الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Many anti-cancer drugs target specific defects of the cancer cell genome. While this has been fruitful in several cancer types, treatment failure rates remain disappointingly high, owing to the immense intra- and inter-tumoral cancer cell heterogeneity. Devising therapies that take advantage of cancer cells’ unique molecular pathologies is a leading approach to drug discovery that spares non-cancer tissues. Cancer cells have invariably higher reactive oxygen species (ROS) levels compared to non-cancer cells; these cellular toxins enable the stepwise progression of malignancy by oxidizing deoxyribonucleotides (dNTPs), thus facilitating the occurrence of mutations. However, cancer cells must maintain their ROS levels within a viable range that enables their malignant behavior all while curbing excessively high levels that would irreversibly damage the cell to the point of cellular demise. The human MTH1 molecule is part of a cancer cell’s toolbox for survival; it prevents the incorporation of oxidized dNTPs into a replicating DNA molecule amid high ROS levels, preempting the introduction of mutagenic events into the genome. Inhibiting the MTH1 molecule has proven to selectively target cancer cell survival while sparing non-cancer cells. To investigate whether this effect could be potentiated by a synergistic combination, dual inhibition of MTH1 and PI3 Kinase was rationalized. PI3K pathway aberrations are involved in virtually all cancer types, and the pathway is activated by a wide number of growth factors/signals. The activation of this pathway by ROS helps mitigate the damaging effects of ROS by promoting cellular growth, survival, and metastasis.
A range of human cancer cell lines were cultured and treated for 72 hours with an MTH1 inhibitor, PI3K inhibitor (LY294002), or both, using a serial dilution of drug concentrations. Cell viability assays were performed upon termination of treatment. Immunocytochemical staining of treated cells was done to detect expression of DNA damage markers γ-H2AX and phosphorylated S10H3, as well as Western blotting for protein expression analysis of the DNA damage markers γ-H2AX, phosphorylated S10H3, and the apoptosis marker cleaved PARP1.
Calculation of synergism indices using CompuSyn software revealed a combination index of less than 1 at several concentrations of TH588+LY294002 and TH1579+LY294002 combination treatment, signifying presence of synergism. Cell viability percentages of cells treated with either single agent (MTH1 inhibitor or PI3K inhibitor) were further reduced, in some instances to 5% or less viability, upon co-administration of both inhibitors. Immunocytochemistry detected 19% of cells that expressed γ-H2AX, compared to 4% for LY294002-only treated cells, and 9% for TH588-only treated cells. Immunoblotting band staining and band thickness was visibly greater for cells treated with combination therapy versus single agent.
The simultaneous targeting of MTH1 and PI3K inhibition seems to offer a molecular one-two punch that could enhance the targeting of cancer cells by mechanisms exclusive to them. Further studies on the mechanisms of synergism beyond DNA damage markers e.g. tubulin polymerization and glycolysis aberrations, would be helpful in further understanding the combination dynamic. The usage of isoform-specific PI3K inhibitors would offer a more selective treatment with milder side effects. Optimization of dosing schedule and duration would need to be investigated before in vivo studies could be advanced, as well as possible other drug candidates that could converge on MTH1 inhibition’s mechanistic pathway(s).