Uveal melanoma (UM) is the most frequent ocular cancer in adults, accounting for ~5% of the total melanoma incidence. Although the primary tumor is well treatable, patients frequently develop metastases for which no curative therapy exists. Highly activated protein kinase C (PKC) is a common feature of UM and has shown potential as therapeutic intervention for UM patients. Unfortunately, PKC inhibition as single treatment appears to have only limited clinical benefit. Combining PKC inhibition with activation of p53, which is rarely mutated in UM, by MDM2 inhibitors has shown promising results in vitro and in vivo. However, clinical studies have shown strong adverse effects of MDM2 inhibition. Therefore, we investigated alternative approaches to achieve similar anticancer effects, but with potentially less adverse effects. We studied the potential of targeting MDMX, an essential p53 inhibitor during embryonal development but less universally expressed in adult tissues compared with MDM2. Therefore, targeting MDMX is predicted to have less adverse effects in patients. Depletion of MDMX, like the pharmacological activation of p53, inhibits the survival of UM cells, which is enhanced in combination with PKC inhibition. Also pan-PKC inhibitors elicit adverse effects in patients. As the PKC family consists of 10 different isoforms, it could be hypothesized that targeting a single PKC isoform would have less adverse effects compared with a pan-PKC inhibitor. Here we show that specifically depleting PKCδ inhibits UM cell growth, which can be further enhanced by p53 reactivation. In conclusion, our data show that the synergistic effects of p53 activation by MDM2 inhibition and broad spectrum PKC inhibition on survival of UM cells can also largely be achieved by the presumably less toxic combination of depletion of MDMX and targeting a specific PKC isoform, PKCδ.
OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.
4'-Thio-beta-D-arabinofuranosylcytosine (OSI-7836) is a nucleoside analogue with structural similarity to gemcitabine and cytarabine (ara-C). Myelosuppression, reversible transaminase elevations, and flu-like symptoms are common side effects associated with human use of gemcitabine and ara-C. Fatigue is also associated with the use of gemcitabine and OSI-7836 in humans. To better understand the toxicity of OSI-7836, subchronic studies were conducted in dogs. OSI-7836 was administered on days 1 and 8 or on days 1, 2, and 3 of a 21-day dose regimen. These schedules attempted to match clinical trial dosing regimens. Routine toxicity study end points demonstrated that OSI-7836 was primarily cytotoxic to the gastrointestinal tract, bone marrow, and testes; the myelotoxicity was mild and reversible. Plasma pharmacokinetics were dose-linear with an elimination half-life of 2.2 h. Follow-up single dose experiments in dogs assessed drug effects on lymphocyte subpopulations and on adrenal and thyroid function. Populations of T and B cells were equally reduced following OSI-7836 administration. There were no adverse effects on thyroid function, but there were marked reductions in circulating cortisol and adrenocorticotropic hormone concentrations suggesting a centrally mediated impairment of the hypothalamic-pituitary-adrenal axis. These findings show a toxicological profile with OSI-7836 similar to other nucleoside analogues and suggest that the beagle is a model for studying one possible cause of OSI-7836-related fatigue, impaired function of the hypothalamic-pituitary-adrenal axis.
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