Arsenoplatin-1 Is a Dual Pharmacophore Anticancer Agent.

Arsenoplatins are adducts of two chemically important anticancer drugs, cisplatin and arsenic trioxide, that have a Pt(II) bond to an As(III) hydroxide center. Screens of the NCI-60 human tumor cell lines reveal that arsenoplatin-1 (AP-1), [Pt(µ-NHC(CH3)O)2ClAs(OH)2], the first representative of this novel class of anticancer agents, displays a superior activity profile relative to the parent drugs As2O3 or cisplatin in a majority of cancer cell lines tested. These activity profiles are important because the success of arsenic trioxide in blood cancers (such as APL) has not been seen in solid tumors due to the rapid clearance of arsenous acid from the body. To understand the biological chemistry of these compounds, we evaluated interactions of AP-1 with the two important classes of biomolecules-proteins and DNA. The first structural studies of AP-1 bound to model proteins reveal that platinum(II) binds the Nε of His in a manner that preserves the Pt-As bond. We find that AP-1 readily enters cells and binds to DNA with an intact Pt-As bond (Pt:As ratio of 1). At longer incubation times, however, the Pt:As ratio in DNA samples increases, suggesting that the Pt-As bond breaks and releases the As(OH)2 moiety. We conclude that arsenoplatin-1 has the potential to deliver both Pt and As species to a variety of hematological and solid cancers.

Beyond Cisplatin: Combination Therapy with Arsenic Trioxide.

Platinum drugs (cisplatin, oxaliplatin, and carboplatin) and arsenic trioxide are the only commercial inorganic non-radioactive anticancer drugs approved by the US Food and Drug Administration. Numerous efforts are underway to take advantage of the synergy between the anticancer activity of cisplatin and arsenic trioxide - two drugs with strikingly different mechanisms of action. These include co-encapsulation of the two drugs in novel nanoscale delivery systems as well as the development of small molecule agents that combine the activity of these two inorganic materials. Several of these new molecular entities containing Pt-As bonds have broad anticancer activity, are robust in physiological buffer solutions, and form stable complexes with biopolymers. This review summarizes results from a number of preclinical studies involving the combination of cisplatin and As2O3, co-encapsulation and nanoformulation efforts, and the chemistry and cytotoxicity of the first member of platinum anticancer agents with an arsenous acid moiety bound to the platinum(II) center: arsenoplatins.

Radiation-enhanced therapeutic targeting of galectin-1 enriched malignant stroma in triple negative breast cancer.

Currently there are no FDA approved targeted therapies for Triple Negative Breast Cancer (TNBC). Ongoing clinical trials for TNBC have focused primarily on targeting the epithelial cancer cells. However, targeted delivery of cytotoxic payloads to the non-transformed tumor associated-endothelium can prove to be an alternate approach that is currently unexplored. The present study is supported by recent findings on elevated expression of stromal galectin-1 in clinical samples of TNBC and our ongoing findings on stromal targeting of radiation induced galectin-1 by the anginex-conjugated arsenic-cisplatin loaded liposomes using a novel murine tumor model. We demonstrate inhibition of tumor growth and metastasis in response to the multimodal nanotherapeutic strategy using a TNBC model with orthotopic tumors originating from 3D tumor tissue analogs (TTA) comprised of tumor cells, endothelial cells and fibroblasts. The 'rigorous' combined treatment regimen of radiation and targeted liposomes is also shown to be well tolerated. More importantly, the results presented provide a means to exploit clinically relevant radiation dose for concurrent receptor mediated enhanced delivery of chemotherapy while limiting overall toxicity. The proposed study is significant as it falls in line with developing combinatorial therapeutic approaches for stroma-directed tumor targeting using tumor models that have an appropriate representation of the TNBC microenvironment.

3D tumor tissue analogs and their orthotopic implants for understanding tumor-targeting of microenvironment-responsive nanosized chemotherapy and radiation.

An appropriate representation of the tumor microenvironment in tumor models can have a pronounced impact on directing combinatorial treatment strategies and cancer nanotherapeutics. The present study develops a novel 3D co-culture spheroid model (3D TNBC) incorporating tumor cells, endothelial cells and fibroblasts as color-coded murine tumor tissue analogs (TTA) to better represent the tumor milieu of triple negative breast cancer in vitro. Implantation of TTA orthotopically in nude mice, resulted in enhanced growth and aggressive metastasis to ectopic sites. Subsequently, the utility of the model is demonstrated for preferential targeting of irradiated tumor endothelial cells via radiation-induced stromal enrichment of galectin-1 using anginex conjugated nanoparticles (nanobins) carrying arsenic trioxide and cisplatin. Demonstration of a multimodal nanotherapeutic system and inclusion of the biological response to radiation using an in vitro/in vivo tumor model incorporating characteristics of tumor microenvironment presents an advance in preclinical evaluation of existing and novel cancer nanotherapies. FROM THE CLINICAL EDITOR: Existing in-vivo tumor models are established by implanting tumor cells into nude mice. Here, the authors described their approach 3D spheres containing tumor cells, enodothelial cells and fibroblasts. This would mimic tumor micro-environment more realistically. This interesting 3D model should reflect more accurately tumor response to various drugs and would enable the design of new treatment modalities.

Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with As2O3 will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses.

Structural and mechanistic basis of zinc regulation across the E. coli Zur regulon.

Commensal microbes, whether they are beneficial or pathogenic, are sensitive to host processes that starve or swamp the prokaryote with large fluctuations in local zinc concentration. To understand how microorganisms coordinate a dynamic response to changes in zinc availability at the molecular level, we evaluated the molecular mechanism of the zinc-sensing zinc uptake regulator (Zur) protein at each of the known Zur-regulated genes in Escherichia coli. We solved the structure of zinc-loaded Zur bound to the P(znuABC) promoter and show that this metalloregulatory protein represses gene expression by a highly cooperative binding of two adjacent dimers to essentially encircle the core element of each of the Zur-regulated promoters. Cooperativity in these protein-DNA interactions requires a pair of asymmetric salt bridges between Arg52 and Asp49' that connect otherwise independent dimers. Analysis of the protein-DNA interface led to the discovery of a new member of the Zur-regulon: pliG. We demonstrate this gene is directly regulated by Zur in a zinc responsive manner. The pliG promoter forms stable complexes with either one or two Zur dimers with significantly less protein-DNA cooperativity than observed at other Zur regulon promoters. Comparison of the in vitro Zur-DNA binding affinity at each of four Zur-regulon promoters reveals ca. 10,000-fold variation Zur-DNA binding constants. The degree of Zur repression observed in vivo by comparison of transcript copy number in wild-type and Δzur strains parallels this trend spanning a 100-fold difference. We conclude that the number of ferric uptake regulator (Fur)-family dimers that bind within any given promoter varies significantly and that the thermodynamic profile of the Zur-DNA interactions directly correlates with the physiological response at different promoters.

Anticancer activity of small-molecule and nanoparticulate arsenic(III) complexes.

Starting in ancient China and Greece, arsenic-containing compounds have been used in the treatment of disease for over 3000 years. They were used for a variety of diseases in the 20th century, including parasitic and sexually transmitted illnesses. A resurgence of interest in the therapeutic application of arsenicals has been driven by the discovery that low doses of a 1% aqueous solution of arsenic trioxide (i.e., arsenous acid) lead to complete remission of certain types of leukemia. Since Food and Drug Administration (FDA) approval of arsenic trioxide (As2O3) for treatment of acute promyelocytic leukemia in 2000, it has become a front-line therapy in this indication. There are currently over 100 active clinical trials involving inorganic arsenic or organoarsenic compounds registered with the FDA for the treatment of cancers. New generations of inorganic and organometallic arsenic compounds with enhanced activity or targeted cytotoxicity are being developed to overcome some of the shortcomings of arsenic therapeutics, namely, short plasma half-lives and a narrow therapeutic window.

Urokinase plasminogen activator system-targeted delivery of nanobins as a novel ovarian cancer therapy.

The urokinase system is overexpressed in epithelial ovarian cancer cells and is expressed at low levels in normal cells. To develop a platform for intracellular and targeted delivery of therapeutics in ovarian cancer, we conjugated urokinase plasminogen activator (uPA) antibodies to liposomal nanobins. The arsenic trioxide-loaded nanobins had favorable physicochemical properties and the ability to bind specifically to uPA. Confocal microscopy showed that the uPA-targeted nanobins were internalized by ovarian cancer cells, whereas both inductively coupled plasma optical mass spectrometry (ICP-MS) and fluorescence-activated cell sorting (FACS) analyses confirmed more than four-fold higher uptake of targeted nanobins when compared with untargeted nanobins. In a coculture assay, the targeted nanobins showed efficient uptake in ovarian cancer cells but not in the normal primary omental mesothelial cells. Moreover, this uptake could be blocked by either downregulating uPA receptor expression in the ovarian cancer cells using short-hairpin RNA (shRNA) or by competition with free uPA or uPA antibody. In proof-of-concept experiments, mice bearing orthotopic ovarian tumors showed a greater reduction in tumor burden when treated with targeted nanobins than with untargeted nanobins (47% vs. 27%; P < 0.001). The targeted nanobins more effectively inhibited tumor cell growth both in vitro and in vivo compared with untargeted nanobins, inducing caspase-mediated apoptosis and impairing stem cell marker, aldehyde dehydrogenase-1A1 (ALDH1A1), expression. Ex vivo fluorescence imaging of tumors and organs corroborated these results, showing preferential localization of the targeted nanobins to the tumor. These findings suggest that uPA-targeted nanobins capable of specifically and efficiently delivering payloads to cancer cells could serve as the foundation for a new targeted cancer therapy using protease receptors.

pH-Responsive Theranostic Polymer-Caged Nanobins (PCNs): Enhanced Cytotoxicity and T1 MRI Contrast by Her2-Targeting.

A PCN theranostic platform comprises a doxorubicin (DXR)-loaded liposomal core and an acid-sensitive polymer shell that is functionalized with Herceptin and GdIII-based MRI contrast agents. In vitro testing reveals a 14-fold increase in DXR-based cytotoxicity versus a non-targeted analogue and an 120-fold improvement in cellular GdIII-uptake in comparison with clinically approved DOTA-GdIII, leading to significant T1 MRI contrast enhancement.

Improved anti-proliferative effect of doxorubicin-containing polymer nanoparticles upon surface modification with cationic groups.

Polymer nanoparticles (PNPs) possessing a high density of drug payload have been successfully stabilized against aggregation in biological buffers after amine modification, which renders these PNPs positively charged. The resulting charge-stabilized PNPs retain their original narrow particle size distributions and well-defined spherical morphologies. This stabilization allows these PNPs to have an improved anti-proliferative effect on MDA-MB-231-Br human breast cancer cells compared to non-functionalized PNPs. As a non-cytotoxic control, similar surface-modified PNPs containing cholesterol in place of doxorubicin did not inhibit cell proliferation, indicating that the induced cytotoxic response was solely due to the doxorubicin release from the PNPs.

A novel nanoparticulate formulation of arsenic trioxide with enhanced therapeutic efficacy in a murine model of breast cancer.

PURPOSE: The clinical success of arsenic trioxide (As(2)O(3)) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As(2)O(3) encapsulated in liposomal vesicles or "nanobins" [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As(2)O(3) would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. EXPERIMENTAL DESIGN: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As(2)O(3) was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As(2)O(3) were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. RESULTS: The NB(Ni,As) agent was much less cytotoxic in vitro than free As(2)O(3) against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As(2)O(3) in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). CONCLUSIONS: Nanobin encapsulation of As(2)O(3) improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors.