- Research article
- Open Access
- Open Peer Review
Inhibition of metastasis, angiogenesis, and tumor growth by Chinese herbal cocktail Tien-Hsien Liquid
© Chia et al; licensee BioMed Central Ltd. 2010
- Received: 7 October 2009
- Accepted: 30 April 2010
- Published: 30 April 2010
Advanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. Chinese herbal cocktail which contains various phytochemicals may target multiple dys-regulated pathways in cancer cells and thus may provide an alternative/complementary way to treat cancers. Previously we reported that the Chinese herbal cocktail Tien-Hsien Liguid (THL) can specifically induce apoptosis in various cancer cells and have immuno-modulating activity. In this study, we further evaluated the anti-metastatic, anti-angiogenic and anti-tumor activities of THL with a series of in vitro and in vivo experiments.
The migration and invasion of cancer cells and endothelial cells was determined by Boyden chamber transwell assays. The effect of THL on pulmonary metastasis was done by injecting CT-26 colon cancer cells intravenously to syngenic mice. The in vitro and in vivo microvessel formation was determined by the tube formation assay and the Matrigel plug assay, respectively. The in vivo anti-tumor effect of THL was determined by a human MDA-MB-231 breast cancer xenograft model. The expression of metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) was measured by gelatin zymography. The expression of HIF-1α and the phosphorylation of ERK1/2 were determined by Western blot.
THL inhibited the migration and invasion ability of various cancer cells in vitro, decreased the secretion of MMP-2, MMP-9, and uPA and the activity of ERK1/2 in cancer cells, and suppressed pulmonary metastasis of CT-26 cancer cells in syngenic mice. Moreover, THL inhibited the migration, invasion, and tube formation of endothelial cells in vitro, decreased the secretion of MMP-2 and uPA in endothelial cells, and suppressed neovascularization in Matrigel plugs in mice. Besides its inhibitory effect on endothelial cells, THL inhibited hypoxia-induced HIF-1α and vascular endothelial growth factor-A expression in cancer cells. Finally, our results show that THL inhibited the growth of human MDA-MB-231 breast cancer xenografts in NOD-SCID mice. This suppression of tumor growth was associated with decreased microvessel formation and increased apoptosis caused by THL.
Our data demonstrate that THL had broad-spectra anti-cancer activities and merits further evaluation for its use in cancer therapy.
- Human Umbilical Vein Endothelial Cell
- Matrigel Plug
- Radix Astragalus
- Cordyceps Sinensis
- Suppress Tumor Angiogenesis
Metastasis, or the spread of cancer from its primary site to a distant organ, is the main cause of death in patients with malignancy . Metastasis of cancer cells involves multiple processes and various cytophysiological changes . To metastasize, cancer cells first lose the ability to adhere to neighboring tumor cells and gain migratory and invasive capabilities. Cancer cells can then permeate the basement membrane, invade surrounding tissues and gain direct access to blood and lymphatic vessels via which cancer cells can disseminate throughout the body. During this process, degradation of the extracellular matrix and components of the basement membrane by proteases, such as matrix metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA), plays a critical role in tumor invasion and metastasis [3–6]. Patients with metastatic cancers can no longer be cured by local therapy alone and usually die after painful chemotherapy. Thus control of cancer metastasis is an important issue in tumor treatment.
Angiogenesis, the process of new blood vessel formation, plays a crucial role in the growth and metastasis of tumors . Tumor growth and progression require angiogenesis because in its absence tumor growth is restricted to a few millimeters in diameter due to the physical constraint set by simple diffusion of nutrients and oxygen. In addition, angiogenesis and vascularization allows metastatic tumor cells escape into the circulation and lodge in other organs [7, 8]. As a tumor expands, local hypoxic conditions induce a molecular response in tumor cells, leading to the activation of a key transcription factor, the hypoxia-inducible factor (HIF) . This transcription factor induces the expression of pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which in turn bind to and activate their respective receptors on the surface of endothelial cells, leading to angiogenesis [10, 11]. Since angiogenesis plays a prominent role in tumor growth and metastasis, inhibition of angiogenesis is considered to be an important strategy for cancer therapy [12, 13].
Advanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. The fact that chemotherapy using cytotoxic anti-cancer drugs has significant side effects and offers little survival benefit for patients with advanced malignancies has prompted the use of alternative medicine in cancer treatment. Chinese/Oriental herbal medicine, an ancient and complete medicinal system based on empirical observations, has long been used for treatment of malignancies. Whereas single herbs are seldom used alone for cancer treatment, herbal cocktails containing extracts from several herbs are often used. A number of herbal cocktails have been reported to have anti-tumor activities [14–19] and some of them have been used by cancer patients for many years. However, herbal remedies are yet to be integrated into main stream medicine mainly due to lack of experimental and clinical studies on their safety, efficacy, and pharmacological mechanisms . Careful in vitro and in vivo studies will be essential and necessary to evaluate their efficacy and safety before clinical trials can be contemplated.
Herbal cocktail may target multiple cellular pathways to correct the dys-regulated cellular functions accompanying different stages of cancer development. It is believed that a properly formulated herbal cocktail which takes advantage of synergy and interactions among a myriad of phytochemicals present in the different herbs may achieve better therapeutic efficacy than single herbs. The Chinese herbal cocktail, Tien-Hsien Liquid (THL, prepared by China-Japan Feida Union Co., Ltd., Hong Kong), is a herbal mixture that has been used as an anticancer dietary supplement for more than 15 years and has been used by many cancer patients with favorable results in over 15 countries. Moreover, our previous studies indicate that it has immuno-modulating activity [21, 22] and can specifically induce apoptosis in a wide variety of cancer cells . THL is an aqueous preparation of herbal mixture and consists mainly of extracts from 14 Chinese medicinal herbs: Cordyceps sinensis (CS), Oldenlandia diffusa (OD), Indigo pulverata levis (IPL; also known as Indigo Naturalis), Polyporus umbellatus (PU), Radix astragali (RA), Panax ginseng (PG), Solanum nigrum L. (SNL), Pogostemon cablin (PC), Atractylodis macrocephalae rhizoma (AMR), Trichosanthes radix (TR), Clematis radix (CR), Margarite (M), Ligustrum lucidum Ait (LLA), and Glycyrrhiza radix (GR) [21–23]. Among these constituent herbs, the following herbs or their components have been shown to have anti-tumor activity: CS [24–27], OD [28, 29], IPL [30, 31], PU [32, 33], RA [34–37], PG [38–40], SNL , TR [42, 43], CR , LLA , and GR [45, 46]. Moreover, whereas CS [27, 47], PG [48, 49], GR [50, 51] and OD  have been shown to inhibit tumor metastasis, PG [39, 48, 49], GR  and SNL  have been shown to have anti-angiogenic activity. These results suggest that THL may have inhibitory effect on tumor growth, metastasis and angiogenesis. In this study, we evaluated the anti-metastatic, anti-angiogenic and anti-tumor effects of THL with a series of in vitro and in vivo pre-clinical experiments. Our data indicate that THL had anti-metastatic, anti-angiogenic, and anti-tumor activities. These results support the merit of this herbal cocktail for therapy of various cancers.
The human lung carcinoma cell line, H1299, and the mouse colon carcinoma cell line, CT-26, were routinely grown in Dulbecco's modified Eagle medium (GIBCO BRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) in 5% CO2. The human breast adenocarcinoma cell line, MDA-MB-231, was cultured in DMEM/F-12 1:1 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS in 5% CO2. The human prostate adenocarcinoma cell line, PC-3, was cultured in RPMI-1640 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS in 5% CO2. The human microvascular endothelial cell line-1 (HMEC-1) was cultured in MCDB131 medium (GIBCO BRL Life Technologies) supplemented with 10% FBS, 10 ng/ml epidermal growth factor (Becton Dickinson, San Jose, CA), and 1 mg/ml hydrocortisone (Sigma-Aldrich, Inc., St. Louis, MO) in 5% CO2. Primary human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cord as described  and maintained in medium 199 (GIBCO BRL Life Technologies) supplemented with 20% FBS, 30 μg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), 15 μg/ml heparin (Leo Pharmaceutical Product, Ballerup, Denmark), and 1 mM pyruvate in 5% CO2.
Handling of THL
For the in vitro experiments, the culture medium containing THL was prepared as follows. THL (obtained from China-Japan Feida Union Co., Ltd., Hong Kong) was centrifuged to remove insoluble ingredients, and the supernatant was added to the appropriate culture medium to the final concentrations of 0.1, 0.25, 0.5, 0.75 or 1% (v/v). For the in vivo pulmonary metastasis experiment, THL was used directly (without centrifugation before use). For the breast cancer xenograft experiment, THL was centrifuged to remove insoluble ingredients, and the supernatant was diluted with phosphate-buffered saline (PBS) at 1:1 (v/v) ratio.
Preparation of conditioned medium of MDA-MB-231 cancer cells (231-CM)
2 × 106 MDA-MB-231 cells were incubated for 24 h in 5 ml of serum-free medium. The medium was then collected, filtrated to remove cell debris, and stored at -20°C until use. For mouse Matrigel plug assays, the conditioned medium was concentrated 50 fold before use.
Wound healing migration assay
Cancer cells were seeded at a density of 1-5 × 105cells/well in 12-well culture plates and allowed to form a confluent monolayer. The layer of cells was then scraped with a 20-200 μl micropipette tip to create a wound of ~1 mm width. Cells were then washed twice with fresh medium, and replaced with medium containing indicated concentration of THL. After incubation at 37°C for 20 h, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.5% Coomassie Brilliant Blue. Images of the wounds were captures at 0 h and 20 h after scraping at 100-fold magnification and the average distance of the wound was calculated by using Image Pro Plus software (Media Cybernetics, Bethesda, MD). The ability of the cells to close the wound, that is, their motility, was calculated in the following way: (average distance of the wound at 0 h - average distance of the wound at 20 h/average distance of the wound at 0 h) × 100.
Migration and invasion assays
The in vitro cell migration and invasion assays were performed by using a modified Boyden chamber inserted with polyethylene terephthalate filter membrane containing 8-μm pores in 24-well plates (Millipore, Billerica, MA). For cell invasion assays, the filter membranes were coated with Matrigel (30 μg, Becton Dickinson, San Jose, CA).
Cells (1 × 105) suspended in 200 μl of serum-free medium were seeded onto the upper compartment of the transwell chamber. The lower chamber was filled with serum-free medium containing chemoattractants (10% FBS for migration and invasion of cancer cells; 231-CM for migration and invasion of endothelial cells) and various concentrations of THL. After incubation for 6 h (for migration assays) or 24 h (for invasion assays), the medium in the upper chamber was removed and the filters were fixed with 70% ethanol for 10 min. The cells remaining on the upper surface of the filter membrane were then completely removed by wiping with a cotton swab, and the cells on the opposite surface of the filter membrane were stained with 0.5% Coomassie Brilliant Blue for 10 min. The migrated/invaded cells were then visualized and counted from six randomly selected fields (× 200 magnification) under an inverted microscope.
Production of MMPs and uPA by cancer or endothelial cells were analyzed by gelatin and plasminogen-casein zymography, respectively. In MMP gelatin zymography, cells were cultured in serum-free medium with various concentration of THL for 24 h (cancer cells) or 6 h (endothelial cells), and conditioned media were collected, filtrated, and concentrated (~50 fold). Equal amount of conditioned medium samples were mixed with SDS sample buffer containing 2% SDS without β-mercaptoethanol and applied, without boiling, to 7.5% SDS polyacrylamide gels copolymerized with 2 mg/ml gelatin (Sigma-Aldrich, Inc., St. Louis, MO). After electrophoresis, gels were washed for 30 min at room temperature with gentle agitation in renaturing buffer (2.5% Triton X-100 in H2O) to remove SDS. The gels were then equilibrated in developing buffer (40 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM CaCl2) at room temperature with gentle agitation for 30 min. After removing the old developing buffer, the gels were incubated in fresh developing buffer at 37°C overnight. The gels were then stained with 0.5% Coomassie Brilliant Blue and destained. The MMP activities were visualized as clear bands against the blue background of the stained gels. The uPA zymography was performed as described in the MMP gelatin zymography, except that the SDS polyacrylamide gels containing 1 mg/ml casein (MP Biomedicals, Inc., Irvine, CA) and 1 U/ml plasminogen (MP Biomedicals, Inc.) were used.
Pulmonary metastasis assay
All animal experiments in this study were performed following the Guidelines for Animal Experiments in National Taiwan University and were approved by the Institutional Animal Care and Use Committee in College of Medicine, National Taiwan University (IACUC Approval No: 20060184). Balb/c female mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University (Taipei, Taiwan) and given food and water ad libitum. The mice were oral fed with either THL or water (200 μl; twice a day) throughout the experimental duration. On Day 8 of treatment, the mice were injected intravenously (via tail veins) with 2 × 105 mouse CT-26 colon cancer cells to establish pulmonary metastasis. Mice were killed 15 days after tumor cell injection and the metastatic nodules on the surface of the lungs were counted. The lungs were fixed with formalin. Thin sections were stained with hematoxylin and eosin. Representative fields (at × 40 or × 100 magnification) for each group were photographed.
Tube formation assay
Tube formation of HUVEC and HMEC-1 endothelial cells on Matrigel was performed as described . Matrigel (200 μl) was added to 24-well plates and allowed to solidify for 30 min at 37°C. Endothelial cells (5 × 104 cells/well) suspended in 500 μl of complete medium or 231-CM containing various concentration of THL were seeded on the solidified Matrigel. After incubation for 5 h, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet in 20% methanol. Randomly chosen fields were photographed at × 100 magnification and the closed networks of vessel-like tubes were counted.
Mouse Matrigel plug assay
Female NOD-SCID mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University and housed in pathogen-free condition. The mice were subcutaneously injected with 500 μl of Matrigel containing concentrated 231-CM (10 μl), heparin (10 U) and either THL (5 μl) or water (5 μl). Fourteen days later, mice were killed and the Matrigel plugs were removed. To quantitate the formation of functional blood vessel, the amount of hemoglobin was measured using the Drakin's reagent kit (Sigma-Aldrich, St Louis, MO).
Human MDA-MB-231 breast cancer xenograft model
Female NOD-SCID mice (6-8 weeks old) were purchased from Laboratory Animal Center at College of Medicine, National Taiwan University and housed in pathogen-free condition throughout the experimental duration. Mice were given free access to commercial rodent chow and water. MDA-MB-231 cancer cells (3 × 106, suspended in 100 μl of PBS) were injected subcutaneously into both flanks of the mouse. One week after tumor cell inoculation, the mice were randomly divided into two groups. The weight of the mice in these two groups was similar at this time point. One group was intraperitoneally injected with 100 μl of PBS-diluted THL [1:1 (v/v) dilution] once a day until the end of the experiment (It should be noted that the dosage of THL was optimized in our preliminary experiments and that the THL dose used here showed no toxic effect to the mice). The other group was administrated with PBS using similar protocol as described above. The mouse body weight and tumor size were measured at different time points following tumor implantation, and the tumor volume was calculated according to the following formula: 1/2 (Length × Width2). The tumors were removed at Day 36 after tumor implantation, photographed, and weighed. The tumors were snap-frozen in liquid nitrogen for immunohistochemical (IHC) and TUNEL analyses.
IHC and TUNEL analyses
Cryostat sections of frozen tumors were fixed with 4% paraformaldehyde, washed with PBS, and the endogenous peroxidase activity was blocked with Dako Dual endogenous enzyme block (Dako, Glostrup, Denmark). After washing with PBS, the sections were blocked with 5% FBS in PBS. To detect CD31-positive stained microvessels, the sections were probed with rat anti-CD31 antibody (Becton Dickinson, San Jose, CA), and then incubated with horseradish peroxidase-conjugated secondary antibody by using Rat on Mouse HRP-Polymer Kit (Biocare Medical, Concord, CA). Following color development by using Dako DAB reagent (Dako), the nuclei were stained with hematoxyline. The sections were then sealed with glycerol-gelatin (Sigma-Aldrich, Inc., St. Louis, MO) for microscopic observation. Randomly chosen fields were photographed at × 200 magnification and the number of CD31-positive stained blood vessels was counted. For TUNEL assay, the cryostat sections were fixed in 4% paraformaldehyde, washed with PBS and permeated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate in PBS). The sections were then labeled with TUNEL reaction mixture according to the protocol provided by the manufacturer (Roche Applied Science, Mannheim, Germany) to detect apoptotic cells. Following TUNEL reaction, the sections were rinsed three times with PBS, and incubated in Hoechest 33258 solution to label nuclear DNA. The sections were then sealed with mounting medium (Sigma-Aldrich, Inc.) and subjected to fluorescence microscopy. Randomly chosen fields were photographed at × 200 magnification and the number of TUNEL-positive cells was counted.
Data are present as the mean ± SD. The significance of the difference between groups was evaluated with the Student's t-test, p < 0.05 was considered significant.
THL inhibits the migration ability of various cancer cells
THL inhibits the invasion ability of cancer cells
We next tested whether THL could inhibit the invasion ability of cancer cells. The invasion property of MDA-MB-231, H1299, PC-3, and CT-26 cancer cells was analyzed in the Matrigel-coated Boyden chamber in the presence of various concentrations of THL. In all the cell lines tested, the number of cells invaded through the Matrigel-coated filter was reduced by THL dose-dependently (Fig 1D), indicating that THL can inhibit the invasion ability of these cancer cells.
The extracellular signal-regulated kinase (ERK) signaling pathway is known to up-regulate the expression of MMPs . We thus tested the effect of THL on the ERK signaling pathway. We found that THL could inhibit the phosphorylation of ERK1/2 but not that of AKT (Fig 2D) in CT-26 cancer cells, indicating that THL can inhibit the ERK signaling pathway but not the phosphatidylinositol-3-OH kinase (PI3K)/AKT signaling pathway.
THL inhibits the pulmonary metastasis of mouse CT-26 colon cancer cells in mice
THL inhibits the migration and invasion ability of endothelial cells
MMP-2 and uPA are known to play important roles in endothelial cell invasion. We thus tested whether THL could inhibit the secretion of MMP-2 and uPA in endothelial cells. Zymography assays indicated that THL dose-dependently inhibited the secretion of MMP-2 (Fig 4C) and uPA (Fig 4D) in HMEC-1 cells. VEGF is known to be able to induce MMP-2 expression in endothelial cells . We thus tested whether THL could inhibit VEGF-induced MMP-2 expression in endothelial cells. The data shown in Fig 4E indicate that this is indeed the case.
THL inhibits tube formation by endothelial cells
THL inhibits angiogenesis in the Matrigel plug model
THL inhibits the expression of HIF-1α and VEGF-A in MDA-MB-231 breast cancer cells
THL inhibits the growth of human MDA-MB-231 breast cancer xenografts in SCID mice
Advanced cancer is a multifactorial disease that accumulates many genetic and epigenetic alterations affecting multiple distinct regulatory circuits within cells. To treat advanced cancer, there is a growing belief that combination therapy using multiple drugs targeting various cellular pathways would yield better outcomes than monotherapies. In this respect, herbal cocktail which contains various phytochemicals targeting multiple dys-regulated pathways in cancer cells may provide an alternative/complementary way to treat cancers. In this study, we demonstrated that the Chinese herbal cocktail THL not only could inhibit the in vitro migration and invasion ability of various cancer cells but also could inhibit the metastasis of colon cancer cells to lung in mice. Angiogenesis plays a critical role in the growth and metastasis of tumors. Our data indicate that THL not only could inhibit the migration, invasion, and tube formation ability of endothelial cells but also could inhibit cancer cells to secrete the pro-angiogenic factor VEGF-A. The in vivo anti-angiogenic activity of THL was also demonstrated in the Matrigel plug model and tumor xenograft in NOD-SCID mice. Together, these data indicate that THL is a potential cancer therapeutic agent and merits further evaluation for preventive and therapeutic application to human cancers.
The formation of distant metastasis is the main cause of morbidity and mortality in patients with cancer. In recent years, much effort has been taken to develop drugs that can inhibit metastasis. However, till now promising anti-metastatic agents are still lacking . In this study, we demonstrated that THL could inhibit the in vitro migration and invasion ability of breast, lung, prostate, and colon cancer cells (Fig 1) and suppress the pulmonary metastasis of colon cancer cells in mice (Fig 3). Our data indicate that several activities of THL may account for its inhibitory effect on cancer metastasis. Firstly, THL could inhibit, in cancer cells, the expression and secretion of MMP-2, MMP-9, and uPA (Fig 2A, C), which involve in degradation of extracellular matrix and play important roles in cancer cell migration and invasion [5, 6, 63]. Secondly, THL could inhibit the activity of MMP-2 directly (Fig 2B). Thirdly, THL could inhibit, in cancer cells, the activity of ERK1/2, key molecules of the ERK signaling pathway that has been shown to promote tumor invasion and metastasis [61, 64] (Fig 2D). Fourthly, THL could inhibit, in cancer cells, the expression of HIF-1α (Fig 7A), a transcription factor that promotes metastasis by regulating the expression of metastasis-related genes . Together, these data suggest that THL has multiple anti-metastatic activities and has the potential to be developed into an anti-metastatic agent. This argument is further supported by the observation that THL could inhibit angiogenesis, a critical process for cancer cells to spread to other organs.
We demonstrated that THL could suppress tumor angiogenesis in immunodeficient NOD-SCID mice (Fig 8E). We also demonstrated that THL could inhibit in vivo neovascularization in Matrigel plugs loaded with cancer cell-derived conditioned medium (231-CM) (Fig 6). At least two activities of THL may contribute to its anti-angiogenic effect in tumors. Firstly, THL could directly inhibit the migration, invasion and tube formation of endothelial cells (Fig 4 and 5). Secondly, THL could inhibit the secretion of pro-angiogenic factor by cancer cells (Fig 7). With regard to the direct inhibitory effect on endothelial cells, we have found that THL could inhibit the expression of MMP-2 (Fig 4C) and uPA (Fig 4D) in endothelial cells. MMPs and uPA are known to play a critical role in endothelial cell migration and invasion [8, 63]. The inhibition of MMPs and uPA expression in endothelial cells can lead to suppression of endothelial cell migration and invasion. With regard to the effect of THL on cancer cells, we have found that THL could inhibit the secretion of VEGF-A by cancer cells (Fig 7B). At least two mechanisms may account for THL inhibition of expression of VEGF-A in cancer cells. Firstly, we found that THL could inhibit the hypoxia-induced expression of HIF-1α which is a transcriptional activator of the vegf-A gene , in cancer cells (Fig 7A). Secondly, we found that THL could inhibit, in cancer cells, the activity of ERK1/2 (Fig 2D), which phosphorylates the transcription factor Sp1 and causes the recruitment of Sp1 to the vegf-A promoter . Our finding that THL can inhibit cancer cells to express HIF-1α and to secrete VEGF-A is important in terms of suppression of tumor angiogenesis. To induce neovascularization in tumors, tumor cells must secrete pro-angiogenic factors, such as VEGF-A, which attracts and guides sprouting neovessels into oxygen-depleted regions of the tumor mass. The blocking of HIF-1α and VEGF-A expression in tumor cells can thus lead to inhibition of neovessel formation in tumors.
This study has identified multiple biological pathways as potential targets of the anti-tumor activities of the THL formula. It will be of interest to identify the active chemical compounds in the formula that target these pathways. We have started to fractionate THL by ethyl acetate partition and silica gel column chromatography. Our preliminary results indicated that several fractions of THL could inhibit the migration/invasion ability of H1299 cancer cells. Among them, fraction 4 had the strongest activity in inhibiting H1299 migration/invasion ability (data not shown). Experiments using high-performance liquid chromatography to isolate active components of fraction 4 are in progress. Conceptually, it will be interesting and important to determine whether the anti-metastatic and anti-tumor activities of THL can be reconstituted by a few active chemical compounds identified from THL.
Chinese herbal cocktails designed to maximize the synergistic and minimize the antagonistic interactions among various phytochemicals present in different herbs may have therapeutic efficacy against multifactorial diseases such as cancer. In this study, we demonstrated that the Chinese herbal cocktail THL could inhibit cancer metastasis and angiogenesis by targeting multiple biological and pathological processes in cancer cells. We also demonstrated that THL, which was delivered one week after tumor implantation, could exert anti-tumor effects but had no adverse effect on body weight in immuno-compromised mice, implicating that THL has potential to be used as a therapeutic agent for established tumors. Moreover, our preliminary results also indicated that oral delivery of THL could inhibit tumor growth and induce anti-tumor immunity in immuno-competent mice (J-L Du and W-B Wang, data not shown). Together these data suggest that THL is a promising cancer therapeutic agent and merits further investigation.
We thank Dr Czau-Siung Yang for critical reviewing the manuscript. This work was supported by a grant from Ching-Hsing Medical Foundation, Taipei, Taiwan.
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