Perspectives on the Role of Isoliquiritigenin in Cancer.

ISL is a flavonoid with a simple chalcone structure. The structure of ISL and its metabolites are shown in Figure 1 . The previous studies demonstrated the six metabolites detected in phase I [ 5 , 6 , 7 ], including liquritigenin (M1), 2′,4,4′,5′-tetrahydroxychalcone (M2), sulfuretin (M3), butein (M4), davidigenin (M5), and cis -6,4′-dihydroxyaurone (M6). Among the six metabolites, butein is the more active metabolite in the liver and in HT22 cells, with significant distribution on M1, M3, and M4 ( Figure 1 ) [ 5 , 6 , 8 ]. Moreover, the previous study reported that the dominant metabolites of ISL are THC (2,4,2′,4′-tetrahydroxychalcone) and naringenin chalcone in lung cells [ 9 ]. In vivo absorption of ISL occurs in the intestines, transported to the liver for phase II biotransformation [ 7 ]. In phase II metabolism, liquiritigenin, glucuronidated ISL, glucuronidated liquiritigenin, and glucuronidated ISL are produced. Only glucuronidated liquiritigenin is predominant [ 10 ]. Many studies have suggested that secondary metabolites are involved in different biological activities and pharmaceuticals [ 5 , 7 , 8 , 11 ]. Therefore, these metabolites may differ in various cell lines or organs; however, they all share a similar structure to that of chalcone, which contains two aromatic rings connected by an unsaturated carbon chain, resulting in interconnected biological activities. Evaluation of the safety of ISL is necessary for future clinical applications. Therefore, many studies, through different routes of administrations, including intravenously (IV), via hypodermic (IH) or intraperitoneal (IP) injection, and orally, have indicated that ISL exhibits a robust absorption capacity (absorption rate: ~60–90 min; oral absorption: >90%) with a strong elimination ability (t 1/2 : 2–4.9 h) [ 10 , 12 , 13 , 14 ]. Moreover, the data showed similar trends among different analytic methods, including high-performance liquid chromatography (HPLC), HPLC–MS/MS, and fluorescence spectrometry (SFS) [ 10 , 12 , 13 ]. This means that the absorption of ISL is quickly and widely distributed throughout the body [ 10 , 12 , 13 , 14 ]. Concentrations of ISL may vary in different tissues, including the heart, liver, lungs, spleen, kidneys, brain, muscles, and fat. ISL distribution mainly relies on the blood circulation, with the brain showing the lowest level of ISL due to the blood–brain barrier (BBB). These results imply that ISL is able to penetrate the BBB and exhibits neuroprotective activity in a male middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia rat model and high fat diet (HFD)-induced ICR mice model [ 15 , 16 ]. Interestingly, only after oral administration does [ISL] plasma exhibit a double-peak of ISL [ 14 , 17 , 18 , 19 ], the possible mechanism for which has been proposed as enterohepatic recycling. As a matter of fact, oral administration has become the most advanced application route. Generally speaking, poor bioavailability, rapid degradation, fast metabolism, and systemic elimination are the essential factors that lead to insufficient bioavailability. Insufficient bioavailability of ISL means that its efficacy is far less than 20% [ 10 , 14 ]. The term insufficient bioavailability implies that patients show intolerance to bulk administration of ISL to reach the desired effect, thereby highlighting the need to improve its effectiveness. To improve solubility, enhancing its bioavailability and distribution, encapsulated ISL nanoparticles or nano-ISL have been developed. Below, we summarize various ISL nanoparticles applied in preclinical studies, for example, polymer nanoparticles, liposomes, micelles, solid lipid nanoparticles (SLNs), and polymer conjugates. Nanosuspension: ISL is milled with HPC (hydroxypropyl cellulose) SSL and PVP (polyvinylpyrrolidone) K30 to form a lamelliform or ellipse shape of the nanosuspension. HPC SSL and PVP K30 act as stabilizer. These two nanosuspension particles (size: 238.1 ± 4.9 nm with SSL; 354.1 ± 9.1 nm with K30) do not only improve the solubility issue, but also enhance the cytotoxicity a 7.5–10-fold [ 20 ]. Nanoencapsulation: Mesoporous silica nanoparticles (MSNs) are a solid material, acting as a biodegradable nanoscale drug carrier. When MSNs are encapsulated with ISL, they improve the efficacy of ISL in vitro and in vivo [ 21 ]. Lipid–polymer hybrid nanoparticle system: 3.1. iRGD hybrid NPs: The composition of lipid–polymer hybrid nanoparticles (NPs) include lactic-co-glycolic acid (PLGA), lecithin, and a hydrophilic poly-ethylene-glycol (PEG). ISL-loaded hybrid NPs are composed of an inner PLGA core with an outer lipid layer (PEG, lecithin, and iRGD peptides). iRGD peptides (CRGDK/RGPD/EC, a tumor-homing peptides), can deliver drugs to a tumor. In vitro, ISL–iRGD NPs show stronger inhibition effects and induce apoptosis effects. In vivo, ISL–iRGD NPs show stronger effects in the viability of tumor cells. Herein, iRGD-modified lipid–polymer NPs showed better solubility, bioavailability, and targeting distribution [ 22 ]. 3.2. Hydrophilic polyanion solid lipid nanoparticles (SLNs): SLNs are composed of natural lipids such as lecithin or triglycerides that remain solid at 37 °C. SLNs can protect labile compounds from chemical degradation and can improve bioavailability. Low-molecular-weight heparins (LMWHs) are fragments of heparin showing hydrophilic polyanions that can improve the efficacy of ISL [ 23 ]. iRGD hybrid NPs: The composition of lipid–polymer hybrid nanoparticles (NPs) include lactic-co-glycolic acid (PLGA), lecithin, and a hydrophilic poly-ethylene-glycol (PEG). ISL-loaded hybrid NPs are composed of an inner PLGA core with an outer lipid layer (PEG, lecithin, and iRGD peptides). iRGD peptides (CRGDK/RGPD/EC, a tumor-homing peptides), can deliver drugs to a tumor. In vitro, ISL–iRGD NPs show stronger inhibition effects and induce apoptosis effects. In vivo, ISL–iRGD NPs show stronger effects in the viability of tumor cells. Herein, iRGD-modified lipid–polymer NPs showed better solubility, bioavailability, and targeting distribution [ 22 ]. Hydrophilic polyanion solid lipid nanoparticles (SLNs): SLNs are composed of natural lipids such as lecithin or triglycerides that remain solid at 37 °C. SLNs can protect labile compounds from chemical degradation and can improve bioavailability. Low-molecular-weight heparins (LMWHs) are fragments of heparin showing hydrophilic polyanions that can improve the efficacy of ISL [ 23 ]. Microemulsion: The self-microemulsifying drug delivery system (SEMDDS) was designed for improving the solubility, absorption, and bioavailability of lipophilic drugs. The SMEDDS comprises ethyl oleate (EO; oil phase), Tween 80 (surfactant), and PEG 400 (co-surfactant). ISL-loaded SMEDDS has been proven to improve the solubility and oral in vivo availability [ 17 ]. ISL-loaded nanostructured lipid carriers (ISL-NLCs): NLCs mix solid lipids with spatially incompatible liquid lipids, which leads to a special nanostructure with improved properties for drug loading. ISL-loaded NLCs are constructed by glycerol monostearate (MS) and Mi-glyol-812 as the solid and liquid lipid materials to carry the ISL [ 24 ]. In pharmacokinetic studies, less than 10% of the NLCs remains in the stomach after oral administration, mainly absorbed in the colon [ 19 ]. Moreover, the antitumor effect of ISL-loaded NLCs has been evaluated in sarcoma 180 (S180)-bearing and murine hepatoma (H22)-bearing mice models via IP administration [ 24 ]. A biodistribution study showed that the ISL concentration of ISL-loaded NLCs in the tumor is higher 2.5-fold than free ISL. In a skin permeability study, the previous study suggested NLCs as a promising carrier to deliver the ISL [ 25 ]. TPGS-modified proliposomes: D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) has been selected as an excipient for ISL-loaded TPGS-modified proliposomes (ISL-TPGS-PLP), prepared using the film dispersion method with ISL-loaded proliposomes (ISL–PLP). ISL-TPGS-PLP can enhance the solubility, bioavailability and liver-targeting ability of ISL [ 18 ]. Polymeric micelles: PEO (polyethylene oxide)–PPO (polypropylene oxide)–PEO (polyethylene oxide) triblock copolymers are highly biocompatible and act as surface-active agents. P123 (PEO20–PPO65–PEO20) can remarkably enhance the retention of poorly soluble drugs in the blood circulation. Another important derivative of Pluronic, F127 (PEO100–PPO69–PEO100), possesses high biocompatibility. Therefore, mixed F127/P123 polymeric micelles have been developed, which have remarkably enhanced bioavailability with high encapsulation efficiency and low particle size. ISL-loaded F127/P123 polymeric micelles (ISL-FPM) improve the solubility as well as enhance the bioavailability and antioxidant activity of ISL [ 26 ]. Nanoliposomes (NLs): Drug-loaded PEGylated nanomaterials have shown effective cancer cell-killing ability, PEG2000-DPSE-QUE-NLs (polyethyleneglycol-2000-distearoyl phosphatidyl ethanolamine loaded with querce-tin (QUE)) can efficiently disperse in aqueous media compared to controls, and PEGylated (PEG2000-DPSE) NLs have been found to be effective drug delivery vehicles when simply loaded with ISL. ISL-NLs as tumor-targeted drug carriers are more effective in regulating glycolysis in colon cancer cell lines (CRC: HCT116) [ 27 ]. Hydrogel: Hydrogels are composed of hyaluronic acid (HA) and hydroxyethyl cellulose (HEC), and they can improve the skin permeation of ISL [ 28 ]. As described above, many experiments have been conducted to evaluate the various properties of ISL nanoformulation have been developed to address the problems of bioavailability and solubility. Nanoformulation studies have been conducted in vitro and in vivo ( Table 1 ), demonstrating that ISL nanoformulations improve the bioavailability by 2–10-fold [ 17 , 24 , 26 ]. ISL-derived new compounds offer another solution to improve the bioavailability and water-soluble issues [ 31 , 32 , 33 , 34 , 35 , 36 ]. Considering the chalone structure, the α,β-unsaturated ketone is an important part of its biological activity by modifying on the phenol ring to improve the performance of ISL. We summarized a few new analogues of ISL in below (see Figure 2 ): 4-C-β-D-glucosylated ISL ( Figure 2 a): Glucosylation of low molecular weight compounds have improve water solubility and bioavailability with a good inhibition of aldose reductase (AR) [ 37 ]. Synthetic isoliquiritigenin derivatives (BS5 and BS11 in Figure 2 b,c): The compounds BS5 and BS11 with m-, p-dimethoxy, o-bromo phenyl group shows neuroprotective effects at 3 μM to 6 μM with higher viability (~80–100%) [ 36 ]. Robtein (ISL-derivative #10; Figure 2 d): Robtein exhibited osteoclast differentiation and activation without any significant changes of viability or cytotoxicity [ 34 ]. 2′,4′-dimethoxy-4-hydroxychalcone ( Figure 2 e): shows in vivo antidiabetic activity [ 35 ]. 3′,4′,5′,4″-tetramethoxychalcone (TMC; Figure 2 f): Introducing methylation of hydroxy groups significant increase cytotoxic activity in breast cancer [ 31 ], especially targeting on triple-negative breast cancer (TNBC) [ 33 ]. ISL-17 ( Figure 2 g): A fluorine atom was introduced to the structure of ISL named ISL-17 showed the anti-tumor activities in gastric cancer [ 32 ]. 4-C-β-D-glucosylated ISL ( Figure 2 a): Glucosylation of low molecular weight compounds have improve water solubility and bioavailability with a good inhibition of aldose reductase (AR) [ 37 ]. Synthetic isoliquiritigenin derivatives (BS5 and BS11 in Figure 2 b,c): The compounds BS5 and BS11 with m-, p-dimethoxy, o-bromo phenyl group shows neuroprotective effects at 3 μM to 6 μM with higher viability (~80–100%) [ 36 ]. Robtein (ISL-derivative #10; Figure 2 d): Robtein exhibited osteoclast differentiation and activation without any significant changes of viability or cytotoxicity [ 34 ]. 2′,4′-dimethoxy-4-hydroxychalcone ( Figure 2 e): shows in vivo antidiabetic activity [ 35 ]. 3′,4′,5′,4″-tetramethoxychalcone (TMC; Figure 2 f): Introducing methylation of hydroxy groups significant increase cytotoxic activity in breast cancer [ 31 ], especially targeting on triple-negative breast cancer (TNBC) [ 33 ]. ISL-17 ( Figure 2 g): A fluorine atom was introduced to the structure of ISL named ISL-17 showed the anti-tumor activities in gastric cancer [ 32 ]. However, the poor bioavailability and water-solubility issues remain in clinical applications. Future studies are still needed to elucidate the ISL formulations that would be more suitable for human clinical trials. ISL had been reported to exert diverse biological properties, but the specific molecular interaction that underlies these activities has not been fully unveiled. Based on molecular docking analysis, many studies have proposed that ISL has a direct interaction in different molecules ( Figure 3 ), such as SIRT1 [ 38 ], VEGF2 receptor [ 39 ], GRP78 [ 40 ], FLT3 [ 41 ], EGFR [ 42 ], IKKβ [ 43 ], Toll-like receptors (TLRs) [ 44 ], CK-2 (IC 50 : 17.3 µM) [ 45 ], H2R [ 46 ], COX-2 [ 47 ], aromatase (Ki: 2.8 µM) [ 48 , 49 ], topoisomerase I [ 50 ] and DNMT1 [ 51 ]. These docking results imply that the binding pocket is composed of hydrophobic regions and is stabilized by a hydrogen bond with its neighboring carbonyl group. The hydrogen bond interactions and π–π stacking contribute to a tight interaction with the binding site. These docking results provide valuable information about the binding interactions of ISL and the active site, although more studies are required to approve them. Using a bioassay-guided purification method, suggested that isolated ISL acts as a xanthine oxidase inhibitor (IC 50 : 55.8 µM; Ki: 17.4 µM) to avoid transplantation rejection and ischemia reperfusion damage [ 52 ]. In brief, multiple docking candidates indicate that ISL exhibits multiple biological properties and serves as a potential lead compound for developing new therapy in cancer treatment. In targeting cancers, ISL possesses various biologic activities, such as anti-inflammation, antioxidation, antiviral, antidiabetic, neuroprotective effect, chemopreventive, and antitumor growth properties ( Figure 4 and Figure 5 ). A selective cytotoxicity effect of ISL has been reported ( Table 2 and Table 3 ), and the effective dose in tumor cell lines shows very little cytotoxic effect on normal cells. Most studies have claimed that ISL significantly inhibits the viability of cancer cell but has little toxicity on normal cells. For example, Wu et al. (2017) compared the human endometrial stromal cells (T-HESCs; as a control) and human endometrial cancer cell lines (Ishikawa, HEC-1A, and RL95-2 cells). Their results indicated that ISL inhibits the growth of cancer cells at concentrations below 27 μM, but has little effect on normal cells [ 53 ]. Na et al. (2018) claimed that ISL shows little toxicity on normal hepatocyte cell lines (AML-12); only when applied in concentrations of over 100 μM is ISL harmful to normal hepatocytes [ 54 ]. Most studies have focused on the cytotoxicity between tumor and normal cells, and the effects of ISL on normal cells remain unknown. As Peng et al. (2015) mentioned, further research on the target organ toxicity or side effects of ISL is needed. The safety of ISL is always one of the most important concerns that must be evaluated.

Many previous studies have provided evidence that ISL has anticarcinogenic activity in various types of cancers, including breast cancer, colon cancer, gastrointestinal cancer, lung cancer, ovarian cancer, leukemia, and melanoma. In Table 3 and Table 4 , we summarize the research progress regarding the ISL’s antitumor activity in vitro and in vivo, respectively. From the WHO database ( https://www.who.int/cancer ), breast cancer is the most common cancer among women, impacting 2.1 million women each year. Breast cancer is still regarded as the second leading cause of cancer death in women. In the 2018 cancer statistics, it was estimated that 627,000 women died from breast cancer, which accounted for approximately 15% of all cancer deaths among women. Breast cancer can be triggered by multiple factors such as cancer stem cells (CSCs), the tumor microenvironment, genetic and epigenetic abnormalities, and so on. Most typical types of breast cancer are based on the expression of the ER-positive type of estrogen receptor (ER). Above two-thirds of breast cancers are termed hormone-dependent breast cancers, which rely on estrogen for tumor growth. Hormonal therapy or aromatase inhibitors are commonly applied in ER-positive breast cancer. Aromatase inhibitors may exert tumor-suppressing effects, preventing the conversion of androgen into estrogen. According to the function of aromatase inhibitors, previous studies have strongly suggested that ISL can act as an aromatase inhibitor [ 39 , 48 , 49 , 70 ] for a breast cancer remedy. However, another type of breast cancer, known as basal-like or triple-negative breast cancer (TNBC), does not respond to hormonal therapy. The advanced treatments in breast cancer include radiation, surgical exclusion, and the use of various chemotherapeutic drugs such as paclitaxel, doxorubicin, cisplatin, epirubicin, and 5-FU (5-fluorouracil). However, the incidence of drug resistance and serious side-effects associated with these treatment methods has greatly reduced their therapeutic potential. Therefore, alternative and safer chemotherapeutic strategies are needed. Doxorubicin is one of the most effective agents for a wide spectrum of cancers, including breast cancer. The mechanism of doxorubicin is the inhibition of the DNA topoisomerase I & II and DNMT1, the same as ISL [ 50 , 51 , 141 ]. However, when treated with doxorubicin, patients suffer from serious cardiotoxicity and drug resistance. Lin et al. (2017) demonstrated that treatment with ISL alone or in combination with doxorubicin is highly effective in sensitizing doxorubicin-resistant cancer cells, resulting in the reduced survival of cancer cells [ 142 ]. Moreover, ISL not only inhibits cancer cell growth by inducing apoptosis and autophagy, but can also enhance chemosensitivity [ 40 , 66 , 67 , 69 ]. It has also been reported that doxorubicin triggers an epithelial-to-mesenchymal transition (EMT) in TNBC through mediating the PI3K/AKT pathway. Interestingly, ISL also can inhibit the PI3K/AKT pathway and thus suppresses EMT and increases the antiproliferative effect [ 40 , 66 , 67 , 69 ]. ISL or its derivatives show a greater influence by regulating the miR-374a/BAX axis, the -374a/PTEN/AKT axis, or the autophagy-mediated apoptosis (p62/caspase-8) pathway, especially in TNBC [ 33 , 67 , 69 ]. Recent studies have also demonstrated that ISL causes chemosensitization and induces autophagy following the degradation of the ABCG2 autophagy–lysosome pathway or the miR-25-mediating ULK1 (a kinase involved in autophagy) [ 40 , 56 ]. To further prevent the invasion in breast cancer, upregulating RECK (tumor suppressor gene) and downregulating miR-21 has been reported [ 65 , 68 ]. ISL has been suggested to be a supplement with chemotherapy or an alternative therapeutic agent for clinical trials against breast cancer, thereby warranting further investigation. The other first-line chemotherapies for breast cancer are epirubicin, 5-FU, and Taxol. Remarkably, previous studies have also shown that ISL can interact synergistically with these first-line chemotherapy drugs through mediating cell death (apoptosis) and autophagy and suppressing breast CSCs [ 40 , 56 , 69 ]. In a preclinical study, ISL was able to shed a novel light on reversing the epigenetic changes of Wnt inhibitory factor 1 (WIF-1), which induced the demethylation of WIF-1 promoter and subsequently prevented tumor initiation by inhibiting CSCs [ 51 ]. Based on the research described above, ISL greatly enhances the therapeutic efficacy of different chemotherapy drugs, overcomes drug resistance, and achieves sensitization to radiation ( Table 5 ). Even without combination treatment, ISL alone possesses anticancer activities in multistage carcinogenesis processes, including proliferation suppression, cell cycle arrest, angiogenesis inhibition, metastasis obstruction, apoptosis induction, autophagy induction, and metabolism (arachidonic acid and glucose metabolism). The administration of ISL alone to xenograft animals significantly inhibits lung metastasis in breast cancer and suppresses the expression of matrix metallopeptidase-9/7/2 (MMP-9/7/2), NF-κB, and cyclooxygenase-2 (COX-2) [ 57 , 63 , 64 , 66 ]. Concerning the inhibition of the tumorigenesis and metastasis of breast cancer, ISL can rectify the abnormal PI3K/AKT, NF-kB, and p38 signaling pathways in order to reduce the occurrence of metastasis through correcting the expression of MMP-2, MMP-7, MMP-9, VEGF, and HIF-1α [ 39 , 57 , 65 , 66 , 67 ]. Moreover, ISL hampers breast cancer growth and the neoangiogenesis accompanying suppressed VEGF/VEGFR-2 signaling, which prompts HIF-1α proteasome degradation or directly blocks VEGF-2 ( Figure 3 ) [ 39 ]. ISL inhibited the multiple mRNA expression of phospholipase A2 (PLA2), cyclooxygenases-2 (COX-2), and cytochrome P450 (CYP) in an arachidonic acid (AA) metabolic network, as well as decreased the secretion of prostaglandin E2 (PGE2), 20-hydroxyeicosatetraenoic acid, and phosphorylation of PI3K. Meanwhile, in an in vivo test, ISL interferes with the AA metabolic enzyme to suppress the tumor growth of MDA-MB-231 human breast cancer xenografts in nude mice [ 66 ]. Colorectal cancer (CRC) is a common and lethal disease. In 2020, ~18,000 cases of colorectal cancer were diagnosed in people under 50—the equivalent of 49 new cases daily. Moreover, it is expected that 10 people die from CRC daily [ 150 ]. Generally, CRC develops in the colon or rectum, causing by both environmental and genetic factors such as old age and lifestyle. Some studies have demonstrated that CRC cells show increased proliferation, migration, and invasion in the presence of an acidic tumor microenvironment (TME), which further hinders chemotherapy [ 62 , 151 ]. In an acidic tumor microenvironment, fructose-bisphosphate aldolase A (ALDOA), pyruvate kinase muscle isozyme M2 (PKM2,) and lactate dehydrogenase A (LDHA) are overexpressed in colon cancer, resulting in high acidity of the intracellular environment. LDHA overexpression could engender hypoxia-inducible factor 1-alpha (HIF-1α) stability to enhance the generation of glycolysis [ 152 , 153 ]. To inhibit glycolysis and lactate generation in a tumor, ISL mediates HIF-1α stability and inhibits the AMPK and AKT/mTOR pathway. This phenomenon had been found in colon cell lines and mouse melanoma B16F10 cells [ 27 , 103 ]. More importantly, this downregulation of AA-metabolizing enzymes and the deactivating PI3K/AKT phenomena can also be observed in MDA-MB-231 human breast cancer xenografts in nude mice in vivo [ 66 ]. ISL not only affects the metabolic pathway, but it also inhibits tumor growth via prompting apoptosis and autophagy. In the study of Auyeung et al. (2010) [ 74 ], ISL inhibited tumor growth throughout the downregulation of the antiapoptotic proteins Bcl-2 and Bcl-x(L), arrested in G2. Moreover, ISL remarkably reduces PGE2 and nitric oxide (NO) production to induce apoptosis in mouse and human colon carcinoma cells [ 76 ]. Compared to the chemotherapy treatments in colon cancer, capecitabine, 5-FU, and gemcitabine act as antimetabolites, interfering with DNA synthesis. As mentioned above, ISL can abate the metabolism and possesses a DNA demethylation effect [ 71 ]. However, chemotherapy frequently results in a resistance issue. In a preclinical study, combinations of ISL with other chemotherapy drugs were tested [ 72 , 73 , 75 , 103 ]. Additionally, ISL has been identified as a potential multidrug-resistant (MDR) modulator candidate due to its ability to regulate the expression of the ABCB1, ABCC1, caspase 3, caspase 8, AhR, CYP1A1, and GSTP1 genes in colon-MDR cells [ 11 ]. Thus, a combined treatment in chemotherapy-resistant cells mediates the apoptosis/cell death of resistant cells. Furthermore, it was the first application to combine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with ISL successfully to observe the chemopreventive effects of ISL. Its mechanism is dependent on the amount of death receptor 5 (DR5) protein among the TRAIL receptors. However, this means that the induction of apoptosis primarily relies on the TRAIL function [ 72 ]. In vitro, ISL can also mediate p53, EGRF-MAPK, and NAG-1 expression ( Table 3 ) against colon cancer. Moreover, ISL downregulates ROS, NO-production, NF-κB activity, PGE-2, and COX-2 (see more details in Table 3 ) for tumor suppression. Ovary cancer, the most lethal of all gynecologic malignancies due to the limitation of early detection, presents in postmenopausal women with months of abdominal pain. The overall five-year relative survival rate of invasive epithelial ovarian cancer at diagnosis is approximately 40%. Worldwide, 2.2 million women have developed epithelial ovarian cancer every year [ 154 ]. Some prospective case–control studies have found that genetic mutation, endometriosis, human papillomavirus, perineal talc, and smoking are the risk factors that increase the incidence of ovarian cancer. Surgery and platinum-based cytotoxic chemotherapy are the standard of care for ovarian cancer therapy [ 154 ]. Ovarian cancer can reoccur and cause death due to the high metastatic and spread rates to the organs in the abdominal, brain, or lymph nodes outside of the abdomen. Therefore, controlling ovarian cancer metastasis is considered one of the most effective therapeutic strengths. ISL alone or in combination with other chemotherapeutic agents has been applied for the treatment of ovarian cancer. In an in vitro study, ISL treatment inhibited cell proliferation and induced cell apoptosis in ovarian carcinoma. The IC 50 values of ISL on SKOV-3, OVCAR-5, and ES2 cells were 83.2, 55.5, and 40.1 μM, respectively ( Table 3 ). ISL at 10 µM deterred ovarian carcinoma cells’ epithelial-to-mesenchymal transition (EMT), migration, and invasion through increasing the protein expression of E-cadherin and reducing the levels of ZEB1, vimentin, and TGF-β. ISL at 10 µM can suppress the intraperitoneal xenograft development of ovarian cancers [ 79 ]. Furthermore, ISL also induces ovarian cancer cell apoptosis through inducing oxidative stress, increasing endoplasmic reticulum stress, and leading to excessive intracellular ROS generation. This effect can be alleviated by co-treatment with Z-ATAD, a caspase-12 inhibitor [ 83 ]. Lung cancer, also known as lung carcinoma, is the leading cause of cancer-related deaths worldwide due to the detection at an advanced stage [ 155 , 156 ]. There were more than 230,000 new cases found in U.S. alone in 2018. There are two main types of lung cancer: non-small-cell lung cancer (NSCLC; approximately 80–85%) and small cell lung cancer (SCLC; approximately 10–15%). Long-term tobacco smoking is the predominant risk factor of lung cancer. Previous studies have implied that approximately 80–90% of all cases of lung cancer are caused by cigarette smoking or passive smoking. Other risk factors are chronic obstructive pulmonary disease (COPD), family history, gender (men), and exposure to radon, asbestos, or carcinogens. The mutation of the genes EGFR, KROS, MET, LKB1, BRAF, PIK3CA, ALK, RET , and ROS1 is associated with the development of lung cancer [ 157 ]. EGFR inhibitors possess significant clinical benefit to NSCLC patients. ISL has been found to inhibit cell proliferation and cell cycle arrest in the A549 cell line, a human NSCLC cell line, through the activation of the p21CIP1/WAF pathway (IC 50 = 18.5 or 27.14 µM). This result was comforted by other papers [ 90 , 91 ]. ISL (20 µM for 24 h) inhibits cancer cell migration and induces cell cycle arrest through the inhibition of the mTOR via PI3K/AKT pathway, which is one of the primary anti-apoptotic pathways activated by EGFR. Moreover, ISL downregulates the following protein levels: p21, Bax, Bcl-2, and p53, the most important cell cycle regulator in the A549 cell line. ISL has been found to inhibit E-cadherin P70, cyclin D1, N-cadherin, and vimentin, and thus suppress EMT [ 136 , 158 ]. Furthermore, an in vivo study provided similar results: Tumorigenesis was reduced in six-week-old athymic nude mice after IP injection with ISL (1 or 5 mg/kg, three times per week for two weeks). This phenomenon occurs, at least in part, through targeting with EGFR, thereby reducing the suppressed AKT and ERK1/2 signal pathways [ 42 ]. Inflammation has been demonstrated to play a major role in cancer development. Anti-inflammatory drugs have been considered as cancer therapeutic agents. ISL has been found to possess an anti-inflammatory effect, both in vivo and in vitro [ 86 , 136 , 137 , 138 , 139 ]. The anticancer activity of ISL on leukemia has also been evaluated. In in vitro studies, ISL at 50 µM significantly inhibited lymphocytic leukemia (LCL) cell proliferation after a 24 h administration. This effect occurs, at least in part, through the inhibition of p53 and cell cycle (estimated IC 50 = 40~65 µM) [ 84 ]. Furthermore, ISL also abolishes cell proliferation and induces cell differentiation by the upregulation of antioxidative activity in HL-60 cells (estimated IC 50 = approximately 40.42 µM) [ 85 , 94 , 95 ]. Similarly, ISL induces cell cycle arrest in the G2/M phase in the human T cell leukemia Jurkat and CCRF-CEM cell line (IC 50 = 18.38 μM) [ 96 , 97 ]. In addition, ISL also inhibits DNCB-induced pro-inflammatory cytokines secretion, as well as p38-ERK signaling, in human monocyte model THP-1 cells [ 98 ]. ISL also decreases inflammatory cytokine secretion through the inhibition of the TRIF-dependent pathway in RAW264.7 cell line [ 92 , 93 ]. In in vivo studies, 30-day oral administration of ISL significantly inhibits MV4-11 flank tumor growth and prolongs survival via decreasing cell proliferation and inducing apoptosis [ 41 ]. Oral administration of ISL in experimental AD-like lesion model mice significantly suppresses DNCB-induced IgE and Th2 cytokine upregulation [ 98 ]. ISL possesses an immune-suppressive effect directly on human T cells via covalent binding of IKKβ Cys46 without significant toxicity [ 43 ]. A preclinical study on the T-ALL cell line showed that ISL inhibits the survival of doxorubicin or methotrexate-resistant cell lines. Therefore, ISL may be a valuable adjunct for cancer therapy to treat otherwise drug-resistant tumors [ 96 ]. The anti-inflammatory effect of ISL also implies that it can be applied in allergic asthma patients [ 147 ]. The antitumorigenic effects of ISL on melanoma have also been evaluated extensively. Xiang et al. found that ISL inhibits cell proliferation and induces cell apoptosis through stimulating the expression of C-PARP, Bax, and cleaved-caspase-3 [ 99 ]. It also induces B16F0 melanoma cell differentiation. Three pathways, i.e., the glutathione metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathways, are the most important pathways perturbed by ISL [ 100 ]. Moreover, ISL can activate the mTORC2-AKT-GSK3β signaling pathway, thereby inducing cell cycle arrest, reprogramming A375 melanoma cells (estimated IC 50 = ~48 µM) [ 101 ]. ISL can decrease the expression of mitochondrial protein mitoNEET, thereby decreasing mitochondrial membrane potential, altering ROS content, and subsequently inducing cell apoptosis in A375 cells (estimated IC 50 = ~73 µM) [ 102 ]. In contrast, Wang et al. found that ISL can stimulate ROS, leading to oxidative stress, thereby inducing B16F10 cell apoptosis (estimated IC 50 = ~35 and 22 µg/mL) [ 103 ]. Similarly, ISL increases ROS accumulation and facilitates melanogenesis, thereby stimulating B16F10 cell differentiation [ 104 ]. ISL can inhibit the growth of human as well as murine myeloma cell lines via inhibiting IL-6 signaling ( p -ERK, p -STAT3, etc.), inducing cell apoptosis and cell cycle arrest [ 105 ]. In SCID mice bearing U266, BABL/c bearing MPC-11, or murine myeloma xenograft models, the antitumor activity of ISL has also be found by ISL alone or in combination with Adriamycin via blocking IL-6 signaling [ 105 ]. ISL also induces cell apoptosis in B16 melanoma mouse melanoma via the inhibition of glucose transmembrane transport [ 107 ]. Moreover, ISL exerts antimelanogenic effects through activating the phosphorylation of ERK and inhibiting tyrosinase activity in SK-MEL-2 and HaCaT cells [ 106 ]. Hepatoma, also known as hepatocellular carcinoma (HCC), is the most common primary malignant tumor of the liver in adults. Hepatoma, which is mainly caused by cirrhosis, is the second leading cause of cancer-related death worldwide. Traditional herbal medicines, including licorice, have been widely used for HCC prevention and treatment. ISL, the compound purified from licorice, has been used in hepatoma treatment recently. As depicted in Table 3 , ISL exhibits toxic effects on Hep3B hepatoma cells by inducing cell cycle arrest at the G1/S checkpoints, suppressing migration and metastasis and the PI3K/AKT signal pathway (IC 50 = 42.84 ± 2.01 μM). Upon ISL treatment, the protein expression and kinase activity of the cell cycle regulators are altered in hepatoma cell lines [ 108 ]. Furthermore, ISL induces apoptosis via the MAPK/STAT3/NF-κB/IkB signaling pathway, ROS accumulation, and the p53-dependent pathway and reduces cell cycle-associated protein expression in HepG2 and Hep3B cells [ 109 , 110 , 111 ]. The effects of ISL on the hepatocellular carcinoma cell line Hepa 1c1c7 have been investigated, finding that cell growth decreases and apoptosis is induced in both Hep G2 and PLC/PRF/5 (IC 50 = 36.3 μM) [ 112 ]. ISL has been shown to inhibit liver cancer cells (SK-Hep-1) proliferation (IC 50 = 19.08 μM) [ 113 ]. ISL also inhibits DNA cleavage reaction via inhibiting TOP I activity in the SNU475 cell line [ 50 ]. In a xenograft model in female BALB/c- mice bearing Hep3B cells, when subjected to IP ISL (50 mg/kg/day for three weeks) administration, they showed decreased tumorigenesis and metastasis of HCC due to a reduction in the expression of cyclin D1 and the suppression of the PI3K/AKT pathway [ 108 ]. Similarity, a single IP administration of ISL (10 mg/kg) increases radiosensitization via the inhibition of the Nrf2/Keap1 pathway in four-week-old male athymic BALB/c (nude) mice bearing HepG2 [ 140 ]. Prostate cancer is the most common noncutaneous cancer among men. It is also the second-leading cause of cancer deaths for men in the U.S. Alternative therapies are becoming increasingly popular among patients with prostate cancer. The realization that ISL has a role to play in the chemoprevention of prostate cancer has led to a number of cell line-based investigations aimed at understanding the mechanism of ISL ( Table 2 ). ISL induces cell apoptosis in prostate cancer cells through G2/M cell cycle arrest with concomitant downregulation of cyclin B1, CDK1 (p-Thr14, p-Tyr15, and p-Thr161) (after 48 h of treatment, the IC 50 of ISL on PC-3 and 22RV1 is 19.6 and 36.6 μM, respectively) [ 114 ]. Zhang et al. observed that ISL (IC 50 = 87.0 μM) inhibits the anticancerous effects on C4-2, LNCaP prostate cancer cells, by the reduction of the Psi(m) that triggers apoptosis and the inhibition of the proliferation via the ERK/p38MAPK pathway [ 59 ]. The effects of ISL on prostate cancer cell line DU145 have been investigated, finding that cell cycle arrest in the G2M phase decreases CDC25C and increases p-CDC2 (Tyr15), cyclin B1, and p27 KIP1 [ 115 ]. The anti-invasion and antimetastasis of ISL on the DU145 prostate cancer cell line have been investigated, with the findings suggesting that this mechanism could be achieved through the inhibition of JNK/AP-1 signaling and the downregulation of the reduction of µPA, MPP-9, and AP-1. Moreover, the expression of some proteins, including VEGF, integrin-α2, and ICAM, and VCAM, has also been shown to be reduced [ 116 ]. Furthermore, the inhibition of the PI3K/AKT and HRG-β-induced ErbB3 signaling pathways has also been found in the DU145 cell line [ 117 ]. ISL can induce prostate cancer cell apoptosis via increasing Fas ligand (FasL), Fas, cleaved casapse-8, tBid, cytochrome c, and Smac/Diablo (ISL shows an estimated IC 50 of 13.74, 5.67, and 5.01 µM in the MAT-LyLu cell line treated for 24, 48, and 72 h, respectively; in contrast, it shows an estimated IC 50 of 56.87, 31.49, and 17.60 µM in the MAT-LyLu cell line treated for 24, 48, 72 h, respectively) [ 118 ]. The effects of ISL on prostate cancer cell apoptosis have been investigated, with researchers finding that ISL induces DU145 and LNCaP cell cycle arrest in the G2/M stage through increasing the expression of GADD153 mRNA (estimated IC 50 of ISL on Du145 and LNCaP is 10.56 and 10.78 µM, respectively) [ 119 ]. An antitumor effect of ISL against prostate cancer in an animal model has been reported. In prostate-tumor-bearing animals, i.e., male BALB/c nude mice bearing PC-3, they were treated with 25 or 50 mg/kg/day ISL for 28 days; a decrease in cyclin B1–CDK1 and G2/M arrest and apoptosis was detected [ 114 ]. Cervical cancer is the fourth most common cancer in women worldwide and causes more than one quarter of a million deaths per year. Several studies have also focused on the anti-cervical cancer effects of ISL. ISL induces intrinsic apoptosis and S-phase arrest in Ca Ski, SiHa, HeLa, and C-33A cells. ISL inhibits proliferation and induces cell cycle arrest in the G2/M phase, which may be attributed to the decreased expression of Bcl2 and the increased expression of caspase-related proteins and cell cycle checkpoints, including p53, p21, Bax cyclin B, cyclin A, cdc2, and cdc25C (estimated IC 50 = 39.09 μM in Ca Ski; 53.76 μM in SiHa; 9.8 or 58.10 μM in HeLa; 32.83 μM in C-33A cells) [ 120 , 122 ]. ISL also induces apoptosis in cancer cells through increasing ROS generation, p -eIF2α and GRP78 expression, and caspase-12 activation in HeLa cells (estimated IC 50 = approximately 21.24 μM) [ 121 ]. ROS production is important for the anticancer activity of ISL in HeLa cells. This was proven by Yuan et al., showing that the apoptotic rate is increased after co-treatment of ISL and pro-oxidant, l-buthionine-(S,R)-sulfoximine (BSO). On the contrary, the apoptosis rate is inhibited by co-treatment with free radical scavenger N-acetyl-cysteine (NAC) [ 144 ]. In an in vivo study, in KM mice bearing U14, when administered in combination with cyclophosphamide, ISL enhanced the antitumor activity and decreased the micronucleus formation of DNA strand breaks [ 145 ]. Studies have shown that ISL also induces call apoptosis and/or autophagy in other cancers, including gastric cancer, uterine leiomyoma, osteosarcoma, glioma, bladder cancer, and oral squamous cell carcinomas (OSCC). In gastric cancer, ISL causes cell apoptosis and autophagy in MKN28 cells by suppressing the PI3K/AKT/mTOR pathway and by increasing Beclin-1 (IC 50 = 20.84 µM) [ 123 ]. ISL (5 µM) negatively affects H2R-mediated c-Fos/c-Jun protein expression, acting as an effective H2R antagonist in the MKN-45 cell line [ 46 , 124 ]. ISL (0.11 g/L for 24 h) induces MGC-803 cell apoptosis via the calcium- and Delta psi(m)-dependent pathways [ 125 ]. In uterine sarcoma, the combined treatment of human uterine sarcoma cell line MES-SA cells with ISL and doxorubicin significantly enhances chemosensitivity via inducing apoptosis and autophagy by inhibiting the mTOR pathway [ 142 ]. In uterine leiomyoma, ISL induces cell cycle arrest in the subG1 and G2/M phases by increasing p21Cip1/Waf and reducing Bcl-2, cdk 2/4, and E2F, thereby suppressing the proliferation of primary uterine leiomyoma cells. ISL also induces cell apoptosis through the elevation of FAS ligand and caspase-3 (estimated IC 50 = 39.33 µM) [ 126 ]. In contrast, a low cytotoxicity of ISL has been found in normal myometrium cells (estimated IC 50 = 698.8 µM). In osteosarcoma, ISL has also been shown to cause DNA damage and can initiate apoptosis through increasing Bax and caspase 3 and cell cycle regulators, including p53, p21, and p27, in U2OS and Saos‑2 cells (main dosage of 20 and 30 μM, respectively) [ 127 , 128 ]. In glioma, ISL induces cell apoptosis by the elevation of caspase 3 and the inhibition of TOP I in glioma U87 cells (IC 50 = 6.3 µM) [ 130 ]. In SK-N-BE(2) an IMR-32 neuroblastoma cell line, ISL (>5 µM) has been found to increase the ROS level, thereby inducing cell death. The combined treatment of SK-N-B-E(2) cells with ISL and the anticancer agent cisplatin significantly reduces cell viability compared to cisplatin alone [ 129 ]. ISL inhibits cancer growth and induces apoptosis and autophagy in PC12 cells by dose-dependently downregulating Bcl-2 and Bcl-x and by stimulating caspase-9, caspase-3, caspase-7, Bax, Bim, cytochrome c, Beclin-1, and LC3 expression (IC 50 = 17.8 ± 1.8 μM) [ 131 ]. In bladder cancer, pretreatment with ISL for 24 h enhances cisplatin-induced cell death, ROS production, the upregulation of Bax, Bim, Apaf-1, caspase-9, and caspase-3 levels, and the downregulation of Bcl-2 levels in the T24 cell line. In contrast, it attenuates cisplatin-induced proximal tubular cell (LLC-PK1) injury by upregulation of HO-1 levels [ 132 , 148 ]. In LLC-PK1 porcine kidney cancer cells, pretreatment with ISL induces ER stress and produces hormesis to protect against cisplatin-induced nephrotoxicity [ 149 ]. Oral cancer is defined as any malignant cell growth in the oral cavity. OSCC comprises more than 90% of oral cancer cases, and is the third most prevalent malignancy in developing countries. In our previous study, we found that ISL induces OSCC cell cycle G2/M phase arrest, apoptosis, and DNA damage through the inhibition of ATM signaling. A low dose of ISL (6.25 μM) inhibits OSCC malignancy in vitro. ISL (5 mg/kg) also reduces the tumor size in vivo [ 159 ]. ISL downregulates GRP78 levels, thereby suppressing oncogenicity both in vitro and in vivo. The combination of ISL and cisplatin significantly represses the invasion and colony formation abilities of OSCC cells by downregulating the expression of CSC markers and ABC transporters [ 60 ].

Cancer is one of the leading causes of morbidity and mortality worldwide. Based on GLOBOCAN ( https://gco.iarc.fr ) estimates, approximately 18 million new cancer cases and 9.6 million deaths occurred in 2018 worldwide. Nowadays, it is the second leading cause of death (9.6 million) globally [ 1 ]. Due to the changes in lifestyle habits such as smoking, overweight, physical inactivity, and reproductive patterns associated with urbanization and economic development, the global morbidity and mortality of cancer is predicted to increase quickly over the next few decades. The most common causes of cancer-related death are lung cancer, colorectal cancer, stomach cancer, liver cancer, and breast cancer. Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are the most common strategies to combat cancer [ 2 ]. These therapies are showing more and more limitations because of their poor prognosis and side effects. However, poor prognosis occurs when cancer is resistant to radiotherapy (radioresistance) and chemotherapy (chemoresistance), which presents a challenge in cancer therapeutics. A better therapeutic strategy has the characteristics of low toxicity, high antitumor activity, and specific multi-targeting properties, avoiding the high mortality rate and decreasing the prolonged survival time for metastatic cancer to date. Seeking natural compounds from herbal remedies that possess the high efficacy and low adverse effects associated with cancer or that target cancer themselves is the ultimate cure for cancer. Without any better solution, medicinal plants act as an alternative therapy to improve the unmet needs of cancer survivors. Licorice extracts are one of the most common Chinese herbs widely applied in traditional medicine. Licorice belongs to the genus Glycyrrhiza , and Glycyrrhiza radix is the dried roots and rhizome of licorice. Licorice had been used for diseases since the Former Han dynasty (the second–third centuries B.C.), and has been documented in ancient Egypt, Greek, and Rome. The pharmacological effects of licorice have been demonstrated for peptic ulcers, constipation, coughs, and other diseases, especially in cancer therapy. However, high doses of licorice have a risk of side effects, such as cardiac dysfunction, edema, hypertension and hypokalemic-induced secondary disorders [ 3 ]. It is necessary to identify a more potential candidate from the licorice to improve human health and lifespan. It has been known that one of the most important bioactive candidates in licorice is isoliquiritigenin (2′,4′,4-trihydroxychalcone, ISL). ISL serves as one of the most active components in Glycyrrhiza . For example, Lee et al. (2013) found that ISL remarkably suppresses the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation of murine bone marrow-derived macrophages [ 4 ]. ISL does not only show the same pharmacologic effects as Glycyrrhiza , but also exerts more biological activities, especially in terms of antitumor effects. Clinical trials using ISL alone and in combination against cancer are still in their infancy. However, based on the many in vitro and in vivo studies conducted in various research laboratories across the world, the results are encouraging. In the present review, we summarize the most recent research in the literature on the phytochemical properties and pharmacological applications of ISL to provide further support and evidence for cancer treatments.

ISL exhibits significant anticancer activity through various mechanisms, such as proliferation suppression, apoptosis induction, and/or autophagy, and inhibits migration and invasion in various cancer cells ( Figure 6 ). Licorice Kampo and ISL are not only potential candidates for adjuvant chemotherapy, but also possess anticancer properties. However, clinical trials using ISL against cancer have not been initiated. Undoubtedly, both in vitro and in vivo studies have demonstrated the potential of ISL for the prevention and treatment of different types of cancers ( Table 3 and Table 4 ). With encouraging outcomes in preclinical studies, many studies have strongly emphasized that ISL can increase the chemosensitivity of different kinds of chemotherapies ( Table 5 ). More, the application of ISL in the form of nanoformulations as a novel strategy in order to improve its efficacy ( Table 1 ) is under ongoing development. Overall, the various research works have not only highlighted the significant anticancer activity of ISL in vitro and in vivo, but have also proposed various molecular-based interactions ( Figure 3 ) underlying anticancer mechanisms. Overall, ISL is a promising candidate for a natural product with therapeutic effects and with the ability to alleviate the adverse side-effects in anticancer therapeutics in spite of the suppressive effects of ISL on different cancers in clinical trials being limited still.