Introduction: Hedgehog (Hh) signaling pathway activation is a characteristic molecular feature of medulloblastomas, which arise in infants, older children, and adults. Thus, the pathway is an attractive target for molecular therapeutics. Several cyclopamine analogs, which bind the transmembrane protein Smoothened and block Hh signaling in tumor cells, are currently in clinical trials. Because tumor cells become resistant to cyclopamine analogs by acquiring Smoothened gene mutations, there is a pressing need to develop compounds that block the Hh pathway downstream of Smoothened. We analyzed CPD-31 and CPD-829, two novel Hh pathway inhibitors discovered in a screen for compounds that inhibit proliferation of hepatocytes transformed by hepatitis C virus infection.
Methods: In-vitro proliferation assays on facsimiles of Hedgehog-driven medulloblastomas (DAOY cells) and medulloblastoma precursor cells (SHH-GNP cells) were performed. MTT colorimetric assays assessed the effect of CPD-31 and CPD-829 on proliferation compared with Smoothened inhibitor GDC-0449 and Gli1 transcription factor inhibitor Gant-61, Hh pathway inhibitors. Gli-luciferase assays were used to measure the compounds’ synergistic effect on Hh signaling activity.
Results: CPD-31 effectively inhibited proliferation of DAOY and SHH-GNP cells, but GDC-0449 did not. CPD-829 inhibited SHH-GNP cell proliferation more potently than CPD-31 by a factor of 1.1 and 1.4 at concentrations of 1 µM and 5 µM, respectively. Synergy studies using combinations of CPD-31, GDC-0449, and Gant-61 in Gli-luciferase assays with the Chou-Talalay method show combination index (CI) values <0.3, indicating strong synergy. MTT proliferation studies showed additive effects of CPD-31 and CPD-829 in combination with Gant-61 (CI=1).
Conclusions: Both novel compounds prevented proliferation of Hh medulloblastoma facsimiles. These results, combined with the synergistic and additive effects in our biochemical and cellular proliferation models with known Hedgehog pathway inhibitors, suggest different, complementary targets of action. This is promising for multi-targeted drug therapy regimens against the Hh pathway and warrants further testing in a preclinical animal model.
Patient Care: This research has the potential to be taken from preclinical animal models to part of a multi-targeted drug therapy of medulloblastoma and other hedgehog dependent tumors.
Learning Objectives: -Understand the importance of the hedgehog pathway as a target for molecular target.
-Understand the rationale for multi-targeted drug therapy of the hedgehog pathway.