LT-IIc, A Bacterial Type II Heat-Labile Enterotoxin, Induces Specific Lethality in Triple Negative Breast Cancer Cells by Modulation of Autophagy and Induction of Apoptosis and Necroptosis.

Triple negative breast cancer (TNBC) remains a serious health problem with poor prognosis and limited therapeutic options. To discover novel approaches to treat TNBC, we screened cholera toxin (CT) and the members of the bacterial type II heat-labile enterotoxin family (LT-IIa, LT-IIb, and LT-IIc) for cytotoxicity in TNBC cells. Only LT-IIc significantly reduced viability of the TNBC cell lines BT549 and MDA-MB-231 (IC50 = 82.32 nM). LT-IIc had no significant cytotoxic effect on MCF10A (IC50 = 2600 nM), a non-tumorigenic breast epithelial cell line, and minimal effects on MCF7 and T47D, ER⁺ cells, or SKBR-3 cells, HER2⁺ cells. LT-IIc stimulated autophagy through inhibition of the mTOR pathway, while simultaneously inhibiting autophagic progression, as seen by accumulation of LC3B-II and p62. Morphologically, LT-IIc induced the formation of enlarged LAMP2+ autolysosomes, which was blocked by co-treatment with bafilomycin A1. LT-IIc induced apoptosis as demonstrated by the increase in caspase 3/7 activity and Annexin V staining. Co-treatment with necrostatin-1, however, demonstrated that the lethal response of LT-IIc is elicited, in part, by concomitant induction of necroptosis. Knockdown of ATG-5 failed to rescue LT-IIc-induced cytotoxicity, suggesting LT-IIc can exert its cytotoxic effects downstream or independently of autophagophore initiation. Collectively, these experiments demonstrate that LT-IIc acts bifunctionally, inducing autophagy, while simultaneously blocking autolysosomal progression in TNBC cells, inducing a specific cytotoxicity in this breast cancer subtype.

Targeting the efficacy of a dendrimer-based nanotherapeutic in heterogeneous xenograft tumors in vivo.

Our earlier studies have shown the in vitro and in vivo targeting of a generation 5 (G5) dendrimer-based multifunctional conjugate that contained folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. To clinically apply the synthesized G5-FA-MTX nanotherapeutic, it is important that the anticancer conjugate elicits cytotoxicity specifically and consistently. Toward this objective, we evaluated the large-scale synthesis of a G5-FA-MTX conjugate (Lot # 123-34) for its cytotoxic potential and specificity in vitro and in vivo. The cytotoxicity and specificity were tested by using a coculture assay in which FA receptor-expressing and nonexpressing cells (KB and SK-BR-3 cells, respectively) were cultured together and preferential killing was examined. The in-vitro data were compared with the in-vivo data obtained from a heterogeneous xenograft tumor model. The animal model of the artificial heterogeneous xenograft tumor showed that the nanotherapeutic was preferentially cytotoxic to KB cells.

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe.

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.

Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion.

Nanoemulsion, a water-in-oil formulation stabilized by small amounts of surfactant, is non-toxic to mucous membranes and produces biocidal activity against enveloped viruses. We evaluated nanoemulsion as an adjuvant for mucosal influenza vaccines. Mice (C3H/HeNHsd strain) were vaccinated intranasally with 5 x 10(5) plaque forming units (pfu) of influenza A virus (Ann Arbor/6/60 strain) and a nanoemulsion mixture. The mice were challenged on day 21 after immunization with an intranasal lethal dose of 2 x 10(5) pfu of virus. Animals vaccinated with the influenza A/nanoemulsion mixture were completely protected against infection, while animals vaccinated with either formaldehyde-killed virus or nanoemulsion alone developed viral pneumonitis and died by day 6 after the challenge. Mice vaccinated with virus/nanoemulsion mixture had rapid cytokine responses followed by high levels of specific anti-influenza immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies. Specificity of the immune response was confirmed by assessment of the proliferation and cytokine production in splenocytes. This paper demonstrates that nanoemulsion can be employed as a non-toxic mucosal adjuvant for influenza virus vaccine.