The role of tomatine adjuvant in antigen delivery for cross- presentation.

The aim of this study was to investigate the use of tomatine adjuvant to deliver soluble antigen for crosspresentation by bone marrow-derived dendritic cells (BMDCs). BMDCs were incubated with tomatine adjuvantovalbumin (OVA) complex and analyzed for antigen uptake by flow cytometry. Adjuvant-induced cell death was examined in situ by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. To elucidate the effect of antigen internalization on tomatine adjuvant-mediated antigen presentation, BMDCs were treated with several endocytosis inhibitors, and antigen presentation was analyzed by B3Z activity assay. Our data indicated that tomatine adjuvant enhanced antigen internalization by antigen presenting cells (APCs) and induced significant cell death and leukocyte infiltration at the injection sites. In vitro tomatine adjuvant treatment of BMDCs activated Ova/K(b) restricted B3Z T cell hybridomas, whereas this activation was impaired by pretreatment with brefeldin A, cytochalasin B, wortmannin, or ZnCl2. Our results demonstrated the role of tomatine adjuvant in antigen delivery to antigen presenting cells (APCs) and suggested the involvement of phagocytosis and PI3K signaling during the delivery of soluble antigens in the context of MHC class I.

Anticarcinogenic, cardioprotective, and other health benefits of tomato compounds lycopene, α-tomatine, and tomatidine in pure form and in fresh and processed tomatoes.

Tomatoes produce the bioactive compounds lycopene and α-tomatine that are reported to have potential health-promoting effects in animals and humans, but our understanding of the roles of these compounds in the diet is incomplete. Our current knowledge gained from the chemistry and analysis of these compounds in fresh and processed tomatoes and from studies on their bioavailability, bioactivity, and mechanisms of action against cancer cells and other beneficial bioactivities including antibiotic, anti-inflammatory, antioxidative, cardiovascular, and immunostimulating effects in cells, animals, and humans is discussed and interpreted here. Areas for future research are also suggested. The collated information and suggested research might contribute to a better understanding of the agronomical, biochemical, chemical, physiological, molecular, and cellular bases of the health-promoting effects and facilitate and guide further studies needed to optimize the use of lycopene and α-tomatine in pure form and in fresh tomatoes and processed tomato products to help prevent or treat human disease.

The cytotoxic effect of α-tomatine in MCF-7 human adenocarcinoma breast cancer cells depends on its interaction with cholesterol in incubation media and does not involve apoptosis induction.

In recent years, α-tomatine has been studied for its anticancer activity. In the present study, we focused on the cytotoxic effect of α-tomatine in the MCF-7 human breast adenocarcinoma cell line, its mechanism of action, biotransformation and stability in the culture medium. We observed an inhibition of cell proliferation and viability at concentrations of 6 and 9 µM but then a recovery of cells occurred. The recovery was not caused by the biotransformation of α-tomatine in MCF-7 cells, but by a substantial decrease in the concentration of α-tomatine in the culture medium due to its binding with cholesterol. Regarding the mechanism of action of α-tomatine, we observed no DNA damage, no changes in the levels of the proteins p53 and p21(WAF1/Cip1), and no apoptosis (neither activated caspase-8 and -9, nor sub-G1 peak, or morphological signs). We found a loss of ATP in α-tomatine-treated cells. These results support the conclusion that α-tomatine does not induce apoptosis in the MCF-7 cell line.

The anticancer activity of alpha-tomatine against mammary adenocarcinoma in mice.

AIM: To evaluate the anticancer effect of alpha-tomatine (i.p.) either alone or in combination with doxorubicin (i.v.) in a mouse tumour model. METHODS: We studied the effect of repeated alpha-tomatine (0.1 - 9 mg/kg) and/or doxorubicin (2 mg/kg) on the growth and mitotic activity of the solid Ehrlich tumour in vivo, as well as on the survival of the tumour-bearing mice. RESULTS: Monotherapy with alpha-tomatine had a significant dose-dependent anticancer effect which peaked at 1 mg/kg. This was shown by both slowed tumour growth and reduced tumour cell proliferation. We also provide the first evidence that the combination alpha-tomatine (1 mg/kg) and doxorubicin (2 mg/kg) had a synergistic effect and significantly prolonged the survival of the mice. Neither alpha-tomatine nor doxorubicin influenced the infiltration of tumours with CD3+ lymphocytes; nor were we able to find an in vivo modulation of the key molecules of two regulatory pathways reported in vitro as the principal anti-cancer mechanisms of alpha-tomatine, i.e. iNOS and phosphorylated ERK2. However, alpha-tomatine still led to intracellular DNA inhibition and protein synthesis in Ehrlich tumour cells in a short-term culture ex vivo with IC50 values of 8.7 and 6.6 µM. CONCLUSIONS: The results suggest that ΤΟΜ, especially in combination with doxorubicin, may be a promising agent for the treatment of malignant solid tumours. Despite growing knowledge of the mechanisms of ΤΟΜ action in cancer cells, most aspects remain unclear. Parallel organ toxicity, especially potential liver effects, requires careful attention when performing in vivo studies in the future.

Role of hedgehog signaling in malignant pleural mesothelioma.

PURPOSE: The aim of this study was to assess the activity of hedgehog signaling pathway in malignant pleural mesothelioma (MPM). EXPERIMENTAL DESIGN: The expression of hedgehog signaling components was assessed by quantitative PCR and in situ hybridization in 45 clinical samples. Primary MPM cultures were developed in serum-free condition in 3% oxygen and were used to investigate the effects of smoothened (SMO) inhibitors or GLI1 silencing on cell growth and hedgehog signaling. In vivo effects of SMO antagonists were determined in an MPM xenograft growing in nude mice. RESULTS: A significant increase in GLI1, sonic hedgehog, and human hedgehog interacting protein gene expression was observed in MPM tumors compared with nontumoral pleural tissue. SMO antagonists inhibited GLI1 expression and cell growth in sensitive primary cultures. This effect was mimicked by GLI1 silencing. Reduced survivin and YAP protein levels were also observed. Survivin protein levels were rescued by overexpression of GLI1 or constitutively active YAP1. Treatment of tumor-bearing mice with the SMO inhibitor HhAntag led to a significant inhibition of tumor growth in vivo accompanied by decreased Ki-67 and nuclear YAP immunostaining and a significant difference in selected gene expression profile in tumors. CONCLUSIONS: An aberrant hedgehog signaling is present in MPM, and inhibition of hedgehog signaling decreases tumor growth indicating potential new therapeutic approach.

Tomatidine, a tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting acyl-CoA:cholesterol acyl-transferase (ACAT).

It was previously revealed that esculeoside A, a new glycoalkaloid, and esculeogenin A, a new aglycon of esculeoside A, contained in ripe tomato ameliorate atherosclerosis in apoE-deficent mice. This study examined whether tomatidine, the aglycone of tomatine, which is a major tomato glycoalkaloid, also shows similar inhibitory effects on cholesterol ester (CE) accumulation in human monocyte-derived macrophages (HMDM) and atherogenesis in apoE-deficient mice. Tomatidine significantly inhibited the CE accumulation induced by acetylated LDL in HMDM in a dose-dependent manner. Tomatidine also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-CoA:cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that tomatidine suppresses both ACAT-1 and ACAT-2 activities. Furthermore, the oral administration of tomatidine to apoE-deficient mice significantly reduced levels of serum cholesterol, LDL-cholesterol, and areas of atherosclerotic lesions. The study provides the first evidence that tomatidine significantly suppresses the activity of ACAT and leads to reduction of atherogenesis.

Tomatine adjuvantation of protective immunity to a major pre-erythrocytic vaccine candidate of malaria is mediated via CD8+ T cell release of IFN-gamma.

The glycoalkaloid tomatine, derived from the wild tomato, can act as a powerful adjuvant to elicit an antigen-specific cell-mediated immune response to the circumsporozoite (CS) protein, a major pre-erythrocytic stage malaria vaccine candidate antigen. Using a defined MHC-class-I-restricted CS epitope in a Plasmodium berghei rodent model, antigen-specific cytotoxic T lymphocyte activity and IFN-gamma secretion ex vivo were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunized mice. Further, through lymphocyte depletion it is demonstrated that antigen-specific IFN-gamma is produced exclusively by the CD8(+) T cell subset. We conclude that the processing of the P. berghei CS peptide as an exogenous antigen and its presentation via MHC class I molecules to CD8(+) T cells leads to an immune response that is an in vitro correlate of protection against pre-erythrocytic malaria. Further characterization of tomatine as an adjuvant in malaria vaccine development is indicated.

Protective effect of dietary tomatine against dibenzo[a,l]pyrene (DBP)-induced liver and stomach tumors in rainbow trout.

The potential anti-carcinogenic effects of tomatine, a mixture of commercial tomato glycoalkaloids alpha-tomatine and dehydrotomatine (10:1), were examined in the rainbow trout chemoprevention model. Prior to the chemoprevention study, a preliminary toxicity study revealed that tomatine in the diet fed daily at doses from 100 to 2000 parts per million (ppm) for 4 weeks was not toxic to trout. For the tumor study, replicate groups of 105 trout were fed diets containing dibenzo[a,l]pyrene (DBP) alone (224 ppm), (N = 3), DBP plus tomatine at 2000 ppm (N = 2), tomatine alone (N = 2), or control diet (N = 2) for 4 weeks. The fish were then returned to control diet for 8 months and necropsied for histopathology. Dietary tomatine was found to reduce DBP-initiated liver tumor incidence from 37.0 to 19.0% and stomach tumor incidence from 46.4 to 29.4%. Tomatine also reduced stomach tumor multiplicity. The tomatine-containing diets did not induce mortality, change in fish weights, or liver weights. No adverse pathological effects in the tissues of the fish on the tomatine diets were observed. Dose-response and chemopreventive mechanisms for tomatine protection remain to be examined. This is the first report on the anticarcinogenic effects of tomatine in vivo.

Immunobiology of the Tomatine adjuvant.

Soluble or sub-unit protein vaccines alone are incapable of generating antigen-specific cellular immune responses. This failure can be attributed to the manner in which the immune system processes antigen; endogenous antigens are cycled through the MHC class I pathway to stimulate CD8+ restricted responses and exogenous antigens are processed through the MHC class II pathway to generate humoral immunity. Traditionally sub-unit vaccines have been formulated with adjuvants to enhance immunogenicity, however in the last decade a number of adjuvants have been developed that effectively stimulate the generation of both humoral and cellular immune responses, although the manner in which they exert their effects has not been investigated. Here we describe Tomatine, a glycoalkaloid based adjuvant, capable of stimulating potent antigen-specific humoral and cellular immune responses that contribute to protection against malaria, Francisella tularensis and regression of experimental tumors. Using in vivo models we investigated the manner in which cellular immune responses were generated by Tomatine. We established that Tomatine did not require either lymph node or splenic macrophages to generate cytotoxic T lymphocytes (CTL) and delivered soluble protein into a pathway not dependant on the machinery of the classical MHC class I pathway. We also observed that at the molecular level Tomatine required both CD80 and CD86 costimulation to engender antigen-specific cellular immunity.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen.

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8+ T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2kd class I-binding 9-mer peptide (amino acids 252-260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2kd) mice. Antigen-specific IFN-gamma secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8+ T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

Anti-inflammatory activity of the steroid alkaloid glycoside, toatine.

1. Tomatine, isolated from extracts of crown gall-infected tomato plants or obtained commercially, was tested for anti-inflammatory activity using three different methods.2. Tomatine administered to intact rats intramuscularly in a dose range of 1-10 mg/kg or orally in doses of 15-30 mg/kg exerted a significant dose dependent inhibition of carrageenan induced paw oedema. The inhibitory effect of tomatine when given in a dose of 10 mg/kg intramuscularly to intact rats lasted more than 24 hr.3. In adrenalectomized rats significant dose-related inhibition of paw oedema was obtained with tomatine and the inhibition at each dose level (0.5-10 mg/kg) was found to be greater than that found in intact animals.4. Tomatine administered subcutaneously to intact rats daily for 7 days in doses of 5 or 10 mg/kg exerted a significant, dose dependent inhibition of granulation tissue formation induced by the subcutaneous implantation of carrageenan impregnated cotton pellets.5. Tomatine administered to intact mice in a dose of 10 mg/kg subcutaneously 1 hr before the intraperitoneal injection of acidified saline and intravenous pontamine sky blue significantly decreased the leakage of the protein bound dye into the peritoneal cavity.6. Tomatidine, the aglycone of tomatine, was not effective at dose levels of 10-20 mg/kg in any of the three tests.