Chinese medicine Gecko was purchased from Anhui Bozhou Yonggang Co. Ltd. They were identified Gekko japonicus. The whole dry Gecko were ground to fine powder and diluted in suspension using 0.2% carboxymethyl cellulose (CMC). Cytoxan (CTX) was purchased from Jiangsu Hengrui Medicine Co. Ltd. (Batch No. H32020857). Fifty Kunming mice and ten SD rats were provided by the Medical Experimental Animal Center, Henan Province. All were female. The code number of the animals was 0001350. The human esophageal carcinoma EC9706 cell line was of esophageal carcinoma of fungating type which is well-differentiated squamous cell carcinoma, EC1 cell line was isolated from esophageal carcinoma cell line EC9706 and one subline, and the S180 mouse sarcoma cell line was kindly provided by the Medical College, Zhengzhou University.

The cells were grown in a monolayer culture containing humidified 5% CO₂ in air at 37°C. They were cultured in RPMI-1640 (Sigma, USA) medium supplemented with 10% fetal calf serum, 100 U/mL and penicillin and 100 mg/L streptomycin. The S180 model was established by subcutaneous injection as previously described[9]. Briefly, the S180 mouse sarcoma cells with ascites were harvested, diluted with sterilized saline at a ratio of 1:8 (cell concentration was adjusted to 1.0 × 10⁶/mL), and inoculated subcutaneously into the right armpit region of Kunming mice.

Ten SD rats (aged 10-12 wk and weighing 280 ± 30 g) were randomly divided into five groups: the negative group (NS group), the positive group (CTX group) and three Gecko groups. The CTX group received 50 mg/kg intraperitoneally once a day. Three Gecko groups were treated respectively with oral administration of Gecko at a dose of 13.5 g/kg, 9 g/kg and 4.5 g/kg, twice a day. The NS group received the equivalent amounts of normal saline in the same way. After 1 wk of treatment, according to the method principle of Li Yikui[1011], blood was collected aseptically and serum was collected by centrifugation. It was deactivated at 56°C for 30 min and filtrated for sterilization. Medical serum was stored at -20°C for use.

Cell growth was measured by a modified MTT assay[1213]. The logarithmic cells (EC9706, EC1) were dispersed with 0.02% (w/v) EDTA to prepare 1 × 10⁴/mL cell suspension, and partitioned into 96-well plates at 180 &mgr;L/well for 24 h culture in a 5% CO₂ incubator under 37°C. Five groups were divided, the negative group, the CTX group and the three Gecko groups. Each group had 6 wells. Each well was treated respectively with 20 &mgr;L medical serum prepared as above. Cells were incubated for 24, 48 and 72 h. Then 20 &mgr;L MTT (Sigma, USA) was added to each well and the cells were further incubated at 37°C for 4 h. The supernatant was removed and 200 &mgr;L DMSO (Sigma, USA) was added into each well to solubilize the formazan product. The absorbance also known as A value at wavelength of 470 nm was measured by a microplate reader (Sigma, USA). Triplicate experiments were performed in a parallel manner for each concentration point and the results were presented as mean ± SD. Cell inhibitory rate was calculated by the following formula: inhibitory rate (%) = (1-A treated/A control) × 100%.

EC9706, EC1 cells (1 × 10⁴/mL cell suspension) were plated onto 96-well plates at 180 &mgr;L/well. Each well was treated respectively with 20 &mgr;L medical serum prepared as above and cultured for 7 d. Viable cells were counted under the inverted light microscope by trypan blue dye (Sigma, USA) exclusion method and growth curves were drawn.

Twenty-four hours after establishment of S180 model, the fifty transplanted mice (aged 8-10 wk and weighing 26 ± 2 g) were randomly divided into five groups: the NS group, the CTX group (CTX was proved to have definite effect against mouse S180 sarcoma and frequently used in experiment), and the three Gecko groups. They were treated respectively with oral administration of saline once a day, intraperitoneal injection of CTX 100 mg/kg only once, and oral administration of Gecko at a dose of 13.5 g/kg, 9 g/kg and 4.5 g/kg once a day. After 2 wk of treatment, the anti-tumor activity was evaluated by tumor tissue weighing. The following formula was used: Tumor inhibitory rate (%) = (1-average tumor weighing of administration team/average tumor weighing of the control) ×100%[9].

According to the above-mentioned methods, at the 14th day, 5% chicken-red cells were injected intraperitoneally into each group. After 12 h, the mice were killed and 1 mL peritoneal fluid was drawn for glass slide. After incubated for 30 min, peritoneal fluid was fixed with the mixture of acetone/methanol (1:1, v/v) and dyed with 4% Giemsa stain. Peritoneal macrophages were counted under microscope. The effect of Gecko on phagocytosis of enterocoelia macrophage was evaluated by the chicken-red cell phagocytic index and phagocytic rate[14]. At the same time, thymus and spleen were taken from mice. The impact on immune organ was evaluated based on the thymus index and spleen index[15].

The tumor tissues were fixed with 10% neutral formalin at room temperature for 24 h. The paraffin-embedded specimens were cut into sections with a thickness of 5 &mgr;m. The detection procedure was done as described in Kit protocol (Wuhan Boster Biological Technology Co. Ltd). PBS instead of the first antibodies was used in the negative control. The VEGF and bFGF positive cells were defined when there was an aggregation of brown particles in the cytoplasm of the tumor cells. The immunoreactive score was determined by the sum of extension and intensity as reported previously[16].The intensity of staining was scored on a scale of 0 to 3 (0 = negative staining, 1 = weakly positive staining, 2 = moderately positive staining, and 3 = strongly positive staining). The extent of positivity (“extent of distribution” of positive cells) was estimated on a scale of 0 to 4 (0 = negative, 1 = positive staining in 1%-25% of cells, 2 = positive staining in 26%-50%; 3 = positive staining in 51%-75%; and 4 = positive staining in 76%-100%). The combined staining score (extension + intensity) was considered as positive staining.

Apoptotic cells were detected in situ using TUNEL method according to the manufacturer’s instructions[17]. Slices were treated with proteinase K and 0.3% H₂O₂, labeled with fluorescein dUTP (Wuhan Boster Biological Technology Co.Ltd) in a humid box for 1 h at 37°C, then combined with POD-horseradish peroxidase, stained with DAB and counterstained with methyl green. Controls received the same management except the labeling fluorescein dUTP. Dark brown nucleus represented the positive apoptotic cells. In each section, 5 high -power fields were chosen and the apoptosis index (AI), the percentage of positive cells in the total cells, was calculated. The AI was calculated as follows: AI = number of apoptotic cells/total number × 100%.

Data were presented as mean ± SD and analyzed with SPSS 11.0 software. The one-way ANOVA or Student’s t test was used to analyze data. All comparisons were made with untreated controls and significance of difference is indicated as P < 0.05 and P < 0.01.

As shown in Tables 1 and 2, compared with control group, the growth of cells treated with serum of different concentrations of mice with medicine Gecko was inhibited significantly in a concentration and time-dependent manner. Seventy-two h after treatment, the inhibitory rates were 22.3%-39.1% (EC9706) and 18%-34.1% (EC1). Compared with the NS group, the differences were significant among the groups of Gecko (P < 0.05).

To investigate anti-tumor activity of the Gecko in vitro, tumor cells treated with serum medicine were cultured for 7 d, and then the cell growth curve was drawn. Under the inverted light microscope, obvious difference was observed in the cell morphology among the five groups of cells. Compared with the control group, growth curves of the three Gecko groups gradually moved down in a dose-dependent manner (Figure 1). These results indicated that the serum with medicine Gecko could inhibit EC9706 and EC1 growth and proliferation in vitro.

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Figure 1 Growth curves of EC9706 (A) and EC1 (B) in all groups.

As shown in Table 3, compared with the NS group, the tumors of the Gecko and CTX groups shrank significantly (P < 0.01), but the tumor weight of three Gecko groups had no difference statistically compared with the CTX group (P > 0.05). The tumor inhibitory rates of the CTX group and Gecko groups were 74.6%, 49.8%, 52.8% and 43.1%, respectively. After experiment, there no significant difference was found in mouse weight between the Gecko groups and the NS group (P > 0.05). Compared with the NS groups, the weight of mice in the CTX group decreased significantly (P < 0.05).

As shown in Table 4, compared with the NS group, the thymus index and Spleen index of mice in the CTX group decreased significantly (P < 0.05). However, there was no significant difference between the Gecko groups and the NS group; CTX could decrease the phagocytic index and phagocytic rate very significantly (P < 0.01); Gecko could decrease the phagocytic index and phagocytic rate significantly (P < 0.05). These results indicated that Gecko could not enhance the immune functions.

As shown in Figures 2 and 3, the VEGF and bFGF positive cells were defined when there was an aggregation of brown particles in the cytoplasm of the tumor cells. The proteins of VEGF and bFGF had high expression in the tissue of transplanted sarcoma 180 in mice. Compared with the NS group, the immunoreactive score of expression VEGF and bFGF expression of CTX group and Gecko groups decreased significantly (Table 5), (P < 0.05). It indicated Gecko could decrease VEGF and bFGF protein expression in the tissue of transplanted sarcoma 180 in mice.

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Figure 2 Expression of VEGF on S180 tissues in mice (DAB, x 400). NS group (A), Gecko group (B), CTX group (C).

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Figure 3 Expression of bFGF on S180 tissues in mice (DAB, x 400). NS group (A), Gecko group (B), CTX group (C).

As shown in Figure 4, dark brown nucleus represented the positive apoptotic cells on the tissue of transplanted sarcoma 180 in mice. As shown in Table 5, compared with the NS group, AI of the CTX group and the Gecko groups increased very significantly (P < 0.01), indicating that Gecko could increase AI in the tissue of transplanted sarcoma 180 in mice.

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Figure 4 Apoptotic cells on S180 the tissue in mice (DAB, x 400). NS group (A), Gecko group (B), CTX group (C).

Malignant tumor is always a great menace to human health. Chemical anti-neoplastics have definite effect, but they cause much severe adverse effects. Gecko is a traditional Chinese medicine. It could treat malignant tumors, especially in the tumor of digestive tract. Research on Gecko mainly focuses on anti-tumor compound prescriptions, but the pharmacological studies of Gecko and its mechanisms of anti-tumor action are rare.

Basically, two processing methods of Gecko, living and drying, are used in clinical practice. Questions still remain to be answered such as what is the difference of anti-tumor effect between fresh Gecko and dry Gecko? What kind of tumors can Gecko treat? What is the mechanism of Gecko in treating tumors?

This article describes the effect of the Gecko on esophageal carcinoma cell lines (EC9706 and EC1) in vitro and xenografted sarcoma 180 in vivo. Serum pharmacological method in vitro was used to study the anti-tumor mechanism of Gecko in the aspect of the anti-angiogenic and apoptosis inducing effect and investigate if Gecko had impact on immune organs.

The incidence and mortality of tumors keep ascending all over the world. The study on anti tumor effect and mechanisms of anti-tumor action of Gecko can provide the theoretical evidence in clinical practice. Furthermore, the results may lay a foundation for further research in Gecko’s effective constituent.

Serum pharmacological method is a good approach to study traditional Chinese medicine in vitro. It can expel various interference of traditional Chinese medicine as it is close to the true process of the pharmacological effect inside the body

This is a well-conducted study. This manuscript describes the effect of the Chinese medicine Gecko on esophageal carcinoma cell lines (EC9706 and EC1) and in xenografted sarcoma 180 Kunming mice. The studies have provided data that support the conclusions of the authors. This is an interesting study.