Abstract
BackgroundTo explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC).MethodsHCC cell lines (including Hep3B, Huh7, and PLC5) and HUVECs (human umbilical venous endothelial cells) were tested. The molecular targeting therapy agents tested included NVP-AEW541 (IGFR kinase inhibitor), MK2206 (Akt inhibitor), BEZ235 (PI3K/mTOR inhibitor), and RAD001 (mTOR inhibitor). Potential synergistic antitumor effects were tested by median dose-effect analysis in vitro and by xenograft HCC models. Apoptosis was analyzed by flow cytometry (sub-G1 fraction analysis) and Western blotting. The activities of pertinent signaling pathways and expression of apoptosis-related proteins were measured by Western blotting.ResultsVertical blockade induced a more sustained inhibition of PI3K/Akt/mTOR signaling activities in all the HCC cells and HUVEC tested. Synergistic apoptosis-inducing effects, however, varied among different cell lines and drug combinations and were most prominent when NVP-AEW541 was combined with MK2206. Using an apoptosis array, we identified survivin as a potential downstream mediator. Over-expression of survivin in HCC cells abolished the anti-tumor synergy between NVP-AEW541 and MK2206, whereas knockdown of survivin improved the anti-tumor effects of all drug combinations tested. In vivo by xenograft studies confirmed the anti-tumor synergy between NVP-AEW541 and MK2206 and exhibited acceptable toxicity profiles.ConclusionsVertical blockade of the IGFR/PI3K/Akt/mTOR pathway has promising anti-tumor activity for HCC. Survivin expression may serve as a biomarker to predict treatment efficacy.
Highlights
To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/Phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (Akt)/Mammalian target of rapamycin (mTOR) signaling pathway can improve treatment efficacy for hepatocellular carcinoma (HCC)
The following the optimal dosage were used: NVP-AEW541 once per day for 25 days based on previous studies [22,23], BEZ235 (dissolved in 10% NMP (1-methyl-2-pyrrolidone)/PEG300 90%, 25 mg/kg, gavage) once per day for 25 days based on previous studies [24,25], and MK2206 once per week for 4 weeks based on previous studies [26,27]
Survivin was identified as the candidate molecule because it showed the most consistent inhibition when NVP-AEW541 was combined with MK2206, BEZ235, or RAD001 and correlated with the anti-tumor synergy (Additional file 1: Figure S1)
Summary
To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC). The insulin-like growth factor (IGF) signaling pathway provides an important regulatory mechanism for tumorigenesis and drug resistance in many cancers, including HCC [4,5]. Many previous studies linked IGF signaling activity to HCC pathogenesis, tumor angiogenesis, and drug resistance [6,7,8,9]. Many inhibitors of IGF receptors (IGFR) have been developed and promising antitumor activity has been demonstrated in pre-clinical studies, clinical studies showed very limited efficacy of IGFR inhibitors as single-agent therapy in solid tumors, including HCC [14,15]
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