Part2: Anticancer Activity Of Natural And Synthetic Chalcones

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3. Anticancer Activity

Chalcone derivatives act on various targets, such as aromatase, ATP binding cassette subfamily G member 2(ABCG2), breast cancer resistant protein (BCRP), activated nuclear B cell growth factor (NF-kB), vascular endothelial growth factor(VEGF), and tyrosine kinase receptors (epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET), showing important activities in vitro and in vivo in susceptible and therapy-resistant cancers [161,162]. An important mechanism of antiproliferative activity of chalcones is inhibition of tubulin and interference of these compounds with the assembly of microtubules, elements that are essential for maintaining the shape and function of cells in processes of mitosis and cell replication. Chalcones block the cell cycle and induce apoptosis. The presence of a trimethoxyphenyl residue in chalcone molecules is favorable for the irreversible antimitotic activity of these compounds, which have the ability to interact with cysteine residues in tubulin through a Michael-type addition reaction [163]. Additionally, the replacement of trimethoxyphenyl residue in chalcones with a quinoline or quinazoline is favorable for anti-tubulin activity. These heterocyclic chalcones form hydrogen bonds with the rest of Cys241 and strongly bind to the colchicine binding site (similar to combretastatin A-4, Figure7) [164,165].

3.1. Natural Chalcones with Anticancer Properties 3.1.1.Licochalcones (A-D)

Roots and rhizomes of some species of Glycyrrhiza are used in traditional medicine for the treatment of gastric ulcers, asthma, and inflammation. More than 600 compounds from licorice have been isolated, the main biologically active constituents being saponins and flavonoids. Among flavonoids, a series of retrochalcones, licochalcones A, B, C, D, E, and G, and schematize were identified. There are numerous studies on the biological effects of licorice active compounds, the most important being anti-inflammatory, antimicrobial, antioxidant, antiulcer, cytoprotective, and cytotoxic properties [166,167].

3.1.2. Licochalcone A

Licochalcone A (LA, Tables S1, and S2, Compound 1) is a flavonoid isolated from Glycyrrhiza urakensis, G.glabra, and G.inflata(Fabaceae). It has antitumor, anti-inflammatory, antimicrobial, antiparasitic, anti-obesity, antioxidant, and antiosteoporotic properties, Anticancer effect of LA has been demonstrated for different types of cancer cells, including gastric cancer cells BCG-823, HepG2, OVCAR-3, and SK-OV-3 (ovarian cancer cells) MCF-7, and A549 [168-171]. Studies show that LA induces apoptosis of U87 glioma cells, nasopharyngeal cancer cells, epithelial ovarian carcinoma cells, and bladder cancer cells. Chalcone also has the ability to increase autophagy and block the cell cycle in breast cancer cells. In addition, it induces apoptosis by suppressing specific protein 1 in breast cancer [172]. LA has low cytotoxicity on embryonic lung fibroblast cells. In addition, it has the ability to inhibit tumor growth and attenuate cis-platinum-induced toxicity[173]. Mechanisms by which flavonoids act as anticancer agents include inhibition of Akt activity by suppressing hexokinase-2-mediated tumor glycolysis in gastric cancer, downregulation of metalloproteinase 2 expression and induction of apoptosis in oral cancer cells, increased capacity of miR-144-3p to induce stress in the endoplasmic reticulum and induce apoptosis in human lung cells, reduced PI3K/Akt/mTor activation and decreased autophagy in breast cancer, block-ing G2/M phase of the cell cycle, repressing cell invasion by MEK/ERKand ADAM9signaling pathways in human glioma cells, and inducing caspase-dependent apoptosis in human liver cells [174]. Another mechanism by which LA exhibits a strong cytotoxic effect is ROS-induced apoptosis. For example, chalcone induces oxidative stress and, consequently, apoptosis of T24 cells (cell lines derived from human urinary bladder carcinoma) through mitochondrial-dependent pathways and by inducing oxidative processes in the endoplasmic reticulum [175]. Another study on cytotoxicity and genotoxicity of LA was developed by Bortolotto et al. LA was compared to trans-chalcone (Tables S1 and S2, compound 2). Cytotoxicity of natural chalcones (LA and trans-chalcone) on MCF-7 and 3T3 (embryonic fibroblast cell lines)was determined at 24 and 48 h. Results indicate marked cytotoxic activity after 48 h of treatment. A cytotoxicity MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed a dose-dependent response on MCF-7 cells treated with trans-chalcone and LA. For compounds analyzed, genotoxicity was found to be better on MCF-7 cells compared with 3T3 cells. DNA distortion causes the immune system to activate in order to eliminate destroyed cells. However, activating the immune system to reduce degraded DNA cells contributes to a chronic inflammatory process. Additionally, cellular response to degraded DNA can be corrected by inducing an intrinsic apoptotic pathway. G1 phase is a state that precedes DNA replication, in which factors such as cellular conditions (metabolism, signaling, and cell size) influence the progression of the cell cycle, causing DNA to rebuild in cells or initiating the process of apoptosis.IC50 of LA is 60.46 uM. Trans-chalcone induces cell cycle blockade in the G1 phase and intensifies cellular apoptosis. It has been proposed that treatment with LA and trans-chalcone induces apoptosis through the mitochondrial pathway, considering that induction of crucial genes of this pathway occurs after 24 h, such as protease activator factor 1 of protein apoptosis and protein X associated with Bcl-2. Although MCF-7 cell lines are known to have a caspase 3 deficiency, a study showed that treatment with these chalcones induces cleavage of poly (ADP-ribose) polymerase (PARP), a polymerase whose cleavage into two fragments is an indicator of apoptosis. Repression

of the Bcl-2 gene by LA and trans-chalcone in an experimental PCR analysis showed, at the protein level, a dose-dependent administration to MCF-7 cells and an intrinsic pathway mediation. Cyclin D1 is another protein suppressed on MCF-7 cell lines in presence of chalcones. This protein is crucial for the progression from G1 to the S phase and is an important biomarker for some cancers, including breast cancer. For these reasons, cyclin D1 degradation is an attractive target for the identification of new antitumor agents and is correlated with cell cycle blockade [176].

Qui et al. also examined the effect of LA on lung cancer cells in vitro. Flavonoid treatment significantly decreased viability of A549 and H460 cells(human non-small-cell lung carcinoma）， this change is strongly influenced by dose. A total of 40 μM of licochalcone suppresses the growth of lung cancer cells by 45-80% after 24 or 48 h of treatment. In addition, the compound exhibits low cytotoxicity on normal human lung epithelial cells. To highlight whether one of the mechanisms of inhibition of lung cancer cell growth by LA is cell cycle blockade, cell lines were treated with different concentrations of the compound for 16h then the cell cycle was analyzed by flow cytometry. Results show that, depending on dose, chalcone blocks the G2/M phase on A549 and H460 cells. Subsequently, the role of LAin inducing apoptosis was evaluated using an annexin/propidium iodide colorimetric method. Results show that there is an accumulation of apoptotic cells in the LA-treated group, which is dependent on the concentration used. In addition, levels of proteins associated with apoptosis were examined by the Western blot method. Levels of cleaved PARP and cleaved caspase 3 are elevated, and pre-caspase 3 antiapoptotic proteins, PARP, Bcl-xL, and Bcl-2 are decreased 20 h after chalcone treatment. These results suggest that LA-induced apoptosis is associated with PARP/Bcl-2 pathway[177]. Molecular models showed that LA was docked in ATP binding pockets of EGFR, including exon 19 deletion mutation, L858R single-site mutation, L858R/T790M double mutation, and wild-type. In L858R/790M mutations, LA has the ability to interact with Lys745 through cation-II interaction. Hydrogen bonds are formed between WT EGFRand licochalcone on Met793, Lvs745, and Asp 855. Exon 19 deletion has the ability to change the shape of the pocket in which interaction with licochalcone is considered to occur, forming hydrogen bonds with Met793, Thr790, and Glu762. Data obtained by in silico analyses of LA are an important point for the identification of new, selective EGFR inhibitors on different types of mutations [178].

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3.1.3. Licochalcone B

Anticancer effects of licochalcone B (LB, Tables S1, and S2, compound 3) were demonstrated by analysis on different cell lines, including human cells from bladder cancer T24 and EJ, oral cancer cells HN22 and HSC4, MCF-7, A375(a human melanoma cell line), and A431(squamous cell carcinoma). Studies have shown that LB influences the growth of cancer cells, inhibits the formation of metastases, blocks the cell cycle, and induces apoptosis[161]. Kang et al.investigated the molecular mechanism by which LB induces apoptosis in human melanoma and cells in squamous cell carcinomas. Licochalcone has been shown to induce apoptosis of A375 and A431 cells by both intrinsic and extrinsic pathways. In case of testing the antiproliferative effect of chalcone with trypan blue staining, it was observed that LB induces a significant decrease in cell viability, this decrease is correlated with con-centration After the addition of LB, significant changes in cell characteristics were observed, including cell contraction, rupture of cell membranes, and an increase in the percentage of fragmented nucleus cells. Increased percentages of pre-G1-phase cells and apoptotic cells were also noted [38]. Another study showed that LB significantly blocks cell cycle in the G2/M phase in the case of HepG2-type cancer cell lines, and in the case of bladder and breast tumor cells, compound blocks S phase [179]. Song et al.highlighted the suppressive effect of LB on the growth of JAK2-type esophageal carcinoma squamous cells. Docking studies were performed with Autodock Vina software, which was used to predict binding mode. The structure of the JAK2 receptor with inhibitory potential is available in Protein Data Bank(PDB entry 2B7A, residues 840-1.132). IAK2 plays an important role, being an intracellular mediator of cytokine signaling, and is a tyrosine kinase protein of the JAK family. ATP binds strongly to

magnesium ions in the catalytic domain of tyrosine kinases. In the docking parameter, the size of investigated space includes ATP binding site characterized by residues 855-863 and 822, where ATP was calculated with various potential JAK2 inhibitors. In predictions made, the LB ligand was made with Marvin Sketch software. After docking, the best three possible binding variants were collected, which have similar affinities. Conclusions of predictions were favorable regarding the interaction of LB with ATP binding pocket at JAK2[180,181].

3.1.4.Licochalcone C

Licochalcone C(LC, Tables S1, and S2, compound 4)is known to decrease inflammatory response on monocyte cell lines. This is due to a reduction in iNOS expression and restoration of antioxidant network activity of superoxide dismutase, catalase, and glutathione peroxidase. Kwak et al. conducted a study to describe the relationship between ROS, c-Jun NH2 terminal kinase(INK), and p38mitogen-activated protein kinase(MAPK) and established the impact of LC in inducing apoptosis on KYSE 30 and KYSE450 esophageal cancer cell lines. Previous studies have determined IC50 values for LC treatment(45 ug/mL) after 24 h to inhibit proliferation of A549, MCF-7, and T24 cell lines. Inhibitions of 40,47 and 68% were obtained for three cell lines. Kwak et al. obtained a dose- and time-dependent in vitro inhibition of esophageal cancer cell proliferation. From the five cell types analyzed, KYSE30 and KYSE450, which have common genetic support, had a similar response to LC treatment. In an analysis of anchorage-independent growth in soft agar, results indicate a significant reduction in the ability of KYSE30 and KYSE450 cells to form colonies. Depending on the concentration, chalcones induced apoptosis in both cell lines. The compound also induced an upward regulation of p24 and p27 (negative transition regulators in G1 and S phases of the cell cycle) and regulated downstream cyclin D. LC also increased ROS generation in KYSE30 and KYSE450 cells. ROS activate the mitogen-activated protein kinase (MAPK) pathway and induce cell apoptosis. In addition, the compound increased the level of JNK, c-Jun, and p38 phosphorylation and activated apoptotic pathways [182]. Similar to the docking study by Song et al. for LB[180], Oh et al.highlighted binding interactions between LC and human JAK2 cells. Docking simulation was performed using Autodock Vina. To initiate the docking study, the structure of the JAK2 receptor, which was solvated by an X-ray experiment, was obtained from the Protein Data Bank(PDB entry 2B7A). The structure of the LC ligand was modeled by Marvin Sketch software and optimized by Chimera software. The catalytic site of JAK2 was correlated with a hinge region (residues 929-935), a DFGloop (residues 994-996), and a Ploop (residues 858-865). The loop-shaped hinge region is essential for the recognition of ATP and forms hydrogen bonds with substances. DFGloop contains three amino acids (aspartic acid, phenylalanine, and glycine) and is associated with the binding of a metal required for catalytic phosphorylation. Ploop is useful for stabilizing and forming interactions with ligands. As can be seen, the prediction of a possible binding was made in three functional places. Docking's study showed that LC interacts with ATP binding site toJAK2 and indicated that JAK2 is a direct target of it. Chalcone also suppressed JAK2 autophosphorylation by binding to the ATP pocket of p-JAK2 [183,184].

3.1.5. Licochalcone D

Licochalcone D(LD, Tables S1, and S2, compound 5) is an active flavonoid isolated from Glycyrrhiza inflata. A study was performed to evaluate the ability of LD to inhibit cell proliferation by two targets for lung cancer cells (EGFR and MET)using sensitive and gefitinib-resistant human cells. To understand the direct binding of chalcone to EGFR and MET, gefitinib-sensitive cell lines (HCC827) and gefitinib-resistant cell lines (HCC827GR)were used. Results of evaluations show that flavonoid binds to two receptors, suppressing the activity of EGFR and MET kinases as a competitive inhibitor of ATP. In the EGFR complex, chalcone has two hydrogen bonds formed by Met793 as the main point and lateral corner of Asp855 in the DFG loop.4-Hydroxy-3-(3-methyl but-2-enyl)phenyl group and 3,4-dihydroxy-2-methoxyphenyl group are fixed on the same plane and blocked between hydrophobic residues Leu718, Val726, and Ala743 of P loop and Leu 844. In the Met complex, the keto

group of the chalcone forms a hydrogen bond with Met1160. Tyr1159 as the main point and le1084, Vall092, Ala1108, and Lys1110 of Ploop were covered similarly with a cap. Luis is also strongly supported by side hydrophobic chains of Met1160's main point and Leu1140, Met1211, and Ala1221 of lower ATP pocket.EGFR binding position closely resembles MET binding position, forming hydrogen bonds and hydrophobic interaction. Chalcone is located identically in the binding region for the two receptors. Stabilization of complex can be increased by hydrophobic interaction. Predicted results were compared with experimental data, showing that flavonoid competitively inhibits the two receptors [185].

3.1.6. Xanthohumol

Prenyl chalcones, due to their structural diversity, have different biological properties, including anti-inflammatory, anticancer, and antimutagenic activities [186]. Studies have shown that natural chalcones with prenyl groups have the potential to interfere with p53. For example, treatment of A549 cells with prenyl chalcone xanthohumol (XN, Tables S1, and S2, compound 6) induces apoptotic cell death and blocks cell cycle in the G1 phase. These activities are due to the upregulation of p53 and p21 from the cell cycle and the downregulation of cyclin D1. Apoptosis is induced by the activation of caspase 3 [187].

XN((3'-(3,3-dimethylallyl)-2',A',4-trihydroxy-6'methoxychalcone) is the most abundant prenylated flavonoid(0.1-1% by dry weight) of female hop inflorescences (Humulus lupulus)[180]. XN is also a constituent of beer, a major dietary source of prenylated flavonoids, where it is present in concentrations above 0.96 mg/L.Due to its unique biological activities and its favorable impact on health, prenylchalcone has been widely studied recently [188]. The compound has therapeutic safety and various bioactivities, including anti-cancer, anti-diabetic, anti-inflammatory, antioxidant, and antibacterial properties. In recent years, an increased number of studies have demonstrated a broad spectrum of anticancer activity of XN in lung cancer, hepatocellular carcinoma, breast cancer, leukemia, prostate cancer, pancreatic cancer, colon cancer, pancreatic cancer, and glioblastoma cancer. Exposure of cancer cells to XN inhibits their proliferation, migration, and invasion and modulates autophagy. Chalcone also has the ability to induce apoptosis and block the cell cycle [189-193]. In addition, chalcone induces apoptosis dependent on and independently of caspase activity and inhibits cancer cell invasion and angiogenesis [194]. Its anti-inflammatory, antioxidant, and anticancer properties are correlated with the chemopreventive effect of the compound [195]. Prenylchalcone is also metabolized to 8-prenylnaringenin, the most potent phytoestrogen known to date [196].

Akt (also called protein kinase B or PKB) is a specific serine/threonine-protein kinase and an important point in cellular signaling pathways. Akt activity is altered in many types of cancers and involved in various biological processes, including cell proliferation, apoptosis, transcription, migration, and invasion. To confirm XN's ability to bind to Akt, an in silico docking study was performed using Schrodinger Suite 2015 software. Water molecules were removed, and the pH for hydrogen atoms considered was 7. An ATP bind-ing site was generated for the docking study. XN was prepared for docking in absence of parameters using the LigPrep program. Afterward, docking studies of XN with Aktl and Akt2 were accompanied by absent parameters using the method of additional precision with the Glide program in order to obtain the best structural representations. Results of the docking study show that XN forms hydrogen bonds with Ala230, Glu228, Glu234, and Lys158 of Akt1 and with Glu236, Thr213, and Lvs181 of Akt2. Xenograft (PDX) models were identified for translating basic research studies into clinical applications. To an increased extent, biological and genetic characteristics of donor patients are considered to be preserved by PDX models, which is the major advantage over cell-line-based models. PDX models were used to analyze biomarkers and to predict response to XN therapy in clinical trials. Chemopreventive effects of prenylchalcone were compared based on Akt levels. Results show that tumor models expressing a high level of Akt have a significant decrease in tumor volume and weight when treated with XN [197]. Guo et al. studied in vitro and in vivo the effect of XN in gastric cancer, showing that prenylchalcone induces apoptosis by activating caspases, regulating Bcl-2, and influencing PI3K/Akt/mTOR kinase. XN inhibits the viability of gastric cancer cells in a concentration-dependent manner. On cell lines, flavonoid exerts the best effect on the viability of SGC-7901 cells and does not influence this parameter on GES-1 cells at 6， 8， and 10 μg/mL of chalcone. From flow cytometric analysis， it was observed that prenylchalone significantly increases the number of apoptotic cells in gastric cancer. The effect of XN on pro-and anti-apoptotic proteins was highlighted by Western blot analysis. Bcl-2 and Bcl-XL protein levels decreased after flavonoid administration, this decrease is correlated with administered concentration. XN also increased Bax and Bid protein levels, with the best activity being observed for 10 uM/mL of chalcone. In addition, levels of cleaved caspase 3 and cleaved PARP protein increased significantly in presence of chalcone. For these reasons, it can be stated that flavonoids favorably and significantly influence levels of pro-and anti-apoptotic proteins. A total of 10 uM/mL of XN induces significant apoptosis of SGC-7901 cells. A total of 8 and 6 uM/mL of chalcone induces apoptosis of 34±3% cells and 23±2% cells, respectively. Additionally, flavonoid significantly modifies phosphorylation of PI3K, Akt, and mTOR increases the level of p-PTEM and decreases the level of p-Akt (Thr308), p-Akt (Ser473), and m-Tor (Ser2448). The resulting data indicate that prenylchalcone does not significantly influence Akt, PTEN, GSK-3β, and mTOR levels. Determinations in SGC7901xenograft mice showed that XN treatment decreased tumor volume in a relatively concentration-dependent manner. To confirm suppression of PI3K/Akt signaling in vivo, phosphorylated Akt and mTOR expression on xenographic tumors was evaluated. Pathological examination of hematoxylin and eosin sections revealed significant morphological abnormalities. However, XN reduces concentration-dependent phosphorylated Akt and mTOR levels. Prenylchalcone treatment significantly decreased cell proliferation and increased tumor cell apoptosis compared with control cells [198].

XN, at concentrations higher than 10 umol/L, inhibits the proliferation of pancreatic cancer cells in vitro. At concentrations lower than 5 umol/L, chalcone inhibits NF-kB-dependent angiogenic activity in pancreatic cancer cells. At this concentration, no cytotoxicity was observed on pancreatic cells by WST-1 method. However, the conclusion of the study was that XN influences pancreatic-cancer-induced angiogenesis by downregulating the production of VEGF and IL-8 (an interleukin), which is specific and mediated by NF-kB inactivation [194].To evaluate XN's anticancer activity, HepG2 cell lines were subjected to MTT analysis to determine cell proliferation. Prenylchalcone reduced cell proliferation depending on concentration and time. Zhao et al. observed that exposing cell lines to 200 μM of XN for one day is less effective compared with treating them with 100-200 μM of chalcone for 2-3 days. At 50 uM of chalcone, significant inhibition of HepG2 cell proliferation was observed after 3 days. In the same study, it was shown that prenylchalcone causes a significant increase in caspase 3 activity. Additionally, by Western blot analysis, it was shown that 100-150 uM of XN significantly inhibited the expression of NF-kBprotein on cell lines. By this analysis, it was also observed that prenylchalcone has the capacity to increase the expression of p53 protein, and 20 uM of XN determined an intensification of Bax signaling, this being correlated with time [199]. Studies of the safety profile of XN show that 1000 mg/kg of the compound does not alter the functioning of vital organs and homeostasis in mice. Prenylchalcone has the ability to increase IL-2 production in T cells, which demonstrates its ability to promote a The-mediated immune response. XN also inhibits -12. which indirectly produces differentiation of The cells in the immune system by activating transcription molecules. CytotoxicTlymphocytes are a type of cellular effector crucial to cellular immunity and play an important role in process of antitumor immunology. CD8+T cytotoxic lymphocytes exist as CTL-P, an inactive cell precursor in vivo. This precursor is activated by antigen in presence of Th1 cytokines and then develops into mature cytotoxic T lymphocytes. A significant increase in CD8+/CD25+ was demonstrated, followed by the transition of Th2 to Th1 in tumor microclimate. CD8+/CD25* ratio of T cells is greatly increased when cytotoxic T lymphocytes are activated by CoCl2 on 4T1 cell lines. The function of Th1 and Th2 cells is dependent on the secretion of various cytokines. To investigate the effects of XN on Th1 and Th2 cytokines, Zhang et al. determined serum levels of Th1l and Th2-associated cytokines using ELISA kits. Prenyl derivative has been shown to significantly increase Th1 cytokine expression (including IL-2 and IFN-y) and decrease Th2 cytokine levels (including IL-4 and IL-10). This conclusion is explained by fact that Th1 and Th2 are mutual inhibitors. In addition, the Th1/Th2 ratio was determined by flow cytometry, showing that it is significantly increased by XN. Similar studies have reported this finding for various tumors. Patients with advanced squamous cell carcinomas of the neck and head have low levels of Th1 cytokines compared with patients who are less severe and have high levels of Th2 cytokines. Combination therapy produces the transition of Th2 to Th1 cytokines in the tumor environment. Results of studies indicate a disorder of Th1/Th2 cytokine ratio, this change being observed for several types of tumors, most frequently in terminal stages of cancer. To confirm the potential mechanism of XN on the Thl/'Th2 cytokine ratio, the expression of key factors in the pathway of Th1 and Th2 differentiation was determined. Physiologically, Th0 cells are proportionally differentiated into Thl and Th2 cells. In addition, activation of transcriptional molecules 4 and 6 play a vital role in differentiating Th0 to Th1 and Th2 cells. T-bet and GATA-3 also play two pivotal roles. CpG-ODN(cytosine-phosphorothioate-guanine containing an oligodeoxynucleotide), a potent Th1 adjuvant, decreases GATA-3 expression and activation of transcriptional molecule 6 by activating T-bet and transcriptional molecules 1 and 4 in lung cancer models. XN increases T-bet expression and decreases GATA-3 expression. Activation of transcriptional molecule 4 is increased in the presence of XN, but it does not influence the activation of transcriptional molecule 6. For this reason, it can be stated that activation of transcriptional molecule 4 plays a positive role in regulating the Th1/Th2 cytokine ratio by XN [200].

The notch signaling pathway plays a significant role in breast cancer, which is a therapeutic target for its treatment. It is involved in the initiation and progression of breast cancer, the aberrant activity of this pathway being associated with this pathology. Inhibition of Notch signaling pathway by gamma-secretase inhibitors and by anti-delta-like monoclonal antibody 4 is favorable for treatment of acute lymphoblastic leukemia and solid tumors. Mechanisms of these agents include cell cycle blockage or apoptosis and disruption of angiogenesis. Sun et al. investigated the therapeutic potential of XN on breast cancer cell lines, highlighting its ability to inhibit cell proliferation, block cell cycle, and induce apoptosis in vitro. A reduction in tumor growth in vivo was also determined. In addition, the possibility of prenylchalcone to inhibit the growth of human breast cancer cells by the Notch signaling pathway has been investigated. To determine whether XN targets the Notch signaling pathway, a functionalized Notch 1 method was used, using a gamma-secretase inhibitor (DAPT)as a control. The aim of the study was to evaluate the possibility that prenylchalcone reduces the binding activity of Notch1 to CBF1 transgene. XN has been shown to inhibit proliferation and induce apoptosis by inhibiting the Notch 1 pathway. Additionally, by the MTT method and light microscopy, it was shown that prenylchalcone inhibits cell proliferation on breast cancer cell lines. Previous studies have shown that Notch pathway inhibitors are also inhibitors of EGFR expression, another incriminating element in breast cancer. In addition, XN acts on proteins associated with tumor metastases and inhibits cell migration by increasing the expression of these proteins. A study highlighted the blockade of the cell cycle in the G0/G1 phase and induction of apoptosis for MCF-7 and MDA-MB-231 cells by XN [201].

3.1.7.Panduretin A

Anticancer activity of pandurate A (PA, Tables S1, and S2, compound 7), a cyclohexanylchalcone isolated from Boesenbergia pandurate, has been studied. The plant contains prenyl chalcones and other flavonoids as major bioactive molecules, which are described in the literature to have preferential cytotoxic properties on human pancreatic cell line PANC-1. [202,203] PA is active in melanoma, colon adenocarcinoma, and prostate cancer. Proteomic analyses show that PA has cytotoxicity on melanoma cells that is dependent on denaturation of mitochondrial oxidative phosphorylation process, with the activity of secretory pathway and apoptosis induced by oxidative processes. In this regard, it has been shown that oxidative stress can be the result of stimulating autophagy as a secondary response to elevated ROS [204]. Literature indicates that a concentration of 9 ug/mL of PA completely inhibits the growth of MCF-7 cells and HT-29 cells (a human colon cancer cell line)[205]. The chalcone has anticancer properties on various cell types, including melanoma, colon adenocarcinoma, and prostate cancer [204]. Liu et al. highlighted the cytotoxic effect of the chalcone on MCF-7, T47D (human breast cancer), and MCF-10A (non-tumor breast cells) cell lines. The IC50 values of PA on MCF-7 cells were 15 uM at24h and 11.5 μM at 48 h.In the case of T47D cells， the IC50 was 17.5 μM at 24h and 14.5 μM at 48 h. PA does not influence the proliferation of MCF-10A cells. To identify mechanisms by which the chalcone induces cell cycle blockade in MCF-7 cells in the GO/G1 phase, Western blot analysis was used, which aimed to evaluate the modulation of regulatory proteins in the cell cycle. Results show that PA treatment induces a decrease in cyclin D1 and CDK4expression and increases p21Cip1 and p27 expression, thus explaining the blockade in the G0/Gl phase. Isolated PA from Kaempferia pandurate induces cell cycle blockade in androgen-independent PC-3 (prostate adenocarcinoma) cells and in human DU145 (a human prostate cancer cell line)cells. Internucleosomal DNA fragmentation is a marker of apoptosis. Because low-molecular-weight DNA fragments are extracted by staining cells in aqueous solutions, apoptotic cells can be identified by frequency histograms of DNA content in the form of cells with fractionated DNA content. A sub-G1 phase MCF-7 cell population was analyzed. The G1 phase content of cells was 1.17 ±0.11 and in cells treated with PA（10，15， and 20 μM）it was 1.84±0.18，2.62±0.21， and 4.52±0.28， respectively. The increase in the chalcone treatment was due to the intensification of DNA fragmentation on MCF-7 lines, a fact confirmed by the sub-G1 phase cell population [206].

Among the key proteins of cancer cell invasion and metastasis, induction is matrix metalloproteinases. They degrade components of the extracellular matrix and facilitate the invasion and migration of cells. In addition, overexpression of metalloproteinases may induce epithelial-mesenchymal transition. PA suppresses the secretion and activation of metalloproteinase 2, causing inhibition of endothelial cell migration, invasion, and morphogenesis on human umbilical vein endothelial cell (HUVEC)cells. In addition, subtoxic doses of chalcones are sufficient to downregulate metalloproteinase 2 in lung cancer cells [207].

3.1.8. Cardamonin

Cardamon(CD, Tables S1, and S2, compound 8), a chalcone from Campomanesia adamantium(Myrtaceae), increases DNA fragmentation and decreases NF-kB activity in PC-3 cells. These results indicate the therapeutic potential of chalcone in the treatment of prostate cancer [20]. CD is considered one of the most active antitumor compounds in which activation of Epstein-Barr virus is involved [208]. Anticancer effects of CD are correlated with induction of apoptosis, inhibition of cell proliferation and migration, and an influence on the cell cycle. The chalcone also has the ability to reduce the resistance of cancer cells to therapy. In combination with 5-fluorouracil or cis-platinum, increased antitumor activities are obtained. For example, the CD has the ability to significantly inhibit the resistance to chemotherapy of colon cancer cells, induces apoptosis, activates caspases 3 and 9, facilitates Bax protein expression, significantly inhibits c-myc, and carries specific 50 and NF-kB [209]. Hou et al. investigated the therapeutic potential and molecular mechanisms of CD on 5-fluorouracil-resistant gastric cancer cells. The sensitivity of BGC-823/5-fluorouracil to 5-fluorouracil was confirmed by increasing apoptosis and blocking the cell cycle in the presence of CD. The chalcone increases the sensitivity of cancer cells to 5-fluorouracil by suppressing the Wnt/β-catenin signaling pathway (which plays a significant role in tumorigenesis), and activated mutations in Wnt/β-catenin genes are associated with resistance to anticancer therapy. It inhibits the expression of P-glycoprotein, β-catenin, and TCF-4. Additionally, CDspecifically blocks the formation of the theβ-catenin/TCF-4 complex, thus causing aberrant Wnt/β-catenin signaling [210]. Bedroom et al. investigated the antiproliferative and apoptotic effects of CD on HepG2 cells. The inhibitory action of

the chalcone on HepG2 cell proliferation was significant after 72 h, the cytotoxicity being similar to that of 5-fluorouracil. Values determined for other chemotherapeutic agents used as standards (e.g., sorafenib)were much lower. In addition, the cytotoxic effect of the compound is selective on tumor cells and does not negatively influence normal cells, which is an advantage of CD compared with 5-fluorouracil. Accumulation of CD in the G1 phase of the cell cycle was observed after 72 h and indicates inhibition of HepG2 cell growth by preventing cell division [211].

Comparative docking studies of CD and5-fluorouracil and its interaction with BaxBH3 show that 5-fluorouracil has higher binding energy than a CD. The chalcone forms three hydrogen bonds (Phe30, Val50, and Gln52). Interaction between CD and Bcl is achieved by three hydrogen bonds (Asp15, Gln18, and Ser28), and in the case of 5-fluorouracil is achieved by four bonds. Additionally, in the case of this interaction, the binding energy of CD is lower than in the case of 5-fluorouracil. This can be attributed to aromatic residues in the structure of the chalcone, which are involved in II bonds, which have the ability to stabilize the active pocket and cause a decrease in the binding energy. Results of in silico studies show that 5-fluorouracil has higher binding energies than caspase 3 compared with CD.CDshows two hydrogen bonds in the interaction with caspase 3(Cys163 and Arg64). The chalcone also has II-II bonds with TYR204. The free binding energy of 5-fluorouracil is superior to that of CD, which is explained by the stabilization of the active pocket by two aromatic residues in the structure of the chalcone [212].

3.1.9. Lonchocarpin

Lonchocarpin (Tables S1 and S2, compound 9) is a natural chalcone extracted from Lonchiocarpus sericeus. Cytotoxic effects of this chalcone have been described on neuroblastoma and leukemia cell lines. It is known that 24h after treatment with 50 uM lonchocarpin on SK-N-SH neuroblastoma lines, induction of AMPK phosphorylation takes place, which increases glucose absorption and inhibits protein synthesis. The chalcone also has the ability to decrease cell viability. On colorectal cancer cell lines HCT116, SW480, and DLD, lonchocarpin reduces cell viability by 20μM. Studies show that lonchocarpin has the ability to inhibit H292 lung cancer cells in vitro by the caspase-3-induced cell death that precedes apoptosis. In addition, lonchocarpin has been observed to inhibit Wnt/β-catenin signaling in vivo in embryonic models of Xenopus laevis. The chalcone's injection into a co-injected Wnt8-specific receptor model(SO1234)resulted in 82% suppression of Wnt/β-catenin signaling receptor gene activation [213].

In a study by Chen et al., results of the 3D-QSAR analysis indicate a hydrophobic C-4, C-5, C-11, C-1/, and C-2 interaction in lonchocarpin. This interaction increases the cytotoxic capacity of this compound, having a contribution of 23% in the model. Docking studies for lonchocarpin yielded the same results as the hydrophobic 3D-QSAR model, with the hydrophobic surface in the C-4, C-5, C-11, C-1', and C-2'regions of loncocarpine interacting with the Bcl-2 complex. The hydrophobic loop of the Bcl-2 protein forms a complex with the BaxBH3 peptide, which can be interrupted by synthetic navitoclax or lonchocarpin compounds. This shows that the hydrophobic loop of Bcl-2 family members is a target for loncocarpine-induced apoptosis on H292 cells and thus for activation of caspase 3 [214].

Other natural chalcones with anticancer properties are butein(Tables S1 and S2 compound 10), isoliquiritigenin (Tables S1 and S2, compound 11), flavokawain (Tables S1 and S2, compound 12), and isobavachalcone(Tables S1 and S2, compound 13)[155].

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