GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by insulitis and islet ß-cell loss. Thus, an effective therapy may require ß-cell restoration and immune suppression. Currently, there is no treatment that can achieve both goals efficiently. We report here that GABA exerts antidiabetic effects by acting on both the islet ß-cells and immune system. Unlike in adult brain or islet α-cells in which GABA exerts hyperpolarizing effects, in islet ß-cells, GABA produces membrane depolarization and Ca(2+) influx, leading to the activation of PI3-K/Akt-dependent growth and survival pathways. This provides a potential mechanism underlying our in vivo findings that GABA therapy preserves ß-cell mass and prevents the development of T1D. Remarkably, in severely diabetic mice, GABA restores ß-cell mass and reverses the disease. Furthermore, GABA suppresses insulitis and systemic inflammatory cytokine production. The ß-cell regenerative and immunoinhibitory effects of GABA provide insights into the role of GABA in regulating islet cell function and glucose homeostasis, which may find clinical application.

Impaired negative regulation of homeostatically proliferating T cells.

Acute lymphopenia-induced homeostatic proliferation (HP) of T cells promotes antitumor immunity, but the mechanism is unclear. We hypothesized that this is due to a lack of inhibitory signals that allows activation of T cells with low affinity for self-antigens. Tumors resist immunity in part by expressing inhibitory molecules such as PD-1 ligand 1 (PD-L1), B7-H4, and TGF-beta. In irradiated mice undergoing HP, we found that T cells displayed a severe deficit in the activation-induced expression of inhibitory molecules PD-1 and CTLA-4, and TGF-beta1-induced expression of Foxp3. HP T cells were also less suppressed by B7-H4/Ig and, unlike control T cells, failed to produce IL-10 in response to this molecule. This deficiency in regulation was reversed as normal T-cell numbers were restored. We conclude that T cells are weakly regulated by inhibitory molecules during the acute phase of HP, which could explain their increased effectiveness in cancer immunotherapy.

Tranilast inhibits cell proliferation and migration and promotes apoptosis in murine breast cancer.

The malignant transformation of breast epithelium involves a number of cellular pathways, including those dependent on signaling from TGF beta. Tranilast [N-(3, 4-dimethoxycinnamonyl)-anthranilic acid] is a drug that is used in Japan to control allergic disorders in patients, and its mechanism of action involves TGF beta. In view of the multiple roles of TGF beta in tumor progression, we hypothesized in this study that tranilast impacts cell proliferation, apoptosis, and migration. Using the mouse breast cancer cell line 4T1, our studies showed that tranilast increases AKT1 phosphorylation and decreases ERK1/2 phosphorylation. Alterations in the cell cycle mediators' cyclin D1, p27, cyclin A, pRB, cyclin B, and Cdc2 were observed after exposure to tranilast, favoring cell arrest beyond the G1/S phase. Tranilast reduced tumor cell proliferation even when it was amplified by exogenous TGF beta. TGF beta-neutralizing antibody did not cause a significant decrease in cell proliferation. Tranilast treatment upregulates p53, induces PARP cleavage in vitro, consistent with a promotion of tumor cell apoptosis. TGF beta-neutralizing antibody downregulates endoglin and matrix metalloproteinases (MMP)-9 levels in vitro indicating that the tranilast effect is mediated through TGF beta modulation. Tranilast treatment results in the inhibition of cell migration and invasion. Western blot analysis of tumor lysates from tranilast-treated mice shows decreased levels of TGF beta1, endoglin, and significantly higher levels of p53 and cleaved PARP. Cleaved caspase 3 expression is significantly elevated in tranilast-treated mouse breast tumors. To conclude, tranilast induces cellular and molecular changes in murine breast cancer that can be exploited in preclinical therapeutic trials.

Tranilast inhibits the growth and metastasis of mammary carcinoma.

Tranilast (N-[3,4-dimethoxycinnamonyl]-anthranilic acid) is a drug of low toxicity that is orally administered, and has been used clinically in Japan as an antiallergic and antifibrotic agent. Its antifibrotic effect is thought to depend on the inhibition of transforming growth factor-beta (TGF-beta). It has also been shown to exert antitumor effects, but its mode of action is unclear. Here, we explored the antitumor effects of tranilast in vitro and in vivo. Tranilast inhibited the proliferation of several tumor cell lines including mouse mammary carcinoma (4T1), rat mammary carcinoma stem cell (LA7), and human breast carcinoma (MDA-MB-231 and MCF-7). Tranilast blocked cell-cycle progression in vitro. In the highly metastatic 4T1 cell line, tranilast inhibited phospho-Smad2 generation, consistent with a blockade of TGF-beta signaling. It also inhibited the activation of MAP kinases (extracellularly regulated kinase 1 and 2 and JNK), which have been linked to TGF-beta-dependent epithelial-to-mesenchymal transition and, indeed, it blocked epithelial-to-mesenchymal transition. Although tranilast only partially inhibited TGF-beta production by 4T1 tumor cells, it potently inhibited the production of TGF-beta, interferon-gamma, IL-6, IL-10, and IL-17 by lymphoid cells, suggesting a general anti-inflammatory activity. In vivo, female BALB/c mice were inoculated with syngeneic 4T1 cells in mammary fat pads and treated with tranilast by gavage. Tranilast reduced (>50%) the growth of the primary tumor. However, its effects on metastasis were more striking, with more than 90% reduction of metastases in the lungs and no metastasis in the liver. Thus, tranilast has potential activity as an antimetastatic agent in breast cancer.

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels.

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).

A mutant B7-1/Ig fusion protein that selectively binds to CTLA-4 ameliorates anti-tumor DNA vaccination and counters regulatory T cell activity.

We have shown that a plasmid encoding a B7-1/Ig fusion protein enhanced DNA vaccination against human carcinoembryonic antigen (CEA) more effectively than the plasmid encoding membrane-bound B7-1. However, it was not known if B7-1/Ig acted only by binding CD28 (amplifying a stimulatory signal) or by blocking CTLA-4 on T cells (removing inhibitory signals). Here, we aimed to determine this using a plasmid encoding mutant B7-1/Ig (B7-1wa/Ig), which binds only to CTLA-4 but not to CD28. Our results showed that both the B7-1/Ig and B7-1wa/Ig plasmids, when co-administered with a CEA plasmid, enhanced tumor rejection and the in vitro anti-CEA response. Therefore, B7-1wa/Ig ameliorates DNA vaccination, presumably by binding to CTLA-4. This could result from a number of non-exclusive mechanisms, such as a reduced threshold for T-cell activation, or blockade of CTLA-4/B7-mediated tolerogenic signals in DCs or T cells. We found that, in vitro, a significant fraction of CD3/CD28-activated T cells (in the absence of DCs) expressed CTLA-4 and B7-1. Primed T cells of CTLA-4(+)B7-1(+/-) phenotype acted as regulatory T cells by inhibiting IFNgamma production by re-stimulated CTLA-4(-)B7-1(-) cells, and this was reversed by antibodies against IL-10 or TGF-beta1. Both B7-1wa/Ig and CTLA-4/Ig, which bind to CTLA-4 and B7-1/B7-2 respectively, enhanced IFNgamma production, but not the proliferation or IL-4 release in mixed T-cell populations containing these two cell types. In contrast, CTLA-4(-)B7-1(-) T cells produced IFNgamma which was not affected by B7-1wa/Ig or CTLA-4/Ig. These results suggest that blocking of CTLA-4/B7-1 binding in T cell/T cell interactions blocks negative regulatory signals. This might be the mechanism, at least in part, of the enhancement of anti-tumor immunity by the B7-1wa/Ig and B7-1/Ig plasmids.

Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulate DNA vaccination against carcinoembryonic antigen (CEA).

Vaccination with plasmids encoding an antigen of interest (DNA vaccination) is a new strategy to achieve effective immunization against many agents. DNA vaccination can be ameliorated by co-administration of plasmids encoding a cytokine. Thus far, only plasmids encoding soluble cytokines have been used for this purpose. However, these plasmids can induce release of cytokines into the circulation and could potentially cause many undesirable effects. We undertook this study to determine whether membrane-bound cytokines, which would restrict their localization at the site of administration, can act as immunoadjuvants. We and others have previously shown that plasmids encoding soluble IL-4 and IL-12 are effective adjuvants for DNA vaccination. In this study, we demonstrate that DNA co-vaccination with membrane-bound IL-4 (mbIL-4) or membrane-bound IL-12 (mbIL-12) both enhance anti-CEA immunity, as detected by in vitro and in vivo assays. Mice co-injected with plasmids encoding CEA and either type of membrane-bound cytokine rejected transplanted CEA-positive tumor cells strongly. Notably, unlike secreted IL-4, mbIL-4 was the most effective adjuvant for anti-tumor immunity. This study demonstrates that membrane-bound cytokines are suitable adjuvants for DNA vaccination.