The Cancer Prevention, Anti-Inflammatory and Anti-Oxidation of Bioactive Phytochemicals Targeting the TLR4 Signaling Pathway

Toll-like receptors (TLRs) are a well-known family of pattern recognition receptors that play an important role in a host immune system. TLR triggering leads to the induction of pro-inflammatory cytokines and chemokines, driving the activation of both innate and adaptive immunity. Recently, an increasing number studies have shown the link between TLRs and cancer. Among them, the toll-like receptor 4 (TLR4) signaling pathway is associated with inflammatory response and cancer progression. Dietary phytochemicals are potential modulators of immunological status with various pharmacological properties including anti-cancer, anti-oxidant and anti-inflammatory. Curcumin, 6-gingerol, 6-shogaol, 1-dehydro-10-gingerdione, epigallocatechin gallate (EGCG), luteolin, quercetin, resveratrol, caffeic acid phenethyl ester, xanthohumol, genistein, berberine, and sulforaphane can inhibit TLR4 activation. The aim of the present review is to describe the role of the TLR4 signaling pathway between inflammatory response and cancer progression. We further introduce bioactive phytochemicals with potential anti-inflammation and chemoprevention by inhibiting TLR activation.


Introduction
The immune system defends our body against pathogens, such as viruses, bacteria, and fungus. Monocytes, macrophages, dendritic cells (DCs), neutrophils, and natural killer (NK) cells play an important role in maintaining the innate immune system [1][2][3][4][5]. These immune cells can recognize and respond to invasion, providing efficient protection by specific receptors such as Toll-like receptors (TLRs) [6]. TLRs were named after the toll receptor of Drosophila. The Toll-like protein was shown to induce pro-inflammatory gene expression after ligation with specific pathogen-associated molecular patterns (PAMPs) [7,8]. TLRs are not only expressed in immune cells but also in epithelial cells. Tumor progression involves complex interactions between tumor cells, immune cells, and the tumor microenvironment. TLRs may also cause the development of cancers and inflammatory diseases [9][10][11]. TLRs play anti-tumor or tumor promoter roles in different cancer cells [12].
Several studies have reported that many phytochemicals have anti-inflammatory, anti-oxidation, chemopreventive and anti-cancer properties [13,14]. Recently, some phytochemicals can inhibit pattern recognition receptor (PRR) activation by targeting the receptor or the downstream signaling molecules [15]. In this review, we will discuss the different functions of TLR4 in cancer progression and inflammation. Furthermore, we also summarize and discusses the recent findings of curcumin, 6-gingerol, 6-shogaol, 1-dehydro-10-gingerdione, EGCG, luteolin, quercetin, resveratrol, caffeic acid phenethyl ester, xanthohumol, genistein, berberine, and sulforaphane in inhibiting the activation of TLR4.

Inflammation and Cancer Progression
Physical, chemical, and infectious agents can prompt inflammation. Lipopolysaccharides (LPS) are found in the cell wall of Gram-negative bacteria and can interact with the receptor of immune cells [16]. When inflammation occurs, inflammatory cytokines will be released, including TNF-α, IL-6, and IL-1 from the immune cells [17]. Inflammation can be chronic or acute depending on the characteristics of response and stimulation. Signs of acute inflammation are heat, redness, swelling and pain. Bradykinin, prostaglandins, thromboxanes, and leukotrienes are also involved in the acute inflammatory response. Arachidonic acid is released from the membrane by phospholipase A2. The released arachidonic acid is used as a substrate by the cyclooxygenase (COX) [18]. The prostanoids produced from arachidonic acid cause inflammatory and pain [18].
If the infection/stimulation is not completely cleared by the acute response or it persists for a long time, a chronic inflammation may result. It is believed that inflammation can lead to carcinogenesis [19][20][21]. Inflammation is known to contribute to carcinogenesis by generation of reactive oxygen species (ROS) that can damage DNA, genomic aberrations, and carcinogenesis. It is now established that chronic infection by bacterial or virus infection cause increasing cancer risk. For example, chronic Helicobacter pylori infection is associated with gastric cancer and mucosa-associated lymphoid tissue lymphoma (MALT) [22][23][24]. Chronic hepatitis B virus (HBV) infection increase the risk of hepatocellular carcinoma [25,26]. Tumor-promotion is associated with production of cytokines by immune/inflammatory cells that activate transcription factors, such as NF-κB (nuclear factor kappa B), STAT3 (Signal Transducer and Activator of Transcription 3), and AP-1 (activator protein 1). These transcription factors will upregulate the COX-2/PGE2 (prostaglandin E2) signal pathway in inflammation and cancer cells [27,28]. Inhibition of transcription factors and inflammatory cytokine expression can decrease tumor development and progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) are investigated for the prevention of cancer progression and metastasis particularly in the case of colon cancer. However, long-term NSAIDs treatment develops peptic ulcers [29,30]. Many phytochemicals with anti-inflammatory effects are further examined for their anti-cancer properties.

TLR4, NF-κB in Inflammatory Process and Tumorigenesis
The immune system can control tumor progression and growth. Chronic inflammation and infection can contribute to the development of tumors. TLRs are potent activators of inflammatory response. TLRs activation can result in the production of cytokines, chemokines and interferons and transcription factor NF-κB. NF-κB pathways plays an important role in various diseases by regulation of immunity, proliferation, differentiation, and apoptosis. The NF-κB family consists of five important members including c-Rel, p50, p52, p65/RelA, and RelA. NF-κB is a transcription factor that binds to DNA and activates gene transcription. NF-κB is bound to I kappa B (IκB) in the cytoplasm without stimulation. A variety of stimulation such as ROS and inflammatory cytokines will activate NF-κB by degrading the IkB complex. NF-κB is translocated into the nucleus and binds to DNA and activates the transcription. The role in carcinogenesis depends on the activation of NF-κB and production of related cytokines such as IL-1, IL-2, IL-6, IL-10 and TNF-α [52]. TNF-α and interleukins can be regulated by the NF-κB transcription factor. This will suppress apoptosis, induced cellular proliferation, invasion, metastasis, chemoresistance, and inflammation. It is believed that TLRs are involved in tumor growth and development. However, activation TLRs can have anti-or pro-tumoral effects in tumor growth or metastasis in different cancer cells (Table 1) [12].
Tumorigenesis is a multistep process that can be activated by various factors such as environmental carcinogens, inflammatory mediators, and tumor promoters. The role of TLR4 in tumor progression has been described in many studies. The activation of TLR4 increases IL-8 and IL-6 production in breast cancer [54]. In addition, TLR4 activation increases expression of VEGF and TGF-β1 in prostate cancer cells, which promote tumor development [55]. In colon cancer, the TLR4 stimulation induce nitric oxide and IL-6 production [56]. Moreover, studies also shown that TLR4 overexpression is associated with poor outcome in colon and breast pancreatic cancer [54,56]. The research indicated that the MyD88 pathway of TLR-4 promoted carcinogenesis. However, studies also shown that TLR4 displayed anti-tumor activity in skin cancer [57]. The role of TLR4 should be further examined in the different tumor types. All the above suggest that the release of various cytokines, inflammatory mediators, and chemokines active TLR4 and it could contribute to cancer formation.

Bioactive Phytochemicals Targeting TLR4 and Oxidative Stress
It is well known that plants widely exert various biological properties including anti-inflammatory and anti-cancer effects. The chemical structure of TLR4 antagonist from plants is not similar to LPS. The phytochemicals that inhibit TLR4 activation are from the results demonstrating that phytochemicals decreased LPS-induced expression of cyclooxygenase-2, NF-κB and cytokine production. The pharmacological activity of phytochemicals against inflammation by targeting the TLR4 signaling pathway in many studies have been previously described [58][59][60][61][62].
Free radicals, such as hydroxyl, superoxide alkoxyl, and peroxyl (RO2 • ) radicals, are molecules with unpaired electrons in the outer orbit and are generally unstable and reactive. Excessive levels of ROS directly cause cell membrane, DNA, and protein damage. It will result in inflammation or mutation. The human body has defense mechanisms against ROS-induced damage using enzymes such as catalase and glutathione peroxidase. Excessive levels of ROS can result in adverse effects such as artherosclerosis, inflammation and cancer. A lot of studies have shown that dietary plants have natural antioxidants and intake of these antioxidants can remove free radicals and prevent some diseases. Growing in vitro and in vivo studies have shown that chemopreventive agents can enhance or regulate different signaling pathways such as PI3/AKT, NF-κB, COX-2, apoptotic, cell cycle for the treatment or prevention of different cancer cells.

Zingiberaceae Family
Curcuma longa (turmeric) and Zingiber officinale (ginger) belong to Zingiberaceae family and were used for different purposes for over a thousand years [63,64]. We will introduce the active components of turmeric and ginger.

Curcumin
Turmeric is used in traditional medicine especially in China and India. The polyphenol curcumin is from rhizomes of Curcuma longa and widely used as a food flavoring. Curcumin has many pharmacological properties including anti-oxidant, anti-inflammatory, anti-cancer, antiproliferative, neuroprotective, hepatoprotective, immunomodulatory and chemopreventive effects [65,66]. Curcumin is a hydrophobic molecule and practically insoluble in water. The half-life of curcumin is 10 min in phosphate buffer at physiological pH 7.4 [67]. The curcumin of plasma level is very low even at high dose intake. It has been shown to exhibit anti-inflammatory effects by down-regulating cytokines, such as TNF-α, IL-1, IL-6, IL-8, IL-12, MCP-1, IL-1β, and transcription factors. Studies have shown that curcumin can compete with LPS for TLR4 and inhibition in MyD88-dependent pathway [68][69][70]. Previous studies have shown that curcumin can inhibit LPS-induced inflammation in vascular smooth muscle cells in TLR4-MAPK/NF-κB pathways by blocking ROS production [69]. These studies have shown that curcumin-decreased cytokine production includes TNF-α, IL-6 and IL-1β. Curcumin improves TNBS-induced colitis in rats via the TLR4/NF-κB signaling pathway [71]. Curcumin also possesses neuroprotection activity. Curcumin administration can reduce activation of microglia/macrophages and neuronal apoptosis through a mechanism involving the TLR4/MyD88/NF-κB signaling in experimental traumatic brain injury [70]. Recently, a study has shown that curcumin inhibits DAMP molecule HSP70 and TLR4 signaling in liver cancer cells [72].

Ginger
Ginger is popularly used as spice and traditional medicine in China and Asian countries. It was used in traditional medicine in the treatment of various diseases such as nausea, vomiting, abdominal pain and muscle discomfort. Gingerols, shogaols, gingediols, zingerone, dehydrozingerone, gingerinone, and diarylheptanoids are extracted from rhizomes of ginger. These active constituents of ginger have many pharmacological properties including anti-inflammatory, analgesic, anti-oxidation, and anti-cancer effects [73][74][75].

EGCG
Tea has been popular in China and Asia for nearly five thousand years. Green tea is prepared by steaming freshly harvested leaves. There are many bioactive compounds extracted from Camellia sinensis including polyphenolic substances such as epicatechin-3-gallate (ECG), epigallocatechin (EGC), epigallocatechin-3-gallate (EGCG), and epicatechin (EC). Polyphenols are one of the biggest class of phytochemicals. Polyphenols can divide into two groups: flavonoids and non-flavonoids. The polyphenols chemical structure shares basic polyphenolic structure with a single phenol ring, including phenolic acids and phenolic alcohols. These active bioactive compounds have many pharmacological effects including anti-oxidant, anti-inflammatory and anti-cancer properties [86,87]. It has been previously reported that EGCG has anti-invasive effects and inhibits activation of NF-κB and AP-1 in ECV304 human endothelial cells [88]. Furthermore, EGCG can decrease inflammatory gene expression including COX, NO synthase, and TNF-α [89]. A study also has shown that EGCG inhibits MyD88-dependent signaling pathways and TIR domain-containing adaptor inducing IFN-β (TRIF)-dependent signaling pathways of TLRs in RAW264.7 cells [90]. 67-kDa laminin receptor (67LR) is a nonintegrin cell-surface receptor. The role of 67LR is cell adhesion to the basement membrane and the metastasis of cancer cells. A study has shown that 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor and mediates the anti-cancer effects. EGCG can upregulate Tollip (Toll-interacting protein) and down-regulate of TLR4 expressions via 67LR may be effective in the anti-cancer activity [91,92].

Resveratrol
Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a natural stilbene found in peanuts, grapes, blueberries, rhubarb and wine. Resveratrol has many pharmacological properties including anti-inflammatory, chemopreventive, anti-cancer, cardioprotective, neuroprotective and hepatoprotective properties [98]. Resveratrol induced apoptosis through p53 dependent pathway [99]. Resveratrol decreased the expression of inflammatory markers as COX2, iNOS and NF-κB activation [100]. Several studies have shown resveratrol regulate the expression of TLR4. Therefore, resveratrol can be used for TLR-mediated inflammatory responses and chronic diseases associated with TLR activation. A study indicated that resveratrol decreased NF-κB activation and COX-2 expression in LPS-induced RAW264.7 and inhibited TBK1 and RIP1 in TRIF complex in MyD88-independent signaling pathways [101]. Resveratrol also decreases LPS-induced pro-oxidant effect in AR42J cells via a Myd88-dependent signaling pathway [102]. Together, these results demonstrate that resveratrol displays anti-inflammatory effects in Myd88-dependent signaling pathway or Myd88-independent signaling pathway in different experimental models. Additional study shown that resveratrol can reduce LPS-induced inflammatory responses and the down-regulation of NF-κB activity in human colon cancer cells [103]. Recently, a study further showed that resveratrol has anti-inflammatory effects by attenuating TLR4-TRAF6, MAP kinase and AKT pathways in LPS-induced macrophages [104].

Prospective and Conclusions
Toll-like receptors play an important role in innate immunity. TLRs may serve as a double-edged sword in prompting cancer tumorigenesis, tumor growth, inducing apoptosis, or inhibiting tumor progression in different kinds of cancer cells or resistance to chemotherapy. Much evidence has shown that the activation of TLR4 results in inflammation and carcinogenesis. Recent studies have shown that the anti-inflammatory effects of bioactive compounds may be due to inhibition of the TLR4 pathway through MyD88-and TRIF-dependent signaling pathways. In this review article, we summarize the pharmacological properties of curcumin, 6-gingerol, 6-shogaol, 1-dehydro-10-gingerdione, EGCG, luteolin, quercetin, resveratrol, caffeic acid phenethyl ester, xanthohumol, genistein, berberine, and sulforaphane in interfere with TLR4 (Table 2). These compounds reduce the activation of the TLR4 signal pathway in a MyD88-dependent or TRIF-dependent manner. Phytochemicals interacting with TLR4 could be alternatively considered for the development of new chemoprevention because they have multiple cellular target and pharmacological properties. As we previously described, TLR4 can have pro-or anti-tumor effects in different tumor growth, progression, invasion and metastatic processes. Many pharmaceutical companies are developing TLR4 antagonists or agonists for the treatment of cancers and inflammatory diseases. Most design strategies of TLR4 antagonists use the native ligand as inspiration. Eritoran is a TLR4 antagonist and its chemical structure is similar to LPS [128]. In the future, different TLR agonists and antagonists should be low molecules, improving drug absorption, and more effectively inhibiting or stimulating TLR activity in the human body for targeting various inflammatory conditions including chemoprevention. Table 2. Chemical structure and molecular targets by phytochemicals.

Compound
Chemical Structure Molecular Targets   Curcumin  Table 2. Chemical structure and molecular targets by phytochemicals.