Capítulo 3. El mundo de la indumentaria
3.2. Estrategias de la moda en la actualidad
3.2.1. Marcas como estilo de vida
The effect of pimasertib treatment in combination with gemcitabine on the proliferation of BxPC-3 and PANC-1 cell lines was investigated. A non-constant ratio design employing a fixed concentration of pimasertib (500nM), combined with increasing concentrations of gemcitabine (5nM, 25nM, 50nM, 100nM) was applied. A dose-effect curve of each drug alone was also performed.
Three combination schedules were designed:
1. Simultaneous and continuous exposure to pimasertib with gemcitabine for 48 hours.
2. Sequential combination consisting of 4 hour pre-treatment with pimasertib followed by 48-hour treatment with gemcitabine
3. Sequential combination consisting of 4 hour pre-treatment with pimasertib followed by 24-hour treatment with gemcitabine.
The choice of administering pimasertib 4 hours before gemcitabine was associated with the effect observed upon 4 hours treatment with pimasertib on RRM1 protein expression, which will be discussed in detail in this chapter.
Combination indices (CI), which describe the interaction between drugs, were calculated using the methodology of Chou and Talalay, (Chou 2010) with the Calcusyn Software. This software performs a dose-effect analysis based on the mass-action law based theory (Chou 2006). The CI values generated for each drug combination describe the drug interactions. CI values > 1 indicate antagonism, values =1 indicate additivity and values < 1 indicate synergism.
3.3.4.1 Simultaneous administration of pimasertib with gemcitabine
induces antagonistic or additive antiproliferative effects
BxPC-3 and PANC-1 cells were exposed to increasing concentrations of gemcitabine (5nM, 25nM, 50nM and 250nM) together with a fixed concentration of pimasertib (500nM) for 48 hours continuously. Inhibition of cell proliferation was determined by MTT assay (Figure 3.7A, 3.8A). The effects of the drugs combination was analysed with the CI method. At all concentrations of gemcitabine tested the combination resulted in CI values > 1 in BxPC-3 cell lines indicating a negative interaction and in CI values ≥ 1 in PANC-1 cell lines indicating an additive or negative interaction. A bar chart of CI values obtained from the simultaneous combination of gemcitabine with pimasertib is illustrated (Fig. 3.7B-3.8B).
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Figure 3.7: Effects of simultaneous treatment of pimasertib plus gemcitabine on BxPC-3 cells proliferation.
A. The MTT assay was used to assess the effect of a 48h simultaneous treatment of 500nM pimasertib
with increasing concentrations of gemcitabine (5-25-50-250nM) on the growth of BxPC-3 cells. B. Combination Indices (CI) values describing drug combinations were calculated with the Calcusyn program. Experiments were repeated three times and are represented as mean, Error Bars ± SD.
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Figure 3.8: Effects of simultaneous treatment of pimasertib plus gemcitabine on PANC-1 cells proliferation.
A. The MTT assay was used to assess the effect of a 48h simultaneous treatment of 500nM pimasertib
with increasing concentrations of gemcitabine (5-25-50nM) on the growth of PANC-1 cells. B. Combination Indices (CI) values describing drug combinations were calculated with the Calcusyn program. Experiments were repeated three times and are represented as mean, Error Bars ± SD.
3.3.4.2 Sequential administration of pimasertib with gemcitabine
induces synergistic antiproliferative effects
Since simultaneous administration of pimasertib with gemcitabine did not produce any synergistic antiproliferative effects (Fig. 3.7-3.8), a different time schedule was evaluated in which the MEK inhibitor was administered prior to chemotherapy. Because we found that pimasertib was able to downregulate an important biomarker of gemcitabine resistance after a 4-hour exposure (These results will be described later in the chapter), we decided to adopt the following sequential schedule of administration: BxPC-3 and PANC-1 cells were pre-treated for 4 hours with 500nM pimasertib after which gemcitabine (5nM, 25nM, 50nM, and 250nM) was administered for 48 hours continuously. Inhibition of cell proliferation was determined by MTT assay (Fig. 3.9A, 3.10A). Interestingly, this time schedule of drug administration induced synergistic antiproliferative effects in BxPC-3 and PANC-1 cell lines as indicated by CI values generated from all concentrations of gemcitabine examined being <1. Bar charts of CI values are illustrated in Fig. 3.9B and 3.10B.
Additionally, a 4-hour pre-exposure to pimasertib followed by a short time exposure with increasing concentrations of gemcitabine (24 hours) showed synergistic growth inhibitory activity (Fig. 3.11A), as shown by the CI values generated from all concentrations of gemcitabine examined being < 1 (Fig. 3.11B).
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Figure 3.9: Effects of sequential treatment of pimasertib with gemcitabine on BxPC-3 cells proliferation.
A. The MTT assay was used to assess the effect of a 4h pre-treatment of 500nM pimasertib followed by
continuous exposure for 48h with increasing concentrations of gemcitabine (5-25-50-250nM) on the growth of BxPC-3 cells. B. Combination Indices (CI) values describing drug combinations were calculated with the Calcusyn program. Experiments were repeated three times and are represented as mean, Error Bars ± SD.
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Figure 3.10: Effects of sequential treatment of pimasertib with gemcitabine on PANC-1 cells proliferation.
A. The MTT assay was used to assess the effect of a 4h pre-treatment of 500nM pimasertib followed by
continuous exposure for 48h with increasing concentrations of gemcitabine (5-25-50-250nM) on the growth of PANC-1 cells. B. Combination Indices (CI) values describing drug combinations were calculated with the Calcusyn program. Experiments were repeated three times and are represented as mean, Error Bars ± SD.
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Figure 3.11: Effects of short sequential administration of pimasertib with gemcitabine on BxPC-3 cells proliferation.
A. The MTT assay was used to assess the effect of a 4h pre-treatment of 500nM pimasertib followed by
continuous exposure for 24h with increasing concentrations of gemcitabine (5-25-50-250nM) on the growth of BxPC-3 cells. B. Combination Indices (CI) values describing drug combinations were calculated with the Calcusyn program. Experiments were repeated three times and are represented as mean, Error Bars ± SD.