Abstract
BackgroundTargeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation.MethodsWe evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s) was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs.ResultsSerum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1.ConclusionThere was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents thought to target only one of those pathways. These results indicate that combinations of inhibitors of mTOR and of the B-raf signaling pathways may be more effective as a treatment for melanoma than use of either agent alone.
Highlights
Targeted inhibition of protein kinases is acknowledged as an effective approach for cancer therapy
We show that serum-stimulated melanoma cell proliferation is inhibited by either rapamycin or BAY439006, with B-Raf V599E mutants showing an increased sensitivity to each drug at 10 nM compared to melanoma cells with wild-type B-Raf
BAY43-9006 and rapamycin inhibit proliferation of melanoma cells We examined the serum-dependent proliferation of multiple human melanoma cell lines and the effects of inhibition of B-Raf by BAY43-9006 and of mTOR by rapamycin
Summary
Targeted inhibition of protein kinases is acknowledged as an effective approach for cancer therapy. Cancer therapy has undergone a paradigm shift based on the therapeutic effectiveness of imatinib mesylate (Gleevec) This drug was designed as a specific inhibitor of the BCR-ABL oncogene protein tyrosine kinase, known to be responsible for chronic myeloid leukemia (CML) cells [2]. Pre-clinical and early phase I studies have suggested that BAY 43-9006 may be of therapeutic value in human tumors containing ras gene mutations, and in tumors over-expressing growth factor receptors that activate the Ras/ERK pathway [10]. These studies have not addressed the effects of BAY 43-9006 in combination with any other kinase inhibitors
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