# Research Advances in Antitumor Mechanism of Evodiamine

Authors: Luning Li; Cunxin Zhang; Chen Huang; Xinchen Tian; Wenxue Sun; Shulong Jiang
Journal: Journal of Chemistry
 (2022)

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## Abstract

Evodiamine is a natural alkaloid extracted from Fructus Evodia. This bioactive alkaloid has been reported to have a wide range of biological activities, including anti-injury, antiobesity, vasodilator, and anti-inflammatory effects. In recent years, it has been found that evodiamine has tumor-suppressive effects on a variety of tumors. There is growing evidence that evodiamine can inhibit the rapid proliferation of tumor cells, induce cell cycle arrest at a certain phase, increase the incidence of apoptosis, promote autophagy, inhibit microangiogenesis and migration, and regulate immunotherapy. Evodiamine can inhibit Wnt/β-catenin, mTOR, NF-κB, PI3K/AKT, JAK-STAT, and other signaling pathways in various cancer cells, and it can significantly downregulate the expression of many tumor markers, such as VEGF and COX-2. These facts partially explain the antitumor mechanism of evodiamine. In this article, the antitumor mechanism of evodiamine was reviewed to provide the basis for its clinical application and therapeutic development in the future.


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## Body

## 1. Introduction

The traditional Chinese medicineFructus Evodia is the dry fruit of the plant Evodia rutaecarpa, and it was first mentioned in “Shennong Materia Medica Classic.” It has been found that E. rutaecarpa has analgesic, antidiarrheal, antiemetic, and anti-inflammatory effects in clinical practice [1–5]. The dried, powdered fruit (2.5 kg) of E. rutaecarpa was defatted with n-hexane (2 × 5 L) and extracted with acetone (3 × 5 L) at room temperature to yield an extract (137 g). The crude acetone extract was then eluted with n-hexane: acetone (9 : 1–19 : 1) on a silica gel column to produce evodiamine (6.17 g) [6]. Evodiamine was also purified from Fructus Evodia by a high-performance liquid chromatography method with a gradient elution of acetonitrile-tetrahydrofuran-0.02% phosphoric acid at the detection wavelength of 220 nm [7, 8].Previous studies have shown that evodiamine has many biological activities, including anti-inflammation [9–14], antiobesity [12], analgesia [14], and promotion of vasodilation [15–18]. These activities are related to a wide range of targets, such as caspase 3 and transient receptor potential vanilloid 1 (TRPV1) [19–22]. In recent years, evodiamine has been found to have antitumor activity against a variety of tumor cells, involving various mechanisms that cause cancer cell death, such as induction of cell cycle arrest, promotion of cell apoptosis, promotion of autophagy, and inhibition of tumor invasion and metastasis [23–30] (Figure 1). The signaling pathways involved in the antitumor effect of evodiamine include the classical apoptosis pathway, endoplasmic reticulum stress, MAPK, PI3K-Akt, and JAK-STAT [31–34]. In this article, the antitumor mechanism of evodiamine was reviewed based on research trends in recent years, and the ways through which evodiamine could exert its antitumor effect were summarized to provide a basis for its clinical application and in-depth research.Figure 1
Synopsis of the multifactorial mode of action of evodiamine-type drugs.


## 2. Molecular Mechanisms of Antitumor Effect of Evodiamine

### 2.1. Inhibition of Tumor Cell Proliferation and Cell Cycle

There is evidence that evodiamine reduces the occurrence of tumors by inhibiting the proliferation of cancer cells and changing the cell cycle. Previous studies have shown that evodiamine can inhibit the proliferation of human cancer cell lines, such as breast cancer [35], colon cancer [36], liver cancer [37, 38], cholangiocarcinoma [27], osteosarcoma [39], and melanoma [40] in a dose- and time-dependent manner (Table 1). One of the distinguishing features of tumor cells is their dysregulation of the cell cycle caused by the abnormal expression of cyclins and/or the abnormal replication of DNA. The activity and expression states of CDC25 C, cyclin, and cyclin-dependent kinases (CDKs) regulate the stability of the cell cycle. Many studies have shown that arresting tumor cell cycle progression is an effective strategy to inhibit tumor proliferation [41–45].Table 1
Inhibition of tumor cell activity and proliferation by evodiamine in cancer cell lines.
Cell lineCell typeSpecific resultsMode of actionReferencesMCF-7/ADRBreast cancerThe IC50 values of MCF-7 cells treated with evodiamine for 24, 48, and 72 h were 7.68 μM, 0.64 μM, and 0.30 μM, respectively. The IC50 values of the MCF-7/ADR cells were higher than those of MCF-7, with values of 24.47 μM, 1.26 μM, and 0.47 μM for 24, 48, and 72 h of treatment with evodiamine, respectively.Inhibiting cell activity via the regulation of Ras/MEK/ERK pathway[35]HCT-116/L-OHPColorectal cancerThe cells were treated with evodiamine at 0.4, 0.8, and 1.6μM for 24, 48, and 72 h, respectively. Evodiamine significantly enhanced rhodamine 123 accumulation and caused a significant decrease in the IC50 level in HCT-116/L-OHP cells.Inhibiting the p50/p65 NF-kappaB pathway[36]HepG2, Huh-7, and Hep3BHepatocellular carcinomaThe cells were treated with evodiamine at 0.25, 0.5, 1, 2, 4, 6, and 8μM for 72 h. Evodiamine inhibited the proliferation of HCC cells in a dose-dependent manner under hypoxia (survival fraction: 1.0–0.3).Downregulating HIF-1α under hypoxia[37]HepG2 and Bel-7402Hepatocellular carcinomaEvodiamine treatment at a range of concentrations (0, 0.25, 1, 4, 8, 16, and 32μM) inhibited HepG2 cells and Bel-7402 growth. The IC50 values of HepG2 and Bel-7402 cells at 48 h were 14.7 μM and 16 μM, respectively.Affecting the Hippo-Yes-associated protein signaling pathway[38]HuCCT-1 and TFK-1CholangiocarcinomaTwo CCA cell lines, HuCCT-1 and TFK-1, were treated with evodiamine at different concentrations (0, 5, 10, 20, or 40μM) for different time periods (24, 48, or 72 h). Cell viability: 80%–20% (HuCCT-1); 75%–25% (TFK-1).Inducing IL-6/STAT3 signaling inhibition.[27]143B and MG63Osteosarcoma143B cells were treated with evodiamine at 0.5, 1, 2, 4, 6, 8, 16, and 32μM. MG63 cells were treated with evodiamine at 0.25, 0.5, 1, 2, 4, 6, 8, and 16 μM. Cell viability was measured at 24, 48, and 72 h. Relative growth rate: 95%–15% (143B); 98%–30% (MG63).Inhibiting Wnt/beta-catenin signaling pathway[39]A-375MelanomaThe cells were treated with evodiamine at 5, 7.5, 10, 12.5, and 15μM for 24, 48, and 72 h. Relative cell viability: 1.0–0.2.Inducing reactive oxygen species-dependent apoptosis and necrosis[40]Previous studies have reported that evodiamine may block the cell cycle progression and significantly increase the percentage of G2/M phase cells in tumors (Table2). Zhou et al. found that evodiamine affected the cell cycle progression in human osteosarcoma cells U2OS. The same authors reported that the expression levels of cyclin B1, CDC25 C, and CDC2 changed significantly, which may be related to evodiamine-dependent downregulation of the phosphorylation of MEK and ERK and inhibition of the Raf/MEK/ERK signaling pathway [46]. Moreover, in human lung cancer cells with G2/M phase arrest after evodiamine treatment, cyclin A and cyclin B1 expression levels decreased, whereas those of p-Chk1 and p-Chk2 increased [47]. In addition, evodiamine interferes with the colorectal cancer cell lines COLO205 and HT-29, causing the cell cycle to stagnate in the G2/M phase, which has been shown to be related to changes in cyclin B1/CDC25 C expression and is controlled by JNK [48]. According to the above studies, evodiamine induces the G2/M cycle arrest by affecting signaling factors involved in multiple signaling pathways regulating cyclins, CDKs, and CHK.Table 2
Induction of cell cycle arrest by evodiamine in cancer cell lines in vitro.
Cell lineCell typeSpecific resultsMode of actionReferencesU2OSOsteosarcomaEvodiamine treatment at a range of concentrations (0, 3, 6, and 12μM) triggered G2/M cell cycle arrest (9%–77%) for 24 h.Inhibiting the Raf/MEK/ERK signaling pathway[46]A549Lung cancerEvodiamine treatment at a range of concentrations (0, 1, 2, and 4μM) triggered G2/M cell cycle arrest (15.8%–86.2%) for 24 h.Downregulation of ERK[47]COLO205 and HT-29Colorectal carcinomaEvodiamine treatment at a range of concentrations (0, 1, 2, and 4μM) triggered G2/M cell cycle arrest for 24 h; evodiamine treatment at 2 μM triggered G2/M cell cycle arrest for 6, 12, and 24 h.JNK activation[48]MDA-MB-231Breast cancerExposure to different concentrations of evodiamine (0, 15, 30, 60, 90, and 120 µM) caused G0/G1 arrest of MDA-MB-231 (53.29%–60.12%) for 24 h.Decreasing Bcl-2, cyclin D1, and cyclin-dependent kinase 6 expression[49]MCF-7 and MDA-MB-231Breast cancerCell cycle analyses in monolayer culture (condition with FBS, without FBS), sphere culture condition. Evodiamine (0, 100, and 200 nM) mildly but significantly increased the proportion of cells at the G1 stage.Activating the p53-p21-Rb pathway[50]Evodiamine may also cause some tumor cells to stagnate at the G0/G1 phase (Table2). Interestingly, Du et al. found evidence of evodiamine-induced downregulation of cyclin D1 and CDK6 in human breast cancer MDA-MB-231 cells; these cells stagnated at the G0/G1 phase in the presence of evodiamine treatment [49]. In another study, after using evodiamine, the cells were seeded in the upper well of a Transwell and incubated with 10 µg/mL PGI/AMF and 10 µg/mL PGI/AMF +6 µmol/L evodiamine for 28 h. Normal cells served as the control group. We then used these cells to treat human breast cancer MCF-7 and MDA-MB-231 cells, and tumor cells were arrested at the G0/G1 phase; p53 and p21 were upregulated; and p-Rb, cyclin B, and cyclin A were downregulated [50]. Based on these results, evodiamine keeps the cell cycle in the G0/G1 phase by targeting the p53-p21-Rb signaling pathway and affecting the expression of G0/G1 phase cyclin-related proteins.


### 2.2. Promotion of Apoptosis in Tumor Cells

Apoptosis is a very important and highly conservative cell death model that can effectively inhibit tumorigenesis. The classical process of cell apoptosis involves caspases as the core proteins that activate other proenzymes in a sequence, which involves multiple signaling pathways leading to cellular apoptosis [51–55]. The promotion effects of evodiamine on apoptosis signal transduction pathways can be divided into extrinsic and intrinsic categories. The extrinsic pathway involves the activation of the death receptor pathway, whereas the intrinsic pathway includes the activation of the mitochondrial and endoplasmic reticulum pathways.Evodiamine induces the expression of death receptors on the surface of tumor cells and promotes the binding of death receptors to their ligands, thereby accelerating the formation of DISC and leading to apoptosis (Table3). According to Khan et al., evodiamine can upregulate the death receptors DR4 and DR5 in U87 glioblastoma cells under the synergistic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which leads to the activation of caspases 3 and 8 and the induction of apoptosis in tumor cells [56].Table 3
Induction of apoptosis and autophagy by evodiamine in cancer cell lines in vitro.
Cell lineCell typeSpecific resultsMode of actionReferencesU87GlioblastomaThe cells were treated with evodiamine (10 µM) and TRAIL (50 ng/ml) either separately or in combination for 24 h. TRAIL alone did not induce apoptosis, whereas evodiamine significantly induced apoptosis in U87 glioblastoma cells after 24 h (apoptosis rate: 35%–70%).Sensitizing cells to TRAIL via the death receptor pathway[56]A549 and H1299Lung epithelial cancerEvodiamine (20–40 µM) treatment of A549 cells for 24 h significantly increased the number of apoptotic cells by sixfold.Activating both the intrinsic and the extrinsic apoptosis pathways[57]U937LeukemiaThe cells were treated with evodiamine (0.2–0.8 µmol/L) for 18 h. Evodiamine induced apoptosis in a dose-dependent manner (sub-G1 population: 5%–45%).Activating caspase-dependent and caspase-independent pathways[58]MC3 and HSC4Oral cancerThe cells were treated with various concentrations of evodiamine (0.25, 0.5, and 1μM for MC3 cells; and 0.2, 2, and 20 μM for HSC4 cells) for 24 h. The apoptotic effect of evodiamine was confirmed by 4′-6-diamidino-2-phenylindole (DAPI) staining. Evodiamine-treated cells displayed nuclear condensation and fragmentation—hallmarks of apoptosis.Inhibiting the AKT pathway[59]H446 and H1688Small cell lung cancerThe induction of apoptosis was detected in H446 or H1688 cells after 24 h of treatment with 10μM evodiamine. The apoptosis rate of evodiamine-treated H446 (∼15%) or H1688 (∼11%) cells was much higher than that of the untreated cells (blank control, ∼5% or ∼4%).Activating mitochondria-dependent and endoplasmic reticulum stress-induced pathways[60]U87-MGGlioblastomasThe percentage of autophagy induced by evodiamine (0, 0.1, 6, 10μM) increased to a maximum at 24 h, but not at 48 h, indicating that evodiamine-induced autophagy in a dual-regulated manner.Activating calcium/c-Jun N-terminal kinase and calcium/mitochondria-mediated pathways[61, 62]Panc-1 and SW1990Pancreatic cancerPANC-1 and SW1990 cells were treated with evodiamine (1.0, 5.0, and 10 µM) for 48 h. The exposure of cells to evodiamine led to a dose-dependent increase in apoptosis. Apoptosis rate: 2.5%–17.5% (Panc-1); 4%–18% (SW1990).Inhibiting the PI3K-Akt and the MAPK/ERK signaling pathways[63]Murine Lewis lung carcinomaLung carcinomaThe cells exhibited an increase in MDC fluorescence within 1.5–6 h after evodiamine treatment, while the peak of autophagy activity was observed in 1.5 h treatment. In addition, evodiamine-treated cells displayed a greater number of distinct spots within the cytoplasm or perinuclear regions compared with controls.Activating caspase-independent pathways[64]Evodiamine can transform the tumor cells’ mitochondrial membrane into a state of increased permeability, thereby causing the irreversible release of various apoptosis promoters, including cytochrome C and AIF, into the cytoplasm or the nucleus and promoting the occurrence of apoptosis (Table3). Mohan et al. found that evodiamine significantly increased the mitochondrial membrane depolarization, increased the cell apoptosis rate, and slightly decreased the Bcl-2/Bax ratio in human lung cancer A549 and H1299 cells. It also increased the release of cytochrome C from mitochondria into the cytoplasm, thereby activating the cascade of proapoptotic pathways, including caspases 3 and 9 [57]. In another study, evodiamine was found to promote the mitochondrial release of AIF into the nucleus in human leukemia U937 cells, leading to chromosome agglutination and the induction of cellular apoptosis [58]. In addition, evodiamine can also promote mitochondrial apoptosis of tumor cells by suppressing the expression of the inhibitor of apoptosis proteins. Mcl-1 is an apoptosis inhibitor protein located on the mitochondrial membrane. Sachita et al. found that evodiamine reduced the expression of Mcl-1 during transcriptional modification, and knockout of Mcl-1 significantly increased the expression of active Bax, thereby inducing apoptosis [59]. These studies have shown that evodiamine can effectively regulate the ratio of proapoptotic and antiapoptotic factors to promote changes in mitochondrial membrane that are conducive to apoptosis and promote the occurrence of mitochondria-dependent apoptosis.Endoplasmic reticulum stress occurs when proteins are disrupted during transport from the endoplasmic reticulum (ER) to the Golgi apparatus. When newly synthesized or when the Ca2+ equilibrium state is broken, unfolded or misfolded proteins accumulate in large quantities in the ER. Fang et al. treated human small cell lung cancer H446 and H1688 cells with evodiamine and found excessive accumulation of reactive oxygen species (ROS), which caused endoplasmic reticulum stress and released a large amount of Ca2+. Furthermore, the upregulation of caspase-12 led to the upregulation of caspase-9 and caspase-3 expression, which in turn led to apoptosis [60]. Therefore, evodiamine can disrupt the Ca2+ balance in cells and induce ER stress by activating caspase-12 and other signaling pathways, thereby activating the ER-mediated apoptosis pathway (Table 3).


### 2.3. Induction of Autophagy of Tumor Cells

Autophagy is a process of lysosome phagocytosis and degradation of its own structure, which can remove damaged cellular structures and senile organelles. This process exists in most eukaryotic cells [65–67]. Autophagosome formation and processing of microtubule-associated protein 1 light 3 (LC3) are two major signs of autophagy.The role of autophagy in tumors is controversial. In fact, it may play different roles at different stages of tumor genesis and development [68–73]. Evodiamine induces protective autophagy mediated by the activation of the JNK pathway following extracellular Ca2+ influx. Liu et al. found that evodiamine could induce autophagy in human glioblastoma U87-MG cells. The application of an extracellular calcium scavenger and an antagonist of transient receptor potential vanillin-1 (TRPV-1) reduced the percentage of cells undergoing autophagy. The same phenomenon was confirmed by knocking down the expression of TRPV-1 by small-interfering RNA technology. Moreover, the activation of c-Junn terminal kinase (JNK) by evodiamine was decreased by the TRPV1 antagonist, which further confirmed that evodiamine can promote autophagy through the Ca2+-mediated JNK pathway [61].Although there have been many reports that evodiamine can promote autophagy in tumors, some studies have shown that evodiamine-related induction of autophagy in specific cells antagonizes apoptosis. This observation illustrates the complexity of the antitumor mechanism of evodiamine. Hong et al. found that evodiamine-induced apoptosis of prostate cancer (PC) cells by inhibiting the PI3K-Akt and MAPK/ERK signaling pathways in human pancreatic cancer Panc-1 and SW1990 cells, and by inhibiting the phosphorylation of signal transduction and transcriptional activator 3 to inhibit autophagy [63]. Tu et al. found that evodiamine could improve the expression level of the autophagy-specific genes (Atgs) in Lewis lung cancer cells, accelerate the transformation from LC3-I to LC3-II, and thus promote the formation of autophagosomes (Table 3). Moreover, when combined with the autophagy inhibitor 3-methyladenine (3-MA), autophagy induced by evodiamine was significantly reduced, while apoptosis was increased. This suggests that autophagy induced by evodiamine may have a protective effect on tumor cells and that the inhibition of autophagy can promote the occurrence of apoptosis [64]. Liu et al. further found that evodiamine could induce autophagy mediated by calcium/JNK signals in glioma cells, while treatment with Ca2+ scavenger BAPTA-AM significantly inhibited the activation of the intracellular JNK pathway and decreased autophagy, while apoptosis was increased [62]. These results confirm that evodiamine can promote Ca2+-mediated autophagy in the JNK pathway and that the inhibition of autophagy induced by this pathway can reduce the activity of tumor cells and promote apoptosis.


### 2.4. Inhibition of Tumor Microangiogenesis and Tumor Migration

Vascular endothelial growth factor (VEGF), one of the most important growth factors, plays a key role in promoting angiogenesis (Table4). It can promote mitosis and has antiapoptotic effect on endothelial cells; moreover, it increases vascular permeability and promotes cell migration. Because of these effects, VEGF actively regulates the angiogenesis of normal and pathological blood vessels [78–80]. VEGF and VEGF receptors (VEGFRs) are expressed on both endothelial and nonendothelial cells [81, 82]. Currently, the use of anti-VEGF and anti-VEGF receptor therapies to block angiogenesis in cancer or other pathological processes is considered extremely important.Table 4
Inhibition of microangiogenesis and migration by evodiamine in cancer cell lines in vitro or in vivo.
Cell lineCell type/animal modelsSpecific resultsMode of actionReferencesHepG2, SMMC-7721, H22Hepatocellular carcinoma BALB/c nude mice(1) To evaluate the effect of evodiamine on VEGF-induced angiogenesis, Matrigel plug assays were performed with C57BL/6 mice. The addition of evodiamine (200μg) to the plugs containing VEGF inhibited vascular formation. These plugs displayed a paler appearance, and fewer blood vessels were observed.(2) HepG2 cells and SMMC-7721 cells were exposed to 0, 5, 10, or 20 μmol/L of evodiamine for 24 h. Representative images of cell migration and invasion were assessed with Transwells.Inhibitingβ-catenin and reducing VEGFa expression[74]PC-3 and DU145Prostate cancerPC-3 and DU145 cells were treated with 5 µM evodiamine for 24, 36, and 48 h. Evodiamine substantially downregulated the expression of VEGF.Inhibiting the c-Met/Src/STAT3 signaling pathway[75]HT-29 and HCT-116Colorectal cancer BALB/c nude miceAfter treatment with different concentrations of evodiamine (0 or 1.5μmol/L) for 24 h, the migration rates were detected. Wound-healing assay showed that treatment with evodiamine (1.5 μmol/L) significantly repressed the migration of HT-29 cells and HCT-116 cells.Activating Sirt1[76]HCT-116Colorectal cancerMigration potential was assessed by Transwell assay after the cells had been treated by agents. The cells were seeded in the upper well of a Transwell insert, incubated with 10 µg/mL PGI/AMF and with 10 µg/mL PGI/AMF +6 µmol/L evodiamine for 28 h. Normal cells served as the control group. Evodiamine significantly inhibited PGI-induced migration in HCT-116 cells.Inactivating the JAK2/STAT3 pathway through the downregulation of PGI[77]Hepatocellular carcinoma (HCC) is a highly vascularized tumor, with high microvascular density and high levels of circulating VEGF. Shi et al. found that after the intervention with evodiamine in the subcutaneous H22 cell xenograft model, tumor growth was inhibited, and serum tumor markers,β-catenin, and VEGFa levels were significantly reduced. Evodiamine also blocked angiogenesis in the matrigel plug assay by inhibiting VEGF. In addition, evodiamine inhibited tumor growth and reduced the expression levels of various angiogenesis biomarkers, β-catenin, and VEGFa in the SMMC-7721 HCC cell xenograft model. Evodiamine was shown to have an antitumor effect on HCCs by inhibiting β-catenin, interacting with VEGFa, and reducing VEGFa expression, thereby inhibiting angiogenesis [74]. These results suggest that evodiamine can inhibit cell invasion and migration, block angiogenesis, and act as a potential therapeutic agent for HCC.Signal transduction and activator of transcription (STAT) is activated in many human tumor cell lines and primary tumors. STAT3 plays an important role in angiogenesis in the tumor microenvironment and can regulate the expression of VEGF, matrix metallopeptidases (MMP), and other factors [83–86]. Hwang et al. found that treatment with evodiamine in PC-3 and DU145 cells significantly inhibited cell proliferation, reduced HGF-regulated c-Met/Src/STAT3 phosphorylation, and disrupted nuclear transfer of STAT3 protein. That study also revealed that evodiamine downregulated the expression of several carcinogenic markers, including COX-2, VEGF, and MMP-9. In addition, reduced Src/STAT3 activation was observed in PC-3 and DU145 cells transfected with c-Met small-interfering RNA (siRNA), resulting in reduced evodiamine-induced apoptosis [75] (Table 4). These results indicate that evodiamine can markedly inhibit the activation of the c-Met/Src/STAT3 signaling pathway, thereby inhibiting tumor cell survival, proliferation, and angiogenesis.Metastasis is one of the main hazards and characteristics of malignant tumors, and is a key factor affecting the prognosis and survival status of patients. Matrix metalloproteinases are a large class of zinc-containing endopeptidases that have important biological functions in various events, including metastasis of cancer cells [87–90]. Zhou et al. found that evodiamine inhibited the expression of MMP-9 in vitro and in vivo by inhibiting the acetyl-NF-κB p65 subunit induced by Sirt1 in colorectal cancer HT-29 and HCT-116 cells, and thus inhibited the invasion and metastasis of the tumor cells [76]. Zhao et al. also found that evodiamine inactivated the JAK2/STAT3 signaling pathway by downregulating the expression of phosphoglucose isomerase (PGI), thereby downregulating the expression of MMP3 to inhibit the migration of human colorectal cancer HCT-116 cells [77] (Table 4). According to these studies, evodiamine has a significant inhibitory effect on MMPs, the key regulatory enzymes essential for tumor migration, which makes it suitable for further study as a lead compound for the inhibition of tumor migration.


### 2.5. Antitumor Immunotherapeutic Mechanism of Evodiamine

Inhibitory receptors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD-1), play crucial roles in the inactivation of immune cells in the tumor microenvironment. The activation, amplification, and effector function of CD8+ T cells are significantly inhibited by the interaction between PD-1 and programmed death ligand 1 (PD-L1), which also assists in the immune escape of cancer cells [91–93]. In addition, the interaction between the microenvironment and immune regulation plays a critical role in clinical treatment. It is widely believed that activated CD8+ T cells have anticancer immunity in a variety of tumors and have a significant positive prognostic effect. Furthermore, the survival status of cancer patients is significantly affected by tumor-infiltrating CD8+ lymphocytes (TILs) in the tumor environment, further supporting the strong link between immune escape and the tumor microenvironment [94]. MUC1-C is highly expressed in a variety of tumor cells and regulates a variety of genes, including PD-L1, that are important for the immune escape of tumor cells. Jiang et al. found that, after acting on non-small cell lung cancer cells, evodiamine downregulated the expression of the MUC1-C/PD-L1 signaling pathway at both the transcription and protein levels, reduced the apoptosis of T cells, and significantly increased the level of CD8+ T cells. Evodiamine may therefore be a potential targeted therapy suitable as an adjuvant for immunotherapy [94] (Table 5). By acting on PD-L1 and other inhibitors that suppress T-cell activation, evodiamine improves the tumor microenvironment, increases the activation level of CD8+ T cells, and effectively inhibits immune escape, which has the potential to promote the efficacy of immunotherapy.Table 5
Antitumor immunotherapeutic mechanisms and antitumor drug combinations involving evodiamine.
Cell lineCell type/animal modelsSpecific resultsMode of actionReferencesH1650 and H1975Non-small cell lung cancerEvodiamine (0, 2, 4, 8, 16μM; 24 h) suppressed IFN-γ-induced PD-L1 expression in H1975 and H1650 cells. MUC1-C mRNA and protein expression were decreased by evodiamine (0, 2, 4, 8, 16 μM; 24 h) in NSCLC cells as well. Evodiamine (0, 2, 4, 8, 16 μM; 24 h) downregulated the PD-L1 expression and diminished the apoptosis of T cells. It inhibited MUC1-C expression and potentiated CD8+ T-cell effector function.Elevating CD8+ T cells and downregulating the MUC1-C/PD-L1 signaling pathway[94]A549, H460, H1299, and H1650Non-small cell lung cancerThe cells were treated with evodiamine (3μM), erlotinib (20 μM), or the combination for 48 h. Combining evodiamine with erlotinib successfully inhibited cell proliferation and survival in wild-type EGFR NSCLC cells, characterized as erlotinib-resistant. In addition, evodiamine plus erlotinib significantly increased the apoptotic rate (61.86%) of NSCLC cells, as compared to single-agent treatment alone (evodiamine ∼33.71%; erlotinib ∼21.92%).mTOR/S6K1- mediated downregulation of Mcl-1[95]Caco-2 and HT-29Colorectal adenocarcinomaEvodiamine (10μM) enhanced the effect of berberine (0, 2.5, 5, 10, 20, 40 μM) on cell viability with IC50 values ranging from 38.85 μM to 15.82 μM in Caco-2 cells at 24 h, but evodiamine did not have this effect in HT-29 cells.Attenuating the overexpression of P-gp gene[96]


### 2.6. Other Effects of Evodiamine

In recent years, the development of antitumor drugs has focused on the development of bioactive molecules that can effectively and selectively act on a single molecular target. However, due to the complexity of various regulatory mechanisms in tumor cells, it is difficult for many single-target drugs to achieve lasting and effective control of tumors in the actual development process [97–100]. Therefore, the use of drug combinations is an advanced way to overcome these limitations.Li et al. found that evodiamine combined with erlotinib can successfully inhibit the proliferation and survival of wild-type EGFR non-small cell lung cancer (NSCLC) cells with erlotinib resistance. Furthermore, the combination of evodiamine and erlotinib promoted apoptosis in NSCLC cells more significantly than any single-dose therapy. That study suggested that the combination of evodiamine and erlotinib may downregulate the expression of MCL-1 by regulating the mTOR/S6K1 pathway. Thus, evodiamine can significantly increase the sensitivity of tumor cells to erlotinib, and the combination of erlotinib and evodiamine could be an alternative solution for the problem of erlotinib resistance [95] (Table 5). In addition, chemotherapy can lead to inevitable side effects. Guan et al. found that evodiamine significantly inhibited the overexpression of the p-glycoprotein gene of P-glycoprotein-positive colorectal cancer cells through the synergistic effect with berberine, thus achieving an excellent anticancer effect. However, this synergistic effect was not associated with cell cycle arrest and apoptosis. The same study found that the synergistic effect of evodiamine and berberine also reduced the cardiotoxicity caused by evodiamine, which was closely related to berberine’s regulation of exogenous apoptosis in NRF2-dependent and ROS-independent pathways. Therefore, the combination regimen of berberine and evodiamine is considered to have a better antitumor effect, while it significantly reduces side effects by targeting specific cells [96] (Table 5). The above research provides strong evidence for the effects of evodiamine in antitumor combination drugs and further supports the rationality of its application in tumor chemotherapy.


### 2.7. Structure-Activity Analysis of the Effects of Evodiamine-Related Compounds on the Antitumor Mechanism of Evodiamine

Structure-activity analysis of evodiamine, evodiamine-2, evodiamine-4, evodiamine-7, evodiamine-8, and evodiamine-12 showed that these compounds have the ability to induce DNA ladder formation in some cell lines. The results showed that adding an alkyl group, such as methyl or butyl, to the 14th position of quinazoline was essential for evodiamine to induce apoptosis [48]. According to another report, the researchers performed docking and molecular dynamics simulations on the homology model of TRPV1 to better understand the possible binding mode of evodiamine in the TRPV1 binding pocket [101]. The pharmacodynamic gene model further provided confidence in the effectiveness of docking research. This study revealed for the first time the structural determinants required for the interaction between TRPV1 and evodiamine, and provided new suggestions for the rational design of new TRPV1 ligands.Evodiamine has a free amine group with a medium molecular weight, which is easily converted into active derivatives with medicinal properties [102]. This structure, with a free N-H group and a scaffold with a molecular weight of approximately 350, can be included in structure-based virtual screening (SBVS) studies. In addition, evodiamine shares an “L-shaped” conformation at the active site of the topo1-DNA cleavable complex. Its free indole N-H function is a suitable site for chemical derivatization. Proper functional group transformation to maintain hydrogen bond interaction is an effective method to maintain the binding affinity. When indoleamine is transferred to the amide, its carbonyl group can also form a hydrogen bond with Arg364. In this way, the key N-H group of evodiamine can undergo further chemical reactions and can be converted into active derivatives.


### 2.8. Cardiovascular Side Effects of Evodiamine

The effects of evodiamine on primary cultured neonatal rat cardiomyocytes have been studied in vitro, and the effects of evodiamine on zebrafish have been studied in vivo [103]. In vitro experiments have shown that evodiamine at a concentration of 28.44 µg/mL cocultured with cells for 24 h resulted in a 50% inhibition rate of cell viability. In vivo results have shown that evodiamine at a concentration of 354 ng/mL caused cardiac dysfunction. These findings suggest that cardiac function should be monitored during evodiamine therapy.


## 2.1. Inhibition of Tumor Cell Proliferation and Cell Cycle

There is evidence that evodiamine reduces the occurrence of tumors by inhibiting the proliferation of cancer cells and changing the cell cycle. Previous studies have shown that evodiamine can inhibit the proliferation of human cancer cell lines, such as breast cancer [35], colon cancer [36], liver cancer [37, 38], cholangiocarcinoma [27], osteosarcoma [39], and melanoma [40] in a dose- and time-dependent manner (Table 1). One of the distinguishing features of tumor cells is their dysregulation of the cell cycle caused by the abnormal expression of cyclins and/or the abnormal replication of DNA. The activity and expression states of CDC25 C, cyclin, and cyclin-dependent kinases (CDKs) regulate the stability of the cell cycle. Many studies have shown that arresting tumor cell cycle progression is an effective strategy to inhibit tumor proliferation [41–45].Table 1
Inhibition of tumor cell activity and proliferation by evodiamine in cancer cell lines.
Cell lineCell typeSpecific resultsMode of actionReferencesMCF-7/ADRBreast cancerThe IC50 values of MCF-7 cells treated with evodiamine for 24, 48, and 72 h were 7.68 μM, 0.64 μM, and 0.30 μM, respectively. The IC50 values of the MCF-7/ADR cells were higher than those of MCF-7, with values of 24.47 μM, 1.26 μM, and 0.47 μM for 24, 48, and 72 h of treatment with evodiamine, respectively.Inhibiting cell activity via the regulation of Ras/MEK/ERK pathway[35]HCT-116/L-OHPColorectal cancerThe cells were treated with evodiamine at 0.4, 0.8, and 1.6μM for 24, 48, and 72 h, respectively. Evodiamine significantly enhanced rhodamine 123 accumulation and caused a significant decrease in the IC50 level in HCT-116/L-OHP cells.Inhibiting the p50/p65 NF-kappaB pathway[36]HepG2, Huh-7, and Hep3BHepatocellular carcinomaThe cells were treated with evodiamine at 0.25, 0.5, 1, 2, 4, 6, and 8μM for 72 h. Evodiamine inhibited the proliferation of HCC cells in a dose-dependent manner under hypoxia (survival fraction: 1.0–0.3).Downregulating HIF-1α under hypoxia[37]HepG2 and Bel-7402Hepatocellular carcinomaEvodiamine treatment at a range of concentrations (0, 0.25, 1, 4, 8, 16, and 32μM) inhibited HepG2 cells and Bel-7402 growth. The IC50 values of HepG2 and Bel-7402 cells at 48 h were 14.7 μM and 16 μM, respectively.Affecting the Hippo-Yes-associated protein signaling pathway[38]HuCCT-1 and TFK-1CholangiocarcinomaTwo CCA cell lines, HuCCT-1 and TFK-1, were treated with evodiamine at different concentrations (0, 5, 10, 20, or 40μM) for different time periods (24, 48, or 72 h). Cell viability: 80%–20% (HuCCT-1); 75%–25% (TFK-1).Inducing IL-6/STAT3 signaling inhibition.[27]143B and MG63Osteosarcoma143B cells were treated with evodiamine at 0.5, 1, 2, 4, 6, 8, 16, and 32μM. MG63 cells were treated with evodiamine at 0.25, 0.5, 1, 2, 4, 6, 8, and 16 μM. Cell viability was measured at 24, 48, and 72 h. Relative growth rate: 95%–15% (143B); 98%–30% (MG63).Inhibiting Wnt/beta-catenin signaling pathway[39]A-375MelanomaThe cells were treated with evodiamine at 5, 7.5, 10, 12.5, and 15μM for 24, 48, and 72 h. Relative cell viability: 1.0–0.2.Inducing reactive oxygen species-dependent apoptosis and necrosis[40]Previous studies have reported that evodiamine may block the cell cycle progression and significantly increase the percentage of G2/M phase cells in tumors (Table2). Zhou et al. found that evodiamine affected the cell cycle progression in human osteosarcoma cells U2OS. The same authors reported that the expression levels of cyclin B1, CDC25 C, and CDC2 changed significantly, which may be related to evodiamine-dependent downregulation of the phosphorylation of MEK and ERK and inhibition of the Raf/MEK/ERK signaling pathway [46]. Moreover, in human lung cancer cells with G2/M phase arrest after evodiamine treatment, cyclin A and cyclin B1 expression levels decreased, whereas those of p-Chk1 and p-Chk2 increased [47]. In addition, evodiamine interferes with the colorectal cancer cell lines COLO205 and HT-29, causing the cell cycle to stagnate in the G2/M phase, which has been shown to be related to changes in cyclin B1/CDC25 C expression and is controlled by JNK [48]. According to the above studies, evodiamine induces the G2/M cycle arrest by affecting signaling factors involved in multiple signaling pathways regulating cyclins, CDKs, and CHK.Table 2
Induction of cell cycle arrest by evodiamine in cancer cell lines in vitro.
Cell lineCell typeSpecific resultsMode of actionReferencesU2OSOsteosarcomaEvodiamine treatment at a range of concentrations (0, 3, 6, and 12μM) triggered G2/M cell cycle arrest (9%–77%) for 24 h.Inhibiting the Raf/MEK/ERK signaling pathway[46]A549Lung cancerEvodiamine treatment at a range of concentrations (0, 1, 2, and 4μM) triggered G2/M cell cycle arrest (15.8%–86.2%) for 24 h.Downregulation of ERK[47]COLO205 and HT-29Colorectal carcinomaEvodiamine treatment at a range of concentrations (0, 1, 2, and 4μM) triggered G2/M cell cycle arrest for 24 h; evodiamine treatment at 2 μM triggered G2/M cell cycle arrest for 6, 12, and 24 h.JNK activation[48]MDA-MB-231Breast cancerExposure to different concentrations of evodiamine (0, 15, 30, 60, 90, and 120 µM) caused G0/G1 arrest of MDA-MB-231 (53.29%–60.12%) for 24 h.Decreasing Bcl-2, cyclin D1, and cyclin-dependent kinase 6 expression[49]MCF-7 and MDA-MB-231Breast cancerCell cycle analyses in monolayer culture (condition with FBS, without FBS), sphere culture condition. Evodiamine (0, 100, and 200 nM) mildly but significantly increased the proportion of cells at the G1 stage.Activating the p53-p21-Rb pathway[50]Evodiamine may also cause some tumor cells to stagnate at the G0/G1 phase (Table2). Interestingly, Du et al. found evidence of evodiamine-induced downregulation of cyclin D1 and CDK6 in human breast cancer MDA-MB-231 cells; these cells stagnated at the G0/G1 phase in the presence of evodiamine treatment [49]. In another study, after using evodiamine, the cells were seeded in the upper well of a Transwell and incubated with 10 µg/mL PGI/AMF and 10 µg/mL PGI/AMF +6 µmol/L evodiamine for 28 h. Normal cells served as the control group. We then used these cells to treat human breast cancer MCF-7 and MDA-MB-231 cells, and tumor cells were arrested at the G0/G1 phase; p53 and p21 were upregulated; and p-Rb, cyclin B, and cyclin A were downregulated [50]. Based on these results, evodiamine keeps the cell cycle in the G0/G1 phase by targeting the p53-p21-Rb signaling pathway and affecting the expression of G0/G1 phase cyclin-related proteins.


## 2.2. Promotion of Apoptosis in Tumor Cells

Apoptosis is a very important and highly conservative cell death model that can effectively inhibit tumorigenesis. The classical process of cell apoptosis involves caspases as the core proteins that activate other proenzymes in a sequence, which involves multiple signaling pathways leading to cellular apoptosis [51–55]. The promotion effects of evodiamine on apoptosis signal transduction pathways can be divided into extrinsic and intrinsic categories. The extrinsic pathway involves the activation of the death receptor pathway, whereas the intrinsic pathway includes the activation of the mitochondrial and endoplasmic reticulum pathways.Evodiamine induces the expression of death receptors on the surface of tumor cells and promotes the binding of death receptors to their ligands, thereby accelerating the formation of DISC and leading to apoptosis (Table3). According to Khan et al., evodiamine can upregulate the death receptors DR4 and DR5 in U87 glioblastoma cells under the synergistic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which leads to the activation of caspases 3 and 8 and the induction of apoptosis in tumor cells [56].Table 3
Induction of apoptosis and autophagy by evodiamine in cancer cell lines in vitro.
Cell lineCell typeSpecific resultsMode of actionReferencesU87GlioblastomaThe cells were treated with evodiamine (10 µM) and TRAIL (50 ng/ml) either separately or in combination for 24 h. TRAIL alone did not induce apoptosis, whereas evodiamine significantly induced apoptosis in U87 glioblastoma cells after 24 h (apoptosis rate: 35%–70%).Sensitizing cells to TRAIL via the death receptor pathway[56]A549 and H1299Lung epithelial cancerEvodiamine (20–40 µM) treatment of A549 cells for 24 h significantly increased the number of apoptotic cells by sixfold.Activating both the intrinsic and the extrinsic apoptosis pathways[57]U937LeukemiaThe cells were treated with evodiamine (0.2–0.8 µmol/L) for 18 h. Evodiamine induced apoptosis in a dose-dependent manner (sub-G1 population: 5%–45%).Activating caspase-dependent and caspase-independent pathways[58]MC3 and HSC4Oral cancerThe cells were treated with various concentrations of evodiamine (0.25, 0.5, and 1μM for MC3 cells; and 0.2, 2, and 20 μM for HSC4 cells) for 24 h. The apoptotic effect of evodiamine was confirmed by 4′-6-diamidino-2-phenylindole (DAPI) staining. Evodiamine-treated cells displayed nuclear condensation and fragmentation—hallmarks of apoptosis.Inhibiting the AKT pathway[59]H446 and H1688Small cell lung cancerThe induction of apoptosis was detected in H446 or H1688 cells after 24 h of treatment with 10μM evodiamine. The apoptosis rate of evodiamine-treated H446 (∼15%) or H1688 (∼11%) cells was much higher than that of the untreated cells (blank control, ∼5% or ∼4%).Activating mitochondria-dependent and endoplasmic reticulum stress-induced pathways[60]U87-MGGlioblastomasThe percentage of autophagy induced by evodiamine (0, 0.1, 6, 10μM) increased to a maximum at 24 h, but not at 48 h, indicating that evodiamine-induced autophagy in a dual-regulated manner.Activating calcium/c-Jun N-terminal kinase and calcium/mitochondria-mediated pathways[61, 62]Panc-1 and SW1990Pancreatic cancerPANC-1 and SW1990 cells were treated with evodiamine (1.0, 5.0, and 10 µM) for 48 h. The exposure of cells to evodiamine led to a dose-dependent increase in apoptosis. Apoptosis rate: 2.5%–17.5% (Panc-1); 4%–18% (SW1990).Inhibiting the PI3K-Akt and the MAPK/ERK signaling pathways[63]Murine Lewis lung carcinomaLung carcinomaThe cells exhibited an increase in MDC fluorescence within 1.5–6 h after evodiamine treatment, while the peak of autophagy activity was observed in 1.5 h treatment. In addition, evodiamine-treated cells displayed a greater number of distinct spots within the cytoplasm or perinuclear regions compared with controls.Activating caspase-independent pathways[64]Evodiamine can transform the tumor cells’ mitochondrial membrane into a state of increased permeability, thereby causing the irreversible release of various apoptosis promoters, including cytochrome C and AIF, into the cytoplasm or the nucleus and promoting the occurrence of apoptosis (Table3). Mohan et al. found that evodiamine significantly increased the mitochondrial membrane depolarization, increased the cell apoptosis rate, and slightly decreased the Bcl-2/Bax ratio in human lung cancer A549 and H1299 cells. It also increased the release of cytochrome C from mitochondria into the cytoplasm, thereby activating the cascade of proapoptotic pathways, including caspases 3 and 9 [57]. In another study, evodiamine was found to promote the mitochondrial release of AIF into the nucleus in human leukemia U937 cells, leading to chromosome agglutination and the induction of cellular apoptosis [58]. In addition, evodiamine can also promote mitochondrial apoptosis of tumor cells by suppressing the expression of the inhibitor of apoptosis proteins. Mcl-1 is an apoptosis inhibitor protein located on the mitochondrial membrane. Sachita et al. found that evodiamine reduced the expression of Mcl-1 during transcriptional modification, and knockout of Mcl-1 significantly increased the expression of active Bax, thereby inducing apoptosis [59]. These studies have shown that evodiamine can effectively regulate the ratio of proapoptotic and antiapoptotic factors to promote changes in mitochondrial membrane that are conducive to apoptosis and promote the occurrence of mitochondria-dependent apoptosis.Endoplasmic reticulum stress occurs when proteins are disrupted during transport from the endoplasmic reticulum (ER) to the Golgi apparatus. When newly synthesized or when the Ca2+ equilibrium state is broken, unfolded or misfolded proteins accumulate in large quantities in the ER. Fang et al. treated human small cell lung cancer H446 and H1688 cells with evodiamine and found excessive accumulation of reactive oxygen species (ROS), which caused endoplasmic reticulum stress and released a large amount of Ca2+. Furthermore, the upregulation of caspase-12 led to the upregulation of caspase-9 and caspase-3 expression, which in turn led to apoptosis [60]. Therefore, evodiamine can disrupt the Ca2+ balance in cells and induce ER stress by activating caspase-12 and other signaling pathways, thereby activating the ER-mediated apoptosis pathway (Table 3).


## 2.3. Induction of Autophagy of Tumor Cells

Autophagy is a process of lysosome phagocytosis and degradation of its own structure, which can remove damaged cellular structures and senile organelles. This process exists in most eukaryotic cells [65–67]. Autophagosome formation and processing of microtubule-associated protein 1 light 3 (LC3) are two major signs of autophagy.The role of autophagy in tumors is controversial. In fact, it may play different roles at different stages of tumor genesis and development [68–73]. Evodiamine induces protective autophagy mediated by the activation of the JNK pathway following extracellular Ca2+ influx. Liu et al. found that evodiamine could induce autophagy in human glioblastoma U87-MG cells. The application of an extracellular calcium scavenger and an antagonist of transient receptor potential vanillin-1 (TRPV-1) reduced the percentage of cells undergoing autophagy. The same phenomenon was confirmed by knocking down the expression of TRPV-1 by small-interfering RNA technology. Moreover, the activation of c-Junn terminal kinase (JNK) by evodiamine was decreased by the TRPV1 antagonist, which further confirmed that evodiamine can promote autophagy through the Ca2+-mediated JNK pathway [61].Although there have been many reports that evodiamine can promote autophagy in tumors, some studies have shown that evodiamine-related induction of autophagy in specific cells antagonizes apoptosis. This observation illustrates the complexity of the antitumor mechanism of evodiamine. Hong et al. found that evodiamine-induced apoptosis of prostate cancer (PC) cells by inhibiting the PI3K-Akt and MAPK/ERK signaling pathways in human pancreatic cancer Panc-1 and SW1990 cells, and by inhibiting the phosphorylation of signal transduction and transcriptional activator 3 to inhibit autophagy [63]. Tu et al. found that evodiamine could improve the expression level of the autophagy-specific genes (Atgs) in Lewis lung cancer cells, accelerate the transformation from LC3-I to LC3-II, and thus promote the formation of autophagosomes (Table 3). Moreover, when combined with the autophagy inhibitor 3-methyladenine (3-MA), autophagy induced by evodiamine was significantly reduced, while apoptosis was increased. This suggests that autophagy induced by evodiamine may have a protective effect on tumor cells and that the inhibition of autophagy can promote the occurrence of apoptosis [64]. Liu et al. further found that evodiamine could induce autophagy mediated by calcium/JNK signals in glioma cells, while treatment with Ca2+ scavenger BAPTA-AM significantly inhibited the activation of the intracellular JNK pathway and decreased autophagy, while apoptosis was increased [62]. These results confirm that evodiamine can promote Ca2+-mediated autophagy in the JNK pathway and that the inhibition of autophagy induced by this pathway can reduce the activity of tumor cells and promote apoptosis.


## 2.4. Inhibition of Tumor Microangiogenesis and Tumor Migration

Vascular endothelial growth factor (VEGF), one of the most important growth factors, plays a key role in promoting angiogenesis (Table4). It can promote mitosis and has antiapoptotic effect on endothelial cells; moreover, it increases vascular permeability and promotes cell migration. Because of these effects, VEGF actively regulates the angiogenesis of normal and pathological blood vessels [78–80]. VEGF and VEGF receptors (VEGFRs) are expressed on both endothelial and nonendothelial cells [81, 82]. Currently, the use of anti-VEGF and anti-VEGF receptor therapies to block angiogenesis in cancer or other pathological processes is considered extremely important.Table 4
Inhibition of microangiogenesis and migration by evodiamine in cancer cell lines in vitro or in vivo.
Cell lineCell type/animal modelsSpecific resultsMode of actionReferencesHepG2, SMMC-7721, H22Hepatocellular carcinoma BALB/c nude mice(1) To evaluate the effect of evodiamine on VEGF-induced angiogenesis, Matrigel plug assays were performed with C57BL/6 mice. The addition of evodiamine (200μg) to the plugs containing VEGF inhibited vascular formation. These plugs displayed a paler appearance, and fewer blood vessels were observed.(2) HepG2 cells and SMMC-7721 cells were exposed to 0, 5, 10, or 20 μmol/L of evodiamine for 24 h. Representative images of cell migration and invasion were assessed with Transwells.Inhibitingβ-catenin and reducing VEGFa expression[74]PC-3 and DU145Prostate cancerPC-3 and DU145 cells were treated with 5 µM evodiamine for 24, 36, and 48 h. Evodiamine substantially downregulated the expression of VEGF.Inhibiting the c-Met/Src/STAT3 signaling pathway[75]HT-29 and HCT-116Colorectal cancer BALB/c nude miceAfter treatment with different concentrations of evodiamine (0 or 1.5μmol/L) for 24 h, the migration rates were detected. Wound-healing assay showed that treatment with evodiamine (1.5 μmol/L) significantly repressed the migration of HT-29 cells and HCT-116 cells.Activating Sirt1[76]HCT-116Colorectal cancerMigration potential was assessed by Transwell assay after the cells had been treated by agents. The cells were seeded in the upper well of a Transwell insert, incubated with 10 µg/mL PGI/AMF and with 10 µg/mL PGI/AMF +6 µmol/L evodiamine for 28 h. Normal cells served as the control group. Evodiamine significantly inhibited PGI-induced migration in HCT-116 cells.Inactivating the JAK2/STAT3 pathway through the downregulation of PGI[77]Hepatocellular carcinoma (HCC) is a highly vascularized tumor, with high microvascular density and high levels of circulating VEGF. Shi et al. found that after the intervention with evodiamine in the subcutaneous H22 cell xenograft model, tumor growth was inhibited, and serum tumor markers,β-catenin, and VEGFa levels were significantly reduced. Evodiamine also blocked angiogenesis in the matrigel plug assay by inhibiting VEGF. In addition, evodiamine inhibited tumor growth and reduced the expression levels of various angiogenesis biomarkers, β-catenin, and VEGFa in the SMMC-7721 HCC cell xenograft model. Evodiamine was shown to have an antitumor effect on HCCs by inhibiting β-catenin, interacting with VEGFa, and reducing VEGFa expression, thereby inhibiting angiogenesis [74]. These results suggest that evodiamine can inhibit cell invasion and migration, block angiogenesis, and act as a potential therapeutic agent for HCC.Signal transduction and activator of transcription (STAT) is activated in many human tumor cell lines and primary tumors. STAT3 plays an important role in angiogenesis in the tumor microenvironment and can regulate the expression of VEGF, matrix metallopeptidases (MMP), and other factors [83–86]. Hwang et al. found that treatment with evodiamine in PC-3 and DU145 cells significantly inhibited cell proliferation, reduced HGF-regulated c-Met/Src/STAT3 phosphorylation, and disrupted nuclear transfer of STAT3 protein. That study also revealed that evodiamine downregulated the expression of several carcinogenic markers, including COX-2, VEGF, and MMP-9. In addition, reduced Src/STAT3 activation was observed in PC-3 and DU145 cells transfected with c-Met small-interfering RNA (siRNA), resulting in reduced evodiamine-induced apoptosis [75] (Table 4). These results indicate that evodiamine can markedly inhibit the activation of the c-Met/Src/STAT3 signaling pathway, thereby inhibiting tumor cell survival, proliferation, and angiogenesis.Metastasis is one of the main hazards and characteristics of malignant tumors, and is a key factor affecting the prognosis and survival status of patients. Matrix metalloproteinases are a large class of zinc-containing endopeptidases that have important biological functions in various events, including metastasis of cancer cells [87–90]. Zhou et al. found that evodiamine inhibited the expression of MMP-9 in vitro and in vivo by inhibiting the acetyl-NF-κB p65 subunit induced by Sirt1 in colorectal cancer HT-29 and HCT-116 cells, and thus inhibited the invasion and metastasis of the tumor cells [76]. Zhao et al. also found that evodiamine inactivated the JAK2/STAT3 signaling pathway by downregulating the expression of phosphoglucose isomerase (PGI), thereby downregulating the expression of MMP3 to inhibit the migration of human colorectal cancer HCT-116 cells [77] (Table 4). According to these studies, evodiamine has a significant inhibitory effect on MMPs, the key regulatory enzymes essential for tumor migration, which makes it suitable for further study as a lead compound for the inhibition of tumor migration.


## 2.5. Antitumor Immunotherapeutic Mechanism of Evodiamine

Inhibitory receptors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD-1), play crucial roles in the inactivation of immune cells in the tumor microenvironment. The activation, amplification, and effector function of CD8+ T cells are significantly inhibited by the interaction between PD-1 and programmed death ligand 1 (PD-L1), which also assists in the immune escape of cancer cells [91–93]. In addition, the interaction between the microenvironment and immune regulation plays a critical role in clinical treatment. It is widely believed that activated CD8+ T cells have anticancer immunity in a variety of tumors and have a significant positive prognostic effect. Furthermore, the survival status of cancer patients is significantly affected by tumor-infiltrating CD8+ lymphocytes (TILs) in the tumor environment, further supporting the strong link between immune escape and the tumor microenvironment [94]. MUC1-C is highly expressed in a variety of tumor cells and regulates a variety of genes, including PD-L1, that are important for the immune escape of tumor cells. Jiang et al. found that, after acting on non-small cell lung cancer cells, evodiamine downregulated the expression of the MUC1-C/PD-L1 signaling pathway at both the transcription and protein levels, reduced the apoptosis of T cells, and significantly increased the level of CD8+ T cells. Evodiamine may therefore be a potential targeted therapy suitable as an adjuvant for immunotherapy [94] (Table 5). By acting on PD-L1 and other inhibitors that suppress T-cell activation, evodiamine improves the tumor microenvironment, increases the activation level of CD8+ T cells, and effectively inhibits immune escape, which has the potential to promote the efficacy of immunotherapy.Table 5
Antitumor immunotherapeutic mechanisms and antitumor drug combinations involving evodiamine.
Cell lineCell type/animal modelsSpecific resultsMode of actionReferencesH1650 and H1975Non-small cell lung cancerEvodiamine (0, 2, 4, 8, 16μM; 24 h) suppressed IFN-γ-induced PD-L1 expression in H1975 and H1650 cells. MUC1-C mRNA and protein expression were decreased by evodiamine (0, 2, 4, 8, 16 μM; 24 h) in NSCLC cells as well. Evodiamine (0, 2, 4, 8, 16 μM; 24 h) downregulated the PD-L1 expression and diminished the apoptosis of T cells. It inhibited MUC1-C expression and potentiated CD8+ T-cell effector function.Elevating CD8+ T cells and downregulating the MUC1-C/PD-L1 signaling pathway[94]A549, H460, H1299, and H1650Non-small cell lung cancerThe cells were treated with evodiamine (3μM), erlotinib (20 μM), or the combination for 48 h. Combining evodiamine with erlotinib successfully inhibited cell proliferation and survival in wild-type EGFR NSCLC cells, characterized as erlotinib-resistant. In addition, evodiamine plus erlotinib significantly increased the apoptotic rate (61.86%) of NSCLC cells, as compared to single-agent treatment alone (evodiamine ∼33.71%; erlotinib ∼21.92%).mTOR/S6K1- mediated downregulation of Mcl-1[95]Caco-2 and HT-29Colorectal adenocarcinomaEvodiamine (10μM) enhanced the effect of berberine (0, 2.5, 5, 10, 20, 40 μM) on cell viability with IC50 values ranging from 38.85 μM to 15.82 μM in Caco-2 cells at 24 h, but evodiamine did not have this effect in HT-29 cells.Attenuating the overexpression of P-gp gene[96]


## 2.6. Other Effects of Evodiamine

In recent years, the development of antitumor drugs has focused on the development of bioactive molecules that can effectively and selectively act on a single molecular target. However, due to the complexity of various regulatory mechanisms in tumor cells, it is difficult for many single-target drugs to achieve lasting and effective control of tumors in the actual development process [97–100]. Therefore, the use of drug combinations is an advanced way to overcome these limitations.Li et al. found that evodiamine combined with erlotinib can successfully inhibit the proliferation and survival of wild-type EGFR non-small cell lung cancer (NSCLC) cells with erlotinib resistance. Furthermore, the combination of evodiamine and erlotinib promoted apoptosis in NSCLC cells more significantly than any single-dose therapy. That study suggested that the combination of evodiamine and erlotinib may downregulate the expression of MCL-1 by regulating the mTOR/S6K1 pathway. Thus, evodiamine can significantly increase the sensitivity of tumor cells to erlotinib, and the combination of erlotinib and evodiamine could be an alternative solution for the problem of erlotinib resistance [95] (Table 5). In addition, chemotherapy can lead to inevitable side effects. Guan et al. found that evodiamine significantly inhibited the overexpression of the p-glycoprotein gene of P-glycoprotein-positive colorectal cancer cells through the synergistic effect with berberine, thus achieving an excellent anticancer effect. However, this synergistic effect was not associated with cell cycle arrest and apoptosis. The same study found that the synergistic effect of evodiamine and berberine also reduced the cardiotoxicity caused by evodiamine, which was closely related to berberine’s regulation of exogenous apoptosis in NRF2-dependent and ROS-independent pathways. Therefore, the combination regimen of berberine and evodiamine is considered to have a better antitumor effect, while it significantly reduces side effects by targeting specific cells [96] (Table 5). The above research provides strong evidence for the effects of evodiamine in antitumor combination drugs and further supports the rationality of its application in tumor chemotherapy.


## 2.7. Structure-Activity Analysis of the Effects of Evodiamine-Related Compounds on the Antitumor Mechanism of Evodiamine

Structure-activity analysis of evodiamine, evodiamine-2, evodiamine-4, evodiamine-7, evodiamine-8, and evodiamine-12 showed that these compounds have the ability to induce DNA ladder formation in some cell lines. The results showed that adding an alkyl group, such as methyl or butyl, to the 14th position of quinazoline was essential for evodiamine to induce apoptosis [48]. According to another report, the researchers performed docking and molecular dynamics simulations on the homology model of TRPV1 to better understand the possible binding mode of evodiamine in the TRPV1 binding pocket [101]. The pharmacodynamic gene model further provided confidence in the effectiveness of docking research. This study revealed for the first time the structural determinants required for the interaction between TRPV1 and evodiamine, and provided new suggestions for the rational design of new TRPV1 ligands.Evodiamine has a free amine group with a medium molecular weight, which is easily converted into active derivatives with medicinal properties [102]. This structure, with a free N-H group and a scaffold with a molecular weight of approximately 350, can be included in structure-based virtual screening (SBVS) studies. In addition, evodiamine shares an “L-shaped” conformation at the active site of the topo1-DNA cleavable complex. Its free indole N-H function is a suitable site for chemical derivatization. Proper functional group transformation to maintain hydrogen bond interaction is an effective method to maintain the binding affinity. When indoleamine is transferred to the amide, its carbonyl group can also form a hydrogen bond with Arg364. In this way, the key N-H group of evodiamine can undergo further chemical reactions and can be converted into active derivatives.


## 2.8. Cardiovascular Side Effects of Evodiamine

The effects of evodiamine on primary cultured neonatal rat cardiomyocytes have been studied in vitro, and the effects of evodiamine on zebrafish have been studied in vivo [103]. In vitro experiments have shown that evodiamine at a concentration of 28.44 µg/mL cocultured with cells for 24 h resulted in a 50% inhibition rate of cell viability. In vivo results have shown that evodiamine at a concentration of 354 ng/mL caused cardiac dysfunction. These findings suggest that cardiac function should be monitored during evodiamine therapy.


## 3. Conclusions and Prospects

In recent years, a large number of experiments in vivo and in vitro have shown that evodiamine can inhibit the activity, proliferation, and cell cycle progression in various tumor cells. Evodiamine also promotes apoptosis and induces autophagy in tumor cells, inhibits the formation and migration of tumor microvessels, and participates in the regulation of tumor immunotherapy. In addition, several signaling pathways are modulated by evodiamine, including PI3K/Akt, mTOR, and NF-κB cascades. Moreover, the excellent effect of the combination of other anticancer drugs with evodiamine in antitumor studies has further indicated its value for medicinal development (Figure 2).Figure 2
Probable molecular mechanisms by which evodiamine kills tumor cells.It is predicted that efforts in using evodiamine as a multitarget antitumor drug in the following studies will be directed as follows. Its solubility, stability, and bioavailability need to be further improved so that it can exert its biological activity to a greater extent and more efficiently while reducing adverse reactions. In addition, further studies on the antitumor mechanism of evodiamine need to be conducted. For example, Src nonreceptor tyrosine kinases are important oncogenes that play an important role as an intermediate hub for phosphorylation and regulation of a variety of cytoplasmic downstream signals. In addition, STAT family transcriptional coactivators are important targets of Src. Furthermore, Src mediates the regulatory effects of extracellular signals on cell proliferation, migration, and apoptosis by participating in the transduction of various cellular signaling pathways. Current studies have shown that evodiamine interacts with the c-Met/Src/STAT3 signaling pathway and inhibits tumor cell angiogenesis. However, the molecular target characteristics of its interaction with upstream Src need to be further studied. In addition, the existing experimental results are still limited to in vitro cell and in vivo animal experiments. The lack of clinical research reports related to evodiamine and the adverse reactions to evodiamine need to be further studied in depth.[103]



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