Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer.

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA-approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF-Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF-Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress-mediated apoptosis in multiple IBC cellular models. In vivo, DSF-Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo- and/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization.

Although there is no standard treatment protocol for inflammatory breast cancer (IBC), multi-modality treatment has improved survival. In this study we profiled the NCI approved oncology drug set in a qHTS format to identify those that are efficacious in basal type and ErbB2 overexpressing IBC models. Further, we characterized the sensitivity of an acquired therapeutic resistance model to the oncology drugs. We observed that lapatinib-induced acquired resistance in SUM149 cells led to cross-resistance to other targeted- and chemotherapeutic drugs. Removal of the primary drug to which the model was developed led to re-sensitization to multiple drugs to a degree comparable to the parental cell line; this coincided with the cells regaining the ability to accumulate ROS and reduced expression of anti-apoptotic factors and the antioxidant SOD2. We suggest that our findings provide a unique IBC model system for gaining an understanding of acquired therapeutic resistance and the effect of redox adaptation on anti-cancer drug efficacy.

Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism.

X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflammatory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (K (d) > 1 µM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. A modest increase in TNF-α production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-α addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-α or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant-mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment.

Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines.

BACKGROUND: Crosstalk between malignant hepatocytes and the surrounding peritumoral stroma is a key modulator of hepatocarcinogenesis and therapeutic resistance. To examine the chemotherapy resistance of these two cellular compartments in vitro, we evaluated a well-established hepatic tumor cell line, HepG2, and an adult hepatic stellate cell line, LX2. The aim was to compare the chemosensitization potential of arsenic trioxide (ATO) in combination with sorafenib or fluorouracil (5-FU), in both hepatic tumor cells and stromal cells. METHODS: Cytotoxicity of ATO, 5-FU, and sorafenib, alone and in combination against HepG2 cells and LX2 cells was measured by an automated high throughput cell-based proliferation assay. Changes in survival and apoptotic signaling pathways were analyzed by flow cytometry and western blot. Gene expression of the 5-FU metabolic enzyme, thymidylate synthase, was analyzed by real time PCR. RESULTS: Both HepG2 and LX2 cell lines were susceptible to single agent sorafenib and ATO at 24 hr (ATO IC(50): 5.3 µM in LX2; 32.7 µM in HepG2; Sorafenib IC(50): 11.8 µM in LX2; 9.9 µM in HepG2). In contrast, 5-FU cytotoxicity required higher concentrations and prolonged (48-72 hr) drug exposure. Concurrent ATO and 5-FU treatment of HepG2 cells was synergistic, leading to increased cytotoxicity due in part to modulation of thymidylate synthase levels by ATO. Concurrent ATO and sorafenib treatment showed a trend towards increased HepG2 cytotoxicity, possibly due to a significant decrease in MAPK activation in comparison to treatment with ATO alone. CONCLUSIONS: ATO differentially sensitizes hepatic tumor cells and adult hepatic stellate cells to 5-FU and sorafenib. Given the importance of both of these cell types in hepatocarcinogenesis, these data have implications for the rational development of anti-cancer therapy combinations for the treatment of hepatocellular carcinoma (HCC).

XIAP inhibition and generation of reactive oxygen species enhances TRAIL sensitivity in inflammatory breast cancer cells.

We recently identified superoxide dismutase (SOD) overexpression and decreased induction of reactive oxygen species (ROS)-mediated apoptosis in models of inflammatory breast cancer (IBC) cells with acquired therapeutic resistance. This population of cells has high expression of X-linked inhibitor of apoptosis protein (XIAP), which inhibits both extrinsic and intrinsic apoptosis pathways. We therefore wanted to evaluate the effect of classical apoptosis-inducing agent TRAIL, a proapoptotic receptor agonist that selectively triggers death receptor (DR)-mediated apoptosis in cancer cells, in the IBC acquired resistance model. XIAP levels and subsequent inhibition of caspase activity inversely correlated with TRAIL sensitivity in our models of IBC. These include SUM149, a basal-type cell line isolated from primary IBC tumors and isogenic SUM149-derived lines rSUM149 and SUM149 wtXIAP, models of acquired therapeutic resistance with endogenous and exogenous XIAP overexpression, respectively. Inhibition of XIAP function using embelin, a plant-derived cell permeable small molecule, in combination with TRAIL caused a synergistic decrease in cell viability. Embelin treatment resulted in activation of extracellular signal-regulated kinase (ERK)1/2 and ROS accumulation, which correlated with downregulation of antioxidant protein SOD1 and consumption of redox modulator reduced glutathione in the XIAP-overexpressing cells. Simultaneous treatment with an SOD mimic, which protects against ROS accumulation, reversed the decrease in cell viability caused by embelin + TRAIL treatment. Embelin primes IBC cells for TRAIL-mediated apoptosis by its direct action on the anti-caspase activity of XIAP and by shifting the cellular redox balance toward oxidative stress-mediated apoptosis. Thus, ROS modulators represent a novel approach to enhance efficacy of TRAIL-based treatment protocols in IBC.

ErbB1/2 tyrosine kinase inhibitor mediates oxidative stress-induced apoptosis in inflammatory breast cancer cells.

Overexpression of epidermal growth factor receptors (ErbB) is frequently seen in inflammatory breast cancer (IBC). Treatment with ErbB1/2-targeting agents (lapatinib) mediates tumor apoptosis by downregulating ErbB1/2 phosphorylation and downstream survival signaling. In this study, using carboxy-H(2)DCFDA, DHE, and MitoSOX Red to examine changes in hydrogen peroxide radicals, cytoplasmic and mitochondrial superoxide, respectively, we observed that GW583340 (a lapatinib-analog) increases reactive oxygen species (ROS) in two models of IBC (SUM149, SUM190) that are sensitive to ErbB1/2 blockade. This significant increase in ROS levels was similar to those generated by classical oxidative agents H(2)O(2) and paraquat. In contrast, minimal to basal levels of ROS were measured in a clonal population of GW583340-resistant IBC cells (rSUM149 and rSUM190). The GW583340-resistant IBC cells displayed increased SOD1, SOD2, and glutathione expression, which correlated with decreased sensitivity to the apoptotic-inducing effects of GW583340, H(2)O(2), and paraquat. The ROS increase and cell death in the GW583340-sensitive cells was reversed by simultaneous treatment with a superoxide dismutase (SOD) mimic. Additionally, overcoming the high levels of antioxidants using redox modulators induced apoptosis in the GW583340-resistant cells. Taken together, these data demonstrate a novel mechanism of lapatinib-analog-induced apoptosis and indicate that resistant cells have increased antioxidant potential, which can be overcome by treatment with SOD modulators.

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate.

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.