Insight into norcantharidin, a small-molecule synthetic compound with potential multi-target anticancer activities

Norcantharidin (NCTD) is a demethylated derivative of cantharidin, which is an anticancer active ingredient of traditional Chinese medicine, and is currently used clinically as a routine anti-cancer drug in China. Clarifying the anticancer effect and molecular mechanism of NCTD is critical for its clinical application. Here, we summarized the physiological, chemical, pharmacokinetic characteristics and clinical applications of NCTD. Besides, we mainly focus on its potential multi-target anticancer activities and underlying mechanisms, and discuss the problems existing in clinical application and scientific research of NCTD, so as to provide a potential anticancer therapeutic agent for human malignant tumors.


Background
Since Tu Youyou was awarded the 2015 Nobel Prize in physiology or medicine for the discovery of artemisinin used for malaria treatment, traditional Chinese medicines (TCMs) and natural medicine are getting more attention. A growing body of evidences indicate that TCMs contain anticancer ingredient. Norcantharidin (NCTD), a demethylated derivative of cantharidin which is an active ingredient of TCM-Mylabris [1][2][3], is currently used clinically as an optional anticancer drug in China, because of its relatively synthesized facility, potential anticancer activity, and less side-effects such as myelosuppression, gastrointestinal and urinary tract toxicity [1][2][3][4][5]. Increasing evidences show that NCTD not only effectively inhibited the proliferation of many tumor cells in vitro and in vivo, including hepatoma HepG2 [6][7][8], SMMC-7721 [8,9] and BEL-7402 [10,11], gallbladder cancer GBC-SD cells [12,13], colon cancer CT26 and HT29 cells [14,15], breast cancer cells [16,17], leukemia K562 [18] and HL-60 cells [4,5,19], melanoma A375 cells [20], and oral cancer KB cells [21], but also decreased tumor growth and prolonged survival in animal models in vivo [17,22]. As an efficacious anticancer drug, it has been used to treat hepatic cancer, gastric cancer and leucopenia patients in China for many years. To deepen the understanding of the characteristics and clinical application of NCTD is of great significance for NCTD to work as an anticancer drug in clinic. Here, we review the physiological, chemical, pharmacokinetic characteristics and clinical uses, especially, potential multi-target anticancer activities such as inducing apoptosis, inhibiting proliferation, blocking invasion/metastasis, antiangiogenesis, anti-vasculogenic mimicry, anti-lymphangiogenesis and underlying mechanisms of NCTD, so as to provide a potential anticancer therapeutic agent for human malignant tumors.
In pharmacokinetics, radionuclide and whole-body autoradiography showed that NCTD was rapidly absorbed by intragastric administration in mice with 3 H-norcantharidin, reached a higher concentration within 15 min and 2 h after dosing in the kidney, liver, tumor, stomach, intestines, heart and lung. NCTD was highly distributed in the bile duct, liver, kidney, heart and lung by intravenous administration, reached the peak concentration in liver and cancer tissues within 15 min after dosing. After 6 h, the concentration decreased significantly by being excreted from the urethra. Most of drugs were excreted from the kidneys within 24 h, and were rarely accumulated in the various organs of the body [24]. Thus, NCTD is less likely to cause drug accumulation poisoning.

Clinical uses
As an efficacious anticancer drug, NCTD has been used to treat cancer patients clinically in China for many years. Two thousand years ago, Mylabris (Spanish fly), a traditional Chinese medicine, was used to treat "abdominal Fig. 1 The origin, evolvement and molecular formula of norcantharidin (NCTD). Mylabris, also known as Spanish fly, is a traditional Chinese medicine. Cantharidin (CTD), a 7-oxabicyclo [2.2.1] heptane-2, 3-dicarboxylic acid derivative, a natural toxin and the active ingredient with antitumor properties extracted from a traditional Chinese medicine Mylabris. NCTD (7-oxabicyclo [2.2.1] heptane-2, 3-dicarboxylic anhydride), with a molecular formula of C 8 H 8 O 4 and formula weight of 168.15 g/mol, is the demethylated analog and the low-cytotoxic derivative of CTD with antitumor properties Fig. 2 Synthesis of NCTD by furan and maleic anhydride through Diels-Alder reaction. NCTD can be synthesized by furan and maleic anhydride through Diels-Alder reaction under appropriate conditions mass" in China [1][2][3][4]. Later, an active ingredient of Mylabris-CTD was artificially extracted and be used to treat many human tumors as a natural toxin [1][2][3][4]. Afterwards, in order to alleviate side effects of CTD such as gastrointestinal and urinary tract toxicity, NCTD was extracted from CTD, or was synthesized from furan and maleic anhydride [1][2][3][4]23]. Now, NCTD is clinically used as a routine anticancer drug in China.
Clinical indications of NCTD include: (1) It is used to treat patients with digestive tumors, such as hepatocellular cancer, esophageal cancer, gastric cancer, and colorectal cancer and it shows better curative effect; (2) It is used to treat other cancer patients, such as lung, breast and ovarian cancers and has certain curative effect; (3) Also, it is used as premedication or in combination with other antineoplastic drugs. In addition, NCTD can also be used for hepatitis, liver cirrhosis and leukopenia.
Usage of NCTD includes oral, intravenous administration and local injection. For oral, 5-15 mg (most dose can be added to 30 mg) NCTD is used for one time, 3 times a day, 1 months for 1 courses, generally 3 courses. For intravenous infusion or intravenous drip, 10-20 mg a day, added to the 5% glucose injection 250-500 ml, in a slow drop by intravenous drip; or added to the 5% glucose injection 10-20 ml, by slow intravenous injection; 1 month for 1 treatment course. And for local injection, 20-40 mg/times, once a week, 2-4 times for 1 courses.
Growing clinical evidences demonstrated that NCTD was an efficacious anticancer drug for cancer patients. Table 1 illustrates the clinical uses of NCTD and the related results . No matter NCTD is used alone via oral, intravenous administration, intro-tumor injection, or in combination with chemotherapy, radiotherapy and other therapies such as interventional therapy (IVT), transcatheter arterial chemoembolization (TACE) and TCMs can reduce tumors, improve symptoms and life quality, alleviate side effects, and prolong survival time in most patients with mid-advanced stage tumors such as hepatocellular cancer, esophageal cancer, gastric cancer, lung cancer, ovarian cancer, non-Hodgkin lymphoma and so on . Thus, NCTD is believed as a useful adjunct anticancer drug in clinical treatment of midadvanced stage tumors and in the prevention of postoperational recurrent tumors.

Multi-target anticancer activities and underlying mechanisms
The multi-target anticancer activities and underlying mechanisms of NCTD in treatment of different cancer models and cell lines have been reported. Here, we systematically review the potential anticancer activities and underlying molecular mechanisms of NCTD in vitro and in vivo.

Inhibiting proliferation and inducing apoptosis
In recent years, a large number of researches have been carried out to study the effects of NCTD on inhibiting proliferation and inducing apoptosis in different cancer models (Table 2). NCTD has a cytotoxic effect on a variety of tumor cells. Significant anti-proliferative and apoptotic effects are observed in NCTD-treated tumor cells [7,49,50]. At the same time, relevant studies have confirmed that NCTD has no myelosuppression and can induce hematopoiesis via bone marrow stimulation while exerting its anticancer activity [4,5]. NCTD has no effect on the viability of normal peripheral blood mononuclear cells (MNC) [51,52]. These are incomparable advantages over many traditional anticancer drugs. In addition, NCTD has a synergistic effect with a variety of anticancer drugs, such as cisplatin and gefitinib [53,54].
The anti-proliferation and pro-apoptotic effects of NCTD depend on the complex interactions between different molecules (Fig. 3). On the one hand, the inhibitory effect of NCTD on proliferation is mainly achieved through cell cycle arrest and inhibition of DNA synthesis by inhibiting the expression of cyclins, cyclin-dependent kinases (CDKs) and increasing the expression of cyclindependent kinase inhibitors (CDKIs, such as p21 Cip/ Waf1 , p27 kip1 ); On the other hand, NCTD can also induce apoptosis by increasing the expression of pro-apoptotic protein such as P53, Bax, Caspases, and reducing the expression of anti-apoptotic proteins such as Bcl-2 (B-cell lymphoma-2) and survivin. These mechanisms have been confirmed in a variety of tumor cell lines such as leukemia K562 and HL-60 [18,55], hepatoma HepG2, SMMC-7721 and BEL-7402 [56][57][58], colorectal cancer CT26 and HCT-15 cells [59,60], etc. It is generally believed that serine/threonine protein phosphatases, such as protein phosphatase type 1 (PP1), protein phosphatase-2A (PP2A) and protein phosphatase-2B (PP2B), play important roles in intracellular signal transduction, whose inhibition is an excellent target for the development of novel anti-cancer agents [5,61,62]. Some studies have confirmed that NCTD, as a PP2A inhibitor, can inhibit cancer cell proliferation and induce apoptosis by inhibiting the activity of PP2A [5,62,63]. In addition, DNA replication-initiation protein Cdc6 (cell division cycle protein 6) is an effective target to disturb DNA replication [64]. Other studies have found that NCTD can inhibit cell proliferation by inducing Cdc6 degradation [65,66]. In gallbladder cancer, it was reported that NCTD inhibited the expression of GBC-SD cell proliferation-related gene proteins PCNA (proliferating cell nuclear antigen) and Ki-67, this may be one of the mechanisms by which NCTD inhibit the proliferation and growth of tumor cells [12,67].   In vitro [6] HepG2 Hep3B Huh-7 M-phase cell-cycle arrest; phosphorylation of p21, Cdc25C; regulation of cyclin B1-associated kinase activity; phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-3, -9 [7] SMMC-7721 BEL-7402 Inducing the activation of caspase-9, -3 and the cleavage of PARP, and downregulating the expression of Bcl-2, Bcl-X(L) and Mcl-1. [11]
The Wnt/β-catenin signaling pathway is considered to be another target for antitumor drugs [69]. Some studies have shown that NCTD can reduce the proliferation of leukemia Jurkat cells by inhibiting Wnt/β-catenin signaling [70]. Due to the ability to cross the blood-brain barrier, NCTD can also significantly inhibit the growth of medulloblastoma through Wnt/β-catenin signaling pathway [71]. In addition, NCTD can inhibit the expression of the proliferation-related protein cyclin D1, downregulate the expression of anti-apoptotic protein, and upregulate the expression of pro-apoptotic protein by blocking PI3K (phosphoinositide 3 kinase)/Akt/NF-κB (nuclear factor-kappa B) pathway [72,73]. So, the PI3K/ Akt/NF-κB pathway has been shown to be another signal pathway for the regulation of NCTD-mediated anti-proliferation and pro-apoptosis.

Inhibiting tumor invasion/metastasis
Two major protein families are involved in NCTD against tumor invasion and metastasis, including matrix metalloproteinases (MMPs) and adhesion molecules [74]. The MMP family, particularly MMP-2 and MMP-9, has gelatinase activity and is capable of proteolytic cleavage of plasminogen in extracellular matrix [75]. Cell adhesion molecules such as α-catenin and b-catenin have the function of adhering tumor cells to other cellular and matrix components [76], both of them play an important role in local invasion and distant metastasis.
It has been confirmed that NCTD has anti-invasion and anti-metastasis effects in many kinds of tumor cells (Table 3). Some experiments indicated that NCTD reduces the activity of MMP-2 and MMP-9 by upregulating the transcription factor STAT1 (signal transducers and activators of transcription 1) and inhibiting the transactivation of Sp1 (specificity protein 1), thereby inhibiting the invasion and metastasis of tumor cells [77,78]. Another study showed that NCTD has the ability to reduce the expression of α-catenin and β-catenin in colorectal cancer CT26 cells, suggesting that the anti-invasive and anti-metastatic activity of NCTD may be related to the regulation of these adhesion molecules [75]. Furthermore, epithelial-mesenchymal transition (EMT) is widely involved in the invasion and metastasis of malignant epithelial tumors [79]. NCTD inhibits the EMT process in non-small cell lung cancer, colorectal cancer and hepatocellular cancer cells via the αvβ6-ERK-Ets1 (E-Twenty-Six-1) signaling pathway blocking and NCTDmediated Yes-associated protein (YAP) inhibition [78,80,81]. These regulatory mechanism of NCTD against tumor invasion and metastasis is detailed in Fig. 4.

Anti-angiogenesis and anti-vasculogenic mimicry
Angiogenesis and effective blood supply are basic conditions for tumor growth and metastasis [82]. Multiple angiogenic growth factors and cytokines play important roles in regulating tumor angiogenesis, such as vascular endothelial growth factor (VEGF) and its corresponding receptor, thrombospondin (TSP), angiogenin (Ang), and tissue metalloproteinase inhibitor (TIMP) family. In gallbladder and colorectal cancer, it has been confirmed that NCTD can inhibit angiogenesis, induce apoptosis of vascular endothelial cells, downregulate the expression of angiogenic factors such as VEGF, VEGFR-2 (vascular endothelial growth factor receptor-2), Ang-2, and upregulate the expression of anti-angiogenic factors such as TSP and TIMP-2 [83][84][85][86]. So, NCTD may be a potential anti-angiogenic drug for cancer treatment.
Tumor vasculogenic mimicry (VM) refers to a novel tumor blood supply pattern that occurs in certain highly aggressive malignancies and is associated with poor clinical outcomes and poor prognosis [87]. TIMP-2 has anti-VM activity in some highly aggressive malignancies [88]. Furthermore, the PI3-K (phosphatidylinositol 3-kinase)/ MMPs (matrix metalloproteinases)/Ln-5γ2 (laminin 5γ2) and EphA2 (ephrin type a receptor 2)/FAK (focal adhesion kinase)/Paxillin signaling pathways are two critical pathways for the control of VM formation [89], while MMP-2 and MT1-MMP (membrane type 1-matrix metalloproteinase) are key molecules and important mediators of these two pathways, regulating VM formation in invasive malignant cells [90]. NCTD is believed as a potential anti-VM active drug, its anti-VM mechanisms mainly involves two aspects: NCTD downregulates the expression of MMP-2 and MT1-MMP via inhibiting EphA2/FAK/Paxillin signaling pathway, thereby enhancing the anti-VM activity of TIMP-2; in turn, a decrease in MMP-2 and MT1-MMP activity inhibits PI3-K/MMPs/ Ln-5γ2 signaling and exerts an anti-VM effect on malignant cells [13,[91][92][93].

Anti-lymphangiogenesis
Lymphatic metastasis is one of the important metastatic pathways of tumors, and tumor lymphatic tube formation (lymphangiogenesis) plays an important role in tumor growth, metastasis and prognosis [94]. Lymphatic endothelial growth factors, including two members of the VEGF family, VEGF-C and VEGF-D, as well as their cognate receptor VEGFR-3, are the main regulators of tumor lymphangiogenesis and is of great significance in tumor lymph node metastasis [95][96][97]. In recent years, some researchers have reported that NCTD is an effective lymphangiogenesis inhibitor. The basic mechanism of NCTD anti-lymphangiogenesis refers to directly or indirectly downregulate the expression of VEGF-C, VEGF-D and VEGFR-3 at protein and mRNA levels, which has been proved in human lymphatic endothelial cells (HLECs) and human colonic adenocarcinomas (HCACs) [98][99][100]. In addition, NCTD in combine with sorafenib or mF4-31C1 enhanced the ability of anti-lymphangiogenesis in human colonic adenocarcinomas [100].
The relevant researches and mechanisms of NCTD inhibiting tumor vascularization (Angiogenesis, VM and lymphangiogenesis) are summarized in Table 4 and Fig. 5.

Overcoming multi-drug resistance
Multi-drug resistance (MDR) refers to tumor cells develop resistance to anti-tumor drugs, as well as In vitro In vivo [22] Giant cell tumor of bone (GCTB)

Inhibiting the EMT process
Modulating the miR-30a/MTDH/ AKT cell signaling pathway In vitro [169] producing cross-resistance to other antineoplastics with different structures and mechanisms [101]. As one of the main problems in clinical tumor chemotherapy, MDR directly affects the efficacy of chemotherapy drugs and even lead to treatment failure [102]. In human breast cancer cells, NCTD may overcome MDR through inhibiting sonic hedgehog (Shh) signaling and its downstream MDR-1/P-gp expression [103], which has been shown to increase resistance to a variety of structurally unrelated antitumor drugs [104]. Bcl-2 family proteins Bcl-2 and Bcl-xL are resistant to multiple chemotherapeutic agents in a variety of cell lines [105][106][107], and it was reported that NCTD downregulated the expression of Bcl-2 and Bcl-xL in oral cancer cells [108]. In addition, Bcl-2 family inhibitors ABT-737 and ABT-263 are two promising anticancer agents with anticancer activity against a variety of cancer cells [109,110]. NCTD significantly enhances ABT-263 and ABT-737-mediated anticancer activity, and overcomes the increased ABT-737 resistance caused by elevated Mcl-1 levels in cancer cells [111][112][113]. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are widely used in anti-tumor therapy for non-small cell lung cancer (NSCLC) [114]. HGF (hepatocyte growth factor) overexpression is a major factor contributing to acquired resistance caused by EGFR-TKI [115]. NCTD can overcome HGF-induced EGFR-TKI resistance in EGFRmutant lung cancer cells by inhibition of the Met/PI3K/ Akt pathway [116]. Therefore, NCTD may be a potential agent to reverse MDR (Table 5).

Promoting tumor cell demethylation
Tumorigenesis is a process of interaction between genetic and epigenetic mechanisms. DNA methylation is an important epigenetic regulator closely related to the occurrence and development of tumors [117]. Abnormal DNA methylation is involved in the pathogenesis of tumors. DNA hypomethylation promotes gene expression, while DNA hypermethylation inhibits gene expression [118,119]. Hypermethylation of    In vitro [171] RASSF1A (a tumor suppressor gene) results in loss of function in human tumor cells [120]. It was reported that NCTD can inhibit RASSF1A methylation and inducing its re-expression in hepatocellular cancers [121]. Moreover, the Wnt/β-catenin signaling pathway is closely related to a variety of neoplastic diseases and is activated in tumor formation [122,123]. Wnt inhibitory factor-1 (WIF-1), as a Wnt antagonist, has the function of inhibiting Wnt signal transduction. And due to hypermethylation of the promoter, WIF-1 silencing occurs in some tumor cells [124]. Studies have demonstrated that NCTD can activate WIF-1 to inhibit Wnt signaling pathway through promoter demethylation in NSCLC and glioma cells [125,126] (Table 6).

Modulating immune responses
The immune system plays a very important role in the development of tumors. The inflammatory response is a common and serious complication due to the continued damage to the immune system by the cancer itself and anti-cancer drugs. NCTD positively regulates macrophage-mediated immune responses via the AKT/ NF-κB signaling pathway, helping to clear invading pathogens [127]; NCTD also reduces tissue inflammation by suppressing PBMC (human peripheral blood mononuclear cells) proliferation and cytokine gene expression and production [128]. In addition, the increased production of IL-10 will block the effect of specific T lymphocytes on tumor cells [129], and NCTD inhibits the     In vitro In vivo [23] production of IL-10 in PBMC induced by PHA (phytohemagglutinin) [128] (Table 6).

Others
NCTD has also been reported to have some other anticancer activities, including inhibition of tumor glucose oxidative metabolism [130]; inhibition of NAT (N-acetyltransferase) activity [131]; regulation of macrophage polarization [175]; regulation of leukemia stem cell activity [4] (Table 6). Due to the lack of relevant researches, it is necessary to further verify the relevant mechanisms and applications in the clinic.

Discussion
In recent years, the anti-tumor effect of TCMs has aroused extensive attention. However, due to the complexity of components, difficulty in extraction and high toxicity, the clinical application of many anti-tumor TCMs is limited. NCTD, as a demethylation product of CTD, can be extracted from CTD or synthesized artificially at a low cost. In addition, its physical and chemical properties are clear, so it is convenient for basic and clinical research. These prerequisites are helpful for the promotion of NCTD in clinical practice. On the basis of summarizing the relevant literature, we found that there are two main ways of clinical application of NCTD. First of all, NCTD can be used as an antitumor drug alone in the treatment of liver cancer, gastric cancer and other tumors, especially for advanced malignant tumors that have lost the opportunity of operation. Secondly, it is used as an adjuvant of other anti-tumor drugs, which is currently the most important way for NCTD applied in clinic. Some studies have shown that the combination of NCTD with other anticancer drugs, or as an adjuvant to chemotherapy or interventional therapy, can help to improve the efficacy, increase the tolerance of patients, reduce side effects, and improve the prognosis [28,30,33].
Adverse reactions and serious complications of NCTD are rare. Gastrointestinal symptoms such as nausea and vomiting may occur when the oral dose or injection is excessive. A study has shown that patients with advanced liver cancer who take NCTD more than 45 mg/day will have significant gastrointestinal response [25]. It has also been reported that when the dosage of NCTD reaches 600 mg, the patients may have slight gastrointestinal symptoms, but it will be relieved soon after the drug is stopped or the alkaline agent is taken [27]. A large number of clinical studies have proven that patients treated with NCTD have no obvious symptoms of urinary irritation, no adverse effects on liver and renal function, and no myelosuppression [27,28,32].
Among the three routes of administration, oral administration and intravenous administration are simple and safe. The disadvantage is that the drug is eliminated quickly in the body, resulting in poor anti-tumor effect. It is reported that the half-life of NCTD in blood is short, only about 0.26 h [17]. Local injection is mainly used for some solid tumors, especially for advanced liver cancer which can not be treated by surgery. Compared with the former two, this method has better curative effect. However, due to the invasive operation, there are some risks such as bleeding, cancer rupture and so on.
NCTD has the disadvantages of poor water solubility, short half-life and low tumor targeting efficiency, which limits its clinical application [132,176]. Therefore, a variety of NCTD analogues have been developed to improve the clinical applicability and efficacy. These NCTD analogues can be divided into two categories: new NCTD reagents and drug delivery systems. For example, it has been reported a new type of NCTD conjugate recently, called CNC conjugates (NCTD-conjugated carboxymethyl chitosan). Compared with the same dose of free NCTD, CNC conjugates have higher therapeutic concentration and longer half-life. It can not only enhance the inhibitory effect on cancer cells, but also reduce side effects [177,178]. In addition, some other NCTD derivatives and liposomes, such as NOC15 (N-farnesyloxy-norcantharimide) [179] and SG-NCTD-LIP (NCTD-loaded liposomes modified with stearyl glycyrrhetinate) [176], also can effectively improve the anticancer activity and reduce the toxicity of NCTD. However, although these studies have shown that NCTD analogues have a very broad application prospect, most of the existing NCTD analogues have no obvious selectivity for tumors and targets. And it should be noted that most of the relevant researches are in the stage of basic research at present, whether these NCTD analogues can be applied to clinical needs to be confirmed by a large number of clinical experiments.

Conclusions
Collectively, NCTD, as a demethylation derivative of traditional Chinese medicine, has been clinically used to treat cancer patients, and is gradually believed as a useful adjunct anticancer drug, especially for the patients with mid-advanced and postoperational recurrent cancers. The underlying molecular mechanisms of NCTD anticancer activities maybe "multi-factor", "more targets" and "multi-points priming" mechanisms, include inhibiting proliferation, inducing apoptosis, inhibiting tumor invasion and metastasis, anti-neoangiogenesis (including anti-angiogenesis and anti-VM), anti-lymphangiogenesis, overcoming multiple drug resistance, promoting tumor cell demethylation, modulating immune responses and so on. Numerous clinical applications and drug experiments have also demonstrated that NCTD has effective and "multi-factor" anticancer activities, especially in apoptotic inducement in human cancer cells by "more targets" and "multi-points priming" mechanisms. But other mechanisms of NCTD's anticancer effects such as anti-angiogenesis, anti-VM, anti-lymphangiogenesis as well as overcoming multiple drug resistance are seldom reported. It is necessary to improve the relevant research, which is of great significance for the development of NCTD as a potential chemotherapeutic agent.