Synergistic Induction of Apoptosis in Primary B-CLL Cells after Treatment with Recombinant Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand and Histone Deacetylase Inhibitors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently being investigated as a therapeutic agent for a variety of malignancies, as it triggers apoptosis specifically in transformed cells. However, TRAIL use as a stand alone therapeutic is hampered by the fact that many primary tumor cells are resistant to TRAIL-mediated apoptosis. Here, we investigated the extent to which pretreatment of TRAIL-resistant primary B-cell chronic lymphocytic leukemia (B-CLL) cells with histone deacetylase inhibitors (HDACis) could render them susceptible to killing by TRAIL. We found that HDAC inhibition in B-CLL cells led to increased TRAIL receptor expression, increased caspase activation, decreased expression of antiapoptotic regulators such as Bcl-2, and ultimately, enhanced TRAIL-induced apoptosis. Importantly, untransformed peripheral blood mononuclear cells remained largely resistant to TRAIL, even in the presence of HDACis. These results suggest that combination therapies using HDAC inhibition and TRAIL could prove beneficial for the treatment of B-CLL

BKCa channel inhibitor modulates the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway

In breast cancers, the large conductance Ca(2+) and voltage sensitive K(+) (BKCa) channels have been hypothesized to function as oncoproteins, yet it remains unclear how inhibition of channel activity impacts oncogenesis. We demonstrated herein that iberiotoxin (IbTX), an inhibitor of BKCa channels, differentially modulated the in vitro tumorigenic activities of hormone-independent breast cancer cells. Specifically, in HER-2/neu-overexpressing UACC893 cells and triple-negative MDA-MB-231 cells, IbTX selectively attenuated anchorage-independent growth with concomitant downregulation of β-catenin as well as total and phosphorylated Akt and HER-2/neu. By contrast, HER-2/neu-overexpressing SK-BR-3 cells were insensitive to IbTX. Molecular analyses showed an absence of β-catenin and a dose-dependent upregulation of total and phosphorylated Akt and HER-2/neu in these cells. Taken together, these studies identify β-catenin as a putative modulator of the inhibitory actions of IbTX in sensitive breast cancer cells

Eradication of Metastatic Renal Cell Carcinoma after Adenovirus-Encoded TNF-Related Apoptosis-Inducing Ligand (TRAIL)/CpG Immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC

An Antibiotic-Responsive Mouse Model of Fulminant Ulcerative Colitis

Paul Allen and colleagues describe the development of a mouse model of fulminant ulcerative colitis with multiple genetic hits in immune regulation which can be moderated by anti-cytokine therapy and broad-spectrum antibiotics

Advances in Viral Vector-Based TRAIL Gene Therapy for Cancer

Numerous biologic approaches are being investigated as anti-cancer therapies in an attempt to induce tumor regression while circumventing the toxic side effects associated with standard chemo- or radiotherapies. Among these, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has shown particular promise in pre-clinical and early clinical trials, due to its preferential ability to induce apoptotic cell death in cancer cells and its minimal toxicity. One limitation of TRAIL use is the fact that many tumor types display an inherent resistance to TRAIL-induced apoptosis. To circumvent this problem, researchers have explored a number of strategies to optimize TRAIL delivery and to improve its efficacy via co-administration with other anti-cancer agents. In this review, we will focus on TRAIL-based gene therapy approaches for the treatment of malignancies. We will discuss the main viral vectors that are being used for TRAIL gene therapy and the strategies that are currently being attempted to improve the efficacy of TRAIL as an anti-cancer therapeutic

T cell cytolytic activity in peripheral lymphoid organs and tumors.

<p>DUC18 T cells were transferred as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000821#pone-0000821-g001" target="_blank">Figure 1</a>. A) On day 4, 5×10⁶ each TAMRA⁺ control cells and CFSE⁺ targets were injected i.v. Spleens and dLNs were harvested and analyzed on day 5. Percentages of cells within the indicated gates are shown. Data are representative of 3 experiments. B) CMS5 cells were injected s.c. and allowed to grow for 8 days. At this time, 3×10⁶ each TAMRA⁺ Meth A reference cells and CFSE⁺ CMS5 targets were injected i.t. Tumors were harvested and analyzed on day 9; the percentages of TAMRA⁺ and CFSE⁺ cells in individual tumors were determined by flow cytometry. C) The mean ratio +/− SEM of CFSE⁺ CMS5 to TAMRA⁺ Meth A cells was calculated using data from 13 individual mice. D) Tumor areas were measured on day 8 after CMS5 challenge. Four days later, fluorescently labeled tumor cells were injected as in B. Tumor sizes were measured just prior to i.t. injections and again the following day. Data represent the means +/− s.d. for 12 tumors from 4 independent experiments. No statistical difference is present in the size of control versus FL+ tumors at day 13 (p = 0.11) or in the change in control versus FL+ tumor sizes from day 12 to day 13 (p = 0.40). E) DUC18 T cells were transferred into tumor-bearing mice as in A. On day 4 post-T cell transfer, fluorescently labeled tumor cells were injected as in B. Tumors were harvested and analyzed on day 5. Percentages of cells within the indicated gates are shown. Data from 1 experiment are shown; representative of 10.</p

DUC18 CTL mediate regression of CMS5 tumors.

<p>3×10⁶ CMS5 tumor cells were injected s.c. and were allowed to grow for 8 days prior to transfer of 30×10⁶ in vitro activated DUC18 T cells. This transfer protocol was repeated for all subsequent experiments. A) Tumor sizes for 6 DUC18 T cell⁺ recipient and 4 control mice are shown. Data are representative of 3 experiments. B) Mean tumor sizes +/− SEM for 30 DUC18 T cell⁺ recipient mice.</p

An initial lag phase in cytolytic activity precedes robust T cell mediated killing within tumors.

<p>A) ITC assays were performed beginning on days 2, 4, or 6. For tumors, each point represents the mean percent lysis, derived from 2–3 individual mice, for one experiment. Results from 17 experiments are shown, with overall mean values indicated by bars. For dLN and spleen data, each point represents killing from one mouse, with overall means indicated by bars. B) Thy1.1⁺ DUC18 T cells were transferred on day 0, and organs harvested and analyzed on indicated days. Numbers of live Thy1.1 DUC18 T cells are plotted (for tumors, p values for day 2 versus day 4 = .03, for day 2 versus day 6 = .05; for dLNs, p = 0.03 for day 2 versus day 4, and p = 0.04 for day 2 versus day 6). Points represent individual mice (n = 13–20) from 3 experiments. C) Normalized killing values were calculated by dividing the mean % specific killing by the mean number of live DUC18 T cells present in tumors for each time point. D) Activated DUC18 T cells were transferred into tumor bearing mice on day 0. Organs were harvested on days 2 and 6, and the percentage of cells expressing IFNγ ex vivo was determined by intracellular cytokine staining, after gating on Thy1.1⁺ Vβ8.3⁺ DUC18 T cells. E) Linear regression analyses for % specific killing in tumors versus % Meth A reference cells present for all individual mice used in ITC assays shown in A. The slopes were not statistically different from 0; day 2 p = 0.83, day 4 p = .90, day 6 p = .16).</p