Dulbecco's modified Eagle's medium (DMEM), 0.25% trypsin-EDTA solution, sodium bicarbonate solution, bovine serum albumin (BSA), 3-[4,5-dimethythiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), propidium iodide, ethidium bromide, acridine orange, rhodamine 123, agarose, β-actin antibody and ADC were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) and antibiotic/antimycotic solution were purchased from Gibco (Gibco, USA). Sodium phosphate (monobasic and dibasic), sodium chloride, sodium hydroxide, sodium carbonate, hydrochloric acid and methanol were purchased from Sisco Research Laboratories (Mumbai, India). The primary antibodies against c-Myc, p53 and survivin were purchased from Dako (Amsterdam, Netherlands), ERK and MEK were purchased from Genzyme (San Francisco, CA, USA).

Adipose-derived mesenchymal stem cells (ASCs) (from Dr. Young-il Yang) were isolated from the lipoaspirates of patients undergoing cosmetic liposuction, as described by Zuk et al. [12]. In accordance with a consensus reached by the investigators, the author referred to this adherent cell population as ASCs. Briefly, the tissue was digested for 45 min with 0.075% collagenase I (Gibco, USA). The stromal-vascular fraction was separated from the remaining fibrous material and the floating adipocytes by centrifugation at 300 g. The sedimented cells were filtered through a 100 µm pore filter. Erythrocyte contamination was reduced by performing density gradient centrifugation with Bicoll (Biochrom). High contamination with erythrocytes was found to markedly decrease cell adherence and proliferation. A preceding density gradient separation procedure provided a better yield of adherent cells than did treatment with an erythrocyte lysing buffer. For initial cell culture and expansion of the cells, DMEM with a physiologic glucose concentration (100 mg/dl) supplemented with 10% fetal calf serum (FCS; PAA) was used. The primary cell isolates and cultured cells were characterized as described by Brzoska et al. [13]. The cultured ASCs were CD29+, CD44+, CD49a+, CD73+, CD90+, CD105+, CD166+, and CD14-, CD31-, CD45-. The in vitro differentiation potential of the ASCs was proven by using specific media. For these experiments, the 2nd-5th passage of the ASCs was used.

The ASC line was from Dr. Young-il Yang. The cells were grown in T75 culture flasks containing DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 units/ml streptomycin in a humidified 5% CO₂ atmosphere at 37℃. Upon reaching confluence, the cells were detached using Trypsin-EDTA solution.

Proliferation of the ASCs was assessed by MTT assay. The cells were plated in 24-well plates at a concentration of 5 × 10⁴ cells/well. Twenty-four hr after plating, they were washed twice with 500 µl of serum-free medium and then they were starved by incubation in serum-free medium for an hour at 37℃. After starvation, the cells were treated with ADC at 0.5, 1, 2, and 2.5 um concentrations for 24, 48, and 72 hr. At the end of treatment, the media from the control and ADC-treated cells were discarded and 500 µl of MTT containing DMEM (0.5 mg/ml) was added to each well. The cells were then incubated for 4 hr at 37℃ in a CO₂ incubator. The MTT-containing medium was then discarded and the cells were washed with 1× phosphate-buffered saline (PBS; 1 ml). The crystals were then dissolved by adding 500 µl of solubilization solution and this was effectively mixed by pipetting up and down. The spectrophotometrical absorbance of the purple blue formazan dye was measured using a microplate reader at 620 nm. The optical density of each sample was then compared with the control optical density and the graphs were plotted. Based on MTT assay, the dose of 1 um ADC treatment was selected for further studies because this dose was below the IC50 (50% inhibitory concentration) value of ADC for the ASCs.

The cells were subjected to cell cytotoxic assay against ADC with various dosages of ADC. A 1 uM dosage was found to be most appropriate dosage for this study with using ASC. The appropriate number of the cells (1 × 10⁴) was seeded onto 96-well plates and the cells were treated with 1 uM dose of ADC for 24, 48 and 72 hr. Afterwards the cells were detached with trypsin-EDTA and the plates were rinsed and the cells were collected. The cells were stained with 0.4% trypan blue. The viable cells were calculated under the hemocytometer.

The ASC cells from the same isolation and in the same passage were identically treated. The cells were grown to subconfluence (70%), washed and kept in serum-starved for 2 hr. In brief, the cells were lysed using 10 mM Tris pH 7.4, 0.1% SDS, 0.1% Tween 20, 0.5% TritonX100, 150 mM NaCl, 10 mM EDTA, 1 M urea, 10 mM NEM, 4 mM benzamidine, and 1 mM PMSF and then they were collected by scraping.

The total RNA was isolated from each of the samples after lysis in quanidinium isothiocyanate and phenol extraction using the commercial kit (Trizol, Invitrogen Laboratories, SanDiego, USA).

The cDNA was synthesized from 4 µg of total RNA in a 25 µl reaction mixture containing 6 µl of 5X reverse transcriptase reaction buffer, oligo (dT) (100 pmol/ul) 1µl, 10 mM dNTP 4 µl, 40 unit/µl of RNAsin and 0.5 µl (200 units/L) of Moloney leukemia virus reverse transcriptase (MMLV RTase). The mixture was incubated at 4℃ for 60 min heated to 94℃ for 3 min and then chilled on ice. To test the cDNA integrity, GADPH was amplified in each of the samples. All the oligonucleotide primers were synthesized by and purchased from the Bioneer Co (Taejun, Korea). The oligonucleotide primers were solubilized in TE buffer to the concentration of 100 pmol/µl. The aliquots were diluted to 10 pmol/µl before use. To ensure that the RNA was not degraded, a PCR assay with primers specific for beta-actin cDNA was carried out for each of the cases under the following cycling conditions: 25 cycles of 30 sec at 94℃, 30 sec at 60℃ and 30 sec at 72℃ with pre-denaturation for 2 min at 94℃ and post-extension at 72℃. A 10 µl, aliquot of each reaction mixture was fractionated on 1% agarose gel and the results were visualized with ethiumbromide staining. cDNA was synthesized by random priming using less than 1 µg of total RNA as a template. As an internal standard, a fragment of human G3PDH was amplified by PCR using the forward primer 5'-ACC ACA GTC CAT GCC ATC AC-3' and the reverse primer 5'-TCC ACC ACC CTG TTG CTG TA-3' which gave a PCR product of 450 bp. The PCR amplification was performed in a final volume of 20 µl which was comprised of 1 µl of the reverse transcriptase reaction mix, 3 pmol each of the 5' and 3' primers for the survivin or the G3PDH genes, 160 µM of deoxy nucleotide triphosphate, 1 mM MgSO₄ and 0.3 units of Taq DNA polymerase in reaction buffer. The PCR was performed with an initial denaturation step at 94℃ for 2 min followed by 30 cycles of 20 sec at 94℃ for denaturation, 30 sec at 68℃ for annealing and 45 sec at 72℃ for extension. The final extension step was prolonged to 5 min at 72℃. The PCR products were separated on 2% agarose gels by electrophoresis.

The wizard plus SV miniprep kit (Quazen, Munik, Germany) was used for the template DNA preparation for sequencing after subcloning the RT-PCR products. An automatic DNA sequencer (ABI sequencer 3700, Macrozen, Seoul, Korea) was used for sequencing. The sequence data were analyzed by the NCBI (NIH, USA) Blast search program.

The western blot analysis for the protein expression in the ASC cells was assessed using the following method. Approximately 50 µg of protein was mixed with an equal volume of 2× sample buffer, this was boiled for 5 min at 95℃, cooled, loaded on each lane of an 8-15% polyacrylamide gel, and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at room temperature. The resolved proteins were electrophoretically transferred to nitrocellulose membranes which were then blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 hr at room temperature, and then the proteins were probed with the following primary antibodies c-Myc (dilution 1 : 500), p53 (rabbit polyclonal antibody at a dilution of 1 : 1,000), ERK (rabbit polyclonal antibody at a dilution of 1 : 500), p53 (rabbit polyclonal antibody at a dilution of 1 : 1,000), MEK (mouse monoclonal antibody at a dilution of 1 : 500), survivin (mouse monoclonal antibody at a dilution of 1 : 500), and β-actin (mouse monoclonal antibody at a dilution of 1 : 2,000), this was performed overnight at 4℃. Blots were then extensively washed with TBS-T and they were incubated with the respective (anti-goat, anti-rabbit and anti-mouse) horseradish peroxidase-labeled secondary antibodies (Genei, Bangalore, India) at dilution of 1 : 2,000 for 1 hr at room temperature. After thorough washes in TBS-T, the bands were visualized by treating the membranes with 3,3'-diaminobenzidine tetrahydrochloride (Sisco Research Laboratories). The membranes were photographed and next quantified using image analysis software (NIH, Bethesda, MD, USA). The densitometry data presented in the bar graphs are the 'fold change' as compared with that of the control in each case.

The results were analyzed using SPSS software (version 17, Stanford, SC, USA) and the difference between the protein levels of the treated and untreated groups was evaluated using Student's t-test. Multiple groups were compared using the oneway analysis of variance (ANOVA) test. P values < 0.05 were deemed statistically significant.

The ASCs expressed survivin mRNA before ADC treatment and they still expressed survivin mRNA after treatment, and the level was decreased slightly after 48 hr and it was restored after 72 hr (Fig. 1).

The immunoblotting showed increased levels of the protein expression of survivin up to 48 hr after treatment. However, its level after 72 hr was decreased to lower than that before treatment. The levels of p-MEK and p-ERK were increased time-dependently, however these levels at 72 hr were decreased to lower than that of before treatment. The expression of unphosphorylated ERK was not changed. The expression of c-Myc was elevated after ADC treatment and it was highest at 48 hr after treatment. The expression c-Myc at 72 hr was higher than that before treatment (Fig. 2). The p53 expression was elevated time-dependently up to 72 hr. ADC treatment significantly increased the expression of MEK, ERK, c-Myc, p53 and survivin time-dependently as was evident from the immunoblotting and the corresponding densitometry data (P < 0.05). The survivin expression was significantly correlated with those of c-Myc (P = 0.003) and p-ERK (P = 0.001) (Fig. 3).

ADC inhibited the population growth of the ASCs and the percentage of apoptotic cells was increased at 24, 48, and 72 hr after treatment (Fig. 4). The immunohistochemistry showed that the nuclear expressions of ERK and survivin were increased after ADC treatment (Fig. 5, 6).