Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity.

Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury.

Combination intraperitoneal chemotherapy is superior to mitomycin C or oxaliplatin for colorectal carcinomatosis in vivo.

BACKGROUND: Hyperthermic intraperitoneal (IP) chemotherapy after cytoreduction improves survival in patients with colorectal carcinomatosis of the peritoneal surface. Most protocols use single agents (mitomycin C or oxaliplatin) provided IP. The purpose of this study was to determine whether combination IP chemotherapy is superior to single-agent therapy in a mouse model. METHODS: Nu/Nu mice were injected IP with HT-29 colorectal cancer cells. Animals were treated with single agents or combinations. Primary end point was overall survival. Agents explored included oxaliplatin, mitomycin C, panitumumab, erlotinib, cetuximab, and irinotecan delivered IP as single agents; mitomycin C, panitumumab, and irinotecan in combination IP; and 5-fluorouracil-leucovorin-irinotecan (FOLFIRI) in combination delivered intravenously. RESULTS: Survival of mice receiving irinotecan or mitomycin C IP was greater than controls. Median survival of mice receiving intravenous FOLFIRI was also greater than control. However, survival of mice receiving IP irinotecan or mitomycin C was far greater than mice receiving intravenous FOLFIRI. For combination therapy, a positive interaction was observed with mitomycin C and irinotecan, whereas survival was greater than either agent individually. No interaction was observed between panitumumab and mitomycin C or irinotecan. However, an overall survival benefit was observed with the combination of irinotecan, mitomycin C, and panitumumab; at 120 days after cell injection, 100% of the triagent therapy group survived. CONCLUSIONS: IP therapy with mitomycin C or irinotecan provided a survival benefit compared with intravenous FOLFIRI. Combination IP therapy with mitomycin C, panitumumab, and irinotecan was superior to all other agents tested alone or in combination. This warrants further combination analysis and supports consideration for a phase I application.

Overcoming Wnt-ß-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

Comparative oncology approach to drug repurposing in osteosarcoma.

BACKGROUND: Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans. METHODS: In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS. RESULTS: We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin. CONCLUSIONS: Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation.

A unique class of duocarmycin and CC-1065 analogues subject to reductive activation.

N-Acyl O-amino phenol derivatives of CBI-TMI and CBI-indole2 are reported as prototypical members of a new class of reductively activated prodrugs of the duocarmycin and CC-1065 class of antitumor agents. The expectation being that hypoxic tumor environments, with their higher reducing capacity, carry an intrinsic higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives (tunable N-O bond cleavage) and increasing their sensitivity to the prodrug treatment. Preliminary studies indicate the prodrugs effectively release the free drug in functional cellular assays for cytotoxic activity approaching or matching the activity of the free drug, yet remain essentially stable and unreactive to in vitro DNA alkylation conditions (<0.1-0.01% free drug release) and pH 7.0 phosphate buffer, and exhibit a robust half-life in human plasma (t1/2 = 3 h). Characterization of a representative O-(acylamino) prodrug in vivo indicates that they approach the potency and exceed the efficacy of the free drug itself (CBI-indole2), indicating that not only is the free drug effectively released from the inactive prodrug but also that they offer additional advantages related to a controlled or targeted release in vivo.

Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation.

BACKGROUND: Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition. METHODS: An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay. RESULTS: Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities. CONCLUSION: This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and anti-CSC drug leads.

Drug repurposing for gastrointestinal stromal tumor.

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25 to 30 million people in the United States alone. Given the costs associated with the discovery, development, and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, "drug repurposing" or "repositioning," has emerged as an alternative to the traditional drug development process. In this study, we screened 796 U.S. Food and Drug Administration (FDA)-approved drugs and found that two of these compounds, auranofin (Ridaura) and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs, including imatinib-resistant cells. One of the most notable drug hits, auranofin, an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis, was found to inhibit thioredoxin reductase activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anticancer activity associated with auranofin was independent of imatinib-resistant status, but was closely related to the endogenous and inducible levels of ROS. Coupled with the fact that auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for patients with GIST, particularly in those suffering from imatinib-resistant and recurrent forms of this disease.

Design, synthesis, and evaluation of duocarmycin O-amino phenol prodrugs subject to tunable reductive activation.

A series of N-acyl O-amino derivatives of seco-CBI-indole(2) are reported and examined as prototypical members of a unique class of reductively activated (cleaved) prodrugs of the duocarmycin and CC-1065 family of antitumor agents. These prodrugs were designed to be potentially preferentially activated in hypoxic tumor environments which carry an intrinsically higher concentration of "reducing" nucleophiles (e.g., thiols) capable of activating such derivatives by nucleophilic cleavage of a weak N-O bond. A remarkable range of stabilities and a resulting direct correlation with in vitro/in vivo biological potencies was observed for these prodrugs, even enlisting subtle variations in the electronic and steric environment around the weak N-O bond. An in vivo evaluation of several of the prodrugs demonstrates that some approach the potency and exceed the efficacy of the free drug itself (CBI-indole(2)), suggesting the prodrugs may offer an additional advantage related to a controlled or targeted release.