Acr, Bis, trypsin, RNase A, propidium iodide, MTT and NBT were obtained from Sigma (St. Louis, MO, USA) and bitter melon seeds were obtained from the Institute of Agricultural Science and Technique of Sichuan Province (Chengdu, China). Protein makers were purchased from New England Biolabs (Ipswich, MA, USA). NaH₂PO₄, Na₂HPO4, NaCl, acetic acid, ammonium sulfate, SDS, dimethylsulfoxide, ethanol, phenol, Tris and EDTA were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Coomassie brilliant blue R-250 was purchased from XiYa Company (Chengdu, China). Proteinase K was obtained from Merck Millipore (Darmstadt, Germany). Agarose was purchased from Amresco LLC (Solon, Ohio, USA). Human lung adenocarcinoma epithelial A549 cells and human liver carcinoma HepG-2 cells were provided by the American Type Culture Collection (Manassas, VA, USA). The 96-well plates were purchased from Corning Inc. (Corning, NY, USA) and cisplatin was the product of Qilu Pharma (Jining, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco-BRL (Carlsbad, CA, USA). Bovine serum albumin was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Sulfopropyl (SP)-sepharose fast flow (FF), sephacryl S-100 and macro-Cap-SP were obtained from GE Healthcare (Little Chalfont, UK). The inverted microscope Olympus CKX41SF (Olympus, Tokyo, Japan) and the Unico UV-2012 PC spectrophotometer (Shanghai Unico Instrument Corp., Shanghai, China) were used. BT100M peristaltic pump was purchased from Chuangrui (Baoding, China). POWER-PAC300 electrophores and the Bio-Rad 680 microplate spectrophotometer were obtained from Bio-Rad Laboratories Inc. (Hercules, CA, USA). A JY-02S UV-transilluminator was obtained from YunYi (Beijing, China) and the Cytomics FC 500 flow cytometer was purchased from Beckman Coulter, Inc. (Fullerton, CA, USA).

Unless specifically stated, all steps in the process of purification were performed at 4–6°C. According to the purification method of α-MMC and MAP30, as previously described (18), the powder from bitter melon seeds was extracted with 20 mM pH 6.5 phosphate buffer (PB) with 0.15 M NaCl for 12 h and then centrifuged at 1,000 × g for 30 min. Following adjustment of the pH of the supernatant to 3.6 using acetic acid, it was centrifuged at 1,000 × g for 15 min. The supernatant was then neutralized to pH 7 with 1 M PB and fractionated using precipitation with ammonium sulfate (30–60%). The precipitate was dissolved and dialyzed against 20 mM PB (pH 7.0) and the dialyzed sample was passed through the SP-sepharose FF column, equilibrated with the dialysis buffer and eluted with 20 mM PB (pH 6.5) containing 0.15 M NaCl. The eluted protein was then loaded onto the sephacryl S-100 and macro-Cap-SP columns in sequence and eluted using a linear gradient of 0.05–0.15 M NaCl in 20 mM PB (pH 6.0), with a flow rate of 1 ml/min achieved using a BT100M peristaltic pump which was incorporated into the chromatographic column. The two peaks with 30 kDa proteins were analyzed using 12% SDS-PAGE, the fractions of which were collected and diluted to a volume of ~10 ml and stored at 4°C until further use. The protein concentrations were determined using the method previously described by Lowry (19) and the N-terminal amino acid sequences were determined using the Edman degradation method (20).

An MTT assay was used to determine the effects of α-MMC and MAP30 on cell viability and proliferation in vitro. A549 and HepG-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and maintained in an incubator at 37°C with 5% CO₂. Cells were trypsinized, diluted to 2×10⁴/ml and then seeded into a 96-well plate (100 μl per well). Following incubation at 37°C for 12 h, cells were exposed to various concentrations of α-MMC and MAP30 (0.5, 1, 2, 4 and 8 μM) for 24, 48, 72 and 96 h; controls were administered equivalent amounts of phosphate-buffered saline (PBS). MTT was then added to each well and the plates were incubated at 37°C for 4 h. The medium was removed and 100 μl dimethylsulfoxide was added to each well; optical density (OD) of the solutions was then measured at 490 nm (21). The percentage of inhibition was calculated using the following formula:

A549 cells (2×10⁵ per well) were seeded into a six-well plate and incubated at 37°C for 12 h. Cells were then treated with 4 μM α-MMC and MAP30. Following incubation for 48 h, the cells (both viable and nonviable) were collected, washed twice with cold PBS and then fixed in 70% ethanol at −20°C overnight. Harvested cells were then washed twice with cold PBS and resuspended in 500 μl PBS containing 50 μg RNaseA and 10 μg propidium iodide, followed by incubation at 37°C for 30 min (22). Cell cycle analysis was performed using flow cytometery.

A549 cells were cultured to ~70% confluence and treated with α-MMC and MAP30 at concentrations of 1 and 4 μM for 48 h. Cisplatin was used as positive control at a concentration of 1 μM under identical conditions. Viable and nonviable cells were collected and washed twice with cold PBS, then resuspended in 0.5 ml DNA extraction buffer (100 mM Tris, 100 mM EDTA, 200 mM NaCl and 2% SDS) containing 100 mg/ml proteinase K and incubated at 55°C for 3 h. DNA was extracted using phenol and precipitated with 95% ethanol for 2 h at −20°C. The solution was centrifuged at 10,000 × g at 4°C for 15 min and the pellet was then air-dried and dissolved in 20 μl Tris/EDTA containing 20 μg/ml RNase; DNA was then electrophoresed over 2% agarose gel in Tris-acetate-EDTA buffer. The bands were visualized and images were captured using an ultraviolet (UV) transilluminator (23).

All experiments were performed at 25°C. The initial solution consisted of 10 μl 180 mM fresh pyrogallol solution in 3 ml 0.1 M Tris-HCl (pH 8.2) and the increase in the OD of the mixture was recorded over 3 min at 420 nm in a UV spectrophotometer. In a separate solution, 10 μl 180 mM pyrogallol solution was added to 3 ml 0.1 M Tris-HCl (pH 8.2) containing α-MMC, MAP30 or crude extract solution at the final concentration of 10 μM. The rates of pyrogallol autoxidation in each solution were then measured. SOD activity was defined as the amount of enzyme that inhibited the pyrogallol autoxidation by 50% (24).

The samples of α-MMC, MAP30 and crude extract were loaded onto 4–12% (w/v) gradient polyacylamide gels. Following electrophoresis, half of the gels were stained using 375 μM NBT solution. Following 45 min of incubation in the dark, the gel was then exposed to light in 50 mM PB (pH 7.8) containing 28 μM riboflavin, 28 mM tetramethylethylenediamine and 10 mM EDTA-Na₂ for 30 min (25). Simultaneously, the remaining half of the gels were stained with Coomassie brilliant blue R-250.

SPSS version 15.0 statistical software and Studen’t t-test were used for analysis. Values are presented as the mean ± standard error of the mean or mean value ± standard deviation. P<0.05 was considered to indicate a statistically significant difference between values.

Purification of α-MMC and MAP30 was performed using ammonium sulfate precipitation and acidification as well as SP-sepharose FF, Sephacryl S-100 and Macro-Cap-SP chromatography. A total of 210 mg α-MMC and 100 mg MAP30 were obtained from 200 g starting material seeds. SDS-PAGE was then used to assess the protein fractions of α-MMC and MAP30 (Fig. 1). In addition, Edman degradation analysis revealed that the N-terminal sequences of the purified proteins, N-Asp-Val-Ser-Phe-Arg and N-Asp-Val-Asn-Phe-Asp, were consistent with the theoretical sequences of α-MMC and MAP30.

As shown in Fig. 2, following treatment with α-MMC or MAP30, the viability of A549 and HepG-2 cells was decreased in a time- and dose-dependent manner, with significant cell growth inhibition at 72 and 96 h post-treatment as well as all concentrations ≥2 μM α-MMC or MAP30. In addition, the results indicated that MAP30 exhibited more potent anti-tumor effects compared to those of α-MMC. As shown in Fig. 3, phase contrast microscopy revealed that untreated cells grew well and displayed extended and flat cell shape, whereas cells treated with α-MMC or MAP30 for 72 h demonstrated typical apoptotic morphological changes, including shrinkage, blebbing and loss of membrane asymmetry.

Flow cytometric analysis demonstrated a significantly increased rate of cell cycle arrest of A549 cells in S phase following treatment with 4 μM α-MMC or MAP30 for 48 h (Fig. 4). In addition, the results showed that treated cells had a decreased proportion of cells in G₀/G₁ phase and G₂/M phase; furthermore, the appearance of hypodiploid peaks in the treated groups indicated a large number of apoptotic cells (Fig. 4B and C).

As shown in Fig. 5, following exposure of A549 cells to α-MMC or MAP30 at concentrations of 1 and 4 μM for 48 h, gel electrophoresis did not reveal any typical apoptotic DNA fragmentation pattern in A549 cells. Instead, DNA was shown to be degraded into small fragments, which increased in a dose-dependent manner. However, following treatment of A549 cells with cisplatin, a typical apoptotic pattern was observed.

The rate of pyrogallol autoxidation was 0.056 min⁻¹ in the reaction system without RIPs and 0.028 min⁻¹ in the same reaction system containing 330 μg/ml total protein of crude extract, which indicated that the SOD activity of crude extract was ≤75 U/ml. However, when the concentrations of α-MMC or MAP30 were the same as that of crude extract in the reaction system, the rate of pyrogallol autoxidation was 0.056 min⁻¹, indicating that α-MMC and MAP30 did not have SOD activity (Table I).

As shown in Fig. 6, following staining with NBT, the lane of the crude extract from Momordica charantia revealed an SOD activity band; however, a SOD activity band was not observed in the sites corresponding to α-MMC and MAP30.