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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 67  |  Page : 169-176  

Cordyceps militaris fraction inhibits angiogenesis of hepatocellular carcinoma in vitro and in vivo


1 Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou, China
2 Biotechnological Institute of Chinese Materia Medica, Jinan University, Guangzhou, China
3 Department of Natural Product Chemistry, College of Pharmacy, Jinan University, Guangzhou, China
4 Biotechnological Institute of Chinese Materia Medica; Department of Natural Product Chemistry, College of Pharmacy, Jinan University, Guangzhou, China

Date of Submission02-Aug-2019
Date of Decision22-Aug-2019
Date of Web Publication11-Feb-2020

Correspondence Address:
Liyan Song
Department of Pharmacology, College of Pharmacy, Jinan University, Guangzhou, 510632
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_347_19

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   Abstract 


Background: Cordyceps militaris fraction (CMF) was found to inhibit the proliferation of chronic myeloid leukemia K562 cells, oral squamous carcinoma KB cells, and the metastasis of lung cancer cells. This study focuses on the activity of CMF against angiogenesis of hepatocellular carcinoma (HCC). Objectives: The objective of the study is to research the antiangiogenic activity of CMF in HCC cells and the underlying mechanism in vitro and in vivo. Materials and Methods: Transwell migration and invasion assays were used to measure the effects of CMF on migration and invasion of SMMC-7721 cells and human umbilical vein endothelial cells (HUVECs). Tube formation and rat aortic ring assays were used to assess the antiangiogenic potential of CMF. The antiangiogenic mechanism was detected by immunofluorescence and western blot analysis. The nude mice xenografted with SMMC-7721 cells were used to study the antiangiogenic activity of CMF and its underlying mechanism in vivo. Immunohistochemistry analysis was used to evaluate the effect of CMF on the expression of CD31, and western blot analysis was also performed to detect the expression of vascular endothelial growth factor (VEGF) and other related proteins in tumor tissues. Results: CMF inhibited the migration and invasion of SMMC-7721 cells and HUVECs and suppressed VEGF-induced tube formation of HUVECs and the formation of aortic ring capillaries of rats in a concentration-dependent manner. CMF attenuated the phosphorylation of VEGF receptor 2 (VEGFR2), Akt, and ERK in SMMC-7721 cells and HUVECs. CMF significantly inhibited tumor growth in nude mice xenografted with SMMC-7721 cells. CMF reduced the expression level of CD31, western blot analysis indicated that CMF downregulated the expression of VEGFR2 and attenuated the phosphorylation of Akt and ERK in the tumor tissues, which was consistent with the results obtained from in vitro study. Conclusion: CMF can inhibit the angiogenesis of HCC and the mechanism is associated with suppression of the VEGF/VEGFR2 signaling pathway.

Keywords: Antiangiogenesis, Cordyceps militaris fraction, human umbilical vein endothelial cells, SMMC-7721, vascular endothelial growth factor receptor 2


How to cite this article:
Li Z, Guo Z, Zhu J, Bi S, Luo Y, Yu R, Huang W, Song L. Cordyceps militaris fraction inhibits angiogenesis of hepatocellular carcinoma in vitro and in vivo. Phcog Mag 2020;16:169-76

How to cite this URL:
Li Z, Guo Z, Zhu J, Bi S, Luo Y, Yu R, Huang W, Song L. Cordyceps militaris fraction inhibits angiogenesis of hepatocellular carcinoma in vitro and in vivo. Phcog Mag [serial online] 2020 [cited 2020 Feb 24];16:169-76. Available from: http://www.phcog.com/text.asp?2020/16/67/169/278017



SUMMARY

  • Cordyceps militaris fraction inhibited the migration and invasion of human umbilical vein endothelial cells and SMMC-7721 cells.
  • Cordyceps militaris fraction inhibited vascular endothelial growth factor-induced tube formation of human umbilical vein endothelial cells and formation of rat aortic ring capillaries.
  • Cordyceps militaris fraction significantly inhibited tumor growth in nude mice xenografted with SMMC-7721 cells.
  • The antiangiogenic mechanism of Cordyceps militaris fraction might be through suppression of the vascular endothelial growth factor/vascular endothelial growth factor receptor 2 signaling pathway.




Abbreviations used: CMF: Cordyceps militaris fraction; HCC: Hepatocellular carcinoma; HUVECs: Human umbilical vein endothelial cells; VEGF: Vascular endothelial growth factor; VEGFR2: Vascular endothelial growth factor receptor 2.


   Introduction Top


Liver cancer is one of the most common cancers in the world, and it is also the third lethal cancer. Liver cancer has maintained a sustained increase in morbidity and mortality. According to the latest report, the mortality rate of liver cancer in male and female were 15.2% and 8.9%, respectively, in 2018.[1]

Hepatocellular carcinoma (HCC) is a vascular malignant tumor, and the process of its occurrence and development is closely related to the formation of tumor blood vessels.[2] Angiogenesis is essential for organ growth, repair, and imbalance in this process can lead to many malignant outcomes such as inflammation, ischemia, infection, and immune disease.[3] Antiangiogenesis can effectively inhibit the occurrence, development, and metastasis of liver cancer and provide important ideas and methods for clinical treatment.

Cordyceps militaris (L.) Link is an entomopathogenic fungus species. As a valuable Chinese herbal medicine, C. militaris has a long history in traditional Chinese medicine. The components of C. militaris have a variety of pharmacological activities including immune stimulation, antibacteria, antioxidation, antiaging, antiviral, antitumor, antisteroidogenesis, antihypoglycemia, hypolipidemic, antiangiogenesis, antidiabetes, antifatigue, neuroprotection, liver protection, adrenal protection, and lung protection.[4],[5] Cordycepin isolated from C. militaris inhibited melanoma in mice and prolonged survival of tumor-bearing mice.[6]C. militaris polysaccharide promoted the proliferation of murine splenic lymphocytes,[7] and also played an immunomodulatory role in mice by regulating related genes.[8] Fermented C. militaris extract ameliorated hepatosteatosis via activation of fatty acid oxidation.[9]C. militaris hot water extract inhibited the lipopolysaccharide-induced inflammatory response in porcine alveolar macrophages by regulating mitogen-activated protein (MAP) kinase signaling pathway.[10] Constituents isolated from C. militari s suppressed the inflammatory mediator's production and human cancer cell proliferation, and one of them inhibited the proliferation of HepG2 cells.[11]C. militaris aqueous extract was reported to exert the cytotoxic effect on MCF-7 and HepG2 cells through the caspase-dependent mitochondrial pathway.[12]

The vascular endothelial growth factor (VEGF) family plays a crucial role in angiogenesis. As a multifunctional cytokine, VEGF has direct or indirect regulation in angiogenesis and lymphangiogenesis. It can enhance the proliferative capacity of endothelial cells, accelerate the process of angiogenesis, and increase the permeability of blood vessels. VEGF receptor 2 (VEGFR2) is the most important VEGF receptor and mediates VEGF signaling by activating various cellular signaling intermediates. The angiogenesis of liver cancer can be suppressed by inhibiting the phosphorylation of VEGFR2 and regulating the VEGF/VEGFR2 signal transduction.[13] The downstream phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways play the important roles in the growth, proliferation, metastasis, and invasion of tumor cells. It was found that inhibition of the PI3K/Akt/mTOR pathway induced apoptosis and inhibited proliferation of hepatoma cells.[14] It was also reported that suppression of the Ras/Raf/MEK/ERK signaling pathway by sorafenib inhibited tumor angiogenesis and induced tumor cell apoptosis in the HCC model.[15]

Cordyceps militaris fraction (CMF) is the total nucleoside constituents isolated from the artificial culture of C. militaris. Previous studies have found that CMF was resistant to lung cancer metastasis,[16] induced apoptosis in oral cancer cells,[17] and inhibited proliferation of multiple liver cancer cells. As a continuous investigation, we currently report the antiangiogenic ability of CMF in liver cancer.


   Materials and Methods Top


Fraction preparation and reagents

Cultured C. militaris was purchased from Honghao Biological Company of Jiangmen (Guangdong, China). CMF was prepared as per the previous report.[18] CMF was dissolved in DMEM (Gibco, New York, USA) or RPMI-1640 (Gibco, New York, USA) medium with penicillin-streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (Gibco, New York, USA) and store at −20°C. The antibodies for Akt, p-Akt, CD31, ERK, p-ERK, VEGFR2, p-VEGFR2, β-actin, HRP-linked goat anti-rabbit antibodies were obtained from Cell Signaling Technology Inc. (Boston, MA, USA). VEGF was purchased from PeproTech (Rocky Hill, USA).

Cell lines and animals

Human HCC SMMC-7721 cells and human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China), maintained in DMEM and RPMI-1640 medium with penicillin-streptomycin (Biosharp, China) and 10% fetal bovine serum (FBS). The cell lines were incubated at 37°C in a humidified atmosphere of 5% CO2.

Sprague-Dawley rats (SCXK 2013-0002, 130–150 g, 5–7 weeks old) and BALB/c-nu/nu mice (SCXK 2013-0002, 18–20 g, 6–8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center. All animal programs were approved by the Institutional Animal Care and Use Committee of Jinan University in Guangzhou.

Cell migration and invasion assays

The cell migration and invasion assays were performed according to the reference [2] with minor modifications. The migration of SMMC-7721 cells and HUVECs was evaluated by the use of Transwell chambers with 8-μm pore filters (Corning, New York, USA). Cells (2 × 105/mL) were plated on the upper chambers in serum-free medium with CMF. DMEM or RPMI-1640 medium containing 10% FBS was added to the lower chambers as a chemoattractant. After incubation with the different concentrations of CMF for 24 h at 37°C, non-migrating cells were removed with cotton swabs. Cells that migrated to the bottom of the membrane were fixed with methanol, stained with crystal violet (Aladdin Biotech, Shanghai, China) solution for 30 min, and washed twice with PBS (Dingguo Biotech, Beijing, China). Stained cells were visualized under a microscope (×10), and the numbers of cells counted in three random fields were averaged. To assess the effects of CMF on cell invasion, 2 × 105/mL cells with the different concentrations of CMF were added to upper chambers that had been coated with 30 μL of Matrigel (Corning, New York, USA). DMEM or RPMI-1640 medium containing 10% FBS was added to the lower chambers. Cells were incubated with different concentrations of CMF for 24 h at 37°C and non-invading cells were removed with cotton swabs. Invading cells were fixed, stained, and counted.

Tube formation assay

An assay for the formation of endothelial cell tubes in three-dimensional Matrigel cultures of HUVECs was used to assess angiogenic potential.[2] Matrigel (100 μL) containing 10 ng/mL VEGF with 10% FBS was added into 96-well culture plates. Cells (1 × 105/well) were seeded into these plates and treated with various concentrations of CMF for 6 h. Tube formation was examined in photographs under a microscope.

Rat aortic ring assay

The rat aortas isolated from Sprague-Dawley rats were cleaned of fibroadipose tissue and collateral vessels and cut into approximately 1-mm long rings.[2] The aortic rings were cultured in serum-free DMEM for 12 h, randomized and cultured in Matrigel with VEGF (100 ng/ml) coated in 6-wells, and sealed with a 50 μL overlay of Matrigel with VEGF (100 ng/ml). Medium (150 μL) containing VEGF (100 ng/ml) along with different concentrations of CMF was added. The preparations were incubated at 37°C under 5% CO2 for 7 days. At the end of incubation, the sprouting microvessels were fixed and photographed. The numbers of sprouts were assessed semi-quantitatively by Image-Pro Plus Version.

Western blot analysis

Western blot assay was performed as described in the literature.[17] The SMMC-7721 cells and HUVECs (2 × 106 cells per well in 6-well plates) were treated with different concentrations of CMF for 24 h. The cultures were washed twice with PBS, the cells were collected and lysed with RIPA lysis buffer on ice for 10 min and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China). The tumor tissues were cut into small pieces and lysed with RIPA lysis buffer on ice for 10 min and the protein concentration was determined using a BCA protein assay kit. The proteins were loaded onto a 10% or 8% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Germany). The membranes were blocked in 5% BSA for 3 h and incubated overnight with primary antibodies in 4°C. Then, all of the membranes were subsequently incubated with HRP-linked goat anti-rabbit antibodies. The blots were washed with TBS-T and processed with an ECL detection kit (Tanon Crop, Shanghai, China).

Immunofluorescence

The SMMC-7721 cells or HUVECs (1 × 104 per well) were seeded into the wells and cultured at 37°C in a humidified incubator with 5% CO2 for 24 h. The different concentrations of CMF were added to the wells and incubated at 37°C for 24 h. The cells were washed twice with PBS. Then, 4% paraformaldehyde was added and incubated for 20 min and finally adding 0.2% Triton X-100 for 5 min. Then, the cells were treated with 5% bovine serum albumin (Solarbio, Beijing, China) for 2 h. After that, p-VEGFR2 antibody was added to Confocal laser overnight at 4°C and then incubated with FITC goat anti-rabbit antibody (Invitrogen, Grand Island, NY, USA) for 2 h. Subsequently, the dishes were washed three times with PBS, incubated with DAPI for 5 min, and then, finally observed using a Leica TCS SP2 microscope (LeicaBiosystems Nussloch GmbH, Heidelberg, Germany).

Antitumor assay in nude mice

A nude mouse xenograft model was established by using 4–6-week-old male BALB/c nude mice. Mice were housed in sterilized cages at a constant temperature and humidity and fed a regular autoclaved chow diet with water ad libitum. After 4 × 106 SMMC-7721 cells were inoculated subcutaneously into the flanks of 24 nude mice, six nude mice were orally administered with CMF at a daily dose of 260 mg/kg, six mice were orally administered with CMF at a daily dose of 130 mg/kg, six nude mice were intraperitoneally administered with cyclophosphamide (CTX) at twice a week dose of 25 mg/kg and the last six mice were orally administered with sterile water. The tumor volumes were determined by measuring two dimensions, with tumor volume = length × width × width × 0.5. After 20 days of treatment, mice were sacrificed by cervical dislocation under anesthesia with ether and the tumor tissues were collected. Immunohistochemistry (IHC) analysis was conducted.

Statistical analyses

Data are expressed as mean ± standard deviation of the number of independent experiments shown. The significance of the differences between the groups was evaluated by Student's t-test and one-way analysis of variance test. P <0.05 was considered statistically significant.


   Results Top


Cordyceps militaris fraction suppresses migration and invasion of human umbilical vein endothelial cells and SMMC-7721 cells

The process of migration and invasion of tumor cells generally refers to the single tumor cells falling off from the primary tumor and then migrating into the lymphatic vessels or blood vessels, with the blood flow reaching the distant organs and colonizing the organs.[19] The migration and invasion of endothelial cells and tumor cells play the important roles in tumor angiogenesis.[2],[20] Therefore, Transwell migration and invasion assays were used to investigate the effect of CMF on migration and invasion of HUVECs and SMMC-7721 cells. The experimental results showed that the cells treated with CMF migrated significantly less than the untreated cells [Figure 1]a. Similarly, the invasion of HUVECs and SMMC-7721 cells were also suppressed by CMF with a concentration-dependent manner [Figure 1]b.
Figure 1: Cordyceps militaris fraction inhibits the migration and invasion of human umbilical vein endothelial cells and SMMC-7721 cells. (a) Images and numbers of migrating human umbilical vein endothelial cells and SMMC-7721 cells, and (b) Images and number of invading human umbilical vein endothelial cells and SMMC-7721 cells were assessed by Transwell assay. The cells were exposed to 0, 0.625, 1.25, 2.5, 5 or 0, 1.25, 2.5, 5, 10 μg/mL of Cordyceps militaris fraction for 24 h and then stained with crystal violet solution for 30 min. Stained cells were visualized under a microscope (10×) and the numbers of cells counted in three random fields were averaged. *P < 0.05 and **P < 0.01 versus the non-Cordyceps militaris fraction group

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Cordyceps militaris fraction inhibits vascular endothelial growth factor-induced tube formation of human umbilical vein endothelial cellsin vitro and microvessel sprouting ex vivo

HUVECs can form a three-dimensional capillary structure spontaneously on Matrigel, so the tube formation experiment is a simple, reliable, and powerful assay to study antiangiogenesis.[21] To investigate whether CMF has the antiangiogenic capability, the tube formation of HUVECs was performed in the experiments. As shown in [Figure 2], CMF disrupted the tube formation of HUVECs in a concentration-dependent manner. Due to further determine the antiangiogenic effect of CMF, the rat aortic rings were used in the experiment. The results showed that CMF significantly inhibited the formation of aortic ring capillaries and the inhibitory effect increased with the increase of CMF concentrations. When CMF concentrations were 2.5 and 5 μg/mL, the formation of aortic ring capillaries was almost inhibited completely [Figure 3]a and [Figure 3]b. The above results indicated that CMF has the strong antiangiogenic activity.
Figure 2: Cordyceps militaris fraction inhibits vascular endothelial growth factor-induced tube formation of human umbilical vein endothelial cells in vitro. (a) Tubular structures in human umbilical vein endothelial cells were photographed (10×). (b) Numbers of tube in human umbilical vein endothelial cells were measured. The cells were incubated with vascular endothelial growth factor (10 ng/mL) in the presence of 0, 0.625, 1.25, 2.5, or 5 μg/mL of Cordyceps militaris fraction for 6 h, and then, the cells were fixed. **P < 0.01 versus vascular endothelial growth factor -treated group

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Figure 3: Cordyceps militaris fraction inhibits vascular endothelial growth factor-induced microvessel sprouting ex vivo. (a) Aortic rings were photographed (4×). Arrows pointed to microvascular sprouting of rat aortic rings. (b) Numbers of sprouts were assessed semi-quantitatively by Image-Pro plus Version (IOD integrated optical density). The aortas isolated from standard deviation rats were placed in the Matrigel-coated plates, overlayed with Matrigel, and then treated with vascular endothelial growth factor (10 ng/mL) in the presence of 0, 0.625, 1.25, 2.5, or 5 μg/mL of Cordyceps militaris fraction for 7 days. At the end of incubation, the sprouting microvessels were fixed. **P < 0.01 versus vascular endothelial growth factor-treated group

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Cordyceps militaris fraction exerts antiangiogenesis through vascular endothelial growth factor/vascular endothelial growth factor receptor 2 in vitro

To explore the anti-HCC angiogenesis mechanism of CMF in vitro, western blot analysis was used to detect the expression of proteins in the relevant signaling pathway. The VEGF/VEGFR2 signaling pathway plays an important role in tumor angiogenesis, so the expression of related proteins in this pathway was tested in HUVECs and SMMC-7721 cells. The result showed that CMF attenuated phosphorylation of VEGFR2 in HUVECs and SMMC-7721 cells [Figure 4]a. At the same time, phosphorylation of Akt and ERK in HUVECs and SMMC-7721 cells were also attenuated by CMF [Figure 4]b and [Figure 4]c.
Figure 4: Effects of Cordyceps militaris fraction on expression of singling pathway-related proteins in human umbilical vein endothelial cells and SMMC-7721 cells. (a) vascular endothelial growth factor receptor 2/p-vascular endothelial growth factor receptor 2 in human umbilical vein endothelial cells and SMMC-7721 cells, (b) PI3K/Akt/mTOR in human umbilical vein endothelial cells and SMMC-7721 cells and (c) Raf/MEK/ERK in human umbilical vein endothelial cells and SMMC-7721 cells. Human umbilical vein endothelial cells and SMMC-7721 cells were exposed to 0, 0.625, 1.25, 2.5, 5 or 0, 1.25, 2.5, 5, 10 μg/mL of Cordyceps militaris fraction for 24 h and cell lysates were analyzed by Western blot analysis

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Western blot assay demonstrated that CMF attenuated the phosphorylation of VEGFR2, followed by immunofluorescence experiments to further examine the effect of CMF on VEGFR2 phosphorylation. The result showed CMF significantly reduced VEGFR2 phosphorylation of HUVECs and SMMC-7721 cells [Figure 5]a and [Figure 5]b.
Figure 5: Cordyceps militaris fraction reduces phosphorylation of vascular endothelial growth factor receptor 2 in human umbilical vein endothelial cells (a) and SMMC-7721 (b) cells. Human umbilical vein endothelial cells and SMMC-7721 cells were exposed to Cordyceps militaris fraction for 24 h and the cells were fixed and immunostained with phosphovascular endothelial growth factor receptor 2 antibody at the end of incubation. Nuclei were stained with 4′,6-diamidino-2-phenylindole and were taken under ×40 objective by laser scanning confocal microscope

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Cordyceps militaris fraction suppresses tumor growth in nude mice xenografted with SMMC-7721 cells

The above results indicate that CMF has an antiangiogenic effect in vitro. To study the antiangiogenic effect of CMF in vivo, a SMMC-7721 nude mouse xenograft model was established. As a result, compared with the model group, CMF significantly inhibited tumor growth and the tumor volume of the CMF group (260 mg/kg) was close to that of CTX positive control group [Figure 6]a. The same result was found in the weight of the tumor [Figure 6]b and [Figure 6]c. CD31 is a hallmark protein for tumor angiogenesis, whereas the expression of CD31 indicates neovascularization in IHC experiments. In the study, IHC experiments were performed on the tumor tissues of nude mice to detect the expression of CD31. CD31 expression was significantly reduced in the CMF and CTX groups compared with the model group [Figure 6]d and e], indicating that CMF could reduce tumor angiogenesis. In addition, the expression of related proteins in tumor tissues was also detected to explore the mechanism of anti-HCC angiogenesis by CMF in vivo. The results demonstrated that CMF reduced the expression of VEGFR2 and CD31 and decreased the phosphorylation of Akt and ERK. The results above demonstrated that the anti-HCC angiogenesis mechanism [Figure 6]f of CMFin vivo was consistent within vitro study.
Figure 6: Cordyceps militaris fraction inhibits tumor growth in nude mice xenografted with SMMC-7721 cells. (a) Mouse tumor volumes were determined every 4 days during the dosing period. (b) Tumor tissues of nude mice were photographed. (c) Tumor weights of nude mice were measured with an electronic balance. (d) The level of CD31 was measured by IHC analysis (10×). Positive cells were stained brown and negative cells were stained blue.(e) Microvascular IOD was counted by Image-Pro plus Version.(f) Western blots were used to detect protein levels of vascular endothelial growth factor receptor 2, CD31, Akt/p-Akt, and ERK/p-ERK in tumor tissue sections. After 4 × 106 SMMC-7721 cells were inoculated subcutaneously into the flanks of 24 nude mice, the mice were dosed intragastrically with Cordyceps militaris fraction (130 and 260 mg/kg) daily or intraperitoneally with CTX twice a week for 20 days. Data are presented as means ± standard deviation *P < 0.05 and **P < 0.01 versus the model group, n = 6

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   Discussion Top


Liver cancer is one of the five most common cancers in the world. It has maintained a sustained increase in morbidity and mortality. Clinical treatment methods include hepatectomy, liver transplantation, local ablation, hepatic artery embolization, and chemotherapy.[22] With the deepening of antitumor drug research, many oncogenes and signaling pathways have been identified to be closely related to tumorigenesis and development. Our results demonstrated that CMF could suppress the migration and invasion of SMMC-7721 cells and HUVECs inhibit the tube formation of HUVECs and the formation of rat aortic capillaries. Furthermore, CMF significantly inhibited tumor growth in nude mice xenografted with SMMC-7721 cells. The results of western blot analysis suggested that CMF inhibited the angiogenesis of HCC and their possible mechanisms are associated with suppression of the VEGF/VEGFR2 signaling pathway bothin vitro and in vivo.

The VEGF family plays a crucial role in angiogenesis. VEGF can induce lymphangiogenesis, regulate physiological angiogenesis, and even participate in the development and progression of angiogenic diseases.[23] The lumen formation of HUVECs was often used in antiangiogenesis research, and the VEGF plays a crucial role in this process.[20] We found that VEGF could induce lumen formation of HUVECs and CMF could also inhibit the occurrence of this process. VEGF can induce the sprouting of rats aortic ring capillaries ex vivo.[24] Moreover, our experimental result demonstrated that CMF could inhibit VEGF-induced formation of aortic ring capillaries of rats.

The PI3K/Akt/mTOR signal transduction pathway plays an important role in regulating cell survival, proliferation, and migration. Inhibition of PI3K/Akt/mTOR signaling pathway induces apoptosis in liver cancer.[14],[25] CMF could reduce phosphorylation of PI3K/Akt/mTOR in both SMMC-7721 cells and HUVECs. The expression levels of Akt and mTOR phosphorylation were reduced in SMMC-7721 cells and HUVECs after treated with CMF for 24 h. Akt, also known as protein kinase B, is a central node for growth signaling, cytokines, and other cellular signaling downstream of cells stimulation. Abnormal or altered activation of Akt is the basis for the pathophysiological properties of a variety of complex diseases, including type 2 diabetes and cancer. The activation of Akt also activates its downstream signaling pathway, which regulates cell survival, growth, proliferation, metabolism, migration, and invasion. In addition, Akt is also associated with tumor angiogenesis.[26] In contrast, the level of phosphorylated PTEN was increased after treated with CMF. As a unique tumor suppressor, PTEN negatively regulates cells and extracellular matrix interactions to maintain cell sensitivity to apoptosis. The cells will be protected from apoptosis without PTEN.[27]

The Ras/Raf/MEK/ERK signaling pathway is associated with the growth, proliferation, metastasis, and invasion of tumor cells. The inhibition of Ras/Raf/MEK/ERK signaling pathway can effectively inhibit the proliferation of hepatoma cells,[15],[28] migration, and invasion.[29] Raf-1 has been shown to be a cross-linking point in various signaling pathways and plays an important role. Raf-1 overexpression can be considered as an independent prognostic biomarker in HCC and predicts early tumor recurrence and death in HCC patients. It can be used to stratify the risk of poor patient prognosis and to help the selection of the appropriate HCC treatment regimen.[30] The ERK1/2 MAP kinase plays a key role in the mitogenic signal transduction pathway and is an important component of the MAP kinase cascade, including MEK (MAP kinase kinase) and Raf-1. The ERK1/2 has been observed in almost half of known human tumor cell lines and a large number of human primary tumors.[31] When ERK1/2 is activated, it activates downstream signaling pathways to promote hepatoma cell metastasis and invasion.[32] CMF can attenuate ERK1/2 phosphorylation, and it suggested that antiangiogenic effect of CMF may also be related to the suppression of Ras/Raf/MEK/ERK signaling pathway.

VEGF binds to VEGFR and activates the downstream signal transduction cascade, including ERK and the PI3K/Akt/mTOR signaling pathway.[33] Moreover, VEGF-D induces PI3K-mediated Akt activation and stimulates angiogenesis.[34] VEGF could elevate phosphorylation VEGFR2 protein and promoted cholangiocarcinoma cell proliferation, migration, and invasion via downstream Raf/MEK/ERK and PI3K/AKT pathways.[35] Therefore, the antiangiogenic activity of CMF is associated with suppression of VEGF/VEGFR and its downstream signal transduction.


   Conclusion Top


In the present study, the antiangiogenic properties and mechanism of CMF were investigatedin vitro and in vivo. CMF inhibited migration and invasion of HUVECs and SMMC-7721 cells as well as tube formation of HUVECs and the formation of rat aortic capillaries. Antiangiogenic effect of CMF was also provedin vivo in nude mice xenografted with SMMC-7721 cells. Therefore, we could make a conclusion that CMF can inhibit the angiogenesis of HCC, and its possible mechanism is through the suppression of VEGF/VEGFR2-mediated PI3K/Akt/mTOR and RAF/MEK/ERK signaling pathways bothin vitro and in vivo.

Acknowledgements

The authors would like to thank Dr. Dongbo Yu of The University of Chicago, Illinois, USA, for proofreading our manuscript.

Financial support and sponsorship

The present study was supported by the Major National Science and Technology Products/Significant New Drugs Creation (grant no. 2011ZX09102001-033) and National Natural Science Foundation of China (Grant no. 81374015 and 81673646).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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