H I T S  on anticancer metal-based compound discovery

NAMI-A prevents ERK phosphorilation and inhibits c-myc expression in ECV304 cells

by Pintus et al., University of Sassari, Italy

Time course of the effect of NAMI-A on ERK1/2 phosphorylation. Growing cells were stimulated for the indicated time points in the presence of medium M199, containing 10% (v/v) FCS in the absence or presence of 200µg/ml NAMI-A. Equal amounts of protein (10 µg) from each sample were subjected to 10% SDS-PAGE and analysed by immunoblotting against ERK1/2 (Panel B) and phospho ERK1/2 (panel A). The upper part of the panels shows representative autoradiograms of four separate immunoblot experiments corresponding to ERK1/2 and phospho ERK1/2 immunoreactivity. The lower part of the panel reports the quantitative analysis of ERK1/2 and phospho ERK1/2 immunodensity, expressed as % of control (time 0). Data are expressed as means values ± S.E.M (n=4). *, significantly different from untreated.

Time course of the effect of NAMI-A on c-myc mRNA expression. Growing cells were stimulated with medium M199 containing 10% (v/v) FCS in absence () or presence () of 200µg/ml NAMI-A. At the indicated time points, total RNA was extracted and processed for RT-PCR analysis. (Upper part), representative ethidium bromide–stained gels of the reaction products obtained using 5 µl of the RT products after 30 cycles of PCR amplification for GAPDH and c-myc. (Lower part), expression of c-myc mRNA levels detected by [alpha^-32P]dCTP-PCR (30 cycles) using 5µl of RT product. Data represent the mean ± S.E.M. of four different experiments. *, significantly different from untreated.

Time course of the effect of PD98059 on c-myc mRNA expression. Growing cells were stimulated with medium M199 containing 10% (v/v) FCS in absence () or presence () of 50 µM PD98059. , cells were treated with 200µg/ml NAMI-A + 50 µM PD98059 for 3h h. At the indicated time points, total RNA was extracted and processed for RT-PCR analysis. The expression of c-myc mRNA levels was detected by [alpha^-32P]dCTP-PCR (30 cycles) using 5µl of RT product. Data represent the mean ± S.E. of four different experiments. *, significantly different from untreated. In panels A-B, individual results were normalised to GAPDH mRNA detected in each sample and expressed as a ratio to GAPDH.
The present results show the capacity of NAMI-A to drastically reduce c-myc mRNA expression and indicate c-myc gene expression as a part of the molecular patterning affected by this new metastasis inhibiting compound.