LDH assay   LDH assay was performed in culture medium of untreated confluent cells by using a commercial kit (Sclavo Diagnostics, Siena) based on the transformation of pyruvate to lactate by LDH, at pH 7.5, in the presence of NADH coenzyme. The transformation of NADH to NAD+ is accompanied by a decrease in absorbance (A) at 340 nm, which correlates with the LDH activity. The change of absorbance, in the absence or presence of different doses of Rottlerin, was recorded over a 0.5- to 4.5-min period, and the relative ΔA/min was calculated. The change in absorbance was converted to LDH international units per liter (U/l) by the following calculations: ΔA/min ⋅ (tV ⋅ 1,000/EMC ⋅ l ⋅ sV), where tV is the total volume, EMC is the NADH extinction micromolar coefficient (6.22 cm² μmol at 340 nm), l is the light pathlength (1 cm), and sV is the sample volume.

Evaluation of nucleotide content   Nucleotides were evaluated as previously described (12). The cells (5 × 10⁶) were homogenized in 2.7 N perchloric acid in 0.5-ml tubes using a Sigma nylon motor pestle. Extracts were then centrifuged (12,000×g for 10 min) in a cooled microfuge. The supernatant was neutralized with 2.7 N KOH; potassium perchlorate was removed by a subsequent centrifugation at 12,000×g for 3 min. Aliquots of the extracts were analyzed by capillary zone electrophoresis (CZE), as afterward reported. The method permits to determine high-energy phosphates (ATP, ADP, and AMP) from which it is possible to calculate the energy charge of adenylates.

Capillary zone electrophoresis   For electrophoretic separations a Beckman Coulter P/ACE MDQ instrument (Beckman Coulter, Fullerton, CA, USA) was used. Analysis were performed in a Beckman Coulter eCAP™ uncoated fused-silica capillary (65.0 cm × 75 μm i.d.), with the window at a distance of 55.0 cm. The results were read over the range 190–300 nm and analyzed at 254 nm. Between runs capillary was washed with 0.1 mol/l NaOH for 30 s and running buffer for 60 s. The background electrolyte was borate buffer (20 mM), containing SDS (30 mM). The conditions were pH 10.00, 20 kV, and 1.0 psi for 5 s of pressure injection at 25°C, for sample and standard solutions. The electric field was 306 V/cm with a current of approximately 120 μA.

Statistical analysis   The significant difference between control and treated cells was statistically analyzed by paired Student’s t test (p < 0.05).

Results

Rottlerin interference in the MTT assay

Consistently with the decrease in [³H]-thymidine incorporation into DNA previously observed (5), 5 and 20 μM Rottlerin caused a time and a dose-dependent decrease in cell number, although the dose of 5 μM did not cause statistically significant changes before 24 h (Fig. 2a). However, as shown in Fig. 2b, the MTT colorimetric assay strongly underestimated the growth inhibitory activity of 20 μM Rottlerin and absolutely reversed the result, suggesting a direct Rottlerin action on the tetrazolium salt. Rottlerin (5 μM) exhibited the same trend of 20 μM although the results were not statistically significant at any time point. When the intensity of the reduced product color at 570 nm was normalized to cell number (absorbance/cell number), the stimulation of MTT reduction by Rottlerin was evident (Table 1 ). Rottlerin, however, did not reduce MTT in vitro at doses up to 100 μM and for prolonged times (24 h), in several incubation conditions, i.e., in different culture media and in the presence or absence of serum, NAD or NADH (not shown).

Fig. 2 Effect of Rottlerin on cell number and viability assessed by direct cell counting and MTT assay, respectively. MCF-7 cells were treated with 5 and 20 μM Rottlerin (R5 and R20) before adding MTT. a Between 1 and 24 h later, cell number was determined, and b cell viability was measured as described in “Materials and methods”. Results are expressed as % of the control (100%). Values are the average of three separate experiments in quadruplicate and are expressed as mean ± SD. *p < 0.05.