Enhanced efficacy with reduced toxicity of chemotherapy drug 5-fluorouracil by synergistic treatment with Abnormal Savda Munziq from Uyghur medicine

Abnormal Savda Munziq was purchased from Xinjiang Qikanghabo Uighur medicine research co. Ltd. (Urumqi, Xingjiang, China). 5-Fluorouracil was purchased from Tianjin Jinyao Pharmaceutical Industries Co. Ltd. (Tianjin, China). TNF-α was purchased from Boster Biological technology (Wuhan, Hubei, China). AST and ALT were purchased from Nanjing Jiancheng Biological Technology (Nanjing, China). 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beijing Cellchip Biotechnology Corporation (Beijing, China).

Forty-eight Kunming mice with a body weight of 18-22 g (male and female) were provided by Xinjiang Medical University Laboratory Animal Center (Certificate No. SCXK (Xin) 2011–0004). A mouse strain of cervical carcinoma (U27) was provided by the cell bank of Wuhan University.

The dried Lavandula angustifolia Mill and Foeniculum vulgare Mill were extracted with 10 times water by steam distillation to get the volatile oil, then collecting the liquid after filtration. After mixing the volatile oil and β-cyclodextrin, the inclusion were dried, grind and filtered. The dried crude powder of Ziziphus jujuba Mill, Anchusa italica Ret, Alhagi pseudalhagi (Bieb) Desv and Cordia dichotoma G Forst were mixed and extracted with 10 times water under reflux. After filtration through filter paper, followed by concentration in vacuum and centrifugation, and the supernatant were collected. The dried raw powder of Glycyrrhiza glabra L, Euphorbia humifusa Willd Euphorbia maculata L, Melissa officinalis L and Adiantum capillus-veneris L were extracted with 10 times ethanol (65%), and the filtrate were collected after filtration. All the liquid of water extracts were mixed and concentration in vacuum to obtain the extractum with relative density of 1.20–1.25. After dried and grinded, the extractum were mixed with the volatile oil and lactose. With PVP (5%, in 90% ethanol), added the mixture were knead and through a 14 mesh sieve. The granules were obtained after dried and granulated.

The U27 cells growing in logarithmic phase were seeded in 96-well plates at 1.0 × 10⁴ cells per well and allowed to adhere overnight, after which a series of concentrations of ASMq (1.25, 2.5, 5, 10, and 20 mg/mL) and/or 5-FU (3.125, 6.25, 12.5, 25 and 50 mg/L) were added to the well for 24, 48 or 72 h. Controled experiments were carried out by adding ethanol without ASMq to U27 cells. After 24, 48 or 72 h of incubation, 20 μL MTT (5 mg/mL) was added to each well and the cells were re-incubated for another 4 h at 37 °C. After removaling of the supernatant gently, 200 μL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the purple formazan crystals completely. Absorbance values at 570 nm were measured with a microplate reader and were reported as a percentage of growth with respect to the control. Inhibition rate of cell growth was measured using the formula: inhibition rate (%) = [1- OD₅₇₀ (experiment group)/OD₅₇₀ (control group)] × 100. All experiments were repeated in triplicate. The drug concentration that produced 50% inhibition of cell proliferation (IC₅₀) was calculated and analyzed for 5-FU, ASMq and the combination.

Kunming mice housed in groups of four and given five days to acclimate to the housing facility. Environmental conditions were a temperature of 20 ± 2 °C, humidity of 55 ± 5%, 12-h light and 12-h dark cycle with lights on at 08:00 and off at 20:00. Animals were housed in 290 × 178 × 160 mm cages and given ordinary food and water. At the start of the experiments animals weighed 20 ± 2 g.

Forty-eight Kunming mice were randomly divided into the following groups: 1) control group, 2) model group, 3) 5-fluorouracil (5-FU) alone, 4) 5-fluorouracil combined with a high dose of Abnormal Savda Munziq (5-FU + ASMq.H) group, 5) 5-fluorouracil combined with a medium dose of Abnormal Savda Munziq (5-FU + ASMq.M) group, and 6) 5-fluorouracil combined with a low dose of Abnormal Savda Munziq group (5-FU + ASMq.L) group. There were 8 mice in each group, four male and four female. Except the control group, the U27 cell-induced tumor model was established in all mice in the other 5 groups. Methods: healthy mice were selected from which to aspirate ascites under aseptic conditions. U27 cells was diluted into 1.0 × 10⁷ cfu/mL using normal saline and added to the ascites suspension. Then 0.2 mL of ascites suspension was subcutaneously inoculated into the axilla of the left forelimb of every mouse. The entire process was completed within 30 min. After 24 h, intervention was performed according to the weight of mice: 1) control group: intraperitoneal injection of normal saline (NS) 0.2 mL/10 g once every other day, and intragastric administration of NS 0.2 mL/10 g once every day; 2) model group: intraperitoneal injection of NS 0.2 mL/10 g once every other day, and intragastric administration of NS 0.2 mL/10 g once every day; 3) 5-FU group: intraperitoneal injection of 5-FU 30 mg/kg once every other day, and intragastric administration of NS 0.2 ml/10 g once every day; 4) 5-FU + ASMq.L group, 5) 5-FU + ASMq.M group and 6) 5-FU + ASMq.H group: intraperitoneal injection of NS 0.2 mL/10 g once every other day, and intragastric administration of Abnormal Savda Munziq 2 g/kg, 4 g/kg and 8 g/kg, respectively, once every day for 10 days.

After continuous treatment for 10 days, orbital blood was collected and kept at room temperature for 30 min, then centrifuged at 3000 rpm for 20 min before the supernatants were collected. The expression levels of TNF-α, ALT and AST in mouse serum were determined according to the protocols of the corresponding kit.

The length (A) and the shortest(B) diameter of the tumor were measured once every three days, and the tumor volume was calculated according to the formula V = AB [2]/2. The tumor growth curve was according to each group of tumor-bearing nude mice. After continuous treatment for 10 days, the mice were terminated. The thymus, spleen, liver and kidneys were immediately separated. The surrounding connective tissue and fat were excluded. The organs were dried of surface moisture using filter paper, and weighed. Viscera indexes for the thymus, spleen, liver and kidney were calculated by the following formula: organ index = organ weight (mg)/body weight (g). The inhibition rate for each group was obtained using the following formula: inhibition rate of tumor = (average tumor weight in model group - average tumor weight in experimental group)/average tumor weight in model group.

Denaturation of protein samples and gel electrophoresis.

A 10% separation gel (4 mL) and a 4% stacking gel were prepared for gel electrophoresis. The frozen protein samples were thawed on ice immediately prior to use. The protein content was quantified using and the appropriate volume of protein sample was mixed with 5× sample buffer, denatured at 95 °C for 10 min, and loaded onto the gel. The electrophoresis apparatus was set to constant voltage of 80 V to allow the samples to run from stacking gel to separating gel at a rate of about 8 V/cm. Then the voltage was increased to 120 V.

Gel transfer membrane and detection.

The polyvinylidene fluoride(PVDF) membrane was immersed in 100% methanol for 2-3 min, The water cleaned membrane rinsed with transfer buffer twice for 2 min and then soaked in transfer buffer. Six layers of filter paper were cut to the same dimensions as the gel and soaked in transfer buffer. The gel containing the samples was washed once with transfer buffer, then the transfer membrane was placed in blocking buffer at room temperature and incubated for 1 h. The primary antibody was diluted with blocking buffer and incubated with the membrane overnight at 4 °C. The reacting membrane was put into a dish and washed three times with Tris Buffered Saline with Tween(TBST) for 10 min, then incubated with secondary antibody by shaking for 1 h at room temperature. The membrane was then washed in TBST to remove the free secondary antibody, exposed, developed and fixed in the darkroom. Relative expression levels were calculated using GAPDH as a reference protein. The experiment was repeated 3 times, and the mean expression was calculated.

To investigate the synergistic effect of ASMq and 5-FU on U27 cells, inhibitory effects on cell proliferation were determined through a MTT assay. First, growth inhibition was measured in U27 cells treated with each of the two compounds individually. The results demonstrated that ASMq and 5-FU could markedly inhibit the proliferation of U27 cells in a significant time- and dose-dependent manner (Fig. 1). In these tests, the ASMq or 5-FU administered for various durations of time had IC₅₀ values of 52.45 mg/mL (ASMq: 24 h), 36.78 mg/mL (ASMq: 48 h), 21.12 mg/mL (ASMq: 72 h), 110.12 mg/L (5-FU: 24 h), 81.42 mg/L (5-FU: 48 h) and 35.42 mg/L (5-FU: 72 h). When the two compounds were used in combination, the IC₅₀ values for ASMq fell to 18.13 mg/mL, 12.34 mg/mL, and 6.24 mg/mL for incubations of 24, 48 and 72 h, respectively. As the data in Fig. 1 reflect, ASMq can notably strengthen the inhibitory effect of 5-FU on the growth of U27. The IC₅₀ values for 5-FU in the presence of ASMq sharply dropped, reaching 46.03 mg/mL, 26.73 mg/mL and 14.21 mg/mL after 24, 48 and 72 h, respectively. An evaluation of the interaction between ASMq and 5-FU and its effect on U27 cell growth (Table 1) yielded a combination index (CI) <1 for different time points (24 h, 48 h and 72 h), suggesting a synergistic effect between the two drugs.

We observed mice behaviour, autonomic activities, ingestion, drinking, hairs, faeces and urine daily. There was no secretion in eyes, ears, nose and mouth.

Comparing with the control group, the thymus index and spleen index of mice in the model group, the 5-FU group, 5-FU + ASMq.H group and 5-FU + ASMq.L group decreased significantly (P < 0.05); the liver index of mice in the 5-FU group and different doses groups of 5-FU + ASMq were increased significantly (P < 0.05); the kidney index of mice in the different does groups of 5-FU + ASMq were decreased significantly (P < 0.05). Comparing with the model group, the thymus and spleen index of mice in the 5-FU group, 5-FU + ASMq.H group and 5-FU + ASMq.L group were decreased significantly (P < 0.05); the thymus index and spleen index of mice in the 5-FU + ASMq.M group increased significantly (P < 0.05); the liver index in mice in the 5-FU group, 5-FU + ASMq.H group and 5-FU + ASMq.L group were increased significantly (P < 0.05); the kidney index in the 5-FU group was increased significantly (P < 0.05); the kidney index in the 5-FU + ASMq.H group was decreased significantly (P < 0.05). Comparing with the 5-FU group, the thymus index and spleen index of mice different doses groups of 5-FU + ASMq were increased significantly (P < 0.05); the liver index and kidney index of mice in different doses groups of 5-FU + ASMq were decreased significantly (P < 0.05). These results were summarized and shown in Table 2.

Table 3 shows that the inhibition rate of tumors in mice in the 5-FU + ASMq.H group(80.64%), 5-FU + ASMq.M group(90.67%), 5-FU + ASMq.L group(72.03%) and 5-FU group(66.89%), which clearly indicated that the effects of tumor inhibition. Comparing with the model group, the tumor weight in mice in the 5-FU group and groups of 5-FU + ASMq at different doses, decreased significantly (P < 0.05). Comparing with the 5-FU group, the tumor weight in mice in the 5-FU + ASMq.H group and 5-FU + ASMq.M group were decreased significantly (P < 0.05).

As shown in Fig. 2, after treated with ASMq and 5-FU, the mean average tumor volume was smaller than the group which treated only with 5-FU, and the difference was statistically significant (P < 0.05).

Comparing with the control group, the TNF-α expression in mice serum in the model group and 5-FU group were decreased significantly (P < 0.05); TNF-α expression in mice in the 5-FU + ASMq.H group and 5-FU + ASMq.M group were increased significantly (P < 0.05); the expression of ALT and AST in mice in the 5-FU group and different doses groups of 5-FU + ASMq were increased significantly (P < 0.05). Comparing with the model group, TNF-α expression in mice in the 5-FU group was decreased significantly (P < 0.05); TNF-α expression in mice in different doses groups of 5-FU + ASMq were increased significantly (P < 0.05); the content of ALT and AST in mice in the 5-FU group and different doses groups of 5-FU + ASMq were increased significantly (P < 0.05). Comparing with the 5-FU group, TNF-α expression in mice in different doses groups of 5-FU + ASMq were increased significantly (P < 0.05); the expression of ALT and AST in mice in different doses groups of 5-FU + ASMq were decreased significantly (P < 0.05). These results were summarized shown in Table 4.

Compared with the model group, the level of protein expression of HPV16 E2 and TGF-β1 in tumors in the 5-FU group and different doses groups of 5-FU + ASMq were decreased significantly (P < 0.05). Compared with the 5-FU group, the protein level of HPV16 E2 and TGF-β1 in tumors in different doses groups of 5-FU + ASMq were also decreased significantly (P < 0.05). These results were shown in Fig. 3, Fig. 4 and Table 5, respectively.

The pathological changes in the liver from all experimental groups were analyzed by HE staining. From the histological sections in Fig. 5. In the control group, the liver tissue structure was normal, and no pathological changes were found under low power lens or high power lens. In the model group, the liver tissue structure was not distinguished under low power lens, and the liver cells generally swelling to be granular or eosinophilic change. According to high power lens, the liver structure was not distinguishable, and the liver cells generally swollen and changed into granules. In the 5-FU group, the liver tissue structure was not distinguishable, and the cells were generally swollen and changed into granules, or had diffuse eosinophilic change, and the central vein was dilated. Under high power lens, the structure of liver cells was not distinguishable, and the cells were generally swollen and changed into granules, or having diffuse eosinophilic change. In the 5-FU + ASMq.H group, under low power lens, the structure of liver cells was not distinguished, and the cells were generally swollen and changed into granules, or having diffuse eosinophilic change. Under the high power lens, the structure of liver cells was not distinguishable, and the cells were generally swollen and changed into granules. In the 5-FU + ASMq.M group, under low power lens, the liver tissue structure was normal, and the liver cells showed slightly swollen and changed into granules, or diffuse eosinophilic change. Under high power lens, swelling of the liver cells showed granular degeneration or scattered eosinophilic degeneration, and liver sinus were not distinguished. In the 5-FU + ASMq.L group, under the low power lens, the liver tissue structure was not distinguished, and the cells generally swelled and became granular or scattered eosinophilic change, and focal necrosis were found. Under the low power microscope, the liver structure is not clear, cell swelling or granular scattered eosinophilic degeneration, and liver sinus were not distinguishable.