The effect of inhibited glycolysis with XN treatment was also observed in CRC xenograft. In addition, XN induced apoptosis in vitro via release of cyt c and suppression of EGFR/Akt signaling pathway.¹²⁸ Furthermore, the treatment of XN decreased the cell viability, induced ROS and repressed the OXPHOS complexes and sirtuin 1 in SW620 colon cancer cells.⁹² Additionally, the treatment of HCT-15 cells with XN induced cytotoxicity by inhibiting DNA topoisomerase I and genes involved in drug efflux such as ABCB1 (MDR1), ABCC1 (MRP1), ABCC2 (MRP2), and ABCC3 (MRP3).¹⁵⁴

8.7 Leukemia

Leukemia originates from the blood or bone barrow cells.³⁶ It is the most frequent cancer in the children and is associated with high mortality. It is classified into acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML).^193-195 Accumulating lines of evidence have shown the remarkable potential of XN in the prevention and treatment of this cancer. This compound was shown to inhibit cell growth in HL-60 cells by enhancing CCAAT-enhancer-binding protein (C/EBP) and Grp78/Bip, ER stress, p38 MAPK signaling which leads to paraptosis.¹⁹⁵ Furthermore, induction of ER stress and apoptosis with XN treatment was mediated through unfolded protein response (UPR) in chronic lymphocytic leukemia (CLL).¹³¹ Additionally, this compound was shown to suppress cell migration and invasion in ALL cells through the modulation of Akt, FAK and NF-κB pathways.¹⁰⁸ Furthermore, the treatment of XN in Bcr-Abl expressed-K562 cells was shown to induce apoptosis and inhibit invasion, metalloprotease production, and adhesion to the endothelial cells by upregulating p21 and p53 and downregulating survivin.¹⁶¹ This compound was also found to manifest anti-tumor and anti-angiogenic activities by blocking Akt/NF-κB pathway in both in vitro and in vivo settings.¹¹⁶ Furthermore, the treatment of XN in cells derived from B-chronic lymphocytic leukemia (B-CLL) patients decreased cell growth and induced apoptosis through PARP cleavage.¹⁶⁰ Additionally, a randomized crossover trial was investigated in 22 human subjects, provided with beverages containing 12 mg XN for 2 weeks. It was shown to decrease DNA damage initiated due to ROS and oxidative stress. This effect was also confirmed by a follow up randomized study in 10 patients which showed similar protection against DNA damage.¹⁹⁶ Another study was performed to analyze the DNA protective activity of XN (12 mg for 2 weeks) in the lymphocytes collected from the blood samples of 22 participants. This study showed that XN decreased DNA damage in lymphocytes by antagonizing the effect of carcinogens such as nitrosodimethylamine (NDMA), benzo(a)pyrene (B(a)P) and 2-amino-3-methylimidazo(4,5-f) quinoline (IQ).^{197, 198} The protective behavior of XN might be due to the induction of alpha glutathione S-transferase (α-GST) which was observed only in treated group as compared to placebo group.¹⁹⁷

8.8 Liver cancer

Liver cancer is one of the fatal malignancies, and due to its late-stage diagnosis, it is associated with poor prognosis.^{9, 158, 159, 199} The effect of XN was also investigated in liver cancer. One of the studies reported that XN downregulated NF-κB expression and induced p53 and caspase-3 that lead to apoptosis in liver cancer cells. It was also observed that the treatment of HepG2 cells with XN reduced the expression of X-linked inhibitor of apoptosis (XIAP) and Bcl-2/Bax, resulting in suppression of cell survival and proliferation.²⁰⁰ Moreover, inhibition of Notch1 and Hes-1 by XN in HepG2, Hep3B, and SK-Hep-1 cells also inhibited cell proliferation.¹⁵⁷ Furthermore, the suppression of proliferation and migration by XN treatment were also observed in HepG2 and Huh7 cells. XN also downregulated TNF-α-induced NF-κB activation and IL-8 in HepG2 and Huh7 cells.¹⁰⁹ Notably, XN was reported to selectively target liver cancer cells without affecting the normal murine cell lines.²⁰¹

8.9 Lung cancer

Lung cancer is one of the most prevalent and recurrent malignancy reported worldwide.^{121, 202-204} A study on the anti-cancer activity of XN and its related compounds, IXN and 8-prenylnaringenin, showed that these compounds induced cytotoxicity in A549 lung adenocarcinoma cells by inhibiting DNA topoisomerase I which lead to the inhibition of growth-promoting genes.¹⁵⁴ Furthermore, XN was demonstrated to suppress G1 and S phase cell cycle proteins and simultaneously activated caspases-3, -8, and -9.¹⁶² Furthermore, XN inhibited cell growth and proliferation by targeting ERK1/2 and p90RSK kinases, followed by the activation of CREBP and interference of Ras/MEK/ERK signaling pathway in lung cancer cell lines. Additionally, tumor-suppressive proteins such as p53 and p21 were upregulated and cyclin D1 was downregulated upon XN treatment in A549 and H1563 lung cancer cells.¹¹³ Furthermore, the treatment of HCC827, H1975 and H23 lung cancer cells with XN suppressed the ERK1/2-Fra1-cyclin D1 pathway, leading to the inhibition of cell proliferation.⁷⁷ In addition, the treatment of this compound was shown to decrease tumor growth in HCC827 xenograft model.⁷⁷

8.10 Pancreatic cancer

Pancreatic cancer is one of the most lethal malignancies associated with least 1-year survival.^205-208 The treatment with XN resulted in suppression of NF-κB-mediated angiogenesis and its regulated proteins, VEGF, and IL-8 both at transcriptional and translational levels in AsPC-1, BxPC3 and MIA PaCa-2 pancreatic cancer cells in vitro and BxPC3 in vivo.¹⁰⁵ Furthermore, XN also induced cell cycle arrest and apoptosis in PANC-1 and BxPC-3 cells through the suppression of cyclin D1, survivin and Bcl-xL.¹⁶³ In addition, the treatment of XN in different pancreatic cell lines resulted in inhibition of cell growth and proliferation via inhibition of Notch1 signaling and associated proteins like Hes-1 and survivin.¹⁶⁴

8.11 Prostate cancer

Prostate cancer is one of the commonly diagnosed malignancies in men globally.^{4, 30, 167, 179, 209} The treatment of the localized tumor involves surgery and radiation; however, advanced condition of prostate cancer (hormone-independent) is not approachable to the suggested treatment.^210-212 Therefore, development of novel agents for the treatment of this cancer become imperative. XN was shown to induce apoptosis by altering the integrity of mitochondrial membrane followed by release of cytochromes and other cytolytic proteins that eventually induced cell death in BPH-1 prostate cancer cells.²¹³ Furthermore, the treatment of prostate cancer cells with XN induced apoptosis and inhibited proliferation by activating caspases and suppressing Akt/NF-κB pathways.¹⁶⁸ It was also observed that the doses of 5 and 10 μM XN inhibited growth, motility, and invasion in DU145 and PC3 cells through modulation of the FAK/Akt/NF-κB pathways.¹⁶⁹ Furthermore, treatment of XN induced apoptosis in BPH-1 and PC3 cells and inhibited the NF-κB expression.¹⁶⁵ Moreover, XN was also shown to induce caspase-3-independent cell cytotoxicity in PC-3 and DU145 cells.¹⁶⁶ In addition, XN was found to induce the function of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in LNCaP cells which lead to apoptosis mediated by enhanced expression of caspases-3, -8, -9, Bid and Bax.¹⁷⁰ Furthermore, XN is one of the important agents that sensitizes prostate cancer cells to TRAIL that enhances cytotoxicity and apoptosis.¹⁶⁷

8.12 Other cancers

As XN is a pleiotropic agent with wide arrays of anti-cancer properties. Therefore, the effect of XN was also investigated in other cancers such as larynx cancer, oral cancer, melanoma, and thyroid cancer.^132-134 For example, the treatment of RK33 and RK45 larynx carcinoma cells with XN inhibited proliferation through the suppression of ERK1/2 phosphorylation, cyclin D1 and Bcl-2, and upregulation of p53, p21/WAF1, and caspases-3, -8, and -9.¹³³ Furthermore, XN treatment suppressed the proliferation and expression of achaete-scute complex-like 1 (ASCL1) while enhancing phosphorylated ERK1/2 in thyroid cancer cells.¹³² Additionally, the treatment of CAL27, SCC15, SCC25, and SCC9 oral cancer cells with XN inhibited survivin and Akt-Wee1-cyclin-dependent kinase (CDK1) pathway and activated the mitochondrial apoptotic pathway.¹³⁴ Furthermore, the treatment of KYSE70, 450, and 510 esophageal cancer cells with this compound increased apoptosis and suppressed proliferation by inducing G1 cell cycle arrest.¹⁵⁶ In addition, XN inhibited MDR genes such as MDR1, MRP1, MRP2, and MRP3 in ovarian cancer and melanoma cells. This compound also decreased DNA topoisomerase I activity and induced cytotoxicity in SKOV-3 and SK-MEL-2 ovarian cancer cells.¹⁵⁴ Furthermore, the treatment of B16 melanoma cells with XN inhibited melanogenesis by targeting the cAMP-dependent pathway and inhibiting MITF which regulated the melanogenic enzymes.¹³⁵ Furthermore, XN inhibited U266 and RPMI 8226 myeloma cells proliferation and blocked the cell cycle by inhibiting cyclin D1 and inducing p21. XN also induced both intrinsic and extrinsic pathways of apoptosis by modulating p53, Bax, and Bcl-2 and activating caspases.¹¹⁸ In addition, XN induced apoptosis and suppressed proliferation in NGP, SH-SY-5Y, and SK-N-AS neuroblastoma cells by increasing caspase -3/-7, BAX, and PARP degradation, and decreasing Bcl-2. It also reduced the level of death receptor (DR) and inhibited PI3K/Akt pathway. The combined treatment of XN and TRAIL induced synergistic reduction in neuroblastoma cell growth.¹¹⁴ Thus, the given studies suggest the chemopreventive and therapeutic efficacy of XN in the prevention and treatment of different cancers.

9 CHEMOSENSITIZATION AND RADIOSENSITIZATION

The development of chemoresistance and radioresistance are the two prime drawbacks in cancer treatment.^{10, 214} However, studies have reported that various natural compounds and polyherbal formulations can inhibit drug resistance and radioresistance in cancer cells.^{37, 206, 215} In accordance with this, XN could induce anti-cancer activity by increasing response to DNA damage and apoptosis and activates the ataxia telangiectasia mutated (ATM) pathway in colon cancer. The ability of XN in DNA repair resulted in the chemosensitization of CRC cells to chemotherapeutic agent SN38 (known as 7-ethyl-10-hydroxycamptothecin).¹⁴⁴ Another study reported the chemosensitizing potential of XN in breast cancer MCF-7/ADR cells resistant to adriamycin.²¹⁶ Additionally, XN could efficiently sensitize MCF-7/ADR cells to doxorubicin, indicating a potential candidate for the treatment of breast cancer.¹⁴⁸ The pre-treatment of MCF-7/ADR cells with XN also sensitize these cells to radiation, and induced apoptosis. Furthermore, the treatment of XN was shown to inhibit MDR1, EGFR, and STAT3. XN treatment also increased the expression of DR4 and DR5 in MCF-7/ADR cells.²¹⁶ Additionally, XN treatment also reduced radioresistance in CAL27- and SCC25-induced oral cancer xenograft tumors through the suppression of Ki67, p-Akt, and survivin.¹³⁴

10 PHARMACOKINETICS STUDIES ON XN

The study on pharmacokinetics, dynamics and biotransformation are essential to define the efficacy of the drug. Accordingly, the biotransformation study of XN was investigated in rat liver microsomes, where three polar metabolites were identified as chalcones and XN with additional ─OH group at B ring. Another nonpolar metabolite was also identified as dehydrocycloxanthohumol.²¹⁷ Subsequently, another study on the biotransformation of XN in microbial models, Pichia membranifaciens, a mammalian mimic model, generated three metabolites of methoxychalcone which was identical with the previously identified metabolites in rat liver microsomes.²¹⁸ Additionally, in a study, the rats were fed with Hop-derived XN where 20 modified chalcone metabolites and two flavanones were identified in the feces of rats at 24 and 48 hr. However, around 89% of the flavone compounds recovered from the feces were in their unchanged form, that is, XN.²¹⁹ The resorption and metabolism of XN were also studied in rats. In this study, the rats in the first experiment were administered with XN either through oral or intravenous route, and in second experiment only oral administration with different concentrations of XN were given to examine the bioavailability. The content of XN was analyzed in the plasma, urine and feces. It was observed that after an intravenous administration the level of XN in the plasma falls within an hour. However, no traces of XN were found in the plasma after oral administration in both experiments. It was also observed that XN is excreted from the body, mainly in the feces in rats.²²⁰ Subsequently, another group studied the in vitro phase II metabolic fate of XN using recombinant UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT), which resulted in the glucuronidation and sulfation of XN. This led to identifying three mono-glucuronides and three mono-sulfate metabolites.²²¹ Similarly, phase I and II metabolites of XN in rats were identified to undergo the process of oxidation, demethylation, and dehydrogenation for phase I, and glucuronidation and sulfatation for phase II metabolites.²²² Furthermore, in a study to determine the uptake, movement, and accumulation of XN in Caco-2 intestinal cells demonstrated that 70% of the apical XN was accumulated in the cells. More than 90% was accumulated in the cytosol due to its specific binding to cytosolic proteins. This binding of XN to the cytosolic proteins in intestinal cells suggests the poor bioavailability of this compound.²²³ Similarly, the uptake and distribution kinetics of XN was studied in various cell lines such as hepatocytes, hepatic stellate cells (HSC), HuH-7, and Caco-2 cells, where it was observed that XN highly accumulates and binds to cellular factors in these cells.²²⁴

The paper strip extraction (PSE) method was also used to analyze the level of XN in plasma and urine samples. The PSE method has high advantage over liquid–liquid extraction (LLE) due to its high sample throughput, and found that the recovery was high and losses only around 35% or less XN and its metabolites. The recovery of XN was higher in the urine samples with this technique.²²⁵ A study on the pharmacokinetics of XN in human subjects administered with 20, 60, or 180 mg of XN resulted in a unique form of biphasic absorption where XN and IXN were the major circulating metabolites. The mean half-life for XN was observed at 20 and 18 hr for the 60 and 180 mg dose, respectively.²²⁶ Furthermore, the extract containing XN was observed to have slow absorption, contributing to longer half-lives of the compound. The compound also undergoes glucuronidation, and the glucuronides were detected in serum and urine. A secondary peak of the compound was also identified, signifying enterohepatic recirculation, and cyclization of XN to IXN was also observed.²²⁷ Another study determining the PK parameters of XN in male Sprague–Dawley (SD) rats suggests that the bioavailability of XN in both the intravenous and oral gavage methods depends on the concentrations of administered doses.²²⁸

Furthermore, the influence of human intestinal bacteria was studied on the bioavailability of XN in germ-free (GF) and human microbiota-associated (HMA) rats where XN and its conjugates were found to be available in the blood plasma. The total excretion of XN and its metabolites occurred within 48 hr, and it was observed as 4.6 and 4.2% of the administered dose in GF and HMA rats, respectively. The feces form is the major route of excretion for XN.²²⁹ Furthermore, in a study determining the distribution and bioavailability of XN in Wistar rats, the intravenous administration of XN were widely distributed and eliminated after a long time, where enterohepatic recirculation might play a role. In case of oral administration, some fractions of XN reached the plasma; however, XN gets saturated and less absorbed.²³⁰ Thus, various factors play an important role in the biotransformation, absorption, dynamics, and bioavailability of XN in cancer cells.

11 TOXICITY STUDIES

Along with the efficacy of the compound, its safety profile is also an important factor in determining the anti-cancer activity. With regard to this, the oral administration of XN in mice was observed to have no side effect in the functioning of normal organs and tissues. Furthermore, no changes in the metabolism of proteins, lipids, carbohydrates, and uric acid were observed in mice.²³¹ In accordance to this study, the female BALB/c mice were fed with a standard diet and 1,000 mg XN/kg body weight (b.wt.) for 3 weeks which was reported with no toxicity in the functioning and homeostasis of major organs such as liver, kidney, colon, lung, heart, spleen and thymus to XN compared with control.²³² Furthermore, no variations were observed in the body weight or intake of food between the control mice and XN-treated mice. The content of hepatic glycogen such as CYP2E1 and CYP1A2 was also unaltered; however, CYP3A11 levels were reduced by nearly 30%.²³² Furthermore, a study in female SD rats administered with 0.5% XN in the diet or 1,000 mg XN/kg b.wt., resulted in low hepatotoxicity and less developed mammary glands. The treatment of XN with 100 mg/kg b.wt. per day for 28 days, before or during mating and nursing, did not induce any adverse side effects on female reproduction or offspring development.²³³ Additionally, in another study high dose of Hop extract containing XN, 8-PN and IXN, in menopausal women suggest that these compounds have no effect on sex hormones or blood clotting and do not induce acute toxicity, and are safe estrogenic and proestrogenic agents.²²⁷ In addition, another study with the combination of red clover and hops extract (RHEC) treated with different doses 125, 250, and 500 mg/kg, per os (p.o) for 12 weeks, was evaluated in ovariectomized rat to treat menopausal symptoms. It was demonstrated that RHEC was safe and effective in improving various markers of menopausal symptoms such as blood lipid profile, bone metabolism, the status of antioxidants and vasorelaxants.²³⁴ Thus, these studies identify XN as a safe and well-tolerated compound and can be a promising therapeutic agent for the treatment of various diseases.⁶³

12 ANALOGUES OF XN

Despite the potent anti-cancer activity of XN against various cancers, it is still a mandate to synthesize analogues of XN to improve the efficacy of the compound for cancer treatment. In line with this, Zhang et al., synthesized a series of 43 analogues of XN, out of which an analogue 13n (Figure 1c) exhibited highest efficacy against HeLa cells with the IC₅₀ (1.4 μM) 30 times more efficient than XN (40 μM). The compound 13n mechanistically inhibits thioredoxin reductase (TrxR), a component that regulates redox balance, cell survival, and proliferation. This inhibition leads to induction of ROS and apoptosis in HeLa cells.⁵⁸ Furthermore, in another study different analogues of XN were prepared and the compounds 11 (Figure 1d) and 12 (Figure 1e) were observed to protect the PC12 cells from hydrogen peroxide or 6-hydroxydopamine-induced injuries through the activation and nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and induction of antioxidants.⁵⁹ The structural derivatives of XN such as naringenin (Figure 1f), chalconaringenin (Figure 1g), and 4-hydroxy-4′-methoxychalcone (Figure 1h) also showed potential anti-microbial activities against Staphylococcus aureus and Rhodotorula rubra.⁶⁰ Furthermore, to evaluate the effect of the modifications of XN, new analogues were designed and synthesized by replacing phenolic group on B ring of the structure with different halogens, methoxy, and nitro groups. Thirteen new analogues of XN were synthesized and was tested for the formation of capillary-like structures, proliferation, migration and invasion in human umbilical-vein endothelial cells (HUVECs) at 10 μM concentration. Among these analogues, the compound number 13 (Figure 1i) with para-methoxy and fluorine groups on B ring exhibited the highest anti-angiogenic and anti-cancer activities.⁶¹ Another study reported the synthesis of two biotinylated derivatives of XN prepared through esterification and its anti-cancer activity was analyzed in different cancer cell lines. The highest efficacy was observed in MCF-7 and 4T1 breast cancer cells and the analogue compound with double biotinylated XN (Figure 1j) showed highest activity in MCF-7 and 4T1 breast cancer cells and HepG2 liver cancer cells, followed by the efficacy of other analogue, 4-O-biotinyl XN (Figure 1k) and original compound XN.⁶² Thus, XN and its analogues are effective compounds against cancer and the modification of the structure of XN paves the way to improve the efficacy of the compound in the prevention and treatment of cancer.

13 FORMULATIONS OF XN

Though there are several studies reported for the anti-cancer property of XN, its solubility minimizes the bioavailability and gastrointestinal absorption.¹⁰¹ For instance, the poor bioavailability of XN limits its activity against inflammation.²³⁵ In the same study, the investigation of anti-inflammatory activity of the micellar solubilized formulation of XN revealed that this formulation exhibited better improvement in arthritic models.²³⁵ Furthermore, the micellar solubilized XN induced better bioavailability and efficacy in a mouse model of obesity, diabetes and non-alcoholic fatty liver disease.²³⁶ Another study investigated the effect of PEGylated graphene oxide nanosheet formulation of XN (PEG-GO@XN) in MDA-MB-231 and MDA-MB-436 breast cancer cells. The PEG-GO@XN is reasonably stable and biocompatible and also selectively kill cancer cells.²³⁷ In this study, the PEG-GO@XN was shown to inhibit metastasis of breast cancer cells to lung by downregulating the TGF-β1-induced suppression of E-cadherin and upregulation of N-cadherin. PEG-GO@XN also inhibits metastasis of breast cancer cells by selectively targeting the OXPHOS and EMT.²³⁷ Therefore, development of novel more bioavailable formulations of XN has significant potential in the treatment of several diseases including cancer.

14 CONCLUSION

The herbs, plants, and spices are an ultimate source for many phytochemicals that have potential in the prevention and treatment of several diseases.^238-245 Hop is one of the vital plant used in brewing industry, which also has remarkable properties in the treatment of various diseases, including cancer. The principal compound, XN, is well proven as one of the most efficient phytochemicals with versatile activities isolated from the Hop plant. Studies have revealed the pleiotropic actions of XN on various molecular targets that have proven to obliterate different cancer types. XN exerted inhibition on cell survival, growth, proliferation, angiogenesis, invasion, and metastasis in different cancer cells. Furthermore, XN induced cell cycle arrest and apoptosis in the cancer cells. In addition, XN was reported to target various cell signaling pathways such as NF-κB, Akt, STAT3, Notch, ERK, and AMPK pathways by modulating several proteins like MMPs, cyclin D1, caspases, EMT markers, angiogenic markers, and tumor suppressor proteins. Furthermore, XN induced sensitivity in cancer cells to chemotherapeutic drugs and radiation. XN also modulates the expression of different miRNAs that lead to the regulation of different processes of cancer. Additionally, XN is also reported for its slow absorption and longer half-life and is safe and efficacious as observed in various in vitro and in vivo models. The compound XN was also observed to bind with the cytosolic proteins which limits its bioavailability; however, studies have shown the formulations of XN to overcome these drawbacks. Furthermore, the analogues of XN also exhibits similar anti-cancer activities in different cancer cell lines. Therefore, all the given studies suggest that XN is an important compound that could be used as an alternate strategy for the prevention and treatment of various cancers. Notably, most of the studies on XN till date are mostly conducted in in vitro and in vivo models. However, these studies have shown high potential of the compound in the prevention and treatment of different cancers as discussed. Thus, these studies have shown the importance of this compound against various malignancies and necessitates further preclinical and clinical investigation in future to explore the enormous therapeutic values.

ACKNOWLEDGMENTS

This work was supported by BT/556/NE/U-Excel/2016 awarded to Prof Ajaikumar B Kunnumakkara by Department of Biotechnology (DBT) Government of India and the HOD/01 fund awarded by the Department of Biotechnology (DBT), Government of India to the Department of Biosciences and Bioengineering (BSBE), Indian Institute of Technology Guwahati (IITG), India. This work was also supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2021R1I1A2060024).

CONFLICT OF INTEREST

The authors declare no conflict of interest.