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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 5  |  Page : 65-69

The inhibitory effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma and its mechanism research


1 Department of Pharmacy, Zhengjiang Cancer Hospital, Hangzhou, Zhejiang, China
2 Department of Pharmacy, Zhejiang Cancer Research Institute, Zhengjiang Cancer Hospital, Hangzhou, Zhejiang, China

Date of Web Publication30-Aug-2014

Correspondence Address:
Hong-yan Zhang
Zhengjiang Cancer Hospital, Hangzhou 310022
China
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Source of Support: This study was supported by Traditional Chinese medicine scientific research fund project of Zhejiang province. Equipment used in this study was provided by Zhejiang Cancer Research Institute, Conflict of Interest: None


DOI: 10.4103/0973-1482.139765

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 > Abstract 

Background: The incidence of hepatocellular carcinoma (HCC) is very high in the world. However, a safe and effective strategy is still under research.
Aims: Our aim was to demonstrate the inhibitory effect of Shaoyao Ruangan Formmula (SRF) on the tumor of H22-bearing mice and explore its antitumor mechanisms.
Settings and Design: Corresponding physiological indexes of H22-bearing mice treated with SRF were compared with that of saline treated mice, which could reflect the tumor-suppressing effect of SRF.
Materials and Methods: After treatment, tumor weight, survival time, related gene expression levels etc., were recorded or detected.
Statistical Analysis: Data analyzed using a computer SPSS program.
Results and Conclusions: Comparing with blank control group, the tumor inhibitor rate (IR) of low, middle and high dose group of SRF was 17.72%, 33.99% and 23.73%, respectively. IR of CTX was 43.95%. The results also showed that each group of SRF could prolong the life span of H22-bearing mice to some extent. In addition, reverse transcription polymerase chain reaction (RT-PCR) results revealed that SRF was able to influence related genes expression in the tumor tissues of H22-bearing mice. The expression of TGF-β receptor type II (TBRII) gene was significantly upregulated in each SRF group comparing with normal saline group. On the contrary, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was significantly downregulated in each SRF group comparing with normal saline group. In summary, SRF showed tumor-suppressing effect on mice with transplanted H22 hepatocarcinoma. The mechanism of antitumor effect may induced by upregulating TBRII expression and down-regulating NF-κB expression.

Keywords: Anti-tumor effect, HCC, Shaoyao Ruangan formula


How to cite this article:
Zhang Hy, Jiang Jw, Ni Mw, Peng Ys, He Fg. The inhibitory effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma and its mechanism research. J Can Res Ther 2014;10, Suppl S1:65-9

How to cite this URL:
Zhang Hy, Jiang Jw, Ni Mw, Peng Ys, He Fg. The inhibitory effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma and its mechanism research. J Can Res Ther [serial online] 2014 [cited 2021 Feb 24];10:65-9. Available from: https://www.cancerjournal.net/text.asp?2014/10/5/65/139765


 > Introduction Top


Hepatocellular carcinoma (HCC) is one of the 10 most common tumors in the world and also regarded as one of the most malignant tumors. According to the data of GLOBOCAN 2012 of World Health Organization (WHO), the incidence of liver cancer has raised to the sixth and tenth most common type of malignancy tumors of male, and female respectively. Moreover, liver cancer is the third death causes of tumors of male, and the ninth of female. It has been reported that over 50% of new cases and deaths are discovered in China. [1],[2] Therefore, there is an urgent need for the development of a novel drug or prevention role of agent which is safe and effective for patients to fight against HCC. However, the quest for novel therapeutic agents and strategies for liver cancer is still an enormous challenge because liver cancer has been discovered to be highly resistant to existing chemotherapy drugs. [3],[4],[5],[6],[7],[8]

Traditional Chinese Medicine (TCM) based on the holistic concept and syndrome differentiation has been utilized to diagnose and treat disease for thousand years. TCM has been demonstrated to be efficient in improving the clinical symptoms, prolonging life span, improving the quality of life, and preventing recurrence and metastasis. [9] The prescription of TCM is mainly comprised by many herbs, thus comparing with chemotherapy drugs, the advantages of herbs lay in less resistance, synergy and attenuation. Shaoyao Ruangan Formula (SRF), a prescription for advanced liver cancer of Zhejiang Cancer Hospital, contains 19 different herbs (Oldenlandia diffusa, Scutellaria barbata D. Don, Rhizoma Paridis, Tetrastigma hemsleyanum Diels ET Gilg, and etc.). It has been used in the adjuvant treatment of hepatitis, HCC and hepatic cirrhosis for many years. In previous clinical application, SRF was conferred beneficial effects on the chemically induced hepatic damage. [10] In addition, it was also found that the combination of SRF and other chemical drugs could improve the life quality and prolong the survival time of patients. However, the antitumor effect of SRF on HCC and the corresponding mechanism has been barely reported so far. In the present study, therefore, the antitumor effect of SRF was investigated at both body and molecular levels, and the mechanism was illustrated as well.


 > Materials and methods Top


Animals and cells

Imprinting control region (ICR) mice (SPF, 4-6 weeks old) weighing 18-22 g were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science, China (Certificate No. SCXK (Hu)-2007-0005). H22 hepatoma cell lines were provided by Department of Pharmacology of Zhejiang Chinese Medical University. Mouse H22 HCC cells were presented by Pharmaceutical College of Zhejiang Chinese Medical University. All animal handling procedures were performed in strict accordance with the PR China legislation of the use and care of laboratory animals.

Preparation of drugs and reagents

The formulation and preparation technique of SRF were performed by Zhejiang cancer hospital. The concentration of SRF was calculated by the weight of crude drugs. The final concentration was 1 g/ml, and it could be diluted to target concentration for further study. Cyclophosphamide (CTX, 200 mg/bottle, lot No. 9F586E) was purchased from Baxter Oncology Gmbh (Frankfurt am Main, Germany). The expression levels of TGF-β receptor type II (TBRII) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were detected by fluorescence quantitative real-time polymerase chain reaction (RT-PCR) detection kit, which was purchased from Dahui Biotechnology Co. Ltd., Guangzhou.

Grafted tumor growth model

H22 cells were transferred into the abdomen cavity of an ICR mouse. After 6-8 days, the ascites were taken from the mouse and diluted with physiological saline to 1 × 10 7 cells/ml, 0.2 ml of 1 × 10 7 ml -1 H22 cells were inoculated through subcutaneous injection at the right armpit of mouse to establish a solid tumor model. After injection, fifty ICR mice were randomly divided into five groups (n = 10): (1) blank control group (saline), (2) low-dose SRF group, (3) middle-dose SRF group, (4) high-dose SRF group and (5) positive control group (CTX). At 24 h after H22 tumor cells inoculation, mice in (1) (2) (3) (4) groups were administered by gavage once a day for 8 days with the following treatments: (1) 0.2 mL/10 g, (2) 20g/kg, (3) 40 g/kg, (4) 80 g/kg. Meanwhile, mice in group (5) were injected intraperitoneally (i.p.) daily for 8 days with the following treatment: CTX (20 mg/kg). On the 9 th day, all of mice in each group were sacrificed. The segregated tumors were immediately weighed and then stored in liquid nitrogen for further research.

Data collection and detection

Tumor inhibitory rate: The mice weights were recorded before and after each drug administration. The segregated tumors were weighed as described above. The tumor inhibitory rate was calculated by the following formula: Tumor inhibitory rate (%) = (1 - W Treated /W Control ) ×100%. W Treated and W Control were the average tumor weight of the treated and blank control group mice, respectively.

Life prolongation rate: For the survival analysis, 60 mice were inoculated with tumor cells, and then randomly divided into five groups (n = 12) as described above. Mice in different groups were administered with corresponding drugs once a day for 10 days. We kept monitoring and feeding the mice when drug administration was stopped on the 11 th day. General physiological situations, including dietetic state, weight, appearance, behavior, secretion, excrement, poisoning manifestation, death, etc., were recorded during the observation. The life prolongation rate was calculated by the following formula: Life prolongation rate (%) = (D Treated - D Control )/D Control × 100%. D Treated and D Control were the average survival days of the treated and blank control group mice, respectively.

Expression levels of proteins in tumor tissues of H22 hepatoma mice: Briefly, tumor tissues (50-100 mg) were placed into a tube (1.5 mL) and homogenized in 1 mL Trizol. After incubation at room temperature (RT) for 5 min, 0.2 mL chloroform was added. After mixing, the phases were separated by centrifugation (12,000 rpm, 15 min). The aqueous phase was transferred to a new tube and 0.5 mL isopropanol was added. After mixing and incubation for 10 min at RT, RNA was precipitated by centrifugation (12,000 rpm, 10 min). The precipitant was washed twice with 70% ethanol. After removal of the ethanol and 15 min drying, the RNA was resolved in DEPC treated H2O. Ultraviolet spectrophotometry was performed to detect the quantity and quality (OD, 260 nm) of RNA. Then, the RNA was stored at -70 ° C for further study. Reverse transcription (37°C, 60 min) and real-time fluorescent quantitative PCR (93°C denaturing for 3 min, 93°C for 45s, 55°C for 60s, 26 cycles) were performed according to the manufacturer's recommendation.

Primer sequence

TBRII:

Forward Primer: 5'- GGACGACACCCAGCGTTTA-3'

Rverse Primer: 5'- AGATCGCTCCCATAGTTCACC-3'

Probe Primer: 5'- CTCGCAGTGAGATGCCGGAGCCAC-3'

NF - κB:

Forward Primer: 5'- ACGTCCAAAGCTGGCAGC-3'

Rverse Primer: 5'- TCAGGGCGAGGACCAGAG-3'

Probe Primer: 5'- CAGTGCCGGAGGCGCTCAGTAC-3'

Statistical analysis

Statistical analysis was performed using SPSS, version 16.0 (SPSS, Chicago, IL, USA). The data were expressed as the mean ± SD, and significant differences were assessed using Student's t-test. P <0.05 was considered statistically significant.


 > Results Top


Effect of SRF on H22 tumor growth

The tumor-inhibition effect of Shaoyao Ruangan Formula on mice with transplanted H22 hepatocarcinoma has been demonstrated. Results showed that SRF and CTX could significantly inhibit tumor growth; the tumor inhibitor rate (IR) of low, middle, high dose group of Shaoyao Ruangan Formmula and CTX treated group was 17.72%, 33.99%, 23.73% and 43.95% respectively. As shown in [Table 1], significant differences (P < 0.01) was shown through the comparison of the tumor weight between all groups, including blank control group, different dose SRF groups and CTX group.
Table 1: The tumor - inhibition effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma


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Effect of SRF on survival time of H22-bearing mice

The results demonstrated that each group of SRF could prolong the life span of H22-bearing mice to some extent. As shown in [Table 2] and [Figure 1], the life prolongation rate of middle dose SRF group (16.95%) comparing with normal saline group displayed a significant difference (P = 0.014).
Figure 1: Effect of SRF on survival time of H22-bearing mice; *P < 0.05, **P < 0.01, comparing with blank control group

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Table 2:


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Effect of SRF on the expression of TBRII in H22-bearing mice

The standard curve of fluorescent quantitative RT-PCR in [Figure 2]: Y = -3.8371LgX + 44.2261, R 2 = 0.998. The standard curve was complied with the manufacturer's recommendation (R 2 > 0.96). As shown in [Table 3], TBRII had the highest expression level in middle dose SRF group, followed by high dose SRF group, CTX treated group, and low dose SRF group, when compared with normal saline group (P < 0.01). Corresponding fluorescence intensity curves were showed in [Figure 3].
Figure 2: The standard curve of the expression of TRBII in H22-bearing mice

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Figure 3: The fluorescence intensity curves of TBRII in H22-bearing mice tumors

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Table 3:

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Effect of SRF on the expression of NF-κB in H22-bearing mice

The standard curve of fluorescent quantitative RT-PCR in [Figure 4]: Y = -3.6864LgX + 44.4837, R 2 = 0.997. The standard curve was complied with the manufacturer's recommendation (R 2 > 0.96). Corresponding fluorescence intensity curves were showed in [Figure 5]. As shown in [Table 4], NF-κB had the highest expression level in normal saline group, followed by low dose SRF group, middle dose SRF group, CTX group and high dose SRF group. The expression levels of NF-κB in all treatment groups comparing with normal saline group displayed a significant difference (P < 0.01).
Figure 4: The standard curve of the expression of NF-κB in H22-bearing mice

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Figure 5: The fluorescence intensity curves of NF-ƒÈB in H22-bearing mice tumors

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Table 4: The fluorescence quantitative RT-PCR value of NF-κB in each group (x°s; n =10)


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 > Discussion Top


The molecular mechanism of the progression of hepatic carcinoma, which is closely in correlation with the overexpression of several growth factors (IGF -II, VEGF, TGF-β1, NF-κB and HGF) in liver tissues, is very complicated. Transforming growth factor-β, (TGF-β), one kind of cytokines, plays an important role in cell proliferation and differentiation. TGF-β1 could suppress tumor by inhibiting cell growth and promoting differentiation. [11] It has been reported that the expression level of TGF-β1 in the plasma of hepatic carcinoma patient was significantly reduced. [12] The activated TGF-β1 works by its corresponding receptor mediation. For example, the expression level of TGF-β receptor type II (TBRII) in hepatic carcinoma tissues was significantly lower than that in normal tissues. As thus, returning the expression level of TBRII could be a potential therapeutic choice for the treatment of hepatocellular carcinoma. [13] Furthermore, NF-κB involved in numerous aspects of the development of hepatic carcinoma, such as the expression of some related genes (block apoptosis, promote cell proliferation, angiogenesis, metastasis, and etc.), increasing invasion ability of tumor cell, improving the spread of cancer, the development of drug resistance, and etc., It also has been found that the expression level of NF-κB gene was significantly higher in hepatic carcinoma tissues than that in normal tissues, indicating the importance of NF-κB for the promotion of hepatic carcinoma development. [14] The results showed that the expression of TBRII gene was significantly upregulated, while the expression of NF-κB gene was significantly down-regulated. It revealed the anti-tumor mechanism of SRF to some extent at molecular level. In addition, the results of survival experiments demonstrated all groups treated with different dose SRF could extend the survival days of H22 hepatoma ascites tumor mice.

In summary, SRF could not only significantly inhibit the growth of H22 hepatoma cell in mice, but also markedly prolong the survival time of H22-bearing mice. It could be a safe and effective strategy for human to fight against HCC.

 
 > References Top

1.Jemal A, Bray F, Melissa M, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.  Back to cited text no. 1
    
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3.Abou-Alfa GK. Hepatocellular carcinoma: Molecular biology and therapy. Semin Oncol 2006;33:S79-83.  Back to cited text no. 3
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6.Lau WY. Future perspectives for hepatocellular carcinoma. HPB (Oxford) 2003;5:206-13.  Back to cited text no. 6
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7.Lopez PM, Villanueva A, Roayaie S, Llovet JM. Neoadjuvant therapies for hepatocellularcarcinoma before liver transplantation: A critical appraisal. Liver Transpl 2006;12:1747-54.  Back to cited text no. 7
    
8.Midorikawa Y, Makuuchi M, Tang W, Aburatani H. Microarray-based analysis for hepatocellular carcinoma: From gene expression profiling to new challenges. World J Gastroenterol 2007;13:1487-92.  Back to cited text no. 8
    
9.Li X, You JL. Development of studies on treatment of primary liver cancer with TCM. J Chin Med 2010;25:221-3.  Back to cited text no. 9
    
10.He FG, Zhong HJ. The prevention of Zhonggan oral liquid on 21 cases of liver damage by chemotherapy. Zhangjiang Cancer Hosp 1998;4:131.  Back to cited text no. 10
    
11.Bedossa P, Peltier E, Terris B, Franco D, Poynard T. Transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 1receptors in normal, cirrhotic, and neoplastic human livers. Hepatology 1995;21:760-6.  Back to cited text no. 11
    
12.Okumoto K, Hattori E, Tamura K, Kiso S, Watanabe H, Saito K, et al. Possible contribution of circulating transforming growth factor-b1 to immunity and prognosis in unresectable hepatocellular carcinoma. Liver Int 2004;24:21-8.  Back to cited text no. 12
    
13.Kitisin K, Ganesan N, Tang Y, Jogunoori W, Volpe EA, Kim SS, et al. Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation. Oncogene 2007;26:7103-10.  Back to cited text no. 13
    
14.Sakurai T, Maeda S, Chang L, Karin M. Loss of hepatic NF-kappa B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proc Natl Acad Sci USA 2006;103:10544-51.  Back to cited text no. 14
    


    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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