The following cells, reagents and supplies were purchased from commercial sources: MA and CPT-11 from Aladdin Reagents Co., Ltd. (Shanghai, China); human colon cancer HT29 cells from Cell Bank of the Chinese Academy of Sciences (Shanghai, China); RPMI-1640 medium and fetal bovine serum (FBS) from HyClone Laboratories, Inc. (Logan, UT, USA); TOPO I, Bax, Caspase-3, GAPDH primary antibody, and HRP-labeled secondary antibody from Proteintech Group, Inc. (Wuhan, China); cell lysis solution, BCA protein concentration assay kit, and Annexin V-FITC apoptosis detection kit from Beyotime Institute of Biotechnology (Nantong, China). This study was approved by the Ethics Committee of The First Hospital of Lanzhou University.

HT29 cells were incubated in RPMI-1640 medium containing 100 U/ml of penicillin, 100 µg/ml of streptomycin and 10% FBS at 37°C with 5% CO₂. When the cell growth reached the log growth phase, the cells were treated with trypsin to obtain a single cell suspension, and then were seeded into different culture plates or culture dishes for group experiments according to the experimental requirements.

The study was divided into the experimental group and the normal control group. MA and CPT-11 were dissolved in DMSO. HT29 cells in the logarithmic growth phase were collected and seeded into a 96-well plate at 200 µl of 1×10⁴ cells/ml cell suspension. After 24 h incubation, the experimental group was further divided into 12 subgroups for different treatments: 4 subgroups for MA treatment alone with final MA concentrations of 0.25, 0.5, 1.0 and 2.0 mg/ml; 4 subgroups for CPT-11 treatment alone with final CPT-11 concentrations of 1.25, 2.5, 5.0 and 10.0 µg/ml; and 4 subgroups for combination treatment with final concentrations of 0.25 mg/ml MA + 1.25 µg/ml CPT-11, 0.5 mg/ml MA + 2.5 µg/ml CPT-11, 1.0 mg/ml MA + 5 µg/ml CPT-11, and 5.0 mg/ml MA + 10.0 µg/ml CPT-11. Five wells was set for each concentration. Equal volumes of culture medium were added to wells for the normal control group. After 24 h, the culture medium was discarded, and wells were washed 3 times with PBS, followed by addition of 100 µl of MTT (5 mg/ml) per well and incubation for 4 h. DMSO (100 µl) was added to each well, followed by agitation for 10 min in the dark and measurement of absorbance value (OD value) at 570 nm by ELISA. The cell inhibition rate was calculated using the formula: inhibition rate (%) = 100 × (OD value of normal control group - OD value of experimental group)/OD value of normal control group.

Based on the MTT assay results, an appropriate drug concentration was chosen that provided a 20–30% inhibition rate (IC₂₀-IC₃₀) for the experiment. Cells were divided into 4 groups: the normal control group (control), the MA group (concentration 0.5 mg/ml), the CPT-11 group (concentration 1.25 µg/ml), and the combination group (MA 0.5 mg/ml + CPT-11 1.25 µg/ml). HT29 cells in the logarithmic phase were seeded into a 6-well plate. After attachment, the cells were treated with different drugs at the selected concentration described above. After 24 h of incubation, the cells were washed 3 times with PBS, followed by trypsin digestion and centrifugation. We closely followed the instructions provided in the apoptosis detection kit by the manufacturer. The collected cells were re-suspended in 0.3 ml binding buffer, followed by the addition of 5 µl Annexin V and 5 µl PI. After 15 min of incubation at room temperature in the dark, samples were diluted with 0.2 ml of binding buffer and analyzed by flow cytometry (Becton-Dickinson, New York, NY, USA).

Cells were treated and collected according to the method explained above. Cell pellet was immersed in 2.5% glutaraldehyde and left at 4°C for 48 h. After cell fixation, the sample was treated with osmium tetroxide for 30 min and then dehydrated in acetone at a series of concentrations. The sample was then embedded in embedding resin, followed by cutting into ultrathin sections with a microtome and lead-uranium double staining. The prepared sections were observed and photographed under a transmission electron microscope (TEM) (JEOL, Tokyo, Japan).

Cells were treated and collected according to the method explained above. Collected cells were lysed using cell lysis buffer and centrifuged at 10,500 × g for 15 min at 4°C to collect the supernatant. The concentrations of extracted proteins were determined using BCA kit. A sample containing 40 µg of proteins from each group was applied to SDS-PAGE electrophoresis and transferred to the membrane using a wet system. After the membrane was blocked with 10% skimmed milk powder, the TOPO I, Bax, Caspase-3 and GAPDH (dilution, 1:1,000) primary antibodies were added respectively, followed by overnight incubation at 4°C. Then HRP-labeled secondary antibody (dilution, 1:2,000) was added, followed by incubation at room temperature for 2 h, ECL darkroom development, and scanning.

The experimental data were expressed as mean ± standard deviation, and were processed using SPSS 13.0 software (IBM Corp., Armonk, NY, USA). Differences between groups were analyzed using ANOVA single factor analysis of variance, and the difference was considered statistically significant at P<0.05. The drug-drug interaction in the combination treatment was tested using the Jin (Zhengjun) formula: q = E_a + _b/(E_a + E_b – E_a × E_b), where E_a + _b represents the inhibition rate of the combination treatment, and E_a and E_b represent the inhibition rates of MA and CPT-11 treatments alone, respectively. If 0.85<q<1.15, it indicates the total effect was the sum of their individual effects. If q>1.15, it indicated a synergistic effect between the two drugs. If q<0.85, it indicated that the two drugs had an antagonistic effect.

The effects of MA and CPT-11 treatment alone and in combination on the proliferation of HT29 cells were examined using MTT assay. The results indicated that proliferation of HT29 cells was inhibited by MA and CPT-11 treatment alone and in combination (Table I). Additionally, the inhibition rate of the combination treatment was significantly higher than those of MA and CPT-11 treatment alone (P<0.01) at the same concentrations. Combination treatments of MA (1 mg/ml) with CPT-11 (5 µg/ml) and MA (2 mg/ml) with CPT-11 (10 µg/ml) had synergistic effects (q>1.15).

In order to study the pro-apoptotic effect of drugs on HT29 cells and determining the underlying mechanism, a drug concentration at which cell proliferation is partially inhibited was chosen. Thus, drug concentrations providing a 20–30% inhibition rate (IC₂₀-IC₃₀) were considered appropriate for this experiment. Based on MTT results, the concentrations of 0.5 mg/ml for MA and 1.25 µg/ml for CPT-11 were chosen to evaluate the effects of the combination treatment on apoptosis of HT29 cells. Fig. 1 shows that the apoptosis rates in the normal control group, the MA group, the CPT-11 group and the combination treatment group were 2.82, 25.24, 22.93 and 45.03%, respectively. Apoptosis rate in the combination treatment group was significantly higher than those registered in individual drug alone groups (P<0.01).

TEM results are shown in Fig. 2. Cells in the normal control group maintained cell membrane integrity. Microvilli were present on the membrane surface, while chromatin was distributed uniformly. Cells in the MA and the CPT-11 treatment alone groups underwent cell membrane blebbing, cell shrinkage, and chromatin condensation, which are characteristics for cells in apoptosis state. Cells in the combination treatment group suffered further membrane damage, and experienced nuclear fragmentation and formation of apoptotic bodies in the cells, showing a more severe apoptotic state.

Western blot analysis results are presented in Fig. 3. MA treatment alone at 0.5 mg/ml was able to upregulate the expression of TOPO I significantly in HT29 cells. When MA was used in combination with CPT-11, the upregulation of TOPO I expression was even higher (P<0.01). Compared with the normal control group, groups of MA treatment alone, CPT-11 treatment alone and combination treatment all had significantly higher levels of Bax and Caspase-3 expression (P<0.01). Bax and Caspase-3 expression levels in the combination treatment group were higher than the individual drug treatment alone groups (P<0.01).