SIN (C₁₉H₂₃NO₄·0.3 CHC_l3) and 5-FU (C₄H₃FN₂O₂) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, United States) and dissolved at 100 mmol/L in dimethylsulfoxide (DMSO) for storage at -20 °C. The esophageal carcinoma cell line Eca-109 was obtained from China Center for Type Culture Collection. The growth medium consisted of DMEM (Gibco BRL Gaithersburg, MD, United States) containing 10% fetal bovine serum. Cell cultures were maintained at 37 °C in a 95% humidified atmosphere of 5% CO₂ in air.

Cell proliferation in vitro was determined using the WST-8 Cell Counting Kit-8 (Beyotime Biotechnology, Jiangsu, China) as previously described[14]. Generally, cells were seeded in 96-well micro plates at a density of 5 × 10³/well in 0.1 mL growth medium. After exposure to different concentrations of SIN (40, 80, 160, 320, 640 μmol/L), 5-FU (40, 80, 160, 320, 640 μmol/L) or SIN + 5-FU for 48 h, 10 μL CCK-8 solution was added to each well. The plates were then incubated for an additional 2 h at 37 °C. The optical density at 450 nm was measured by a microplate reader (BIO-RAD iMark). The procedure was performed in triplicate.

The amount of phosphotidylserine exposed on the extracellular membrane of the apoptotic cells was quantified by the Annexin V-FITC kit. Following incubation with SIN (160 μmol/L) and 5-FU (160 μmol/L) alone or combined for 48 h, adherent cells were harvested by mild trypsinization, washed twice with cold PBS and resuspended in 500 μL binding buffer. After adding 5 μL Annexin V-FITC conjugate and 10 μL Propidium Iodide (PI), the cells were incubated for 15 min at room temperature in the dark. Flow cytometric analysis was performed immediately with FACSCalibur using the CellQuest software.

The morphological features of apoptotic cells were detected by Hoechst 33258 staining following the manufacturer’s protocol (Beyotime Biotechnology, Jiangsu, China). Cells were seeded on sterile cover glasses placed in the 6-well plates. After exposure to SIN (160 μmol/L), 5-FU (160 μmol/L) alone or combined for 48 h, the cells were fixed, washed and stained with 0.5 mL Hoechst 33258 staining solution for 5 min at room temperature in the dark. Then washed twice with PBS and stained nuclei were scored and categorized according to the condensation and staining characteristics of chromatin under a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Ten random fields per dish were observed and counted.

Whole protein extracts were prepared and analyzed by western blotting using a wet transfer system (Bio-Rad, Hercules, CA, United States). Protein samples (20 μg) were resolved by electrophoresis of SDS-polyacrylamide gels and electro-transferred to nitrocellulose membranes. Transfer efficiency and homogeneous loading was assessed by Ponceau stain. Membranes were blocked for 1 h in 10% non-fat milk, incubated with titrated primary antibody overnight at 4 °C and then labeled with the horseradish peroxidase conjugated secondary antibody for 2 h at ambient temperature. The following antibodies were used: Bax, Bcl-2, GAPDH (Santa Cruz Biotechnology, CA, United States) 1:1000. Antibody reaction was revealed using an enhanced chemiluminescence system (Millipore, Bedford, MA, United States) and then exposed with Kodak X-ray film. Protein band intensity was determined using the CMIASWIN video imaging system (Bio-Rad, Hercules, CA, United States).

Animal experiments, which complied with the NIH Guide for the Care and Use of Laboratory Animals, were approved by the committee on Animal Experiment of Wuhan University. Esophageal carcinoma tumors were established in male BALB/c nude mice (4-6 wk, Laboratory Animal Central of Wuhan University) by serial subcutaneous transplantation of Eca-109 cells (1 × 10⁷ cells/mouse). Eighteen mice bearing xenografted tumors were obtained and a further six mice inoculated with saline acted as controls. When the tumors reached approximately 100 mm³ in size, the mice were divided into four groups (P < 0.05): SIN group (25 mg/kg), 5-FU group (12 mg/kg), combination group, and saline control group. Treatment was administered via intratumoral injection every 3 d. Tumor size was measured 2-3 times per week in two dimensions by calipers, and the volumes were calculated using the following formula: volume (mm³) = [length × (width)²×π/6].

For histologic analysis, tumor tissues were prepared according to a standard protocol, and the paraffin-embedded tumor tissue sections were subsequently stained with hematoxylin and eosin (HE). For TUNEL assay, an in situ apoptosis detection kit (Roche, Branchburg, NJ, United States) was used to detect apoptotic cells in tumor tissue sections. The apoptotic cells were identified, counted (nine random fields per slide), and analyzed under light microscopy (Olympus, Japan).

Blood samples were collected by cardiac puncture. The biomarkers of liver and renal injury, such as the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (Cr), were detected using an OLYMPUS AU5400 analyzer (OLYMPUS, Japan).

All data were expressed as the mean ± SE of the mean, and then subjected to the unpaired Student’s t test. Statistical significance was defined as a value of P < 0.05.

The inhibition of Eca-109 cell proliferation by SIN and 5-FU was assessed by the WSK-8 cell viability assay after 48 h of drug exposure, following 24 h culture in a drug-free medium. As shown in Figure 1, the growth of Eca-109 cells was significantly inhibited in a dose-dependent manner (P < 0.05). Significant differences were observed for each concentration of the combined treatment (SIN:5-FU ratio was 1:1) compared to SIN or 5-FU given individually (P < 0.05), especially at lower concentrations.

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Figure 1 The individual and combined effects of sinomenine and 5-Fluorouracil on the proliferation of esophageal carcinoma cells. The proliferation of Esophageal carcinoma (Eca-109) cells was inhibited by SIN and 5-FU in a dose dependent manner (P < 0.05). SIN and 5-FU had significant synergistic effects on inhibiting the proliferation of Eca-109 cells especially at lower concentrations. SIN: Sinomenine; 5-FU: 5-Fluorouracil.

Apoptosis induced by SIN and 5-FU was confirmed by Annexin V/PI staining to quantify the amount of phosphotidylserine exposed on the apoptotic cellular membrane. As shown in Figure 2, the percentage of Annexin V-positive/PI-negative cells increased progressively in Eca-109 cells incubated at low concentrations of SIN (160 μmol/L) and 5-FU (160 μmol/L) for 48 h. SIN and 5-FU alone significantly induced apoptosis, as compared with the control group (P < 0.05); furthermore, the combined treatment effect was stronger than that of SIN and 5-FU given individually (P < 0.05, Figure 2).

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Figure 2 Quantitative flow cytometric measurements of G₂/M arrest and apoptosis in esophageal carcinoma cells treated with sinomenine and 5-Fluorouracil alone or in combination. The density plots illustrate four cell populations (live, apoptotic, necrotic, and late apoptotic/dead) defined by their fluorescence profiles. SIN and 5-FU alone significantly induced apoptosis compared with the group control (P < 0.05), and the combined effects were stronger than those of SIN or 5-FU alone (P < 0.05). SIN: Sinomenine; 5-FU: 5-Fluorouracil.

The morphological features of apoptotic cells were detected by Hoechst 33258 staining. The apoptotic nuclei were assessed by changes revealed by Hoechst staining and were identified by condensed chromatin as well as nuclear fragmentation with formation of apoptotic bodies. Ten random fields per dish were observed and counted under a fluorescence microscope. The mean values are expressed as the percentage of apoptotic nuclei per field. Significant changes were detected in the number of apoptotic cells, and the percentage of apoptotic cells induced by SIN and 5-FU combined or alone were significant when compared with the group control (P < 0.05, Figure 3); furthermore, the apoptotic rate of the combined treatment was greater than those of the individual treatments (P < 0.05, Figure 3).

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Figure 3 Apoptosis of esophageal carcinoma induced by sinomenine and 5-Fluorouracil alone or in combination. A: Control group; B: SIN group; C: 5-FU group; D: Combined group; apoptotic features were assessed by observing chromatin condensation and fragment staining. SIN: Sinomenine; 5-FU: 5-Fluorouracil.

To further investigate the essential mechanism underlying SIN- and 5-FU-induced apoptosis, their effects on the mitochondrial pathway were examined. As shown in Figure 4, SIN (160 μmol/L) and 5-FU (160 μmol/L) treatment combined or alone caused an increase in Bax/GAPDH protein levels and a decrease in Bcl-2/GAPDH levels in Eca-109 cells, which led to a decrease in the antiapoptotic/proapoptotic (Bcl-2/Bax) protein ratio. The apoptotic effect in the combined treatment group was significantly greater than that of the individual treatment groups.

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Figure 4 Sinomenine and 5-Fluorouracil alone or in combination induced apoptosis in esophageal carcinoma cells through the mitochondrial pathway. Western blotting analysis results of Bcl-2 and Bax protein after SIN (160 μmol/L) and 5-FU (160 μmol/L) treatment. GAPDH was used as a loading control. SIN: Sinomenine; 5-FU: 5-Fluorouracil; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

The effects of SIN and 5-FU on the growth of primary tumor xenografts in nude mice were examined. None of the mice died during the course of the experiment, and all 24 mice successfully grew tumor xenografts. After 14 d growth, the tumor xenografts reached a mean size of 100 mm³. The mice were then randomly divided into four treatment groups, and no significant differences were observed in tumor size in the different groups at the start of the study. The results demonstrated that the tumor volumes and weights in the treatment groups were both significantly reduced compared with the saline control group (P < 0.05, Tables 1 and 2), while the extent of tumor reduction differed for each group. The combination treatment induced greater tumor growth suppression than did SIN or 5-FU alone (P < 0.05, Table 1), and a difference in tumor weight was also observed between groups (P < 0.05, Table 2). When compared with the control group, the tumor volume inhibition rate for the combination group was 91.22%, whereas the inhibition rates for the SIN and 5-FU groups were 64.68% and 71.68%, respectively (Table 1). When compared with the control group, the tumor weight inhibition rate for the combination group was 83.38%, whereas the inhibition rates for the SIN and 5-FU groups were 63.10% and 61.97%, respectively (Table 2). The results suggest that SIN combined with 5-FU exhibits significant anti-tumor potential in vivo.

HE staining and TUNEL analysis of the subcutaneous primary tumor sections indicated that SIN and 5-FU individually or in combination induced significant apoptosis of Eca-109 cells in vivo, as compared with the saline control group (P < 0.05, Figure 5), although the degree of apoptosis differed for each group; the apoptotic rate in the combination treatment group was greater than that in the SIN alone and 5-FU alone groups.

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Figure 5 Detection of apoptotic cells in xenograft tumor tissue by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. A: Control group; B: SIN group; C: 5-FU group; D: Combined group; SIN and 5-FU induced significant apoptosis of esophageal carcinoma cells in vivo compared with the saline control group (P < 0.05), and the apoptotic rate of the combination treatment group was more significant than that of the SIN alone and 5-FU alone groups. SIN: Sinomenine; 5-FU: 5-Fluorouracil.

The above data indicated that the effects of SIN combined with 5-FU against esophageal carcinoma in vivo were superior to that of SIN and 5-FU used individually.

At the end of the experiment, the nude mice were sacrificed and necropsied. No obvious metastasis, hemorrhage, or injury of the liver and kidneys was observed by visual examination.

Blood samples collected by cardiac puncture were used to monitor hepatic and renal toxicity. As biomarkers of liver and renal injury, the activity of ALT and AST, as well as the urea and Cr values, were determined to evaluate potential toxicity. The hepatic and renal toxicity induced by SIN and 5-FU alone or in combination is shown in Table 3. Compared to the control group, there were no significant increases in the values of ALT, AST, urea, and Cr (P > 0.05, Table 3), and no significant differences were observed between the SIN alone, 5-FU alone and combination treatment groups (P > 0.05, Table 3). At necropsy, the livers and kidneys of the mice from the different treatment groups appeared smooth and normal in color. There was no significant difference in the liver and renal volume or weight between the treated groups and the control group, and the histological pathology examination showed no obvious lesions.

Esophageal carcinoma is one of the most refractory and common malignant diseases and is associated with poor outcome. To enhance the effect of systemic chemotherapy and reduce the toxicity, combined treatments with several regimens are often used. 5-Fluorouracil (5-FU) is universally used as an anti-cancer agent in esophageal carcinoma. Sinomenine (SIN) is an immunosuppressive compound extracted from the Chinese medicinal plant Sinomenium acutum; this compound has a wide range of significant pharmacological actions and has been hypothesized to enhance the sensitivity of various cancers to anti-cancer drugs.

As an alkaloid derived from a medical plant, SIN has been used in traditional Chinese medicine for over 2000 years. In the area of SIN treatment for cancer, the crucial areas of research are to confirm its effects, investigate the agent’s mechanism of action and identify combined systemic chemotherapy regimens to prevent the growth of cancer.

Few studies have described the anti-inflammatory, anti-rheumatic, anti-cancer, and anti-angiogenesis effects of SIN. The results of this study suggest that the combined effects of SIN and 5-FU on the growth of esophageal carcinoma are superior, with no observed increase in side effects. The essential mechanism underlying SIN- and 5-FU-induced apoptosis, as investigated by Western blotting, involved the mitochondrial pathway.

In this study, the combined effects of SIN and 5-FU, as well as their essential mechanism were investigated in esophageal carcinoma. The combined use of these two medicines generated a superior anti-cancer effect without increased side effects. This finding may help to provide novel combined systemic chemotherapy in the treatment of esophageal carcinoma.

SIN: SIN is an alkaloid derived from the stem of the Chinese medical plant Sinomenium acutum, which has been used in traditional Chinese medicine for over 2000 years to treat various rheumatoid diseases. The chemical structure of SIN has been clarified, and its potential value in the treatment of immunological and other diseases is recognized by Western medicine.

This is a valuable original study in which the authors examine the inhibitory effects of SIN combined with 5-FU on esophageal carcinoma Eca-109 cells in vitro and in vivo. The results are exciting and suggest that the combined use of SIN and 5-FU is superior to the effects of the individual agents. This study is important for enhancing the efficacy and reducing the toxicity of chemotherapy regimens for patients with esophageal cancer.