Capítulo 3. El mundo de la indumentaria
3.4. Perfumes semiselectivos
To explore the molecular mechanism behind the synergistic activity observed between pimasertib and gemcitabine we investigated alterations of expression of genes involved in gemcitabine metabolism, transport or activity following pimasertib or gemcitabine treatment.
Cytidine Deaminase (CDA) is responsible for deactivating gemcitabine triphosphate (dFdCTP) by converting it into its inactive form difluorodeoxyuridine (dFdU) (Nakano et al. 2007). Preclinical work on pancreatic cancer mouse models has identified a mechanism by which nab-paclitaxel sensitizes to gemcitabine therapy, as a result of a ROS-mediated reduction in the protein levels of CDA (Frese et al. 2012). The effects of pimasertib and gemcitabine on CDA protein expression were explored. PANC-1 and BxPC-3 cells were treated for 24 hours with 1μM pimasertib, 50nM gemcitabine or their combination. Pimasertib alone caused a reduction of CDA protein expression in PANC-1 cells and to lesser extent in BxPC-3 cells; whereas gemcitabine increased CDA protein expression in BxPC-3 cells (Fig. 3.16). CDA protein levels were not altered upon the combination treatment (Fig. 3.16).
Figure 3.16: Effects of pimasertib on CDA protein levels.
BxPC-3 and PANC-1 cells were treated for 24 hours with 1µM pimasertib, 50nM gemcitabine alone or in combination with 1µM pimasertib. Total cell lysates were analysed by immunoblotting. Calnexin was used as loading control.
We then treated BxPC-3 cells for 24 hours with 1μM pimasertib, 50nM gemcitabine or their combination. The protein levels of RRM1, RRM2 and dCK were analysed by immunoblotting analysis. dCK protein expression was upregulated upon pimasertib and gemcitabine administration in BxPC-3 cells (Fig. 3.17A). RRM2 expression was reduced (Fig.3.17A), and this could be explained by the fact that its protein levels are low in the G1 phase of the cell cycle (Nordlund and Reichard 2006) as pimasertib induces a G1 arrest (Fig. 3.6). A slight decrease in RRM2 protein expression was observed with gemcitabine. Interestingly, treatment with pimasertib alone and in combination with gemcitabine reduced RRM1 protein levels (Fig. 3.17A). Bar graphs showing the quantification of RRM1 immunoblots are illustrated in Figure 3.17B.
A B
Figure 3.17: Pimasertib reduces RRM1 protein levels.
A. BxPC-3 cells were treated for 24 hours with 1µM pimasertib, 50nM gemcitabine alone or in
combination with 1µM pimasertib. Total cell lysates were analysed by immunoblotting. Calnexin was used as loading control. B. Densitometric analysis of the immunoblots presented in Figure 3.17A. Results are presented as mean ± SD of three independent experiments. Image J software was used for the quantitation of the western blot bands. Fold decrease of RRM1 was calculated by normalization to the loading control and comparison to the untreated sample.
Downregulation of RRM1 protein was also observed in PANC-1 cell lines after 24h treatment with pimasertib alone or in combination with gemcitabine (Fig. 3.18) but no change in dCK or RRM2 protein expression was found upon gemcitabine or pimasertib (Fig. 3.18A). Bar graphs showing the quantification of RRM1 immunoblots are illustrated in Figure 3.18B.
A B
Figure 3.18: Pimasertib reduces RRM1 protein levels.
A. PANC-1 cells were treated for 24 hours with 1µM pimasertib, 50nM gemcitabine alone or in
combination with 1µM pimasertib. Total cell lysates were analysed by immunoblotting. Calnexin was used as loading control. B. Densitometric analysis of the immunoblots presented in Figure 3.19A. Results are presented as mean ± SD of three independent experiments. Image J software was used for the quantitation of the western blot bands. Fold decrease of RRM1 was calculated by normalization to the loading control and comparison to the untreated sample.
Next, we investigated whether pimasertib induces RRM1 downregulation in other human pancreatic cancer cell lines. SUIT-2 and MIAPaCa-2 cell lines, which had been previously tested for gemcitabine sensitivity, were treated with 1μM pimasertib, 50nM gemcitabine or their combination for 24 hours and protein lysates were analysed by immunoblotting. Pimasertib alone and in combination with gemcitabine reduced RRM1 protein levels in both cell lines tested (Fig. 3.19).
Previous studies have shown that RRM1 protein expression was upregulated in gemcitabine resistant cells (Nakahira et al. 2007). In this present study, gemcitabine increased RRM1 protein levels in PANC-1 (Fig. 3.18), which displayed the highest resistance to gemcitabine, but also in MIAPaCa-2 and SUIT-2 sensitive cell lines (Fig. 3.19). No upregulation of RRM1 was observed in BxPC-3 cells after gemcitabine treatment.
Figure 3.19: Pimasertib reduces RRM1 protein levels in other human pancreatic cancer cell lines.
SUIT-2 and MIAPaCa-2 cells were treated for 24 hours with 1µM pimasertib, 50nM gemcitabine alone or in combination with 1µM pimasertib. Total cell lysates were analysed by immunoblotting. Calnexin was used as loading control.
To explore whether the effect induced by pimasertib on RRM1 expression occurred with other MEK inhibitors, BxPC-3 cells were treated with AS703988 a potent, allosteric MEK1/2 inhibitor. AS703988 inhibited ERK phosphorylation at 1µM and showed the identical effect on RRM1 expression after 24 hours treatment (Fig. 3.20A). Inhibition of ERK phosphorylation and RRM1 protein expression was also observed when AS703988 was combined with gemcitabine (50nM) (Fig. 3.20A). Bar graphs showing the quantification of RRM1 immunoblots are illustrated in Figure 3.20B.
A B
Figure 3.20: The MEK1/2 inhibitor AS703988 alters RRM1 protein expression.
A. BxPC-3 cells were treated for 24 hours with 1µM AS703988, 50nM gemcitabine alone or in
combination with 1µM pimasertib. Total cell lysates were analysed by immunoblotting. Calnexin was used as loading control. B. Densitometric analysis of the immunoblots presented in figure 3.20A. Results are presented as mean ± SD of three independent experiments. Image J software was used for quantitation of the western blot bands. Fold decrease of RRM1 was calculated by normalization to the loading control and comparison to the untreated sample.