Tumor Inhibition Effects and Mechanisms of Angelica sinensis and Sophorae flavescentis ait Decoction Combined with Cisplatin in Xenograft Mice

Malignant tumors seriously endanger people’s health, even life itself, cause physical and psychological pain and economic burden to the patients and their families. DDP (cis-diamine-dichloroplatinum II; C-DDP) is a kind of widely-used and highly effective anticancer agent, also used in anti-cancer experiment as positive control drug. However, the risk of its side effects and the resistance of cancer cells to it (Yu et al., 2012) frequently interrupt the use of higher doses that could maximize its anti-neoplastic effects. In traditional Chinese medicine, the concept of malignant tumor belongs to the category of lumps resulted from blood stasis which then causes the defense system to become weak in overseeing the internal environment. Angelica sinensis and Sophorae flavescentis pill was first seen in Yizongjinjian. The prescription pill consists of Angelica sinensis and Radix Sophora flavescens, has the functions of nourishing the blood and making blood


Introduction
Malignant tumors seriously endanger people's health, even life itself, cause physical and psychological pain and economic burden to the patients and their families.DDP (cis-diamine-dichloroplatinum II; C-DDP) is a kind of widely-used and highly effective anticancer agent, also used in anti-cancer experiment as positive control drug.However, the risk of its side effects and the resistance of cancer cells to it (Yu et al., 2012) frequently interrupt the use of higher doses that could maximize its anti-neoplastic effects.
In traditional Chinese medicine, the concept of malignant tumor belongs to the category of lumps resulted from blood stasis which then causes the defense system to become weak in overseeing the internal environment.

Tumor Inhibition Effects and Mechanisms of Angelica sinensis and Sophorae flavescentis ait Decoction Combined with Cisplatin in Xenograft Mice
De-Qi Yan, Yong-Qi Liu*, Ying-Dong Li, Dou Li, Xiao-Li Cheng, Zhi-Wei Wu peaceful, clearing internal heat and drying dampness, melting phlegm and scattering clot.Radix Sophora flavescens combined with Angelica sinensis is commonly used to treat skin disease with heat and dampness (Chen et al., 2013;Jin et al., 2013).When the cancer patients are in a state of internal stasis caused by heat and dampness, ASSF can be applied and get good effects.A lot of studies showed that some chemical composition in Radix Sophora flavescens was able to inhibit tumor cells in vitro (Zhang et al., 2012;Pu et al., 2013).
Hypoxia is a very common phenomenon in solid tumors and leads to aggressive phenotype and treatment failure.Hypoxia-inducible factor 1α (HIF-1α) regulates more than 5% of total human genes, Overexpression of HIF-1α is detected in many kinds of cancers via different kinds of mechanisms, including decreased oxygen concentration, hyperactivation of protein kinase signaling pathways, mutant oncogene activation, function loss of tumor suppressor gene, etc. Genes regulated by HIF-1α involve in many pathological processes such as drug efflux, metabolic switch, cell proliferation, angiogenesis, especially anti-apoptosis, metastasis and differentiation (Lee et al., 2014), which ultimately leads to drug resistance and tumor growth.Novel strategies have been developed to inhibit HIF activity in targeting hypoxic tumors.HIF-1α protein is often over-expressed in multiple types of human cancer and the major cause of resistance to drugs and radiation, involved in hypoxia-induced tumor progression and chemoresistance (Iovine et al., 2014).
This subject discussed the actual effect of ASSF to inhibit tumor growth, to reduce the side effect of DDP and increase its anti-tumor effect, also revealed the influence of ASSF on serum LDH, AST, ALT, AKP, and HIF-1α, and some apoptotic mechanism, in order to provide experimental basis for the clinical application of ASSF decoction united with DDP to treat cancer disease.

Cell lines
H22 liver cancer strain preserved in Institute of integrative medicine of Gansu College of Traditional Chinese Medicine.

Preparation of ASSF
Put 7.5g Angelica sinensis and 15.0g Sophora flavescens into the clay-made medicine pot.Add 250mL distilled water to immerse the herbs for 30min, then boil for 1h.Pour the boiled liquid into a cup.Add another 250mL distilled water into the clay medicine pot immersing for 30min, boiling 1h.Pour the boiled liquid into the above cup.Heat the mixed liquid in 70℃water bath to 50mL, for gavages on the same day.Drug concentration calculated according to crude herb was 0.45g/mL.

Preparation of DDP solution
Use 50mL physiological saline to wash 10mg DDP bottle and get 10mg/50mL DDP solution, which can be used for 5kg mice, the dosage being 2.0 mg/kg.

Preparation of animal model
174 KM mice were fed for 4 days to adapt the new environment.12 female and 12 male mice were randomly selected into normal control group.The remaining mice were used for modeling.Inoculate 0.1 mL -1 suspension with 105 H22 cancer cells into the subcutaneous right armpit of the ungrouped KM mice in the SPF animal laboratory.To observe the tumor growth of the mice inoculated with H22 cell during 5-7 days after inoculation.On the 7th day after inoculation, the nodules near the inoculation site could be palpable, 5 female and 5 male mice were randomly selected for pathological examination of nodules.All the 10 nodules diagnosed as tumor tissue could suggest that inoculation modeling was successful.

Model animal grouping and treatment
On the eighth day from inoculation, eliminate the mice with too small, or too large nodules, randomly select 48 female mice and 48 male mice with nodules of moderate size, and randomize them into model control group, DDP control group, ASSF group and ASSF-DDP group, 12 female and 12 male mice in each group.Weight the mice after grouping, gavage 0.4ml/20g and intraperitoneally inject 0.2ml/20g physiological saline respectively into the model control group; gavage 0.4ml/20g physiological saline and intraperitoneally inject 0.2ml/20g DDP solution respectively into DDP control group; gavage 0.4ml/20g ASSF and intraperitoneally inject 0.2ml/20g physiological saline respectively into ASSF group; gavage 0.4ml/20g ASSF and intraperitoneally inject 0.2ml/20g DDP solution into ASSF -DDPgroup.The administration of medicine consists for 14 days, once a day.

Tumor inhibition rate and q value
Tumor inhibition rate (%)=(mean tumor weight of model control group -mean tumor weight of experiment group) /mean tumor weight of model control group.q value can be used to determine the type of combined effect of ASSF decoction and DDP.q=EAB/ (EA+ EB -EA *EB) .Where EA is the tumor inhibition rate of ASSF decoction group, EB the tumor inhibition rate of DDP group, EAB is the tumor inhibition rate of ASSF-DDPgroup.q<0.85 means antagonist, q between 0.85~1.15means additive, q>1.15 means synergism (Liu et al., 2011).DOI:http://dx.doi.org/10.7314/APJCP.2014.15.11.4609Tumor Inhibition by Angelica sinensis and Sophorae flavescentis Ait with Cisplatin in Xenograft Mice

Preparation and observation of pathological sections
Acquisition and fixation of the tissue: 8 male and 8 female mice in each group were randomly selected to isolate thymus.Thymus were fixed in 4% poly formaldehyde over16h.
Section cutting, pasting, staining, closing: Cut paraffinembedded tissue blocks into 4μm thick sections.Gently spread on the 43℃ surface of water, flattening it on the middle of a slide.Remove the water from slides and put it in an incubator chamber at 63℃ baking for 20 minutes.Remove the interstitial paraffin wax.According to hematoxylin eosin staining method, dewax, remove benzene, rehydrate, dye, dehydrate, make transparency, and finally close the sections.
Observation and scoring method of pathologic sections: Observe HE pathological sections by inverted fluorescence microscope which magnify the object to 10×40 times, using Cellsense software to acquire picture.Three pathologists respectively select 5 typical visual field for each section, independently grade the picture according to the following standards.The average grading value of each section was calculated according to 15 typical field grading from three experts.The number of different grading values of 16 sections of each group was used in rank sum test analysis.
Observe thymus cells and reticular cells of thymus cortex and medulla of each group, and give them scores in accordance with the thymus cell numbers.0: normal number of thymocytes.1: less number of thymocytes than normal group.2: a serious lack of thymus cells compared with normal group.

Detection of serum LDH, AST, ALT, AKP and HIF-1α
The blood were collected through eye socket and centrifuged to acquire serum for biochemical detection of LDH, AST, ALT, AKP and ELISA method detection of HIF-1α.

Detection of mutant p53 and caspase-3 protein expression in the tumor tissue by immunohistochemistry
5 male and 5 female mice in each tumor-bearing group were randomly selected to isolate tumor.The tumors were cut into 1.0cm×1.0cm×0.3cmpieces, fixed in 4% poly formaldehyde over 16h., processed and embedded in paraffin, then made into slices.P53 and caspase-3 protein expression were detected by immunohistochemistry according to product instruction.Dewax sections in xylene, restore the antigen by microwaving in 0.01 M sodium citrate buffer pH 6.0.Incubate slices with 3% H 2 O 2 for 8 min.Wash 3 times with PBS for 3 min each time.Add agent A and incubate for 12 min at room temperature, then let agent A flow out.Add diluted Rabbit anti-p53  or Rabbit anti-caspase-3 (H277) and incubate for 2.5h at 37℃.Wash with PBS 3 times for 3min each time.Add agent B and incubate for 12 min at 37℃.Add agent C and incubate for 12 min at 37℃.Wash with PBS 3 times for 3min each time.Immerse the slices in diaminobenzidine hydrochloride (DAB).Wash the slices with tap water then mount them.
The mean density and mean grey scale of mutant p53 and caspase-3 slices were calculated by Image-Pro Plus 5.1 (USA) image analysis software.Each group was measured 10 sections representative for 10 tumors respectively.

Detection of bcl-2 and bax mRNA expression in the tumor tissue by qRT-PCR
10 tumors from 5 male and 5 female mice in each group were stored in liquid nitrogen.Homogenize tumor tissue with a homogenizer respectively.Total RNA was extracted from the homogenate by SV total RNA Isolation System(lot: 0000014521, promega coporation).The mRNA was then reversely transcribed into cDNA by Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA).The cDNA was used as a template for quantitative real-time PCR analysis.Sequences for primers were obtained from Genbank.Primers were designed using Primer 5 and synthesized at BGI Tech (Shenzhen, China) (Table 1).For qRT-PCR reactions, 25 μL mixtures were made by using SYBR Premix Ex TaqTMII (DRR820A, Takara, Japanese), containing 12.5 μL Tli RNaseH Plus, 1.0 μL of sense and 1.0 μL of antisense primers, 8.5 μL RNAase-free water and 2μL cDNA.Reaction conditions were set to 3 min at 95℃ (pre-degeneration), 10 s at 95℃ (degeneration) and 30 s at Tm of a specific primer pair (annealing) followed 72℃ for 10 s (extension) for 44 cycle in Thermal Cycler (C1000, BIO RAD, USA).mRNA expression was analyzed for bcl-2 and bax genes, and β-Actin was used for internal control gene.With 2-ΔΔCt assay, the results were analyzed.

Statistical analysis
Bodyweight, tumor weight , tumor volume of experimental data and tumor cell mitosis , mean density and mean grey scale of mutant p53 and caspase 3 can be expressed with (x _ ±s) and analyzed with SPSS17.0 statistical software for single factor analysis of variance.The pathological grade data can be scored according to above standards and analyzed by SPSS17.0 statistical software for rank sum test.p<0.05 was significant.

Pathological examination of nodules
All the 10 nodules examined before treatment showed nuclear atypia, cell mitosis, which suggested that the inoculation modeling was successful: 1) Bodyweight growth curve:          blood and make blood peaceful, dissolve blood stasis and produce fresh blood, so it can maintain good circulation and homeostasis, protect the normal function of the normal cells and kill the cancer cell by immune system and blood flow, reduce DDP-induced damage to the organs and tissues.Currently, extracts from Angelica sinensis possess anti-cancer and anti-oxidant activities, which activate the Nrf2 pathway to mediate the expression of many cellular anti-oxidative stress genes, protect organs and tissues against oxidative stress (Saw et al., 2013); extracts from Angelica sinensis can also suppress the growth of malignant brain tumor cells, up-regulate expression of p53 protein, cyclin kinase inhibitors p16, decrease the phosphorylation of Rb proteins, leading to arrest at the G0-G1 phase and activation of apoptosisassociated proteins in p53 pathway, significantly prolong patient survival (Lin et al., 2013).Angelica sinensis can decrease the adhesive, invasive and migratory ability of human lung adenocarcinoma A549 cells, down-regulate the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) at both the protein and mRNA levels, inhibit the enzymatic activity of MMP-2 and MMP-9, possesses anti-growth and antimetastasis activity against lung cancer cells (Gao et al.,

Discussion
Hepatoma is currently one of the severe malignant diseases with the highest morbidity and mortality worldwide.DDP is the most commonly used chemical drug in the the comprehensive therapy of liver cancer, killing tumor cells mainly through DNA cross-linkage, which impedes cancer cell division, starts apoptotic signaling pathways.However, the clinical application of DDP is limited due to its serious toxic effects to normal cells, causing bone marrow suppression (Karima et al., 2012), liver injury (Bentli et al., 2013), gonad inhibition (García et al., 2012) , nephrotoxicity (Hoda et al., 2013), ototoxicity (Duval et al., 2012).Therefore, Chinese decoction combined with DDP has been applied in clinic, which can improve the therapeutic effects, reduce the clinical doses and toxic effects of DDP.
In traditional Chinese medicine, cancer occurs due to influent qi, blood and fluid stasis.Because stasis can produce accumulating heat and dampness, cancer is often treated through clearing heat and dampness, promoting blood circulation and removing blood stasis, resolving hard lump.
Angelica sinensis is sweet and warm, can nourish .Angelica sinensis polysaccharides can stimulate the human peripheral blood mononuclear cells (MNCs) to secret GM-CSF and IL-3 and protect the hematopoietic function of CD34(+) cells from myelosuppressive agent (Lee et al., 2012), induce CD34+CD38 cell senescence through up-regulation of p53, p16, p21, and Rb genes and repression of telomerase activity, effectively inhibit human acute myelogenous leukemia (AML) CD34+CD38 stem cell proliferation without suppressing normal hematopoietic stem and progenitor cells (Liu et al., 2013).Angelica Sinensis has clinical efficacy in treating radiotherapy-induced pneumonitis and fibrosis through down-expression of TNF-α and TGF-β1 both at mRNA and protein levels (Xie et al., 2006) Radix sophorae flavescentis ait is bitter and cold in traditional Chinese medicine, can clear heat and eliminate dampness, kill parasite, promote urine to be formed, and treat accumulated lump in traditional Chinese medicine.Extracts from Radix sophorae flavescentis ait can inhibit tumor cell growth through up-regulation of caspase 3, down-regulation of bcl-2 at mRNA and protein levels (Wang et al., 2013).Matrine from Radix sophorae flavescentis can cause bid-mediated AIF nuclear translocation inducing caspase-independent apoptosis of human hepatocellular carcinoma cells (Zhou et al., 2014).Matrine can significantly inhibit the proliferation and promote the apoptosis of breast cancer MCF-7 cells in death receptor related pathway through up-regulation of Fas protein, supression of telomerase activity, downregulation of VEGF protein resulting in inhibition of the tumor vascular formation (Li et al., 2013).Matrine can induce the activation of caspase-3 and -9 and the release of mitochondrial cytochrome C (Cyto C) to the cytosol.The upregulation of Bax and downregulation of Bcl-2 can cause apoptosis of human colon cancer cells in mitochondrial apoptotic pathway (Chang et al., 2013).Our previous Western blotting results showed matrine can inhibit the protein expression of bcl-2, VEGF, HDAC1 and increase the protein expression of bax, down regulate the ratio of bcl-2/bax, promoting apotosis of A549 cancer cells, inhibiting the proliferation of A549 cancer cells (Liu et al., 2014).Kuraridin and nor-kurarinone isolated from Sophora flavescens can also induce apoptosis of human gastric adenocarcinoma SGC-7901 cells (Rasul et al., 2011).The present study showed ASSF decoction could decrease P53, bcl-2 and increase bax and caspase-3 in the tumor of KM mice, which led to the apoptosis of tumor cells and inhibition of tumor growth.
Ionizing radiation and chemotheraputic drug both can cause hypoxia which further lead to higher expression of HIF-1α resulting in radioresistance and chemotherapy resistance of mice bearing xenografts (Song et al., 2014;Zhang et al., 2014).The present study showed ASSF decoction could decrease HIF-1α, which resulted in enhanced sensitivity of tumor cells to DDP.
Cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD), resulted in increased leakage of LDH, AST, ALT, AKP, which could be attenuated by resveratrol extracted from herb (Valentovic, 2014).The increase in serum aldehyde dehydrogenase 1(LDH1) is correlated with poor prognosis such as higher pathologic grade and stage, as well as increased rate of recurrence (Hossein et al., 2014).The present study showed ASSF decoction could decrease high level of LDH, AST, ALT, AKP induced by DDP, reduce the toxicity of DDP to organs and tissues.
Caspase 3 and bax play important roles in inducing tumor cell apoptosis (Jung et al., 2014) , while mutant p53 and bcl-2 play important roles in inhibiting tumor cell apoptosis.Inducing tumor cell apoptosis is a good way for cancer theratpy.ASSF can increase caspase 3 and bax expression significantly, but cannot decrease p53 and bcl-2 to the same degree, which may be because ASSF plays strong roles in promoting expression of protein and mRNA, plays weak roles in inhibiting expression of molecules.
In this study, using KM mice inoculated hepatocarcinoma H22 cell line as model, we demonstrated that when ASSF was combined with DDP, the tumor inhibitory effect was significantly stronger than that when ASSF decoction or DDP was used alone.Our results suggest ASSF decoction combined with DDP could reduce the toxic effects of DDP and obtain better curative effects.
In conclusion, ASSF can inhibit tumor growth and has synergistic effect with DDP in treating tumor bearing mice.The synergistic effect may work through protection of thymus to make immune system function properly, decrease blood HIF-1α released by tumor tissue and may be correlated to the up-regulation of pro-apoptotic molecules such as caspase 3 and bax.ASSF can reduce serum LDH, AST (Palipoch et al., 2014), ALT (Palipoch et al., 2014), AKP whose increase might be related with DDP application 2) Tumor weight and inhibition rate comparison between each group Pathological comparison of thymus tissue Results of serum LDH AND HIF-1α, AST, ALT, AKP .955 a,b 236.106±37.112a,b 132.018±30.238a,b AFSPG 2.017±0.536b,c 108.252±33.741a,b,c 68.126±22.175b,c AFSP-DDPG 2.214±0.534c 141.540±30.273a,c 76.382±23.366a,b,c *Compared with Normal Control Group, a P<0.01; compared with Model control group, b P<0.01; compared with DDP control group, c P<0.01 Figure 1.Bodyweight Growth Curve (n=12) of, 1a) Female Mice; 1b) Male Mice

Table 3 . Pathological Injury Score Comparison of Thymus Tissue
*Compared with DDP control group, *p<0.01(ranksum test); Because there are thymus pictures, so the editors may cancel this table 3 and relative notes

Table 4a
a P<0.01: compared with Model control group; b P<0.01: compared with DDP control group; c P<0.

Table 5 . The Mean Density and Mean Grey Scale of Mutant p53 Protein in tumor tissue
Compared with ASSF group, d P <0.01.Protein expression are increased with the increase of mean density and decreased with the increase of mean grey scale

Table 6 . The Mean Density and Mean Grey Scale of Caspase3 in Tumor Tissue Expressed as (x _ ±s, n=10)
*Compared with model control group, b P<0.01;compared with DDP control group, c P<0.01;compared with ASSF group, d P <0.01 .

Table 7 . Relative Expression of bcl-2 mRNA in tumor tissue
*The result indicated no obvious difference between DDP control group and ASSF-DDP group.2-ΔΔCt indicated the relative times of bcl-2 mRNA of treatment group divided by that of model control group

Table 8 . Relative Expression of of Bax mRNA in the Tumor Tissue
*Compared with model control group, b P<0.05.compared with DDP group,