Inhibition of Tumor Growth in Vitro by a Combination of Extracts from Rosa Roxburghii Tratt and Fagopyrum Cymosum

Lung, stomach and esophageal cancers are the first, second and sixth common causes of death worldwide, respectively (Parkin et al., 2005). In China, 5 year survival rates for lung cancer is less than 20%, for stomach and esophageal cancers is only 5%-20% after surgery, and most patients will suffer postoperative recurrence and metastasis (Parkin et al., 2005; Jemal et al., 2010). Chemotherapy still plays an important role in this setting, although long term survival rate of reported regimens is unstatisfactory. It is imperative and critical to search for chemotherapicutic agents with high effectiveness and low adverse reactions. Traditional Chinese herbal medicine has begun to attain great popularity for disease prevention as well as being used as complementary medicines for a variety of diseases worldwide. In Southwest regions of China, traditional herbal medicine soup which contained Rosa roxburghii Tratt and Fagopyrum cymosum was used to cure malignant tumors. However, the mechanism is not well described.


Introduction
Lung, stomach and esophageal cancers are the first, second and sixth common causes of death worldwide, respectively (Parkin et al., 2005).In China, 5 year survival rates for lung cancer is less than 20%, for stomach and esophageal cancers is only 5%-20% after surgery, and most patients will suffer postoperative recurrence and metastasis (Parkin et al., 2005;Jemal et al., 2010).Chemotherapy still plays an important role in this setting, although long term survival rate of reported regimens is unstatisfactory.It is imperative and critical to search for chemotherapicutic agents with high effectiveness and low adverse reactions.
Traditional Chinese herbal medicine has begun to attain great popularity for disease prevention as well as being used as complementary medicines for a variety of diseases worldwide.In Southwest regions of China, traditional herbal medicine soup which contained Rosa roxburghii Tratt and Fagopyrum cymosum was used to cure malignant tumors.However, the mechanism is not well described.

Inhibition of Tumor Growth in Vitro by a Combination of Extracts from Rosa Roxburghii Tratt and Fagopyrum Cymosum
Wei Liu 1 , Su-Yi Li 2 *, Xin-En Huang 3 *, Jiu-Jie Cui 2 , Ting Zhao 2 , Hua Zhang 2   Our objectives were to evaluate the effect and possible mechanism of combination of the extract from Rosa roxburghii Tratt (CL) and the extract from Fagopyrum cymosum (FR) on proliferation inhibition and apoptosis induction of human esophageal squamous carcinoma CaEs-17, human gastric adenocarcinoma SGC-7901 and pulmonary adenocarcinoma A549 cell lines in vitro.

Drugs and chemicals
The extract from Rosa roxburghii Tratt (CL) and the extract from Fagopyrum cymosum (FR) were obtained from Tongjitang Chinese Medicine Co. (Shenzhen, China).MTT was purchased from Sigma (St. Louis, MO, USA).

Cell lines
The human esophageal squamous carcinoma CaEs-17, human gastric adenocarcinoma SGC-7901 and pulmonary adenocarcinoma A549 cell lines were obtained from Shanghai Institute of Cell Biology (Shanghai, China).

MTT assay
CaEs-17, SGC-7901 and A549 were seeded into 96-wells micro plate at concentrations of 5×10 4 /well and incubated for 24 h in 10% FCS medium before treatment.The cells were then treated with different concentrations of CL or FR for 48 h.Cells incubated in serum-free medium were as control.After incubation for 48 hours at 37 ºC, 20 μl of MTT solution (5 mg/ml in PBS) was added to each well for another 4 h at 37 ºC.Then, 150 μl of dimethyl sulfoxide (DMSO) was added into each well for 2 h at 37 ºC.The optical density (OD) was determined by spectrophotometer (Bio-Rad, Hercules, CA, USA) at 570 nm.Growth inhibition was calculated as percentage ratio between number of treated cells and number of untreated cells.Each assay was performed triplicate.The MTT level of cells before drug-treatment was measured as T 0 , T C and T D represented O.D. readings of control or drug-treated cells.Inhibition rate%=1-(T D -T 0 /T C -T 0 )×100%

Experimental groups
IC 30 (30% concentration of inhibition) of CL and FR were obtained from the previous MTT assay.Tumor cells was divided into four groups according to the treatment as follows: control group with no exposure of CL or FR; CL group with the exposure of IC 30 CL; FR group with treatment of IC 30 FR; CL+FR group with the treatment of 1/2(IC 30 CL + IC 30 FR).

Apoptosis Assays by flow cytometry
Cells were plated at 1×10 6 in 6-well dishes (Grier) and were allowed to attach after 24 h.After treated according to the experimental groups for 24 h, cells were harvested with trypsinase and washed twice with PBS.Following the manufacturer's instruction, cells were stained with Annexin V-FITC and PI then processed by flow cytometry.Data acquisition and analysis were done on a BD (Becton Dickinson) FACSCaliber using CellQuest software (BD Biosciences).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis
After treated for 24 h, the expressions of Bcl-2, Bax and Ki-67 mRNAs were assessed by RT-PCR analysis.Total RNA was extracted from different treated cells as above using Trizol Reagent (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer recommendations.First-strand cDNA synthesis was performed on 1 μg of total RNA.PCR was performed using 1 μl cDNA as template, 10 pM of each primer (showed in Table 1), 10 mM deoxynucleoside triphosphates (dNTPs), 1.25 U TaqDNA polymerase (Takara, Japan), 1 × reaction buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.01% gelatin in 25 μl reaction volume.The amplification protocol involved denaturation at 94 ºC for 1min, annealing at 55 ºC for 1 min, and extension at 72 ºC for 1 min.This cycle was repeated for 30 times.The PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide (EB).The mRNA bands were then visualized by UV light.

Western blot analysis
After treated for 24 h, CaEs-17, SGC-7901 and A549 cells were rinsed twice with icecold PBS, then were extracted and the protein concentration was determined by Lowry method.Protein lysates (40 μg) from each sample were subjected to SDS-PAGE on 10% acrylamide gel and the separated proteins transferred to a PVDF membrane.Blots were incubated for 1 hour with 5% nonfat dry milk to block nonspecific binding sites and then incubated with mouse anti-human monoclonal antibody against Bax, Bcl-2, and Ki-67 (Santa Cruz Biotechnology) at 4 ºC overnight.Membranes were then incubated in horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature and washed five times in 0.1% Tween-20 in TBS before antibody binding was detected by using a SuperSignal Chemiluminescent Detection Kit (Pierce, USA).After washing with buffer, the PVDF membrane was rehybridized with a primary antibody for β-actin (Santa Cruz Biotechnology), followed by the same procedures as described above.

Cytotoxicity of CL and FR on the growth CaEs-17, SGC-7901 and A549
The growth curves of human cancer cells after exposures of various dosages of the extract from Rosa roxburghii Tratt (CL) or the extract from Fagopyrum  The CL concentration that caused 30% inhibition of growth (IC 30 ) in CaEs-17, SGC-7901 and A549 was approximately 100 μg/ml, 70 μg/ml, and 75 μg/ml, respectively.The FR concentration that caused 30% inhibition of growth (IC 30 ) in CaEs-17, SGC-7901 and A549 was approximately 120 μg/ml, 70 μg/ml and 80 μg/ml, respectively.
The inhibition effects of cell growth treated by IC 30 CL, IC 30 FR and 1/2(IC 30 CL + IC 30 FR) were shown in Table 2. Compared with CL or FR group, the CL + FR group showed significant inhibition of cell growth in cancer cells of CaEs-17, SGC-7901 and A549 after exposure for 48 hours (P<0.05).

Effects of CL and FR on the cell apoptosis of CaEs-17, SGC-7901 and A549
As showed in Figure 2, compared with the control group, the CL, FR and CL+FR group all significantly increased the apoptosis of CaEs-17, SGC-7901 and A549 (P<0.05).The CL+FR group showed a significant increase on the apoptosis of CaEs-17, SGC-7901 and A549 compared with CL or FR treatment group (P<0.05).

Effects of CL and FR on the expression of Bax, Bcl-2, Ki-67 in CaEs-17, SGC-7901 and A549
RT-PCR and Western blot analysis were used to detect the expressions of Ki-67, Bax and Bcl-2 in mRNA and protein levels.As Figure 3 showed that after exposure to CL or FR alone, the mRNA expressions of anti-apoptotic gene Bcl-2 and proliferation gene Ki-67 decreased, and the expression of pro-apoptotic gene Bax increased in CaEs-17, SGC-7901 and A549.
The down-regulation of Bcl-2 and Ki-67 and the upregulation of Bax exposed to CL or FR alone were also confirmed in protein level in CaEs-17, SGC-7901 and A549 by Western blot analysis (Figure 4).In addition, there was significant decrease in the Bcl-2 and Ki-67 expression and significant increase in the Bax expression between CL or FR alone group and CL + FR group on CaEs-17, SGC-7901 and A549.

Discussion
Traditional Chinese herbal medicines have been used in China in medical practice widely.Rosa roxburghii Tratt and Fagopyrum cymosum two conventional medicinal plants which both have the functions of improving immune response, enhancing digestive ability and anti-aging effect recorded in the Compendium of Materia Medica.Current studies show that extract from Rosa roxburghii Tratt (CL) displays potent effects of anti-aging, deintoxication and anti-mutation (Zhang et al., 1996;Qiang et al., 2001;Xu et al., 2006) and extract from Fagopyrum cymosum (FR) has activity of anticancer (Samel et al., 1996;Chen et al., 2001;Pui-Kwong et al., 2003).We estimated that the combination of the two active extractions would enhance the inhibition of proliferation of tumor cells.
Our experiment combined CL with FR to interfere in the growth of human esophageal squamous carcinoma CaEs-17, human gastric carcinoma SGC-7901 and pulmonary carcinoma A549 cell lines in vitro.We assessed the inhibitory rates of proliferation by MTT assay and early apoptosis rates using flow cytometry.The inhibition rates of CL or FR alone on the proliferation of CaEs-17, SGC-7901 and A549 all increased as the dosage rose and demonstrated dose-dependent manner; meanwhile, the early apoptotic rates also augmented with the rise of dosage of CL or FR alone, and the combined group showed statistically significance compared to CL or FR group alone.
We found that the induction of apoptosis of CL and FR were aimed at some specific proteins.The expressions of Bcl-2 and Bax genes are associated with the inhibition and induction of apoptosis in a variety of cancer cells (Matsumoto et al., 2004;Wang et al., 2004;Zhou et al., 2008).Bcl-2 protein involves in the inactivation of an inner mitochondrial permeability transition pore and the regulation of matrix Ca 2+ , pH and voltage.Bax protein however acts as the pro-apoptotic regulator through the way of inducing the opening of the mitochondrial voltagedependent channel.The quantities of mRNA or protein of Bcl-2 or Bax thus could demonstrate the apoptotic activities in CaEs-17, SGC-7901 and A549 cell lines.Ki67 protein is a cellular marker for proliferation and is strictly associated with cell proliferation (Takeuchi et al., 2003).It often correlates with the clinical course of cancer and predicts the prognosis, recurrence and metastasis for the carcinomas of the prostate and the breast.Our results showed that after exposure to CL, FR or CL+FR, they all showed the phenomena of up-regulation of the expression of Bax and down-regulation of the Bcl-2 and Ki67.Moreover, compared with CL or FR group alone, the expressions of Bax, Bcl-2 and Ki67 were significantly increased in the combined group in CaEs-17, SGC-7901 and A549 cell lines.Dai ZK et al proposed that CL an extract of Rosa roxburghii Tratt had the inhibitive effect on the growth of gastric carcinoma cell line in vitro (Dai et 2005).Chen et al. (2006) showed that FR induced apoptosis and downregulated the telomerase activity HL-60 cells in vitro (Chen et al., 2006).To our knowledge, this is the first time combining CL and FR to assess their inhibitory effects on the growth of human esophageal squamous carcinoma, gastric carcinoma and pulmonary carcinoma cell lines.We found that both CL and FR had potent ability to inhibit the proliferation and induce the apoptosis of the three different cell lines in a dose-dependent manner in vitro.Our results also showed that compared with the CL or FR alone, the combined group had statistically significance on the prevention of growth of human esophageal squamous, gastric and pulmonary carcinoma cells and the synergistic effects of combination of CL and FR were identified.
In conclusion, our study showed that the extractions from traditional Chinese herbal medicines were promising alternative treatments for cancer.Our results also reached an excellent agreement on the synergistic effects of the combination of CL and FR on the growth inhibition of the three cancer cell lines on the levels of cell apoptosis rates, mRNA and protein in vitro.