A promising anti-cancer agent that functions as a dual target inhibitor of SphK1 and microtubule dynamics
Open Access
- Author:
- Dick, Taryn Eve
- Graduate Program:
- Molecular Toxicology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 31, 2016
- Committee Members:
- Jong Kak Yun, Dissertation Advisor/Co-Advisor
Jong Kak Yun, Committee Chair/Co-Chair
Victor J Ruiz Velasco, Committee Member
Shantu G Amin, Committee Member
Dhimant Desai, Committee Member
Richard Robert Young, Committee Member
Jeremy Andrew Hengst, Special Member - Keywords:
- Sphingosine Kinase
Cyclin-dependent kinase 1
cell cycle arrest
mitotic arrest
mitotic cell death
microtubules - Abstract:
- An extensive amount of data points to sphingosine kinase 1 (SphK1) and aberrant regulation of sphingolipids as important components of tumorigenesis. We previously developed SKI-178 as a novel small molecule, SphK1-selective inhibitor that is cytotoxic toward a broad panel of cancer cell lines. Nonetheless, the mechanism underlying SKI-178 induced apoptotic cell death had not yet been elucidated. Therefore, the overarching goal of this research study was to uncover the detailed mechanism-of-action (MOA) of SKI-178 induced apoptosis. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. We further demonstrate that the sustained activation of cyclin-dependent kinase 1 (CDK1) during SKI-178 mediated prolonged mitosis leads to the simultaneous phosphorylation of pro-survival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Based on evidence in the literature suggesting a link between SphK1 activity and mitotic arrest, we originally attributed much of the MOA to SphK1 inhibition. However, in light of recent reports claiming that SphK1 inhibition alone is not sufficient to induce apoptotic cell death; we considered the possibility that SKI-178 may have additional targets that contribute to its cytotoxicity. Based on the striking similarly between the MOA of SKI-178 and various agents that disrupt microtubules, we considered the possibility that SKI-178, in addition to being a SphK1 selective inhibitor, also affects normal microtubule dynamics. We hypothesize that SKI-178 mediated apoptosis is due to its ability to work as a microtubule targeting agent (MTA), and that its concomitant inhibition of SphK1 sensitizes cells to this effect. Herein we present evidence that SKI-178 does indeed function as MTA, preventing the polymerization of the microtubule network. Furthermore, we demonstrate that independent of SKI-178, inhibition of SphK1 alone sensitizes cells to vincristine, a MTA known for its ability to disrupt microtubule polymerization. We also definitively confirm that in cells, SKI-178 is able to directly target engage both SphK1 and tubulin proteins at concentrations known to induce apoptotic cell death in a panel of cancer cell lines. Moreover, multi-drug resistance, mediated by multidrug resistant protein-1 and/or pro-survival Bcl-2 family member over-expression, did not affect the sensitivity of cells to SKI-178. Together, these results establish SKI-178 as a dual target inhibitor with promising chemotherapeutic potential for a wide variety of cancer types including those know to be multi-drug resistant.