This animal study has been approved by the Animal Research Ethics Committee of Xiangya Hospital, Central South University (Changsha, China). All procedures requiring animal manipulation followed the animal experiment guidelines of the Animal Center of Central South University. The approval number of this project is CSU3009-2011-BC009.

IL-32 was obtained from He’nuo Biotech (Changsha, China). The MCF-7 cell line was purchased from Gongji Biotech (Shanghai, China), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) with 100 mg/ml penicillin (Sigma Corporation, Shanghai, China) and 10% fetal bovine serum (Sigma Corporation) at 37°C in 5% CO₂. For 12 h glucose withdrawl, the glucose was removed from the medium for 12 h, with all other components maintained. At the end of the withdrawl period, the glucose-containing medium was used again.

In order to examine the cell viability, an MTT assay was performed. A total of 6,000 cells were seeded into 96-well plates and cultured for 24 or 48 h in the presence of IL-32 at 10, 100 or 500 ng/ml.

Following incubation for 24 or 48 h, 150 μl 5 mg/ml MTT solution was added for 2 h. The supernatant was removed following centrifugation at 5,000 × g for 2 min at 4°C and 150 μl DMSO was added to each well for absorbance reading at a wavelength of 490 nm using a plate reader (Bio-Rad 680; Bio-Rad Laboratories, Shanghai, China). The assay was repeated six times.

In order to measure the levels of cell apoptosis, the Caspase-3 activity assay (Roche Diagnostics, Indianapolis, IN, USA) was used. Additionally, the TUNEL kit (Roche Diagnostics) was adopted for staining and cell counting, according to the manufacturer’s instructions. Following labeling with fluorescein isothiocyanate, the positive cells were counted under a Zeiss Axio Imager 2 microscope (Zeiss, Oberkochen, Germany; magnification, ×40; 10 sites from each experiment, repeated 3 times).

For tumor graft formation, the MDA-MB-231 estrogen-independent breast cancer cell line (Gongji Biotech, Shanghai, China) was employed as previously described (12). In total, 2×10⁷ MDA-MB-231 cells were separated with 170 μl DMEM medium and inoculated subcutaneously into the right flanks of 48 nude mice (weight, 20–30 g; age, 2 months; Animal Research Center, Xiangya Hospital).

For the drug treatment, 0.2 or 1 mg/kg IL-32 was administrated via careful intraperitoneal (i.p.) injection every two days. A total of 16 mice were treated with saline (control group), 16 were treated with 0.2 mg/kg IL-32 and 16 were treated with 1 mg/kg IL-32. An insulin injection syringe (total volume, 0.5 ml; Xiangya Hospital) was used to prevent any harm caused by normal syringe needles.

Tumor size was measured every 5 days for 30 days and the volume was calculated as follows: Volume = 0.5 multiplied by length multiplied by width multiplied by width. On day 30, the mice were sacrificed by CO₂ inhalation and the tumors were harvested and fixed in 10% formalin. The tumors were then cut into 10 μm sections in a Cryostat (Leica CM 3050S; Leica Microsystems, Wetzlar, Germany). A TUNEL in situ cell deah dection kit (Roche Diagostics) was used for staining and cell counting, according to the manufacturer’s instructions. Briefly, the cells were incubated with DNA-labeling solution for 60 min at 37°C, then washed three times with rinse buffer prior to incubation with the antibody-staining solution for 30 min at room temperature in the dark.

Data are presented as the mean ± standard deviation and were analyzed using SPSS software, version 11.0 (SPSS, Inc., Chicago, IL, USA). A t-test and analysis of variance were used to compare differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

The results demonstrated that IL-32 treatment reduced the cell numbers compared with the untreated cells, as detected by light absorbance. After 24 or 48 h, the number of viable cells significantly increased following 100 or 500 ng/ml IL-32 treatment (P<0.01). In addition, this association appeared to be concentration-dependent (Table I).

Given that IL-32 treatment increased cell proliferation, the subsequent step was to investigate whether this treatment leads to a reduction in cell apoptosis after 12 h glucose withdrawal. Using TUNEL staining, the rate of apoptosis in the control group was revealed to be 56.7±8.9%, while those in the 10, 100 and 500 ng/ml IL-32-treated groups were 44.2±6.7% (P<0.01), 37.7±6.9% (P<0.01) and 34.3±5.1% (P<0.01) at the 24 h time point (12 h following the induction of glucose withdrawal).

Following the results of the cell proliferation assay, it was investigated whether IL-32 contributed to tumor growth in vivo. It was revealed that IL-32 treatment at 0.2 mg/kg and 1 mg/kg increased the tumor graft growth compared with treatment with saline only (Fig. 1).