Effect Of Runing II On The Growth And Metastasis Of Transplanted Tumor in Mammary Cancer-bearing Mice And Its Mechanism

Objective:

To study the effect of Runing II (a Chinese herbal preparation for mammary cancer) on the growth and metastasis of transplanted tumors of mammary cancer MA-891-bearing TA2 mice and its mechanism. Methods: The model of mammary cancer MA-891 cell strain transplanted tumor of TA2 mice with lung metastasis was developed to observe the effect of Runing II on the growth and metastasis of the transplanted tumor. The immunohistochemical method and image analysis were adopted to detect the levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), micro-vessel count (MVC), and micro-vessel area (MVA). 

Results: In the Running II group, the tumor weight inhibition rate and the lung metastasis inhibition rate were 37.3% and 65.4% respectively, the tumor growth and lung metastasis were obviously inhibited; And the levels of VEGF and VEGFR, MVC, and MVA were significantly decreased as compared with those in the tumor-bearing control group (P<0.05). Conclusion: The Chinese herbal preparation Running II can inhibit the metastasis of tumors by inhibiting angiogenesis, and the mechanism is possibly related to the down-regulation of VEGF and VEGFR expression.

In addition, at the same time of research, we found that echinacea in Cistanche has an anti-tumor effect. For the application of echinacoside in antitumor drugs, the present invention first adds MTH1, inorganic pyrophosphatase, and dGTP to the reaction solution (100mM pH8.0 Tris-acetic acid, 40mM NaCl, 10mM magnesium acetate, 0.005% Tween20 and 1mMDTT) In the method, the enzyme and substrate were incubated at room temperature for 1 hour and then 25 μl of the malachite green solution was added to terminate the reaction. The absorbance was detected at 630 nm with a BIO‑RAD iMark microplate reader. The results showed that echinacoside had a significant inhibitory effect on the enzyme activity of MTH1; Secondly, the effect of echinacoside on tumor cells at the cellular level was detected by MTT assay, and the results showed that echinacoside could significantly inhibit the growth of SW480 colon cancer cells and U2OS human osteosarcoma cells, and the present invention inhibited the growth of echinacoside MTH1, a specific enzyme that maintains survival in tumor cells, leads to the incorporation of oxidized nucleotides into DNA, causing lethal DNA double-strand breaks in cancer cells, thereby producing anti-tumor effects. The new medical application will lay the foundation for the development of new antitumor drugs in the future.

Click cistanche effects product

Keywords:

mammary tumor; angiogenesis; vascular endothelial growth factor (VEGF); Running II (a Chinese herbal preparation for mammary cancer)

Mammary cancer is a malignant tumor severely threatening female health and the disease incidence ranks first among female malignant tumors, with a tendency to increase year by year. Although its therapeutic methods have made some advances, the long-term survival rate for the patient with mammary cancer has not been raised because of the invasive growth and metastasis of tumors. The angiogenesis of tumors is a basic condition for the growth, invasion, and metastasis of tumors. 

Therefore, inhibition of angiogenesis of tumors is an effective way for preventing the production and development of tumors and prolonging the life of the patient.1 In the present study, the mechanism of the Chinese drug Running II with functions of supplementing qi and nourishing yin, regulating thoroughfare and conception vessels, resolving mass, and detoxicating in inhibiting tumor and anti-metastasis was studied from angiogenesis and its regulatory factors in the transplanted tumor of mammary cancer MA-891 of TA2 mice with lung metastasis.

MATERIALS

Sixty SPF female TA2 mice, weighing (20±2) g, aged 6-8 weeks, were purchased from the Department of Laboratory Animals, Tianjin University of Medical Sciences. The mammary cancer MA-891 cell strain of the mouse was supplied by Prof. Luo Liqin, the Pathological Laboratory, Institute of Tumor, Chinese Academy of Medical Sciences.

Runing II is composed of Sheng Huang Qi (Radix Astragali Mongolici), Shan Ci Gu (Pseudobulbus Cremastrae), Tai Zi Shen (Radix Pseudostellariae), Gou Qi Zi (Fructus Lycii), E Zhu (Rhizoma Curcumae Phaeocaulis), Yi Yi Ren (Semen Coicis), Yin Yang Huo (Herba Epimedii), Dang Gui (Radix Angelicae Sinensis), etc., with dose ratio of 1.67: 1.67: 1.33: 1.33: 1.33: 1.33: 1.33: 1, which were prepared a decoction by the Section of Pharmaceutics, Longhua Hospital, according to a traditional technique, and kept at 4ć for use; Cyclophosphamide (CTX, 200mg/ampule, branch number: 981104) and tamoxifen (TAM) were produced by Shanghai Hualian Pharmaceutical Co. Ltd.; Normal saline was produced by Shanghai Fumin Pharmaceutical Co.

Vascular endothelial growth factor (VEGF) polyclonal antibody (working dilutions 1:25–1:50), vascular endothelial growth factor receptor (VEGFR, working dilutions 1:30–1:50), and FVIIIAg polyclonal antibody (working dilution 1:50) were purchased from Santa Cruz Biotechnology Inc; EnVision second antibody, rabbit anti-rat VEGF antibody, goat anti-rabbit FVIII Ag antibody and goat anti-rabbit VEGFR antibody, and EnVision Kit were purchased from Dako Co., Denmark; 3,3- Diaminobenzidine (DAB) was purchased from Shanghai Huamei Bioengineering Factory.

METHODS

Developing of animal model

The animal model was developed with inference to the methods of Luo and Gao, et al.2,3. In vitro cultured mammary cancer MA-891 cells of the mouse at a logarithmic survival phase were selected and the concentration of cells was regulated as 1x107 /ml. Under the aseptic conditions, the mammalian cancer cells were inoculated into the TA2 mice subcutaneous part of the right axilla (0.2 ml/mouse, i.e. 2X106 cells/ mouse).

Grouping of animals and methods of administration

Two days after inoculation, the mice were randomly divided into 4 groups, 15 mice in each group. 1) Tumor-bearing control group: 0.4 ml normal saline was administrated intragastrically, once each day, five times each week. 2) Runing II group: Runing II was diluted with normal saline and 0.4 ml of this dilution corresponding to 48 g·kg-1·d-1 which was calculated according to the attached table “Conversion Table between Human and Animal Body Surface Areas” in “ Methodology of Studies on pharmacology of Chinese Drugs”4 was given intragastrically, once daily, five times each week. 3) CTX control group: About the dosage used by Jiang Bo,5 CTX was diluted with normal saline, and 0.4 ml corresponding to 50 mg·kg-1·d-1 was given intragastrically, once every other day, thrice each week. 4) TAM control group: TAM was diluted with normal saline and 0.4 ml corresponding to 2.7 mg·kg-1·d-1 was administrated intragastrically, once daily, five times each week. The drugs were given for 3 weeks in all four groups.

Sampling

On the 22nd day after administration, the mice were sacrificed by dislocation of cervical vertebrae and weighed. Then the tumor mass and the lung were taken and weighed respectively. The real body weight of the mouse = the body weight weighted – the tumor weight. Grey nodes on the lung surface, i.e., metastasis focuses, were counted by the naked eye. The tumor mass was placed into a 10% neutral formaldehyde solution for fixation, followed by paraffin embedding, ultrathin sectioning, immunohistochemical Envision two-step straining, and determination of VEGF, VEGFR, microvessel count (MVC), and microvessel area (MVA).

The tumor weight inhibition rate

The tumor inhibition rate = (mean tumor weight of the control group after treatment – mean tumor weight of the medication group after treatment)/mean tumor weight of the control group after treatment × 100%.

The lung metastasis inhibition rate

The lung metastasis inhibition rate = (mean lung metastasis node number of the control group – mean lung metastasis node number of the medication group)/mean lung metastasis node number of the control group x 100%.

Immunohistochemical Envision two-step staining

The paraffin section of 4 μm was taken, followed by routine deparaffin with xylene, gradient dehydration, and washing with PBS, 5 min x 3 times; the section was immersed in 0.01 mol/L, pH6.0 citrate buffer, put in a microwave oven of 98ć for 10 min for antigenic repair and naturally cooled at room temperature after taking from the microwave oven; dripping the first antibody (above-mentioned antibody and dilution of corresponding antibody respectively), 37ć, 90 min, washing with PBS, 5 min x 3 times; dripping Envasion second antibody, at room temperature for 60 min, washing with PBS, 5 min x 3 times; dripping newly prepared DAB color-developing solution, controlling developing color for 3–10 min under a microscope, counterstaining for 45 s with campeachy, rinsing with tap water, drying with a blower, mounting with neutral gum. The cell with the cytoplasm of brown-yellow or black-brown granules was regarded as positive. The known positive section of mammary cancer was used as a positive control, and that in which PBS buffer substituted for the first antibody was used as a negative control.

Image analysis

ACE video camera (resolving power 930 lines) and LEICAQ500W image analyzer and LEICA full-automatic image analysis system were used. Under a microscope x 200, 2 vision fields in each section were randomly selected for the determination of VEGF or 3 vision fields for the determination of VEGFR and MVA, and the percentage of positive area to whole vision field area was calculated in all of the groups. For determination of MVC, 5 regions with the richest blood vessels were selected in each section under a microscope x 100, and then under x 400 the micro-vessels in each vision field of 5 no-overlapping regions were counted, and the mean was used as an average micro-vessel counter (MVC) of the tumor.

Statistical method

Software SPSS8.0 was used and the chi-square test was used for the comparison between the rates, and a t-test or one-way ANOVA was used for the comparison of means.

RESULTS

Tumor-inhibiting and anti-metastasis effects of Running II on the transplanted tumor of mammary cancer MA-891-bearing TA2 mice with lung metastasis

It was shown in Table 1 that the tumor weight inhibition rate was 37.3% in the Runing II group and 86.2% in the CTX control group, both tumor inhibition rates being >30%, with significant difference (both P<0.05) as compared with both tumor-bearing control group and TAM control group, indicating that both Runing II and CTX have a certain tumor-inhibiting effect. And there was a significant difference (P<0.05) between the Runing II group and the CTX control group in the tumor inhibition rate, indicating that the tumor-inhibiting effect of Runing II was lower than that of CTX. The lung metastasis inhibition rate was 65.4% in the Runing II group and 43.9% in the CTX control group, with significant difference (both P<0.05) as compared with a tumor-bearing control group and TAM control group, indicating that both Runing II and CTX have a certain tumor metastasis-inhibiting action. And there was a significant difference between the Runing II group and the CTX control group (P<0.05) in the lung metastasis inhibition rate, suggesting that the anti-metastasis action of Runing II is superior to that of CTX.

Effects of Running II on VEGF and VEGFR expressions in the transplanted tumor of MA-891- bearing TA2 mice 

As shown in Table 2, the expressions of VEGF and VEGFR in both the Running II group and the CTX control group were significantly lower than those in the tumor-bearing control group (P<0.05), with a significant difference between the two. In the Running II group, the VEGF and VEGFR expressions were not significantly different from those in the CTX control group (P>0.05), the VEGF expression was significantly lower than that in the TAM control group (P<0.05), and the VEGFR expression was similar to that in the TAM control group (P>0.05). In the CTX control group, the VEGF and VEGFR expression was lower than those in the TAM control group. In the TAM control group, the VEGF expressions were similar to that in the tumor-bearing control group (P>0.05), and the VEGFR expression was lower than that in the tumor-bearing control group (P<0.05).

Effect of Running II on MVA and MVC of the transplanted tumor in MA-891-bearing TA2 mice

As shown in Table 3, the levels of MVA and MVC in the transplanted cancer in the medication groups were significantly lower than those in the tumor-bearing control group (P<0.05); in the Running II group, the levels of MVA and MVC were significantly higher than those in the CTX control group (P<0.05), the level of MVC was lower than that in the TAM control group (P<0.05), and the MVA level was similar to that in the TAM control group (P>0.05).

DISCUSSION

Traditional Chinese medicine holds that the genesis and development of tumor result from the comprehensive effects of idiopathic and empathic factors but with the stress on the former. In a previous clinical study, Prof. Lu and Tang held that vital-qi deficiency in the interior and disharmony of thoroughfare and conception vessels are the main causes of tumor genesis, and internal invasion, moving and spreading of cancer pathogens are the main conditions of growth and metastasis of mammary cancer.6 Therefore, in treatment, they advocate nourishing vital qi and removing masses. Running II is a Chinese herbal formula that is summed up through the clinical practice of many years according to the treatment principle of strengthening the body resistance first and eliminating pathogenic factors second. In the formula, Sheng Huang Qi (Radix Astragali Mongolici), Tai Zi Shen ( Radix Pseudostellariae), Gou Qi Zi (Fructus Lycii), Yin Yang Huo (Herba Epimedii) are sovereign ingredients, with effects of supplementing qi and nourishing yin, regulating thoroughfare and conception vessels, which conforms to commonly seen characteristics of mammary cancer such as both qi and yin deficiency and disharmony of thoroughfare and conception vessels. Clinically, this formula achieves the obvious effect of prolonging survival time, increasing the quality of life, stabilizing tumor focus, and preventing metastasis of cancer.

The present study established the model of mammary cancer MA-891 cell strain transplanted tumor of TA2 mice with lung metastasis and observed the effect of Running II on solid tumor growth and lung metastasis, and found that there were significant differences in the tumor inhibition rate and the lung metastasis inhibition rate between the Running II group and the tumor-bearing group, and between the Running II group and the CTX control group, showing that Running II has obvious functions of inhibiting tumor growth and anti-metastasis. Additionally, tamoxifen (TAM) does not have the obvious function of inhibiting tumor growth and anti-metastasis in the model mice, being similar to the result that mammary cancer MA-891 is ER-cell strain and the report about direct action of low concentration of TAM abroad.7

The tumor is a typical blood vessel-dependent disease, and its growth, infiltration, and metastasis depend on angiogenesis. At the same time, the neovascularization again supplies a metastasis channel for tumor cells, creating favorable conditions for growth, metastasis, and diffusion of the tumor. 8, 9 VEGF is a pro-angiogenic factor with the strongest, unique, and specific action on vascular endothelial cells, and many tumor cells can secrete VEGF, which increases vascular permeability and directly acting on vascular endothelial cells through binding to the VEGF receptor, stimulating its division and proliferation, inducing angiogenesis of tumor and influencing micro-vessel number, to promote growth and metastasis of tumor.10,11 Most tumor cells have high VEGF expression, while in the normal tissues, only a small number of organs such as the kidney, ovary, etc., have higher VEGF expression. Therefore, some scholars hold that VEGF can be used as markers of tumor metabolism and metastasis12 and more ideal target part of blocking neoangiogenesis in tumors,13 and interfering with vascular regulatory factors of tumors and its acting link, regulating the expression of angiogenesis factors, and inhibiting angiogenesis possibly control growth and metastasis of tumor.

The results in the present study indicate that Runing II can down-regulate expressions of VEGF and VEGFR to possibly block signal conduction of VEGF-induced migration and proliferation of endothelial cells, and further inhibit angiogenesis in tumors, hence exerting antineoplastic and anti-metastasizing effects.

Previous studies showed that in the growth course of malignant tumors, over-fast growth of the tissue inevitably induced serious anoxia of local tissue, and anoxia could stimulate the synthesis of VEGF mRNA and slow down the degradation of VEGF mRNA to induce the genesis of a great number of local new capillary vessels.14,15 In observation of immunohistochemical positive staining cells, the authors found obvious VEGF expression in the cytoplasm of dying necrotic tumor cells, on the contrary, less or no expression of VEGF in tumor cells with active proliferation, which may be related to a higher-level of anoxia inducing factor in tumor cells, conforming to the report of Talks KL, et al.16 Running II contains Dang Gui (ᔧᔦ Radix Angelicae Sinensis), E Zhu (Rhizoma Curcumae Phaeocaulis), Sheng Yi Yi Ren (Semen Coicis), Shan Ci Gu (Pseudobulbus Cremastrae), etc., which function activating blood circulation, resolving phlegm and removing turbid. They inhibit synthesis and secretion of VEGF possibly through improving local microcirculation of the tumor, increasing oxygen supply to tumor cells, hence inhibiting the growth of the tumor to perform anti-metastasis.

REFERENCES

1.Folkman J. Angiogenesis and breast cancer. J Clin Oncol 1994; 12: 441-443.

2. Folkman J. Angiogenesis research: from laboratory to clinic. Forum (Genova) 1999; 9: 59-62.

3. Potgens AJ. Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. Am J Pathol 1995; 5: 775-782.

4.Sauter ER, Nesbit M, Watson JC, et al. The vascular endothelial growth factor is a market of tumor invasion and metastasis in squamous cell carcinomas of the head and neck. Clin Cancer Res 1999; 5: 775-782.

5.Warren RS, Yuan H, Matli MR, et al. Regulation by vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271: 2746-2753.

6. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271: 2746-2753.

7.Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000; 157: 411-421.

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