Direct targeting of amplified gene loci for proapoptotic anticancer therapy.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.

Combination of ebselen and hydrocortisone substantially reduces nitrogen mustard-induced cutaneous injury.

The purpose of the present study was to investigate the vesicant countermeasure effects of hydrocortisone (HC) and ebselen (EB-1), administered as monotherapy or as a combination treatment. The mouse ear vesicant model (MEVM) was utilized and test doses of HC (0.016, 0.023, 0.031, 0.047, 0.063, 0.125 or 0.250 mg/ear), EB-1 (0.125, 0.187, 0.250, 0.375 or 0.500 mg/ear) or the combination of HC + EB-1 were topically applied at 15 min, 4 h and 8 h after nitrogen mustard exposure. Ear punch biopsies were obtained 24 h after mechlorethamine (HN2) exposure. Compared to control ears, ear tissues exposed topically to HN2 (0.500 µmol/ear) presented with an increase in ear thickness, vesication, TUNEL fluorescence and expression of matrix metalloproteinase 9 (MMP-9) and inducible nitric oxide synthase (iNOS). In contrast, HN2 exposed ears treated topically with EB-1 showed a significant decrease in morphometric thickness and vesication vs. HN2 alone. Ear tissues exposed to HN2 and then treated with HC also demonstrated reductions in morphometric thickness and vesication. Combination treatment of HC + EB-1 was found to be the most effective at reducing HN2-induced ear edema and vesication. The combination also dramatically decreased HN2-mediated cutaneous expression of iNOS and MMP-9 and decreased HN2-induced TUNEL staining. Taken together, our study demonstrates that the combination of HC + EB-1 is an efficacious countermeasure to HN2.

Ebselen oxide attenuates mechlorethamine dermatotoxicity in the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard mimetic. Topical exposure to HN2 is associated with tissue blistering. Previous work in our laboratory has shown that ebselen (EB-1) possesses anti-vesicant, anti-inflammatory, anti-bacterial, anti-fungal, and cytoprotective properties, both in vivo and in vitro. We recently reported that ebselen oxide (EB-2), an analog of EB-1 with a tetravalent selenium atom, also possesses anti-bacterial and anti-fungal activity and confers cytoprotection against HN2 in vitro. The purpose of the present study was to determine the vesicant countermeasure potential of EB-2 using the mouse ear vesicant model (MEVM). Compared to control ears, mouse ears exposed to a single dose of HN2 (0.500 µmol/ear) showed an increase in wet weights, ear thickness, hyperplasia, vesication, and inflammatory cell infiltration after 24 h. Fluorescence microscopy of terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-stained sections showed that the occurrence of apoptosis extended from the epidermis of the HN2-treated side, all the way to the contralateral epidermis. In contrast, HN2-exposed ears treated topically with EB-2 at a test dose of 0.250 mg/ear showed a significant decrease in wet weight (12% less vs. HN2 alone), morphometric thickness (13% less vs. HN2 alone), and vesication. In addition, TUNEL staining revealed that HN2 ears treated with EB-2 (0.250 mg/ear) showed a decrease in apoptosis as compared to the HN2 group. EB-2 also reduced the abundance of matrix metalloproteinase-9 (MMP-9) in ear tissues exposed to HN2. Taken together, our study demonstrates that EB-2 is an efficacious countermeasure to HN2.

Assessment of the time-dependent dermatotoxicity of mechlorethamine using the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 µmol/ear) or DMSO vehicle (5 µl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures.