Preclinical evaluation of Laromustine for use in combination with radiation therapy in the treatment of solid tumors

Abstract

Purpose These studies explored questions related to the potential use of Laromustine in the treatment of solid tumors and in combination with radiotherapy. Materials and Methods These studies used mouse EMT6 cells [both parental and transfected with genes for O6-alkylguanine transferase (AGT)], repair-deficient human Fanconi Anemia C and Chinese hamster VC8 (BRCA2−/ −) cells and corresponding control cells, and EMT6 tumors in mice assayed using cell survival and tumor growth assays. Results Hypoxia during Laromustine treatment did not protect EMT6 cells or human fibroblasts from this agent. Rapidly proliferating EMT6 cells were more sensitive than quiescent cultures. EMT6 cells expressing mouse or human AGT, which removes O6 -alkyl groups from DNA guanine, thereby protecting against G-C crosslink formation, increased resistance to Laromustine. Crosslink-repair-deficient Fanconi Anemia C and VC8 cells were hypersensitive to Laromustine, confirming the importance of crosslinks as lethal lesions. In vitro, Laromustine and radiation produced additive toxicities to EMT6 cells. Studies using tumor cell survival and tumor growth assays showed effects of regimens combining Laromustine and radiation that were compatible with additive or subadditive interactions. Conclusions The effects of Laromustine on solid tumors and with radiation are complex and are influenced by microenvironmental and proliferative heterogeneity within these malignancies.

Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W,In VitroandIn Vivo

“…First, they lack detectable O 6 -alkylguanine-DNA alkyltransferase (AGT), which is also known as O 6 -methylguanine DNA-methyltransferase (MGMT) (23, 26, 30). Second, they can be grown as solid tumors in mice, as well as in cell culture (27, 28, 31–32). EMT6 cells were maintained in Waymouth’s Medium with 15% fetal bovine serum and antibiotics.…”

“…Because of this, the dose-response curves shown in Fig. 2 are shown as yield-corrected surviving fractions, which consider the decrease in the numbers of cells in the treated cultures as well as the decrease in the plating efficiencies of the suspended cells (28, 31, 33). In all other experiments, which used shorter incubation times, aerobic incubations, and/or lower concentrations, no changes in the number of cells in treated cultures were observed, and data are therefore shown as surviving fractions.…”

“…Cultures were made severely hypoxic by sealing the bottles with rubber gaskets, inserting needles for the influx and efflux of gases, and gassing for 1 h with a humidified mixture of 95% nitrogen/5% CO 2 certified by the vendor (TechAir, New Haven, CT) to contain <1 ppm O 2 (0.00001% O 2 ). KS119, KS119W or vehicle was injected through the septum of the gasket, without breaking the hypoxia, and cultures were incubated with this agent under continued hypoxia for 2 h as described previously (8, 29, 31, 34). Controls exposed to hypoxia plus vehicle were included in all experiments.…”

“…All protocols used with experimental animals were reviewed and approved by the Yale Institutional Animal Care and Use Committee, and all experiments were performed in full compliance with the regulations and policies of the government, Yale University, and the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and with the principles outlined in the Guide for the Care and Use of Laboratory Animals . Tumors were produced by inoculating 2 × 10 5 EMT6 cells, harvested from exponentially-growing cell cultures, intradermally into the flank, and were treated with KS119, KS119W or radiation approximately 2 weeks later, when the tumor volumes averaged 100 mm 3 (27, 31, 32). Past studies in our laboratory have shown that in EMT6 tumors of this volume the hypoxic fraction (i.e., the proportion of the viable clonogenic tumor cells that are radiobiologically hypoxic as shown by rigorous analyses of paired tumor cell survival curves or tumor growth delay curves) is approximately 30% (1, 2).…”

“…At the time of assay, the mice were euthanized, tumors were explanted sterilely and a single cell suspension was created by mincing and trypsinization as previously described in detail (27, 31, 33). Cells were collected by centrifugation, resuspended in Waymouth’s medium, counted under a phase contrast microscope using trypan blue to determine the number of cells damaged during the suspension process, and plated for colony formation as described above briefly for cells from culture and detailed elsewhere (27, 31, 33). The numbers of cells suspended from untreated and treated tumors were examined and compared to detect any rapid cell loss during treatment; cell loss was not observed in these studies.…”