Herbal Medicine in Uterine Fibroid

3.1 Curcumin

Curcumin is a yellow active natural polyphenol of the perennial herb Curcuma longa,commonly known as turmeric. In addition, curcumin is commonly found as ingredients in food seasoning, cosmetics, or herbal supplements. It has traditionally been used for decades in Asian countries as a medical herb due to its anti-microbial, anti-inflammatory, anti-tumorigenic, and anti-mutagenic properties [111]. Curcumin has many medical effects such as suppression of thrombosis [112], reduction of blood cholesterol [113, 114], and reduction of myocardial infarction [115]. Evidence indicates that curcumin suppresses the growth of several tumor cell lines [116, 117]. All in all, curcumin is effective against a variety of inflammatory illnesses and modulates multiple cell signaling pathways. However, it is still not well understood which binding site or receptor for it. In vitrostudies indicate that curcumin can interact with integral components of cell signaling pathways and therefore may be pharmacologically relevant. However, only limited studies have shown functional consequences of curcumin interaction [118]. The tumor suppression mechanisms of curcumin are accredited by modulation of numerous targets playing important roles in tumor growth [119, 120, 121, 122]. Those targets include transcription factors, receptors, kinases, cytokines, enzymes, and growth factors. Therefore, curcumin has been demonstrated to suppress the growth of several tumor cell lines [123]. It also inhibits the growth of uterine fibroid cells, even though there has yet to be a report describing the precise mechanism of its inhibition.

Curcumin inhibits phorbol ester-induced activation of NF-κB and ERK1/2 [9, 10]. Alternatively, it induces apoptosis via activation of ERK1/2 or SAPK/JNK in cancer cells [11]. Role of ERK1/2 activation in curcumin-treated cancer cells is controversial [124]. Curcumin down-regulates endothelial cell fibrosis and inhibits uterine fibroid cell proliferation via regulation of the apoptotic pathway, and it also reduced production of the ECM component fibronectin (Figure 1). Curcumin has also been shown to attenuate TGF-β-induced endothelial-to-mesenchymal transition [125]. Extracts from Curcuma zedoariainhibits uterine fibroid cell proliferation compared to normal myometrial cells [126]. On the other hand, it stimulates caspase-3 and caspase-9 expression in uterine fibroid cells. Curcumin provides a novel direction for uterine fibroid therapies [127].

Peroxisome proliferator-activated receptor (PPAR) is a ligand-dependent transcription factor of the nuclear hormone receptor superfamily. It is expressed in a tissue-specific manner and plays an important role in the differentiation of adipocytes [128, 129]. PPARγ exerts anti-inflammatory, anticancer, and insulin sensitivity effects and participates in the control, proliferation, and differentiation of the cell cycle. Hepatic stellate cells are the type of hepatocyte responsible for fibrosis in liver damage and can lead to chronic liver damage and cirrhosis. Curcumin induces and activates PPARγ in rat hepatic stellate cells [40]. Curcumin considerably increases the proliferative inhibition of stellate cells by PPARγ. [40]. Besides, curcumin also enhances the activity of PPARγ in human colon cancer cell lines by reducing the expression of cyclin D1 and epidermal growth factor receptor (EGFR), thereby disrupting the cell cycle [130]. These two inhibitory effects depend on PPARγ activation. The study by Takashi Takeda et al. showed that uterine fibroids can share pathogenic characteristics with the development of metabolic syndrome [131]. PPARγ is also virtually involved in insulin signaling. A PPARγ agonist, thiazolidinedione, has been used to treat patients with type II diabetes [129]. Thiazolidinedione may be used to prevent the progression of atherosclerosis and metabolic syndrome [132]. On the other hand, curcumin directly inhibits fibroid proliferation, and curcumin-induced PPARγ activation can also prevent metabolic syndrome and indirectly inhibit fibroid growth [132]. However, since these findings were based on in vitroexperiments, it raised concerns about the observation limitations. Later, Kenji Tsuiji et al. have developed a new in vivouterine fibroid model to study the inhibition of rat leiomyoma (ELT-3) cells by curcumin [132]. The IC50 of curcumin-induced anti-proliferation in uterine fibroid cell lines is 20 μM, however, when patient-matched myometrial cells were exposed to equivalent concentrations of curcumin, there was no statistically significant inhibition of growth [127].

Studies indicated that curcumin absorption rate in the intestine is very low [133, 134, 135, 136]. Similar raising concerns were also in other herb medicines, such as resveratrol discussed in next section. Thus, some modifications of curcumin need to be taken to keep its high blood concentration. Some studies regarding increased absorption and bioavailability of curcumin have been reported. For instance, the co-administration of curcumin and piperine can increase the bioavailability of curcumin [135]. Another strategy is to develop new curcumin analogues with a higher cell growth inhibitory capacity. One such compound, GO-Y030, has been shown to exhibit an 8- to 40-fold greater growth inhibitory potential than curcumin in several cancer cell lines [137, 138]. It may be useful to use the compound to clinically treat uterine fibroids.

3.2 Resveratrol

Polyphenols have been attracting by their anti-oxidative effects during the past years for human chronic diseases involved in inflammation like diabetes mellitus, neurodegenerative diseases, cardiovascular diseases, and cancers [139]. Resveratrol is one of well-studied stilbenes found in peanuts, grapes, and some berries. It is a plant product in response to environmental stress, pathogen infection, and ultraviolet radiation [127]. Resveratrol has been known as chemo-preventive, serving to suppress DMBA-induced ductal breast carcinoma [140] and ultraviolet light (UV)-induced skin cancer [141] in mouse models. Resveratrol induces p53-dependent apoptosis in several human cancer cell lines, including thyroid, prostate, and breast cancer cells [13, 142, 143, 144, 145]. It also induces p53-independent anti-proliferation against cancer cells [14, 146, 147, 148]. Resveratrol has been recommended to be a reversion molecule for multiple drug-resistant breast cancer [149]. Resveratrol is safe and well-tolerated by patients, with common adverse events including nausea, diarrhea, and weight loss [150].

Although surface receptors involved in the resveratrol signal transduction remain to be identified, resveratrol binds to cell surface integrin αvβ3 to activate ERK1/2 and ant-proliferation in cancer cells [13, 145, 151] (Figure 2). Integrin αvβ3 is also involved in AKT phosphorylation [15, 152]. Resveratrol inhibits PI3K-AKT signal pathway to induce anti-proliferation [22] or other biological activities as IL-33-mediated mast cell activation [153].

Overexpression of integrin αvβ3 is observed in several types of solid tumors [154, 155] and highly growing endothelial cells [154, 155]. Our studies indicate that integrin αvβ3 overexpresses in primary uterine fibroid cell lines [12]. The integrin αvβ3 overexpresses in primary human uterine fibroid Case 016 and Case 018 compared to normal Case 003 cells [12]. Therefore, it is a perfect target for resveratrol which has been shown to bind on integrin αvβ3 receptor [13]. Integrins are not classic signaling receptors in that they possess no enzymatic activity. Integrin signaling depends on the allosteric behavior of the receptors, their ability to concentrate into adhesion zones, and the recruitment to these zones of numerous other adhesome components to form complex integrin-based cell adhesions [156, 157]. Many adhesome components are enzymes that interact with classic signaling pathways. Resveratrol regulates signal transduction via integrin αvβ3 in human uterine fibroid cells. Resveratrol attenuates expression of integrin αv and integrin β3 in primary uterine fibroid cell Case 016 and Case 018 but not in normal myometrial cells. Resveratrol induces ERK1/2 activation in uterine fibroid cells [12]. However, the constitutive phosphorylation of AKT in uterine fibroid cells was inhibited by resveratrol.

Integrin signaling and function are heavily dependent on cross-talk with other signaling pathways, especially growth factor signaling pathways [158, 159, 160]. Besides the aberrant integrin expression, IGF-1R highly expresses more in uterine fibroid compared to the normal tissues [161] indicating IGF-1 may also be involved in the abnormal proliferation [92, 162]. IGF-1 binds to IGF-1R to activate downstream AKT which is a target of resveratrol. Resveratrol did not inhibit IGF-1R phosphorylation in primary uterine fibroid cells [12]. These results suggest that the action of resveratrol on IGF-1R-dependent signal transduction is downstream IGF-1R at Akt level [12]. Resveratrol analogue, pterostilbene (3′,5′-dimethoxy-resveratrol) targets mTOR/PI3K/Akt signaling pathway to disrupt mitochondrial membrane potential, and induce apoptosis [163].

IGF-I mRNA is expressed significantly higher in leiomyoma cells than that in myometrial cells [12]. IGF-1 stimulates IGF-1R phosphorylation of but the action is blocked by resveratrol pre-treatment. Growth effect of IGF-1 can possibly reduce by resveratrol [164]. Resveratrol inhibits IGF-1-induced phosphorylated IGF-1R accumulation and proliferation consequently [12]. IGF-1 enhances leiomyoma cell proliferation and thereby accelerates uterine fibroid progression. In summary, resveratrol via a mechanism involved in crosstalk between integrin αvβ3 and IGF-1R-sensitive signal transduction pathways induces anti-proliferation in uterine fibroid.

Steroid hormones and thyroid hormone stimulate TGF-βexpression [165]. Resveratrol blocks TGF-βexpression and functions [166]. Cross talk among TGF-β signaling pathways, integrins, and ECM [88] is essential for uterine fibroid growth. Both Agarwal discussed the ability of resveratrol to infer with ECM formation and deposition in multiple diseases in their review article [53]. Resveratrol suppresses not only expression of fibronectin, fibromodulin, biglycan, and collagen types I and III, but also their protein levels in different cell lines [43]. Resveratrol also reduces MMP-9 protein accumulation but increases TIMP2 protein in ELT-3 cells and healthy uterine smooth muscle cells [43].

Resveratrol inhibits TGF-β signal downstream molecule AKT phosphorylation. Studies of confocal microscopy have shown resveratrol inhibits nuclear pAKT translocation in uterine fibroid cells. Alternatively, resveratrol does not interfere with pAKT nuclear translocation in normal uterine smooth muscle cells even though there are limited pAKT translocated [12]. Resveratrol reduces cellular levels of the phosphorylated/active form of anti-apoptotic kinase AKT in uterine cancer cells [167]. Sequentially, treatment of resveratrol reduces the endogenous cyclooxygenase-2 (COX-2) protein and produces PGE2 and PGF2α [167]. Evidence indicates that endogenous COX-2 is involved in inflammation, therefore, resveratrol inhibits AKT signaling pathway and COX-2 activity to induce anti-inflammation which plays vital roles in uterine fibroid cell growth.

β-catenin modulates and stimulates the stem cell renewal [168]. The regulation of the biologic functions for β-catenin is highly complex and not fully understood. Wnt proteins bind to a special cell-surface receptor, Frizzled, where it promotes activation of a cascade of proteins that leads to decreased β-catenin degradation in the cytosol and reduces nuclear β-catenin levels [7, 95]. The increased β-catenin expression is observed in uterine fibroids compared to the adjacent myometrium samples [169]. Ovarian steroids interact with the Wnt/β-catenin pathway to accelerate tumorigenesis [168]. Our studies also indicate that thyroid hormone increases nuclear β-catenin accumulation, thus β-catenin-dependent gene expression and proliferation [170, 171]. Resveratrol reduces expression and nuclear accumulation of β-catenin.

The expression of resveratrol-induced pro-apoptotic genes such as COX-2and p21induced in uterine fibroid cells. On the other hand, the expression of proliferative (anti-apoptotic) genes was either inhibited such as BCL2, and CDKN2or no changed as Cyclin D1and PCNA. The pro-apoptotic proteins such as caspase 3 and caspase 9, were also increased in resveratrol-treated cells [12]. Resveratrol-induced COX-2 facilitates p53-dependent anti-proliferation [172, 173]. Therefore, resveratrol induces anti-proliferation in uterine fibroid cells [12]. Kim et al. have also shown the extraction of herb medicine, Scutellaria barbata D. Don(Lamiaceae), down-regulates the IGF-I expression [174] and inhibits the proliferation of leiomyoma cells. Scutellaria barbata D. Don(Lamiaceae) induces the uterine smooth muscle cell differentiation markers in uterine smooth muscle cells and uterine leiomyoma smooth muscle cells, such as α smooth muscle actin (α-SMA), calreductin h1, and cyclin p27-dependent kinase inhibitor. In contrast, gene products linked to the G1 phase of the cell cycle, such as cyclin E and cdk2, are not affected by Scutellaria barbata D. Don(Lamiaceae) [175]. These observations agree with our studies. The expression of anti-apoptotic genes, such as BCL2and CDKN2are suppressed or unmodified Cyclin D1and PCNA[12].

Estrogen stimulates proliferation in breast cancer cells [176, 177], endometrial cancer [178, 179] and leiomyoma cells [180]. Estrogen also stimulates cell growth in uterine fibroid cells [12]. Resveratrol can inhibit estrogen-dependent cancer growth [176] and suppresses the proliferation of six sensitive uterine fibroid cases both in the absence and presence of estradiol [12]. These results indicate the suppressing effect of resveratrol on uterine fibroid growth may not go through ER-α.

The immunomodulatory factor, checkpoint PD-1/PD-L1 has been shown to play an important role in uterine fibroid pathogenesis. They also attack attention to be therapeutic targets. Resveratrol suppresses PD-L1 expression. In the presence of thyroid hormone, resveratrol traps PD-L1 in the cytosol, meanwhile, resveratrol-induced COX-2, an inducible transcriptional co-activator [181], is trapped with thyroid hormone-induced PD-L1 in the cytosol [173].

Summarily, in the primary cell culture of patients with resveratrol-sensitive primary uterine fibroids, resveratrol can inhibit uterine fibroid proliferation, induce apoptosis, and transmit integrin-dependent signaling αvβ3. The transduction pathway promotes uterine fibroids cell cycle arrest. Additionally, resveratrol inhibits the activation of IGF-1R dependent signal transduction pathways, which play an important role in uterine fibroid proliferation. Resveratrol may or may not inhibit the expression of proliferation genes. Resveratrol also induces the expression of p21 and COX-2. Analysis of the DNA content of the PI stain indicates that resveratrol induces uterine fibroid cells cell cycle arrest at sub-G1 population [12]. Crosstalk between αvβ3 integrin and IGF-1R plays a crucial role in resveratrol-induced uterine fibroid anti-growth. In addition, resveratrol inhibits signal transduction pathways and gene expression dependent on TGF-β and β-catenin. Therefore, resveratrol can effectively prevent leiomyoma overgrowth and treat uterine fibroids.

3.3 Extract of He-Shou-Wu, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-glucoside

The stilbene glucoside 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) is one of the major bioactive components of Polygonum multiflorumThunb (He Shou Wu). It is glycosylated resveratrol and it has been used as antiaging medicine [182]. THSG suppresses experimental colitis effectively by reducing the level of oxygen and nitrogen free radicals [48]. It also has been shown to exert a protective effect on cardiotoxicity induced by doxorubicin in vitroand in vivo[183]. THSG can also diminish peroxidation levels in the brain of a mouse model with Alzheimer’s disease or cerebral ischemia–reperfusion. Administration of THSG not only prevents learning-memory deficits but also reverses the learning-memory deficit in disease-like mouse models with Alzheimer’s [184].

Mechanism involved in P. multiflorum-induced anti-atherosclerosis may be caused by THSG-induced antagonistic effects on oxidation of lipoprotein, proliferation, and decrease of nitric oxide content of coronary arterial smooth muscle cells [185] which partially explains the antiatherosclerosis mechanism of Polygonum multiflorum. Recently, the pharmacological effects of P. multiflorumon atherosclerosis have been revealed with anti-inflammation and guy microbiota regulation of THSG in ApoE−/− mice [186]. The protective effects of THSG are mediated by modulation of JNK, SirT1, and NF-κB pathways [187] (Figure 2). As resveratrol, THSG can activate signal transduction pathways as AMPK. Treatment with THSG reduces the LPS-induced neuroinflammatory response, and that the mechanism by which THSG induces anti-neuroinflammatory effects may include the Nrf2/AMPK signaling pathways [38]. Consequently, THSG treatment leads to a decrease in the level of iNOS, TNF-α, and IL-6 production [184]. THSG-induced neuroprotective effects are via Akt signaling and TrkB activity [51]. THSG possessed an anti-inflammatory effect that may also be related to the inhibition of COX-2 enzyme activity and expression [188].

As a glycosylated analogue of resveratrol, THSG has similar effects as resveratrol. Studies indicate that resveratrol significantly stimulates cell proliferation of human gingival fibroblasts at low concentration (10 μM) but inhibited cell proliferation at high concentrations (100 and 200 μM) significantly. On the other hand, THSG significantly enhances growth of human gingival fibroblasts when the concentration is over 25 μM and does not show any cytotoxic effect in human gingival fibroblasts [151]. It is evidenced that THSG may not cause cytotoxicity in normal human cells. Although there are no studies regarding effect of THSG on uterine fibroid, it should be more effective than resveratrol. THSG may not enter cells to induce superoxide which causes cytotoxicity to cells. However, studies also indicate that crude extract may cause damage in hepatocellular cells.

3.4 Pycnogenol, French maritime pine bark extract

Pycnogenol is a standardized extract of the bark of French maritime pine. Pycnogenol is composed of flavonoids, mainly proanthocyanidins, and phenolic compounds. It is a known potent antioxidant [49]. Owing to the basic chemical structure of its components, the most obvious feature of pycnogenol is its strong antioxidant activity. In fact, phenolic acids, polyphenols, and in particular flavonoids, are composed of one or more aromatic rings bearing one or more hydroxyl groups. The compositions are hence potentially able to quench free radicals by forming resonance-stabilized phenoxyl radicals [189]. Pycnogenol is a strong antioxidant that may interfere with different pathways, and it plays an important role in diseases associated with oxidative stress. Hyperglycemia is characteristic of diabetic nephropathy and induces renal tubular cell apoptosis. Pycnogenol has been demonstrated to significantly suppress the high glucose-induced morphological changes and the reduction in cell viability associated with cytotoxicity in high glucose-treated renal tubular cells [49]. Pycnogenol is able to protect high glucose-induced apoptosis increasing Bcl2/Bax protein ratio level. Combination treatment of pycnogenol and metformin improves blood glucose levels, vascular reactivity, and left ventricular hypertrophy in induced diabetic rats [33]. Furthermore, combined treatment increases expression of AMPK, glucose transporter 4 (GLUT4), and calcium/calmodulin-dependent protein kinase II (CaMKII) in left ventricle of the hearts. However, the combination of these interventions has failed to possess higher efficacy [33].

Pycnogenol has anti-oxidative and anti-inflammatory efficacy in suppressing lipid peroxidation, total reactive species, superoxide ·O₂, nitric oxide NO·, peroxynitrite (ONOO⁻), pro-inflammatory inducible nitric oxide synthase (iNOS) and COX-2 [49]. It also inhibits NF-κB nuclear translocation [49]. The safety of use of pycnogenol is demonstrated by the lack of side effects or changes in blood biochemistry and hematologic parameters. Therefore, pycnogenol has been recommended both for prevention and treatment of chronic venous insufficiency and related veno-capillary disturbances [190].

3.5 Therapeutic orchid Anoectochilus formosanusextract

Traditional herb medicine, golden thread (Anoectochilus formosanus Hayata) has been used to treat various diseases in Asia. A. formosanusextracts (AFEs) have been reported to possess hepatoprotective, anti-inflammatory, and anti-tumor activates. AFEs reduced blood glucose in hyperglycemic mice while there was no change in control group [191]. AFE and metformin at the same administrated dose of 50 mg/kg showed a similar effect on intraperitoneal glucose tolerance test in hyperglycemic mice. Free-radical scavenger capacity of AFE was concentration-dependent and 200 μg/ml of AFE was able to reduce more than 41% of the free radical [191]. The immunomodulatory protein from A. formosanus(IPAF) stimulated the TNF-α and IL-1β production, upregulated the expression of CD86, MHC II, IL-12, and Th1-associated cytokines/chemokines [28]. It also enhanced the phagocytic activity of macrophages [28]. AFE inhibited constitutive PD-L1expression and its protein accumulation in cancer cells. AFE also induced expression of pro-apoptotic genes but inhibited proliferative and metastatic genes. Furthermore, it induced anti-proliferation in cancer cells. The results suggested that AFE not only reduced blood glucose concentration as metformin but also showed its potential use in cancer immune chemoprevention/therapy via hypoglycemic effect, ROS scavenging, and PD-L1 suppression [191]. In addition, IPAF stimulated expressions of TLR signal-related genes and the activation of NF-κB. IPAF could induce classically activated macrophage differentiation via TLR4-dependent NF-κB activation and had potential of IPAF to modulate the Th1 response [28].

3.6 Epigallocatechin gallate

The green tea polyphenol epigallocatechin gallate (EGCG) has not shown cytotoxicity to normal cells but induces apoptosis and growth inhibition of cancer cells [192, 193]. EGCG inhibits uterine leiomyoma cell growth in vitroand in vivo. The use of a green tea extract with 45% EGCG content has demonstrated clinical activity without side effects in women with uterine fibroid symptoms [194]. However, there are several shortcomings of EGCG including low stability, poor bioavailability, and high metabolic transformations under physiological conditions. All present challenges for its development as a therapeutic agent [194].

The signal transduction pathway by which EGCG exerts cell cycle arrest and induction of apoptosis remains to be clarified. Several mechanisms of cell-cycle arrest by EGCG have been postulated [195]. Transcription factor, p53 regulates downstream genes important in cell cycle arrest, DNA repair, and apoptosis. Loss of p53 in many cancers leads to genomic instability, impaired cell cycle regulation, and inhibition of apoptosis [196]. EGCG-treated HuLM cells exhibited increased expression of several genes that represent p53 pathway such as BAX, p21, transformed 3 T3 cell double minute 2(MDM2) and tumor protein p53 inducible protein 3 (TP53I3) [30]. The NF-κB signal pathway was impaired by EGCG and the expression of bcl2A1, a key factor in NF-κB pathway, was reduced 11.8-fold in 100 μM EGCG-treated uterine fibroid HuLM cells [30] compared to untreated control.

The BCL family includes proapoptotic members and antiapoptotic members, such as BAX and BCL-2, respectively. The effects of apoptosis or anti-apoptosis supplementary depend on the balance between BCL2 and BAX rather than on the BCL2 quantity alone [197]. EGCG treatment causes BCL2 to dramatically decrease while BAX up-regulates [30]. Additionally, the D-type cyclins, through the interaction with CDKs-forming cyclin d1-CDK4/6 complexes, are mainly responsible for driving the cell cycle from G1 to S phase [198]. A significant decrease was observed in the expression of CDK4 and PCNA in EGCG treated uterine fibroid HuLM cells [30].