**9. Inhibitors of EMT**

Numerous tumor EMT inhibitors have been found in this study, and many of which are difficult to apply clinically because of stability and targeting isssues. TGF-β is an important factor in regulating EMT.

Various EMT inhibitors have been found in the TGF-β signaling pathway. Thyroid transcrip‐ tion factor-1 is a protein encoded by the NKX2-1 gene in normal tissues that can inhibit the secretion of TGF-β, which increases the expression of E-cadherin in lung cancer cells.

MMPs are other key factors in the induction of EMT. The inhibitors of MMPs play an important role in blocking EMT development in tumor cells. Several MMP inhibitors, which have been tested in clinical trials, can prevent EMT in cell experiments in vitro and inhibit tumor progression in in vivo animal experiments. Orlistat Plymouth, one of the MMP inhibitors, has a relatively significant effect on non-small cell lung cancer, colorectal cancer, and glioma, both in in vitro cell experiments and in vivo animal experiments. Conducting combination chemo‐ therapy with marimastat, captopril, and Fragmin exerts a certain effect on the treatment of advanced kidney cancer. The human body itself can also synthesize a special kind of MMP inhibitor, a tissue inhibitor of metalloproteinase (tissue inhibitors of metalloproteinases, TIMPs). TIMPs, which are produced using genetic engineering technology, can also be used as targeted drugs to treat tumors caused by the imbalance among MMPs and inhibit the progression of EMT in tumor cells.

The autocrine TGF-β/ZEB/miR-200 signaling regulatory networks that control epithelial and mesenchymal states change in the cells. ZEB1/2 and TGF-β exhibit strong correlation, and negative correlations are detected between TGF-β and miR-200, as well as between ZEB1/2 and miR-200, in invasive ductal carcinomas [88]. ZEB1/2 can induce EMT by inhibiting various

Other miRNAs can directly target EMT transcription factors. MiR-205 in mammary cells maintains epithelial differentiation [90–92]. MiR-29b in prostate cancer inhibits metastasis by regulating the EMT signal. MiR-148a in hepatocellular carcinoma (HCC) cells can negatively regulate Met/Snail signaling and prevent EMT and metastasis [93]. Snail and miR-34 form another double feedback loop. EMT is induced by TGF-β, and an increase in Snail expression can be inhibited by miR-34. A novel miR-203/SNAI1 feedback loop has also been reported in breast cancer. These double feedback loops can enhance the balance between EMT activation and control of the two states of the cell (epithelial and mesenchymal). A novel EMT network that integrates the negative feedback loops, miR-203/Snai1, and miR-200/ZEB has been proposed recently as a control epithelial cell plasticity switches during differentiation and cancer [94]. The expression of miR-10b in metastatic breast cancer cells is shown to be induced by the transcription factor Twist, which binds directly to the putative promoter of miR-10b. Twist-induced miR-10b inhibits the translation of mRNA encoding homeobox D10, which results in an increased expression of RHOC, which is a well-characterized prometastatic gene

Many miRNAs interfere with EMT by targeting the structures of cell components [96–98]. MiR-155 is the direct transcriptional target of TGF-β/Smad 4 signaling [99]. MiR-155 ectopic expression can reduce the RhoA (Ras homolog gene families, member A) protein, a small GTPase protein, to modulate the formation of tight junctions in the formation of stress fibers of the actin cytoskeleton during expression and destruction [100]. The activation of miR-31 in establishing metastases results in the regression of metastasis and the enhancement of the survival of patients. In addition, the induction of miR-31 can reduce the metastatic potential of cancer cells by targeting the RhoA [101]. The upregulation of miR-9 and direct repression of E-cadherin-1 (CDH1) in human breast cancer cells are involved in the regulation of cell-cell adhesion, migration, and the epithelial cell proliferation mechanism of calcium-dependent

Numerous tumor EMT inhibitors have been found in this study, and many of which are difficult to apply clinically because of stability and targeting isssues. TGF-β is an important

Various EMT inhibitors have been found in the TGF-β signaling pathway. Thyroid transcrip‐ tion factor-1 is a protein encoded by the NKX2-1 gene in normal tissues that can inhibit the

secretion of TGF-β, which increases the expression of E-cadherin in lung cancer cells.

protein. CDH1 repression results in increased cell motility and invasion [102].

epithelial genes [89].

228 Tumor Metastasis

[95].

**9. Inhibitors of EMT**

factor in regulating EMT.

Certain phytochemicals or food substances exhibit anti-cancer [103, 104] and anti-EMT properties [105]. For example, AIMs (anthocyanidins) and Morusin (a prenylated flavonoid), which are isolated from the fruits of *Vitis coignetiae* Pulliat (known as meoru in Korea) and the root bark of *Morus australis* (Moraceae), respectively, demonstrate anti-cancer activities by inhibiting EMT through the suppression of nuclear factor (NF)-κB activity [106–108].

AIMs can inhibit NF-κB in a dose-dependent manner, and MMP-9 (EMT marker) can be regulated by NF-κB preferentially [109]. IκBα phosphorylation and GSK-3 activity can also be suppressed by increasing the levels of AIMs [110]. Moreover, AIM downregulates mesenchy‐ mal markers, such as Vim1, N-cadherin, and SNAI, as well as upregulates epithelial markers, such as E-cadherin [110]. The morphological changes induced by TNF-α [110] are also inhibited by AIM. The suppression of the migratory and invasive properties of cervical cells by AIM has also been reported. Cervical cancer contains a heterogeneous population of cells called CSCs. CSCs are cells with chemotherapy- and radiotherapy-resistant properties and are involved in tumor recurrence, metastasis, and high mortality [111, 112]. Morusin, however, are reported to be cytotoxic to several cancer cell lines, including cervical cells. Morusin can inhibit migration and proliferation by inhibiting tumor sphere formation through the inhibition of the NF-kB pathway.

Recent studies in our laboratory have found several EMT inhibitors, including tetracycline and some natural products. The gelatinase inhibitor doxycycline is the prototypical anti-tumor antibiotic. We have investigated the effects of doxycycline on the migration, invasion, and metastasis of human lung cancer cell lines and in a mouse model. We have also measured the effect of doxycycline on the transcription of EMT markers and used immunohistochemistry to determine whether EMT reversal is associated with doxycycline inhibition. Doxycycline dosedependently inhibits the proliferation, migration, and invasion of NCI-H446 human small cell lung cancer cells. It also suppresses tumor growth from NCI-H446 and A549 lung cancer cell xenografts without altering body weight, inhibits Lewis lung carcinoma cell migration, and prolongs survival. The activities of the transcription factors Twist1/2, SNAI1/2, AP1, NF-κB, and Stat3 are suppressed by doxycycline, which reverses EMT and inhibits signal transduction, thereby suppressing tumor growth and metastasis. Our data demonstrate functional targeting of transcription factors by doxycycline to reverse EMT and suppress tumor proliferation and metastasis. Thus, doxycycline selectively targets malignant tumors and reduces their meta‐ static potential with less cytotoxicity in lung cancer patients.

Apigenin is a naturally occurring compound with anti-inflammatory, antioxidant, and anticancer properties. We have investigated the effects of apigenin on migration and metastasis in experimental HCC cell lines in vitro and in vivo. Apigenin dose-dependently inhibits the proliferation, migration, and invasion of PLC and Bel-7402 human HCC cells. It also suppresses tumor growth in PLC cell xenografts without altering body weight, thereby prolonging survival. Apigenin reduces Snai1 and NF-κB expression, reversed increases in EMT marker levels, increases cellular adhesion, regulates actin polymerization and cell migration, and inhibits invasion and migration of HCC cells. Therefore, apigenin may inhibit EMT by inhibiting the NF-κB/Snail pathway in human HCC. **Table 1** shows some EMT inhibitors found in the last 3 years.


**Table 1.** Some EMT inhibitors.
