Carol M. Makepeace

RADIOSENSITIZATION OF HYPOXIC TUMOR CELLS IN VITRO BY NITRIC OXIDE

PURPOSE The effects of nitric oxide (NO) on the radiosensitivity of SCK tumor cells in oxic and hypoxic environments in vitro were studied. METHODS AND MATERIALS NO was delivered to cell suspensions using the NO donors 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), and a spermine/nitric oxide complex (SPER/NO), which release NO at half-lives of 2.1 min and 39 min at pH 7.4, respectively. The cells were suspended in media containing DEA/NO or SPER/NO for varying lengths of time under oxic or hypoxic conditions, irradiated, and the clonogenicity determined. RESULTS Both compounds markedly radiosensitized the hypoxic cells. The drug enhancement ratios (DER) for 0.1, 1.0, and 2.0 mM DEA/NO were 2.0, 2.3 and 3.0, respectively, and those for 0.1, 1.0, and 2.0 mM SPER/NO were 1.6, 2.3, and 2.8, respectively. Aerobic cells were not radiosensitized by DEA/NO or SPER/NO. When DEA/ NO and SPER/NO were incubated in solution overnight to allow release of NO, they were found to have no radiosensitizing effect under hypoxic or oxic conditions indicating the sensitization by the NO donors was due to the NO molecule released from these drugs. At the higher concentrations, SPER/NO was found to be cytotoxic in aerobic conditions but not in hypoxic conditions. DEA/NO was only slightly toxic to the cells in both aerobic and hypoxic conditions. CONCLUSIONS NO released from NO donors DEA/NO and SPER/NO is as effective as oxygen to radiosensitize hypoxic cells in vitro. Its application to the radiosensitization of hypoxic cells in solid tumors remains to be investigated.

Thermosensitization by lowering intracellular pH with 5-(N-ethyl-N-isopropyl) amiloride

It has previously been reported that amiloride, a diuretic drug, sensitizes cells to hyperthermia by inhibiting the Na+/H+ exchange through the plasma membrane and thus decreasing the intracellular pH (pHi), particularly in a low extracellular pH (pHe) environment. In the present study, the efficacy of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an analog of amiloride, to lower the pHi and sensitize tumor cells to hyperthermia was investigated. It was observed that 10 microM EIPA was as effective as 500 microM amiloride to lower the pHi and to increase the thermal sensitivity of SCK tumor cells in vitro. The fact that lowering the pHi and increasing thermal sensitivity of tumor cells by EIPA are more pronounced in acidic medium suggests that the acidic intratumor environment may be exploited to selectively increase the thermal damage in tumors relative to normal tissues by EIPA or its analogs.

Increase in tumor blood flow by pentoxifylline

PURPOSE The effect of pentoxifylline (PTX) on the blood flow in experimental rodent tumors was investigated. METHODS AND MATERIALS When the R3230 AC adenocarcinoma implanted in the leg of Fischer 344 rats grew to about 1 g, the effect of PTX on the blood flow in the tumor and in the skin and muscle was determined with the microsphere method using 85Sr labelled 25 microns diameter microspheres. The SCK mammary carcinoma was induced subcutaneously in the leg or foot of A/J mice and the effect of PTX on the tumors was investigated: the blood perfusion in the leg tumors (7 mm in diameter) was determined with the 86Rb uptake method and that in the foot tumors (5 mm diameter) was determined with the laser Doppler flow (LDF) method. RESULTS The blood flow in the R3230 AC adenocarcinoma significantly increased when measured 30 min after an IP injection of 50 mg/kg PTX while the blood flow in the normal skin and muscle remained unchanged. The 86Rb uptake in the SCK tumor slightly increased 30 min after an IP injection of 50 mg/kg PTX. The LDF in the SCK tumors grown in the foot began to increase 5-10 min after an injection of 25 mg/kg PTX reaching 1.5-2.0 times in 20-30 min and it returned to the original level at 60 min. CONCLUSION The results in the present study together with our previous observation that PTX increases the tumor pO2 in rodent tumors strongly suggest that PTX may be useful for increasing the radiosensitivity of human tumors.

Abstract 1350: Direct pharmacological targeting of Gq/11 in uveal melanoma

Uveal (eye) melanoma is a highly aggressive cancer, in which almost half of patients develop distant metastases that are refractory to therapy. In particular, metastatic uveal melanoma has been clinically unresponsive to the immunotherapeutic agents that have shown success in skin tumors, making the need for novel therapeutic approaches to uveal melanoma all the more urgent. Unlike skin melanomas, which are driven by BRAF and NRAS mutations, uveal melanomas arise typically from mutations that result in constitutive activity of the alpha subunit of the heterotrimeric G-protein, Gq, or its paralog G11. The prevalence of constitutively active Gq/11 in uveal melanoma suggests a dependence of these tumors on Gq/11 activity that could be exploited therapeutically. To address this hypothesis, we are using a potent, bioavailable small molecule that binds to and inhibits Gq/11 to target constitutively active Gq/11 in uveal melanoma cells. This inhibitor functions by sequestering wild type or constitutively active Gq/11 in an inactive state. We first used inositol phosphate accumulation assays and confirmed inhibition of both wildtype and constitutively active Gq/11 by the inhibitor in all uveal melanoma cells. We then assayed the affect of Gq/11 inhibition on overall viability of uveal melanoma cells, and found that uveal melanoma cells with mutant Gq/11 were highly sensitive to the small molecule; whereas, uveal melanoma cells with wildtype Gq/11 showed no loss of viability, even at 1000-fold higher concentrations of inhibitor. In Gq-mutant uveal melanoma cells, Gq/11 inhibition caused cell cycle arrest in G1, and dysregulation of several cell cycle regulatory pathways. Inhibitor treatment also caused Gq/11-driven uveal melanoma cells to become more differentiated, as indicated by increased pigmentation, elevated expression of melanin synthesis and melanosome markers, changes in cell morphology and changes in melanocytic versus melanoma gene programs. None of these phenotypic changes were seen in BRAF-driven uveal melanoma cells treated with the Gq/11 inhibitor, demonstrating that the effects of this inhibitor were exquisitely dependent on the constitutively active Gq/11 oncogene. These results establish that Gq/11 is a druggable target in uveal melanoma cells, and show that Gq/11-mutant uveal melanoma cells are exquisitely sensitive to inhibition by small molecule inhibitors. We are currently transitioning these studies to animal models to establish drug efficacy and toxicity and explore treatment and delivery options. Citation Format: Michael D. Onken, Carol M. Makepeace, Shiqi Wang, Kevin M. Kaltenbronn, S. Michinobu Kanai, Tom J. Broekelmann, John A. Cooper, Kendall J. Blumer. Direct pharmacological targeting of Gq/11 in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1350. doi:10.1158/1538-7445.AM2017-1350