Figure 1.
In vitro anti-proliferative effects of everolimus (RAD) and SN38 combinations in colorectal cancer cell lines.
The combinatorial anti-proliferative effects were evaluated in HT29, HCT116, HCT8 and LS180 cells to assess potential additive or synergistic interactions. Growth inhibition was measured by the sulforhodmine B assay (SRB) following a 72 hour incubation with SN38 (0, 2, 4 or 8 nM) and RAD (0, 2, 20 or 200 nM - indicated by the triangels under the graphs). Data on the graphs represents the mean ± standard deviation of at least 3 separate experiments. The combination index (CI) values were calculated for for all combinations and the values ± the standard deviation are presented in the colored tables below each graph.. The %CV for replicates ranged from 5–40% and averaged ∼20% therefore, we determined that CI values>1.2 are considered antagonistic, 1.2> CI>0.8 are additive, and CI<0.8 are synergistic. Note that SN38 is used in in vitro assays instead of irinotecan since it is the active metabolite.
Figure 2.
Western blot analysis of everolimus and SN38 treatment effects in HCT8, HT29 and HCT116 cells.
Akt, p-Akt, PARP, cleaved PARP, total ribosomal S6 kinase (S6), ribosomal phospho-S6 kinase (pS6), ERK, p-ERK, p21 and α-tubulin were measured in all lines following 24 hours of treatment with RAD (20 nM), SN38 (4 nM) or the combination of the two. Molecular weights are presented next to protein name in parenthesis. Note that SN38 is used in in vitro assays instead of irinotecan since it is the active metabolite.
Table 1.
Pharmacokinetic parameters for everolimusa in humans and mice.
Table 2.
Pharmacokinetic parameters for Irinotecan/SN38 in humans and mice.
Figure 3.
In vivo effects of everolimus (RAD), irinotecan (IRI) and combination of the two agents on CRC tumor xenografts.
(A) HT29 and HCT116 tumor xenograft growth curves. Animals were treated for 28 days with vehicles, RAD, IRI, or the combination of RAD+IRI. Data represents the average ± SEM of 9–12 tumors per group. *P<0.05 versus vehicle. (B) Pharmacokinetic-pharmacodynamic modeling was performed on to quantitatively assess the intensity of the RAD+IRI combination in HT29 and HCT116 tumor xenografts. This is a graphical representation of the interaction term (ψ) for the RAD+IRI combination. Each bar represents the ψ value for an individual tumor. ψ values>1.3 are synergistic, 1.3> ψ>0.7 are additive, 0.7> ψ>0 are less than additive, and ψ<0 are antagonistic.
Figure 4.
Metabolic heat-maps based on quantitative NMR spectroscopic data sets in HT29 and HCT116 xenografts at the end of the study.
1H-, 13C-, and 31P-NMR data are represented as mean ± SEM of 3–6 measurements. The metabolites, their ratios and metabolic fluxes were grouped based on their biochemical relevance. For the control group, all intracellular metabolite levels are given as µmol per gram cell wet weight and metabolite ratios are unitless. Metabolic pathways which were undisturbed by treatment are presented as yellow maps. A decrease in metabolic end-point is indicated by red, while an increase by green spots. Statistical significance for metabolite changes are based on multivariate analysis of metabolic fluxes with p<0.02. The interactive metabolic profile array database was custom-based [23].