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REVIEW Research Progress of Ursolic Acid's Anti-Tumor Actions ZANG Li-li (臧丽丽), WU Bao-ning (吴宝宁), LIN Yuan (林 原), WANG Jun (王 FU Lei (傅 雷), and TANG Ze-yao (唐泽耀)
军),
ABSTRACT Ursolic acid (UA) is a sort of pentacyclic triterpenoid carboxylic acid purified from natural plant. UA has a series of biological effects such as sedative, anti-inflammatory, anti-bacterial, anti-diabetic, antiulcer, etc. It is discovered that UA has a broad-spectrum anti-tumor effect in recent years, which has attracted more and more scholars' attention. This review explained anti-tumor actions of UA, including (1) the protection of cells' DNA from different damages; (2) the anti-tumor cell proliferation by the inhibition of epidermal growth factor receptor/mitogen-activated protein kinase signal or of FoxM1 transcription factors, respectively; (3) antiangiogenesis, (4) the immunological surveillance to tumors; (5) the inhibition of tumor cell migration and invasion; (6) the effect of UA on caspase, cytochromes C, nuclear factor kappa B, cyclooxygenase, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or mammalian target of rapamycin signal to induce tumor cell apoptosis respectively, and etc. Moreover, UA has selective toxicity to tumor cells, basically no effect on normal cells. With further studies, UA would be one of the potential anti-tumor agents. KEYWORDS ursolic acid, anti-tumor, research progress
Ursolic acid (UA) is a sort of pentacyclic triterpenoid carboxylic acid purified from natural plant (Figure 1). It has been discovered recently that it has a series of biological effects such as sedative, anti-inflammatory, anti-bacterial, anti-diabetic, antiulcer, and etc., also its toxicity is low to normal cells. UA can act on a variety of tumor cell lines, including prostate carcinoma, hepatocellular carcinoma, ovarian carcinoma, gastric carcinoma, melanoma and others. It has been proved that UA is involved in several signal transduction pathways to its anti-tumor mechanisms.(1) In recent years, especially in the past three years, the anti-tumor effects of UA have been studied rapidly. Therefore, the anti-tumor effects are summarized as following.
closely related to tumor development. (2) UA can protect DNA from H 2 O 2 -induced oxidative DNA damage in HepG2 cells,(3) Caco-2 cells. Moreover, it is reported that UA not only protects DNA from oxidative damage but also increases repair activity in Caco-2 cells, which contribute to its anticarcinogenic effect. (4) However, there is also a contrary effect as latest study result shows that UA has a vigorous inductive activity of human normal endothelial cell apoptosis because of its activation of P53 protein, Bak, and caspase-dependent cell death pathway. Thus, this phenomenon needs to be confirmed in tumor cells.(5)
Anti-Tumor Cell Proliferations Inhibition of Epidermal Growth Factor Receptor/ Mitogen-Activated Protein Kinase Pathway Activation of mitogen-activated protein kinase (MAPK) allows cell excessive proliferation involved in the carcinogenic process.(6) Subfamilies of MAPK,
Figure 1.
Chemical Structure of UA
Protection of Cells' DNA from Different Damages DNA repair or oxidative DNA damage is
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013 Supported by the National Natural Science Foundation of China ( No. 30772601) 1. Pharmacology Department, Dalian Medical University, Dalian, Liaoning Province (116044), China; 2. Medicine Department, Dalian Third People's Hospital, Dalian, Liaoning Province (1116033), China Correspondence to: Prof. TANG Ze-yao, Tel: 86-411-86110412, E-mail: [email protected] DOI: 10.1007/s11655-013-1541-4
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such as extracellular signal regulated protein kinase (ERK), c-Jun-N-terminal kinase (JNK/SAPK) and p38MAPK play considerable roles in the carcinogenic process of cells.(7) And some kinds of human cancers (such as oral cancer, melanoma, breast cancer, etc.) had been found over-activation of MAPK.(8) Studies have shown that UA can inhibit human endometrial cancer cells and HT-29 cells proliferation by inhibiting the activity of MAPK as well as promoting the phosphorylation of epidermal growth factor receptor (EGFR), ERK1/2, p38MAPK and JNK, and then inhibits the pathway of EGFR/MAPK (9,10) . On the other hand, UA can inhibit the proliferation of breast cancer CAOV3 cells and HT-29 colon cancer cells by suppressing the activity of ERK and the expression of p-ERK1/2.(11)
Inhibition of Transcription Factor-FoxM1 Pathway FoxM1 is a transcription factor belonging to the Fox family which is widely presented in cells with mitotic activity. FoxM1 orchestrates the transcription of genes which are essential for cell cycle progression and cell proliferation. In a great many kinds of tumor cells, the expression of FoxM1 is increased. (12,13) UA can inhibit human breast cancer MCF-7 cell proliferation in time and dose-dependent manner through suppressing the over-expression of FoxM1 activity.(14) In addition, UA can also inhibit cells proliferation of melanoma A375, glioblastoma U87, and anaplastic thyroid cancer (ARO) by inhibiting the endogenous reverse transcriptase activity.(15)
Antiangiogenesis Angiogenesis, the formation of new capillaries from preexisting vessels, is essential for tumor progression. It is confirmed that a variety of active substances can regulate tumor angiogenesis. Angiogenesis factors are mainly a large class of growth factors or cytokines polypeptide. These peptides can directly or indirectly act on vascular endothelial cells, causing blood vessel dilation, endothelial cell deformation and capillaries within the granulation tissue growing into tumor tissues. Among these cytokines polypeptides, vascular endothelial growth factor (VEGF) plays an important role in the occurrence of tumor blood vessels. UA can inhibit the tumor-associated capillary formation in C57BL/6 mice induced by highly metastatic
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B16F-10 melanoma cells. The serum levels of VEGF, nitric oxide (NO), and proinflammatory cytokines of animals treated by UA were significantly reduced compared with those in control animals. Furthermore, the diminished expressions of VEGF and inducible nitric oxide (iNOS) genes in B16F-10 melanoma cells treated with nontoxic concentrations of UA support these observations; the serum tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-2 (IL-2) levels were significantly elevated after the UA treatment.(12) Further study showed that UA could also inhibit VEGF and IL-8 production and expression, maintain glutathione levels, reduce reactive oxygen species (ROS) activity and NO level, delay human hepatoma Hep3B, Huh7 and HA22T cells invasion and migration in a dose-dependent manner. (16) Moreover, matrix metalloproteinases (MMP) also play a very important role to angiogenesis. UA can inhibite MMP-2 and MMP-9 activity so as to antagonize angiogenic effect.(17)
Immunological Surveillance to Tumors Toll-like receptors (TLR) are important protein molecules involved in non-specific immunity (natural immunity) and connect non-specific immunity with specific immunity as a bridge. TLR is an immune adjuvant, playing a key role in enhancing antitumor immunity. Moreover, TLR can recognize endogenous ligands produced by tumors during developing process. Thus, TLR acts significantly in tumor immune surveillance. (18) Surprisingly, UA modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy.(19)
Inhibition of Tumor Cell Migration and Invasion It has been confirmed that MMPs and urokinase plasminogen activator (u-PA) have closely relations with tumor migration and invasion.(20,21) While, the anti-invasive effects of UA on MDAMB231 cells and prostate cancer PC-3 cells might be achieved through the inhibition of Jun N-terminal kinase, Akt and mammalian target of rapamycin (mTOR) phosphorylation and a reduction of the level of nudear factor κB (NF-κB) protein in the nucleus, ultimately leading to down-regulation of MMP-2 and u-PA expression.(22,23) Furthermore, UA could efficiently inhibit the interaction of ZIP/p62 and PKC-zeta which are related to tumor angiogenesis and
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metastasis.(24) Otherwise, UA suppresses the activation of NF-κB and down-regulation of the MMP-9 protein, which in turn contributes to its inhibitory effects on IL-1β or ttumor necrosis factor α (TNF-α)-induced C6 glioma cell invasion.(25) UA suppresses intercellular adhesion molecules-1 (ICAM-1) expression of non-small cell lung cancer (NSCLC) H3255, A549, Calu-6 cells, and significantly inhibits fibronectin expression in a concentration-dependent way. UA significantly suppresses the expression of MMP-9 and MMP-2 and inhibits protein kinase C activity in test cell lines, at the same time, UA reduces cell invasion in a concentration-dependent manner.(26) Some new evidence indicate that Cys-X-Cys (CXC) chemokine receptor 4/CXC chemokine ligand 12 (CXCR4/CXCL12) signaling pathway plays a pivotal role in the process of distant site metastasis that accounts for more than 90% of prostate cancer related deaths in patients. Thus, novel drugs that can down-regulate CXCR4/CXCL12 axis have a great potential in the treatment of metastatic prostate cancer. UA inhibits metastasis in vivo in transgenic adenocarcinoma of mouse prostate (TRAMP) by down-regulating CXCR4 expression in prostate cancer cell in a dose- and time-dependent manner. When investigated for the molecular mechanisms, it has been observed that the down-regulation of CXCR4 is due to transcriptional regulation as indicated by down-regulation of mRNA expression, inhibition of NF-κB activation and modulation of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by UA further correlates with the inhibition of CXCL12-induced migration and invasion in prostate cancer cells. Finally, it has been found that UA can inhibit metastasis of prostate cancer to distal organs, including lung and liver and suppress CXCR4 expression levels in the prostate tissues of TRAMP mice. Overall, UA exerts its antimetastatic effects through the suppression of CXCR4 expression in prostate cancer both in vitro and in vivo .(27) In addition, UA plays a strong role in inhibiting proteases such as urokinase and cathepsin B, while these proteases are involved in tumor invasion and metastasis. Therefore, these results show that UA has the potential role in the chemoprevention and
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treatment of cancer metastasis and invasion.(28)
Induction of Tumor Cell Apoptosis Apoptosis is a progress of genetically controlled programmed cell death. It has a close concern with tumor's occurrence, development, turnover and prognosis. The induction of cell apoptosis is one of the key ways for tumor therapy. Moreover, UA can induce some kinds of tumor cells apoptosis including human breast carcinoma MCF-7 cells, colon cancer HCT116 cells, human hepatoma HuH7 cells, prostate cancer cells, HepG2 cells and etc. While, UA-induced apoptosis of tumor cells is involved in multiple signal pathways.(29-31)
Caspase-Dependent Apoptosis Pathway and Cytochrome C Apoptosis Pathway Caspase is cysteine protease and a key enzyme in apoptosis progress. Caspase exists as a zymogen in normal cells. Once the apoptosic process begins, caspase cascade reaction progresses after caspase is actived, resulting in irreversible apoptosis.(32) UA can initiate apoptosis process of prostate cancer RC-58T/h/SA#4 cells, MCF-7 cells, HuH7 cells and other cells by activating corresponding cysteine aspartase, such as caspase-3, -8 and -9.(29-31) During the induction of apoptosis in human hepatoma HuH7 cells, UA mediates cytochrome C releasing into the cytosol, activates the caspase-3, caspase-9 and then splits the ribose polymerase body which gives rise to apoptosis process.(33) Besides, during the induction of apoptosis of NSCLC H460 cells, UA starts death process through glucocorticoid receptors on cytosol in the way of up-regulating MMP, activating caspase-3 activity, changing the nuclear morphology and leading to the DNA breakage.(34) Additionally, UA can effectively induce the apoptosis of prostate cancer LNCaP, LNCaP-AI cells and leukemia K562 cells, because it is able to activate JNK which leads to Bcl-2 phosphorylation, then induce decrease of caspase activity and the apoptosis of tumor cell. This function make it possible to reduce the resistance to apoptosis of LNCaP-AI cells and leukemia K562 cells.(35,36)
Inhibition of NF-κB Activity Pathway NF-κB is a ubiquitous nuclear transcription factor
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which can regulate expression of cytokines, growth factors, cell adhesion molecules and acute reactive protein gene in eukaryotic cells. NF-κB participates in the transcriptions of target gene to regulate cell proliferation, differentiation, growth and apoptosis. UA can reduce the producing of reactive oxygen species (ROS), prevent the activation of inhibitor of κB kinase (IKK) complex, inhibit subunit of NF-κB (IκBa) phosphorylation, inhibit the degradation of IκBa, repress the translocation of NF-κB nuclear and stop NF-κB binding to DNA. For all these reasons, UA can inhibit excessive activation of NF-κB and regulate the expression of Bcl-2 gene and the activation of caspase-8. (37) Then, UA can inhibit the apoptosis process of TNF-mediated HuH7, prostate cancer, RC58T/h/SA#4, prostate cancer PC-3, LNCaP, human hepatocellular carcinoma SMMC-7721, ovarian cancer CAOV3, leukemia, M4Beu and HaCaT cells.(32,38-40) Moreover, excessive activation of NF-κB can reduce the effects of chemotherapy. However, UA can inhibit the activity of NF-κB, significantly reducing the amount of chemotherapy drugs and enhancing tumor cells' sensitivity to chemotherapeutic drugs. UA anti-tumor sensitivity depends on activating the signal amplification of the endogenous caspase-8, Bid (a member of family Bcl), cytochrome C and caspase-3. Respectively, they achieve their effects by increasing the decomposition of Bid and activating Fas/FasL-caspase-8. UA also shows cell apoptosisinducing effect on human colon carcinoma SW480, SW620 possibly by down-regulating the expressions of apoptosis antagonistic proteins, Bcl-2, Bcl-xL, and surviving. (41) By inhibiting NF-κB activity, UA shows another role of the attenuation of toxicity and augmentation of synergy efficiency of chemotherapy. Using small doses of UA to inhibit NF-κB activity can dilate the therapeutic effects and enhance the sensitivity of tumor cells to chemotherapeutic drugs. Studies have shown that, UA only improve the sensitivity of tumor cells; while normal cells' sensitivity remains the original level. Therefore, without influencing the effects of chemotherapy drugs, adding a dose of UA can reduce the dose of chemotherapeutic drugs but improve the effect of cancer treatment.(42) Signal transducers and activators of transcription 3 (STAT3) is a signal transduction and activator of transcription factor which belongs to signal sensitivity transcription factor family, just like NF-κB,
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presenting in the cytoplasm of normal cells. Variety of cytokines, such as IL-6, TNF, VEGF, etc., can induce the expression of STAT3, then lead normal cells to get malignant transformation (Figure 2). Reversible acetylation of STAT3 can also affect the activity of NF-κB family.(43) It has been confirmed that STAT3 is closely related with various kinds of human tumor cells' growth, proliferation and angiogenesis. Meanwhile Janus protein tyrosine kinase (JAK) is a tyrosine protein kinase which plays an important role in signal transduction. JAK may be relatively activated after cytokine receptors are combined with ligand and further activate other signal proteins. Thereby the activation of STAT3 plays a role in inducing the expression of gene.(44) Inhibiting Cytokines Receptor
Inhibiting
Gene productions
UA
P
Cell membrane
JAK P STAT
STAT Nucleus
P STAT P
DNA
Figure 2. Classical JAK-STAT Signal Conductive Pathway
Notes: JAK may be relatively activated after cytokine receptors are combined with ligand and further activate STAT. When two STAT molecules are combined with each other, the gene expresses. UA can not only inhibit the expression of IL-6induced STAT3 but also down-regulate the STAT3 by regulating gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin (Mcl-1) and VEGF
When dealing with the multiple myeloma, by the way of activating the proto-oncogene-mediated c-Src, JAK1, JAK2, and ERKs, UA can not only inhibit the expression of IL-6-induced STAT3 but also downregulates the STAT3 by regulating gene products, such as cyclin D1, Bcl-2, Bcl-xL, surviving, Mcl-1 and VEGF. Above all, UA can inhibit the proliferation of multiple myeloma cells and induce apoptosis, to arrest cells at G1 phase and G0 phase of cell cycle.(45)
Inhibition of Cyclooxygenase Pathway There is a phenomenon that cyclooxygenase-2 (COX-2) is over-expression in a variety of tumor cells.(46) COX-2 was involved in a resistance mechanism to UAinduced apoptosis in colorectal cancer HT-29 cells and cancer cells SGC7901.(47,48) The significant decrease of the level of COX-2 protein can be found in UAinduced apoptosis of hepatocellular carcinoma HepG2
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cells.(49) It is concluded that UA inhibiting COX-2 overexpression is another way to accelerate the apoptosis of tumor cells.
Activation of the TNF Related Apoptosis Inducing Ligand Pathway Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a kind of apoptotic precursor protein ligand, which is a newly discovered member of the TNF superfamily. It may be associated with the death receptors on cell surface and can activate extrinsic apoptosis pathway which aims at inducing the apoptosis process specifically of a variety of tumor cells, transformed cells or virus infected cells.(50) Therefore, TRAIL may be a new target for antitumor drugs because of its induction of apoptosis selectively.(51) UA can down-regulate cell survival protein and up-regulate the expression of TRAIL receptors, death receptor (DR)-4 and DR-5 on cell surface, both of which need certain activity of ROS. UA can activate ROS through which the TRAIL pathway induces the apoptosis of tumor cells, this effect may be blocked by ROS quencher and JNK inhibitor. In conclusion, through the activation of ROS and JNK, UA can up-regulate DRs and downregulate cell survival protein to enhance the TRAIL pathway and then to induce the apoptosis of tumor cells.(52)
Stimulation of the mTOR Pathway The mTOR signal pathway is involved in energy and nutrient supply of cell growth and division process. The mTOR complex-1 and complex-2 play their roles by regulating other important kinases, such as Akt.(53) Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinases which plays an important role in cell survival and apoptosis. Akt can make a series of proteins phosphorylation, while through a variety of ways inhibit the apoptosis process.(54) Reports have pointed out that after dealing post-menopausal breast cancer MMTV-Wnt-1 cells with UA, UA can regulate the activity of Akt, and then stimulate mTOR signal pathways which leads to terminate cell cycle progression and finally induces apoptosis process.(55)
cancer priming, as well as fight carcinogens such as benzopyrene, aflatoxin Bl-induced mutations.(57) It has been found that UA can inhibit Epstein-Barr virus early antigen (EBV-EA) activation of tumor-promoting agent (TPA)-induced B-cell lymphoma Raji cells and improve the survival rate of Raji cells when some researchers used early antibodies (EBV-EA) activation test type to filter an inhibitor of tumor promoters of skin cancer.(57,58) However, more clinical trials are needed to study in the further.
Outlooks and Prospects Although the high number of plant-derived antitumor drugs, at present, anti-tumor drugs that widely used in clinic directly are just as Changchun bases, camptothecin, Harringtonine bases, podophyllotoxin derivatives and paclitaxel, etc.(59,60) Changchun bases, Harringtonine bases, podophyllotoxin derivatives, and taxol mainly have an effect on tubulin and division of normal cell, preventing proliferation of tumor cells. The camptothecin is through inhibition of DNA topoisomerase I, causing DNA the breaks of single strand, making a killing effect on S-phase, G1 and G 2 phase. And as compared with commonly used herbal medicine in clinic, UA not only inhibits tumor cell proliferation, angiogenesis, induces apoptosis of tumor cells, but also enhances immune function, immune surveillance of tumor cells, attenuates chemotherapeutic drugs, side effects and enhances chemotherapeutic drugs, effects as well as DNA repair (Figure 3). Moreover, UA has selective toxicity to tumor cells, basically no effect on normal cells.(61) UA widely exists in nature, according to incomplete statistics; there are about 27 families and 46 groups, total 62 species of plants to UA in free form or in the form combined with sugar. UA has been extracted and purified from nearly 30 kinds of natural plants.(62,63) However, more animal testing and clinical trials are required in order to make its mechanisms much clearer. In summary, we believe that, UA would be a potential agent with its low toxicity and antitumor activity with the development of systematic study on its pharmacokinetic and pharmacodynamic.
Clinical Application of UA Currently, UA has been used to treat cancer, skin wrinkles in clinical trials.(56) The anti-tumor effect is still in clinical trials. It has been proven by clinical trials that UA can fight against DNA mutations, inhibit
Acknoledgement We thank Dr. XU Bin from Dalian College of Foreign Languages for his hard works on polishing the language.
Chin J Integr Med 2014 Jan;20(1):72-79
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Impeding
Oxidative DNA protection
Increasing Inhibiting
Repair activity EGFR/MAPK pathway Anti-tumor proliferations
Inhibiting Inhibiting
UA
FoxM1 pathway VEGF, NO, ROS, MMP-2, etc. activity
Modulating Inhibiting Activing Inhibiting
TLR↑
Anti-angiogenesis Immunological
MMPs, CXCR4/CXCL12 pathway
Anti-tumor migration and invasion
Caspase, TRAIL, mTOR, etc. pathway Apoptosis NF-κB, COX-2, STAT3 pathway
Figure 3.
UA's Anti-Tumor Actions
Notes: UA has a broad-spectrum anti-tumor effect including (1) the protection of cells' DNA from different damages, (2) the antitumor cell proliferation by the inhibition of EGFR/MAPK signal or of FoxM1 transcription factors respectively, (3) antiangiogenesis, (4) the immunological surveillance to tumors, (5) the inhibition of tumor cell migration and invasion, (6) the effect of ursolic acid on caspase, cytochromes C, NF -κB, COX-2, TRAIL or mTOR signal to induce tumor cell apoptosis respectively, and etc.
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(Received December 3, 2011) Edited by YUAN Lin
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REVIEW Research Progress of Ursolic Acid's Anti-Tumor Actions ZANG Li-li (臧丽丽), WU Bao-ning (吴宝宁), LIN Yuan (林 原), WANG Jun (王 FU Lei (傅 雷), and TANG Ze-yao (唐泽耀)
军),
ABSTRACT Ursolic acid (UA) is a sort of pentacyclic triterpenoid carboxylic acid purified from natural plant. UA has a series of biological effects such as sedative, anti-inflammatory, anti-bacterial, anti-diabetic, antiulcer, etc. It is discovered that UA has a broad-spectrum anti-tumor effect in recent years, which has attracted more and more scholars' attention. This review explained anti-tumor actions of UA, including (1) the protection of cells' DNA from different damages; (2) the anti-tumor cell proliferation by the inhibition of epidermal growth factor receptor/mitogen-activated protein kinase signal or of FoxM1 transcription factors, respectively; (3) antiangiogenesis, (4) the immunological surveillance to tumors; (5) the inhibition of tumor cell migration and invasion; (6) the effect of UA on caspase, cytochromes C, nuclear factor kappa B, cyclooxygenase, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or mammalian target of rapamycin signal to induce tumor cell apoptosis respectively, and etc. Moreover, UA has selective toxicity to tumor cells, basically no effect on normal cells. With further studies, UA would be one of the potential anti-tumor agents. KEYWORDS ursolic acid, anti-tumor, research progress
Ursolic acid (UA) is a sort of pentacyclic triterpenoid carboxylic acid purified from natural plant (Figure 1). It has been discovered recently that it has a series of biological effects such as sedative, anti-inflammatory, anti-bacterial, anti-diabetic, antiulcer, and etc., also its toxicity is low to normal cells. UA can act on a variety of tumor cell lines, including prostate carcinoma, hepatocellular carcinoma, ovarian carcinoma, gastric carcinoma, melanoma and others. It has been proved that UA is involved in several signal transduction pathways to its anti-tumor mechanisms.(1) In recent years, especially in the past three years, the anti-tumor effects of UA have been studied rapidly. Therefore, the anti-tumor effects are summarized as following.
closely related to tumor development. (2) UA can protect DNA from H 2 O 2 -induced oxidative DNA damage in HepG2 cells,(3) Caco-2 cells. Moreover, it is reported that UA not only protects DNA from oxidative damage but also increases repair activity in Caco-2 cells, which contribute to its anticarcinogenic effect. (4) However, there is also a contrary effect as latest study result shows that UA has a vigorous inductive activity of human normal endothelial cell apoptosis because of its activation of P53 protein, Bak, and caspase-dependent cell death pathway. Thus, this phenomenon needs to be confirmed in tumor cells.(5)
Anti-Tumor Cell Proliferations Inhibition of Epidermal Growth Factor Receptor/ Mitogen-Activated Protein Kinase Pathway Activation of mitogen-activated protein kinase (MAPK) allows cell excessive proliferation involved in the carcinogenic process.(6) Subfamilies of MAPK,
Figure 1.
Chemical Structure of UA
Protection of Cells' DNA from Different Damages DNA repair or oxidative DNA damage is
©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2013 Supported by the National Natural Science Foundation of China ( No. 30772601) 1. Pharmacology Department, Dalian Medical University, Dalian, Liaoning Province (116044), China; 2. Medicine Department, Dalian Third People's Hospital, Dalian, Liaoning Province (1116033), China Correspondence to: Prof. TANG Ze-yao, Tel: 86-411-86110412, E-mail: [email protected] DOI: 10.1007/s11655-013-1541-4
Chin J Integr Med 2014 Jan;20(1):72-79
such as extracellular signal regulated protein kinase (ERK), c-Jun-N-terminal kinase (JNK/SAPK) and p38MAPK play considerable roles in the carcinogenic process of cells.(7) And some kinds of human cancers (such as oral cancer, melanoma, breast cancer, etc.) had been found over-activation of MAPK.(8) Studies have shown that UA can inhibit human endometrial cancer cells and HT-29 cells proliferation by inhibiting the activity of MAPK as well as promoting the phosphorylation of epidermal growth factor receptor (EGFR), ERK1/2, p38MAPK and JNK, and then inhibits the pathway of EGFR/MAPK (9,10) . On the other hand, UA can inhibit the proliferation of breast cancer CAOV3 cells and HT-29 colon cancer cells by suppressing the activity of ERK and the expression of p-ERK1/2.(11)
Inhibition of Transcription Factor-FoxM1 Pathway FoxM1 is a transcription factor belonging to the Fox family which is widely presented in cells with mitotic activity. FoxM1 orchestrates the transcription of genes which are essential for cell cycle progression and cell proliferation. In a great many kinds of tumor cells, the expression of FoxM1 is increased. (12,13) UA can inhibit human breast cancer MCF-7 cell proliferation in time and dose-dependent manner through suppressing the over-expression of FoxM1 activity.(14) In addition, UA can also inhibit cells proliferation of melanoma A375, glioblastoma U87, and anaplastic thyroid cancer (ARO) by inhibiting the endogenous reverse transcriptase activity.(15)
Antiangiogenesis Angiogenesis, the formation of new capillaries from preexisting vessels, is essential for tumor progression. It is confirmed that a variety of active substances can regulate tumor angiogenesis. Angiogenesis factors are mainly a large class of growth factors or cytokines polypeptide. These peptides can directly or indirectly act on vascular endothelial cells, causing blood vessel dilation, endothelial cell deformation and capillaries within the granulation tissue growing into tumor tissues. Among these cytokines polypeptides, vascular endothelial growth factor (VEGF) plays an important role in the occurrence of tumor blood vessels. UA can inhibit the tumor-associated capillary formation in C57BL/6 mice induced by highly metastatic
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B16F-10 melanoma cells. The serum levels of VEGF, nitric oxide (NO), and proinflammatory cytokines of animals treated by UA were significantly reduced compared with those in control animals. Furthermore, the diminished expressions of VEGF and inducible nitric oxide (iNOS) genes in B16F-10 melanoma cells treated with nontoxic concentrations of UA support these observations; the serum tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-2 (IL-2) levels were significantly elevated after the UA treatment.(12) Further study showed that UA could also inhibit VEGF and IL-8 production and expression, maintain glutathione levels, reduce reactive oxygen species (ROS) activity and NO level, delay human hepatoma Hep3B, Huh7 and HA22T cells invasion and migration in a dose-dependent manner. (16) Moreover, matrix metalloproteinases (MMP) also play a very important role to angiogenesis. UA can inhibite MMP-2 and MMP-9 activity so as to antagonize angiogenic effect.(17)
Immunological Surveillance to Tumors Toll-like receptors (TLR) are important protein molecules involved in non-specific immunity (natural immunity) and connect non-specific immunity with specific immunity as a bridge. TLR is an immune adjuvant, playing a key role in enhancing antitumor immunity. Moreover, TLR can recognize endogenous ligands produced by tumors during developing process. Thus, TLR acts significantly in tumor immune surveillance. (18) Surprisingly, UA modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy.(19)
Inhibition of Tumor Cell Migration and Invasion It has been confirmed that MMPs and urokinase plasminogen activator (u-PA) have closely relations with tumor migration and invasion.(20,21) While, the anti-invasive effects of UA on MDAMB231 cells and prostate cancer PC-3 cells might be achieved through the inhibition of Jun N-terminal kinase, Akt and mammalian target of rapamycin (mTOR) phosphorylation and a reduction of the level of nudear factor κB (NF-κB) protein in the nucleus, ultimately leading to down-regulation of MMP-2 and u-PA expression.(22,23) Furthermore, UA could efficiently inhibit the interaction of ZIP/p62 and PKC-zeta which are related to tumor angiogenesis and
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metastasis.(24) Otherwise, UA suppresses the activation of NF-κB and down-regulation of the MMP-9 protein, which in turn contributes to its inhibitory effects on IL-1β or ttumor necrosis factor α (TNF-α)-induced C6 glioma cell invasion.(25) UA suppresses intercellular adhesion molecules-1 (ICAM-1) expression of non-small cell lung cancer (NSCLC) H3255, A549, Calu-6 cells, and significantly inhibits fibronectin expression in a concentration-dependent way. UA significantly suppresses the expression of MMP-9 and MMP-2 and inhibits protein kinase C activity in test cell lines, at the same time, UA reduces cell invasion in a concentration-dependent manner.(26) Some new evidence indicate that Cys-X-Cys (CXC) chemokine receptor 4/CXC chemokine ligand 12 (CXCR4/CXCL12) signaling pathway plays a pivotal role in the process of distant site metastasis that accounts for more than 90% of prostate cancer related deaths in patients. Thus, novel drugs that can down-regulate CXCR4/CXCL12 axis have a great potential in the treatment of metastatic prostate cancer. UA inhibits metastasis in vivo in transgenic adenocarcinoma of mouse prostate (TRAMP) by down-regulating CXCR4 expression in prostate cancer cell in a dose- and time-dependent manner. When investigated for the molecular mechanisms, it has been observed that the down-regulation of CXCR4 is due to transcriptional regulation as indicated by down-regulation of mRNA expression, inhibition of NF-κB activation and modulation of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by UA further correlates with the inhibition of CXCL12-induced migration and invasion in prostate cancer cells. Finally, it has been found that UA can inhibit metastasis of prostate cancer to distal organs, including lung and liver and suppress CXCR4 expression levels in the prostate tissues of TRAMP mice. Overall, UA exerts its antimetastatic effects through the suppression of CXCR4 expression in prostate cancer both in vitro and in vivo .(27) In addition, UA plays a strong role in inhibiting proteases such as urokinase and cathepsin B, while these proteases are involved in tumor invasion and metastasis. Therefore, these results show that UA has the potential role in the chemoprevention and
Chin J Integr Med 2014 Jan;20(1):72-79
treatment of cancer metastasis and invasion.(28)
Induction of Tumor Cell Apoptosis Apoptosis is a progress of genetically controlled programmed cell death. It has a close concern with tumor's occurrence, development, turnover and prognosis. The induction of cell apoptosis is one of the key ways for tumor therapy. Moreover, UA can induce some kinds of tumor cells apoptosis including human breast carcinoma MCF-7 cells, colon cancer HCT116 cells, human hepatoma HuH7 cells, prostate cancer cells, HepG2 cells and etc. While, UA-induced apoptosis of tumor cells is involved in multiple signal pathways.(29-31)
Caspase-Dependent Apoptosis Pathway and Cytochrome C Apoptosis Pathway Caspase is cysteine protease and a key enzyme in apoptosis progress. Caspase exists as a zymogen in normal cells. Once the apoptosic process begins, caspase cascade reaction progresses after caspase is actived, resulting in irreversible apoptosis.(32) UA can initiate apoptosis process of prostate cancer RC-58T/h/SA#4 cells, MCF-7 cells, HuH7 cells and other cells by activating corresponding cysteine aspartase, such as caspase-3, -8 and -9.(29-31) During the induction of apoptosis in human hepatoma HuH7 cells, UA mediates cytochrome C releasing into the cytosol, activates the caspase-3, caspase-9 and then splits the ribose polymerase body which gives rise to apoptosis process.(33) Besides, during the induction of apoptosis of NSCLC H460 cells, UA starts death process through glucocorticoid receptors on cytosol in the way of up-regulating MMP, activating caspase-3 activity, changing the nuclear morphology and leading to the DNA breakage.(34) Additionally, UA can effectively induce the apoptosis of prostate cancer LNCaP, LNCaP-AI cells and leukemia K562 cells, because it is able to activate JNK which leads to Bcl-2 phosphorylation, then induce decrease of caspase activity and the apoptosis of tumor cell. This function make it possible to reduce the resistance to apoptosis of LNCaP-AI cells and leukemia K562 cells.(35,36)
Inhibition of NF-κB Activity Pathway NF-κB is a ubiquitous nuclear transcription factor
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which can regulate expression of cytokines, growth factors, cell adhesion molecules and acute reactive protein gene in eukaryotic cells. NF-κB participates in the transcriptions of target gene to regulate cell proliferation, differentiation, growth and apoptosis. UA can reduce the producing of reactive oxygen species (ROS), prevent the activation of inhibitor of κB kinase (IKK) complex, inhibit subunit of NF-κB (IκBa) phosphorylation, inhibit the degradation of IκBa, repress the translocation of NF-κB nuclear and stop NF-κB binding to DNA. For all these reasons, UA can inhibit excessive activation of NF-κB and regulate the expression of Bcl-2 gene and the activation of caspase-8. (37) Then, UA can inhibit the apoptosis process of TNF-mediated HuH7, prostate cancer, RC58T/h/SA#4, prostate cancer PC-3, LNCaP, human hepatocellular carcinoma SMMC-7721, ovarian cancer CAOV3, leukemia, M4Beu and HaCaT cells.(32,38-40) Moreover, excessive activation of NF-κB can reduce the effects of chemotherapy. However, UA can inhibit the activity of NF-κB, significantly reducing the amount of chemotherapy drugs and enhancing tumor cells' sensitivity to chemotherapeutic drugs. UA anti-tumor sensitivity depends on activating the signal amplification of the endogenous caspase-8, Bid (a member of family Bcl), cytochrome C and caspase-3. Respectively, they achieve their effects by increasing the decomposition of Bid and activating Fas/FasL-caspase-8. UA also shows cell apoptosisinducing effect on human colon carcinoma SW480, SW620 possibly by down-regulating the expressions of apoptosis antagonistic proteins, Bcl-2, Bcl-xL, and surviving. (41) By inhibiting NF-κB activity, UA shows another role of the attenuation of toxicity and augmentation of synergy efficiency of chemotherapy. Using small doses of UA to inhibit NF-κB activity can dilate the therapeutic effects and enhance the sensitivity of tumor cells to chemotherapeutic drugs. Studies have shown that, UA only improve the sensitivity of tumor cells; while normal cells' sensitivity remains the original level. Therefore, without influencing the effects of chemotherapy drugs, adding a dose of UA can reduce the dose of chemotherapeutic drugs but improve the effect of cancer treatment.(42) Signal transducers and activators of transcription 3 (STAT3) is a signal transduction and activator of transcription factor which belongs to signal sensitivity transcription factor family, just like NF-κB,
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presenting in the cytoplasm of normal cells. Variety of cytokines, such as IL-6, TNF, VEGF, etc., can induce the expression of STAT3, then lead normal cells to get malignant transformation (Figure 2). Reversible acetylation of STAT3 can also affect the activity of NF-κB family.(43) It has been confirmed that STAT3 is closely related with various kinds of human tumor cells' growth, proliferation and angiogenesis. Meanwhile Janus protein tyrosine kinase (JAK) is a tyrosine protein kinase which plays an important role in signal transduction. JAK may be relatively activated after cytokine receptors are combined with ligand and further activate other signal proteins. Thereby the activation of STAT3 plays a role in inducing the expression of gene.(44) Inhibiting Cytokines Receptor
Inhibiting
Gene productions
UA
P
Cell membrane
JAK P STAT
STAT Nucleus
P STAT P
DNA
Figure 2. Classical JAK-STAT Signal Conductive Pathway
Notes: JAK may be relatively activated after cytokine receptors are combined with ligand and further activate STAT. When two STAT molecules are combined with each other, the gene expresses. UA can not only inhibit the expression of IL-6induced STAT3 but also down-regulate the STAT3 by regulating gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin (Mcl-1) and VEGF
When dealing with the multiple myeloma, by the way of activating the proto-oncogene-mediated c-Src, JAK1, JAK2, and ERKs, UA can not only inhibit the expression of IL-6-induced STAT3 but also downregulates the STAT3 by regulating gene products, such as cyclin D1, Bcl-2, Bcl-xL, surviving, Mcl-1 and VEGF. Above all, UA can inhibit the proliferation of multiple myeloma cells and induce apoptosis, to arrest cells at G1 phase and G0 phase of cell cycle.(45)
Inhibition of Cyclooxygenase Pathway There is a phenomenon that cyclooxygenase-2 (COX-2) is over-expression in a variety of tumor cells.(46) COX-2 was involved in a resistance mechanism to UAinduced apoptosis in colorectal cancer HT-29 cells and cancer cells SGC7901.(47,48) The significant decrease of the level of COX-2 protein can be found in UAinduced apoptosis of hepatocellular carcinoma HepG2
Chin J Integr Med 2014 Jan;20(1):72-79
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cells.(49) It is concluded that UA inhibiting COX-2 overexpression is another way to accelerate the apoptosis of tumor cells.
Activation of the TNF Related Apoptosis Inducing Ligand Pathway Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a kind of apoptotic precursor protein ligand, which is a newly discovered member of the TNF superfamily. It may be associated with the death receptors on cell surface and can activate extrinsic apoptosis pathway which aims at inducing the apoptosis process specifically of a variety of tumor cells, transformed cells or virus infected cells.(50) Therefore, TRAIL may be a new target for antitumor drugs because of its induction of apoptosis selectively.(51) UA can down-regulate cell survival protein and up-regulate the expression of TRAIL receptors, death receptor (DR)-4 and DR-5 on cell surface, both of which need certain activity of ROS. UA can activate ROS through which the TRAIL pathway induces the apoptosis of tumor cells, this effect may be blocked by ROS quencher and JNK inhibitor. In conclusion, through the activation of ROS and JNK, UA can up-regulate DRs and downregulate cell survival protein to enhance the TRAIL pathway and then to induce the apoptosis of tumor cells.(52)
Stimulation of the mTOR Pathway The mTOR signal pathway is involved in energy and nutrient supply of cell growth and division process. The mTOR complex-1 and complex-2 play their roles by regulating other important kinases, such as Akt.(53) Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinases which plays an important role in cell survival and apoptosis. Akt can make a series of proteins phosphorylation, while through a variety of ways inhibit the apoptosis process.(54) Reports have pointed out that after dealing post-menopausal breast cancer MMTV-Wnt-1 cells with UA, UA can regulate the activity of Akt, and then stimulate mTOR signal pathways which leads to terminate cell cycle progression and finally induces apoptosis process.(55)
cancer priming, as well as fight carcinogens such as benzopyrene, aflatoxin Bl-induced mutations.(57) It has been found that UA can inhibit Epstein-Barr virus early antigen (EBV-EA) activation of tumor-promoting agent (TPA)-induced B-cell lymphoma Raji cells and improve the survival rate of Raji cells when some researchers used early antibodies (EBV-EA) activation test type to filter an inhibitor of tumor promoters of skin cancer.(57,58) However, more clinical trials are needed to study in the further.
Outlooks and Prospects Although the high number of plant-derived antitumor drugs, at present, anti-tumor drugs that widely used in clinic directly are just as Changchun bases, camptothecin, Harringtonine bases, podophyllotoxin derivatives and paclitaxel, etc.(59,60) Changchun bases, Harringtonine bases, podophyllotoxin derivatives, and taxol mainly have an effect on tubulin and division of normal cell, preventing proliferation of tumor cells. The camptothecin is through inhibition of DNA topoisomerase I, causing DNA the breaks of single strand, making a killing effect on S-phase, G1 and G 2 phase. And as compared with commonly used herbal medicine in clinic, UA not only inhibits tumor cell proliferation, angiogenesis, induces apoptosis of tumor cells, but also enhances immune function, immune surveillance of tumor cells, attenuates chemotherapeutic drugs, side effects and enhances chemotherapeutic drugs, effects as well as DNA repair (Figure 3). Moreover, UA has selective toxicity to tumor cells, basically no effect on normal cells.(61) UA widely exists in nature, according to incomplete statistics; there are about 27 families and 46 groups, total 62 species of plants to UA in free form or in the form combined with sugar. UA has been extracted and purified from nearly 30 kinds of natural plants.(62,63) However, more animal testing and clinical trials are required in order to make its mechanisms much clearer. In summary, we believe that, UA would be a potential agent with its low toxicity and antitumor activity with the development of systematic study on its pharmacokinetic and pharmacodynamic.
Clinical Application of UA Currently, UA has been used to treat cancer, skin wrinkles in clinical trials.(56) The anti-tumor effect is still in clinical trials. It has been proven by clinical trials that UA can fight against DNA mutations, inhibit
Acknoledgement We thank Dr. XU Bin from Dalian College of Foreign Languages for his hard works on polishing the language.
Chin J Integr Med 2014 Jan;20(1):72-79
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Impeding
Oxidative DNA protection
Increasing Inhibiting
Repair activity EGFR/MAPK pathway Anti-tumor proliferations
Inhibiting Inhibiting
UA
FoxM1 pathway VEGF, NO, ROS, MMP-2, etc. activity
Modulating Inhibiting Activing Inhibiting
TLR↑
Anti-angiogenesis Immunological
MMPs, CXCR4/CXCL12 pathway
Anti-tumor migration and invasion
Caspase, TRAIL, mTOR, etc. pathway Apoptosis NF-κB, COX-2, STAT3 pathway
Figure 3.
UA's Anti-Tumor Actions
Notes: UA has a broad-spectrum anti-tumor effect including (1) the protection of cells' DNA from different damages, (2) the antitumor cell proliferation by the inhibition of EGFR/MAPK signal or of FoxM1 transcription factors respectively, (3) antiangiogenesis, (4) the immunological surveillance to tumors, (5) the inhibition of tumor cell migration and invasion, (6) the effect of ursolic acid on caspase, cytochromes C, NF -κB, COX-2, TRAIL or mTOR signal to induce tumor cell apoptosis respectively, and etc.
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(Received December 3, 2011) Edited by YUAN Lin
