Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos.

ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities - providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. AIM OF THE STUDY: This study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: The toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1-10 µg/embryo (6.7-670 µg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. RESULTS: The cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2-37.7 µg/mL) while the hot-water extracts are non-toxic up to 670 µg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. CONCLUSIONS: The hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Aza-BODIPY-based polymeric nanoparticles for photothermal cancer therapy in a chicken egg tumor model.

A new push-pull aza-BODIPY (AZB-CF3) derivative comprised of dimethylamino groups and trifluoromethyl moieties was successfully synthesized. This derivative exhibited broad absorption in the near-infrared region in the range from 798 to 832 nm. It also exhibited significant near-infrared (NIR) signals in low-polar solvents with emission peaks around 835-940 nm, while non-fluorescence in high-polar environments due to the twisted intramolecular charge transfer (TICT) phenomenon. The nanoprecipitation of this compound with phospholipid-based polyethylene glycol (DSPE-PEG) yielded AZB-CF3@DSPE-PEG nanoparticles (NPs) with a hydrodynamic size of 70 nm. The NPs exhibited good photostability, colloidal stability, biocompatibility, and excellent photothermal (PTT) competence with a conversion efficiency (η) of 44.9%. These NPs were evaluated in vitro and in ovo in a 4T1 breast cancer cell line for NIR light-trigger photothermal therapy. Proven in the chicken egg tumor model, AZB-CF3@DSPE-PEG NPs induced severe vascular damage (∼40% vascular destruction), showed great anticancer efficacy (∼75% tumor growth inhibition), and effectively inhibited distant metastasis via photothermal treatment. As such, this PTT-based nanocarrier system could be a potential candidate for a clinical cancer therapy approach.

Aza-BODIPY based carbonic anhydrase IX: Strategy to overcome hypoxia limitation in photodynamic therapy.

Hypoxia caused by photodynamic therapy (PDT) is a major hurdle to cancer treatment since it can promote recurrence and progression by activating angiogenic factors, lowering therapeutic efficacy dramatically. In this work, AZB-I-CAIX2 was developed as a carbonic anhydrase IX (CAIX)-targeting NIR photosensitizer that can overcome the challenge by utilizing a combination of CAIX knockdown and PDT. AZB-I-CAIX2 showed a specific affinity to CAIX-expressed cancer cells and enhanced photocytotoxicity compared to AZB-I-control (the molecule without acetazolamide). Moreover, selective detection and effective cell cytotoxicity of AZB-I-CAIX2 by PDT in hypoxic CAIX-expressed murine cancer cells were achieved. Essentially, AZB-I-CAIX2 could minimize tumor size in the tumor-bearing mice compared to that in the control groups. The results suggested that AZB-I-CAIX2 can improve therapeutic efficiency by preventing PDT-induced hypoxia through CAIX inhibition.

Hepatoprotective potential of a novel quinazoline derivative in thioacetamide-induced liver toxicity.

Purpose: The compound quinazoline Q-Br, 3-(5-bromo-2-hydroxybenzylideneamino)-2-(5-bromo-2 hydroxyphenyl) 2,3-dihydroquinazoline-4(1H)-one (Q-Br) was evaluated for its antioxidant capacity and potential hepatoprotectivity against sub-chronic liver toxicity induced by thioacetamide in rats. Materials and Methods: Rats were assigned into five groups; healthy (normal) and cirrhosis control groups were given 5% Tween 20 orally, the reference control group was given a Silymarin dose of 50 mg/kg, and low-dose Q-Br and high-dose Q-Br groups were given a daily dose of 25 mg/kg and 50 mg/g Q-Br, respectively. Liver status was detected via fluorescence imaging with intravenous injection of indocyanine green (ICG) and a plasma ICG clearance test. Liver malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were also tested. The degree of fibrosis was determined histologically by hematoxylin and eosin and Masson's Trichrome staining. The immunohistochemistry of liver tissue inhibitor of metalloproteinase (TIMP-1), matrix metalloproteinase (MMP-2), and alpha-smooth muscle actin (α-SMA) was performed. Results: Q-Br recorded mild antioxidant capacity, dose-dependent improvement in the liver status, and inhibition of oxidative stress compared to cirrhosis control. Histopathology notified a remarkable reduction in the degree of fibrosis. Immunohistochemistry revealed an obvious low expression of MMP-2 and α-SMA along with a higher expression of TIMP-1 in Q-Br- and Silymarin-treated livers. Conclusion: Q-Br treatment altered the course of toxicity induced by thioacetamide suggesting significant hepatoprotective potential of Q-Br treatment.

PEGylated Aza-BODIPY Nanoparticles for Photothermal Therapy.

Photothermal therapy is a promising treatment modality in the realm of cancer therapy. Photothermal nanomaterials that absorb and emit in the near-infrared range (750-900 nm) have drawn a lot of attention recently because of the deep penetration of NIR light in biological tissue. Most nanomaterials, however, are produced by encapsulating or altering the surface of a nanoplatform, which has limited loading capacity and long-term storage. Herein, we developed a stable polymer conjugated with aza-BODIPY that self-assembled to form nanoparticles (aza-BODIPY-mPEG) with better hydrophilicity and biocompatibility while retaining the dye's photothermal conversion characteristics. Aza-BODIPY-mPEG with a hydrodynamic size of around 170 nm exhibited great photostability and excellent photothermal therapy in vitro and in ovo. Aza-BODIPY-mPEG exhibits approximately 30% better anti-angiogenesis and antitumor activity against implanted xenograft human HCT116 tumor in the chick embryo compared to parent aza-BODIPY-A, altogether suggesting that aza-BODIPY-mPEG is a promising material for cancer photothermal therapy.

Antibody-dependent cellular phagocytosis of tropomyosin receptor kinase C (TrkC) expressing cancer cells for targeted immunotherapy.

Conventional cancer therapies such as chemotherapy are non-selective and induce immune system anergy, which lead to serious side effects and tumor relapse. It is a challenge to prime the body's immune system in the cancer-bearing subject to produce cancer antigen-targeting antibodies, as most tumor-associated antigens are expressed abundantly in cancer cells and some of normal cells. This study illustrates how hapten-based pre-immunization (for anti-hapten antibodies production) combined with cancer receptor labeling with hapten antigen constructs can elicit antibody-dependent cellular phagocytosis (ADCP). Thus, the hapten antigen 2,4-dinitrophenol (DNP) was covalently combined with a cancer receptor-binding dipeptide (IYIY) to form a dipeptide-hapten construct (IYIY-DNP, MW = 1322.33) that targets the tropomyosin receptor kinase C (TrkC)-expressed on the surface of metastatic cancer cells. IYIY-DNP facilitated selective association of RAW264.7 macrophages to the TrkC expressing 4T1 cancer cells in vitro, forming cell aggregates in the presence of anti-DNP antibodies, suggesting initiation of anti-DNP antibody-dependent cancer cell recognition of macrophages by the IYIY-DNP. In in vivo, IYIY-DNP at 10 mg/kg suppressed growth of 4T1 tumors in DNP-immunized BALB/c mice by 45% (p < 0.05), when comparing the area under the tumor growth curve to that of the saline-treated DNP-immunized mice. Meanwhile, IYIY-DNP at 10 mg/kg had no effect on TrkC-negative 67NR tumor-bearing mice immunized with DNP. Tumor growth suppression activity of IYIY-DNP in DNP-immunized mice was associated with an increase in the anti-DNP IgG (7.3 × 106 ± 1.6 U/mL) and IgM (0.9 × 106 ± 0.07 U/mL) antibodies after five cycles of DNP treatment, demonstrated potential for hapten-based pre-immunization then treatment with IYIY-DNP to elicit ADCP for improved immunotherapy of TrkC expressing cancers.

Differential toxicity and teratogenic effects of the hot water and cold water extracts of Lignosusrhinocerus (Cooke) Ryvarden sclerotium on zebrafish (Danio rerio) embryos.

ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosusrhinocerus (Cooke) Ryvarden is highly valued for its purported medicinal properties. The decoction and macerated materials prepared from the sclerotium are used for treating cancer and other ailments based on extensive traditional knowledge. Scientific evidence from in vitro cytototoxicity, anti-inflammatory and immunomodulatory analyses showed the effectiveness of sclerotial water extracts but toxicity assessment of such preparations has not been reported. AIM OF THE STUDY: This study aimed to compare the differential toxicity and teratogenicity (if any) of the hot water (HW) and cold water (CW) extracts of both wild and cultivated sclerotium on zebrafish (Danio rerio) embryos. MATERIALS AND METHODS: Zebrafish embryos were treated with varying concentrations of the sclerotial HW and CW extracts (0.3-500 µg/mL) for 72 h until hatching. The hatching, mortality and heartbeat rate of the embryos as well as the potential teratogenic effect of the extracts were assessed in embryos post-treatment with the extracts. RESULTS: While the sclerotial HW extracts were nontoxic (LC50 > 500 µg/mL), the sclerotial CW extracts delayed the hatching of the embryos up to 48 h and showed slight toxicity with LC50 values of 398.4 µg/mL and 428.3 µg/mL for the cultivated and wild sclerotium, respectively. The sclerotial CW extracts also induced minor tachycardia in zebrafish larvae. Phenotypic assessment revealed that, while yolk sac edema was observed at high concentrations (300 and 500 µg/mL) of all extracts, curved trunk and bent tail were only observed in the embryos treated with CW extracts of wild sclerotium (300 and 500 µg/mL) but not for CW extracts of cultivated sclerotium at similar concentrations. CONCLUSION: The sclerotial water extracts of L.rhinocerus prepared using different methods have varying degree of toxicity and teratogenicity in zebrafish embryos with the sclerotial CW extracts showed higher toxicity than the HW extracts.

Alternative animal models in predictive toxicology.

Toxicity testing relies heavily on animals, especially rodents as part of the non-clinical laboratory testing of substances. However, the use of mammalians and the number of animals employed in research has become a concern for institutional ethics committees. Toxicity testing involving rodents and other mammals is laborious and costly. Alternatively, non-rodent models are used as replacement, as they have less ethical considerations and are cost-effective. Of the many alternative models that can be used as replacement models, which ones can be used in predictive toxicology? What is the correlation between these models and rodents? Are there standardized protocols governing the toxicity testing of these commonly used predictive models? This review outlines the common alternative animal models for predictive toxicology to address the importance of these models, the challenges, and their standard testing protocols.

Identification of potential candidate genes for lip and oral cavity cancer using network analysis.

Lip and oral cavity cancer, which can occur in any part of the mouth, is the 11th most common type of cancer worldwide. The major obstacles to patients' survival are the poor prognosis, lack of specific biomarkers, and expensive therapeutic alternatives. This study aimed to identify the main genes and pathways associated with lip and oral cavity carcinoma using network analysis and to analyze its molecular mechanism and prognostic significance further. In this study, 472 genes causing lip and oral cavity carcinoma were retrieved from the DisGeNET database. A protein-protein interaction network was developed for network analysis using the STRING database. VEGFA, IL6, MAPK3, INS, TNF, MAPK8, MMP9, CXCL8, EGF, and PTGS2 were recognized as network hub genes using the maximum clique centrality algorithm available in cytoHubba, and nine potential drug candidates (ranibizumab, siltuximab, sulindac, pomalidomide, dexrazoxane, endostatin, pamidronic acid, cetuximab, and apricoxib) for lip and oral cavity cancer were identified from the DGIdb database. Gene enrichment analysis was also performed to identify the gene ontology categorization of cellular components, biological processes, molecular functions, and biological pathways. The genes identified in this study could furnish a new understanding of the underlying molecular mechanisms of carcinogenesis and provide more reliable biomarkers for early diagnosis, prognostication, and treatment of lip and oral cavity cancer.

Active targeted ligand-aza-BODIPY conjugate for near-infrared photodynamic therapy in melanoma.

Active targeting compound, a non-iodinated derivative of IK-IK-I2-azaBODIPY (1a) was previously reported to preferentially bind melanoma over healthy cells. In this study, we evaluate the photodynamic therapy (PDT) efficiency on melanoma cells of 1a, together with its reversed sequence compound KI-KI-I2-azaBODIPY (1b) and a non-targeted control I2-azaBODIPY-NH2 (2). All three test compounds possess absorption wavelengths in the near-infrared (NIR) region (λmax between 678 and 687 nm) which alleviate melanin interference and allow deeper tissue penetration. In vitro studies revealed 1a and 1b are promising photosensitizers with enhanced singlet oxygen generation, have increased uptake by B16-F10 melanoma cells via clathrin-mediated endocytosis and good photocytotoxic efficacies. Ex vivo biodistribution assays showed both 1a and 1b accumulated in the tumour. In B16-F10 tumour bearing-C57BL/6 mice, 10 mg/kg of 1b and light irradiation was found to reduce tumour volume by up to 23% at day-3. Doubling the dosage of 1b (20 mg/kg) enhanced the antitumour effect, showing 96% maximum tumour volume reduction at day-7 and tumour growth suppression for up to 12 days.

Recent strategies to improve boron dipyrromethene (BODIPY) for photodynamic cancer therapy: an updated review.

BODIPYs are photosensitizers activatable by light to generate highly reactive singlet oxygen (1O2) from molecular oxygen, leading to tissue damage in the photoirradiated region. Despite their extraordinary photophysical characteristics, they are not featured in clinical photodynamic therapy. This review discusses the recent advances in the design and/or modifications of BODIPYs since 2013, to improve their potential in photodynamic cancer therapy and related areas.

Doxorubicin-loaded micelles of amphiphilic diblock copolymer with pendant dendron improve antitumor efficacy: In vitro and in vivo studies.

Previously reported amphiphilic diblock copolymer with pendant dendron moieties (P71D3) has been further evaluated in tumor-bearing mice as a potential drug carrier. This P71D3-based micelle of an average diameter of 100nm was found to be biocompatible, non-toxic and physically stable in colloidal system up to 15days. It enhanced the in vitro potency of doxorubicin (DOX) in 4T1 breast tumor cells by increasing its uptake, by 3-fold, compared to free DOX. In 4T1 tumor-bearing mice, the tumor growth rate of P71D3/DOX (2mg/kg DOX equivalent) treated group was significantly delayed and their tumor volume was significantly reduced by 1.5-fold compared to those treated with free DOX. The biodistribution studies indicated that P71D3/DOX enhanced accumulation of DOX in tumor by 5- and 2-fold higher than free DOX treated mice at 15min and 1h post-administration, respectively. These results suggest that P71D3 micelle is a promising nanocarrier for chemotherapeutic agents.

Tropomyosin Receptor Kinase C Targeted Delivery of a Peptidomimetic Ligand-Photosensitizer Conjugate Induces Antitumor Immune Responses Following Photodynamic Therapy.

Tropomyosin receptor kinase C (TrkC) targeted ligand-photosensitizer construct, IYIY-diiodo-boron-dipyrromethene (IYIY-I2-BODIPY) and its scrambled counterpart YIYI-I2-BODIPY have been prepared. IYIY-I2-BODIPY binds TrkC similar to neurotrophin-3 (NT-3), and NT-3 has been reported to modulate immune responses. Moreover, it could be shown that photodynamic therapy (PDT) elevates antitumor immune responses. This prompted us to investigate the immunological impacts mediated by IYIY-I2-BODIPY in pre- and post-PDT conditions. We demonstrated that IYIY-I2-BODIPY (strong response) and YIYI-I2-BODIPY (weak response) at 10 mg/kg, but not I2-BODIPY control, increased the levels of IL-2, IL-4, IL-6 and IL-17, but decreased the levels of systemic immunoregulatory mediators TGF-ß, myeloid-derived suppressor cells and regulatory T-cells. Only IYIY-I2-BODIPY enhanced the IFN-γ+ and IL-17+ T-lymphocytes, and delayed tumor growth (~20% smaller size) in mice when administrated daily for 5 days. All those effects were observed without irradiation; when irradiated (520 nm, 100 J/cm2, 160 mW/cm2) to produce PDT effects (drug-light interval 1 h), IYIY-I2-BODIPY induced stronger responses. Moreover, photoirradiated IYIY-I2-BODIPY treated mice had high levels of effector T-cells compared to controls. Adoptive transfer of immune cells from IYIY-I2-BODIPY-treated survivor mice that were photoirradiated gave significantly delayed tumor growth (~40-50% smaller size) in recipient mice. IYIY-I2-BODIPY alone and in combination with PDT modulates the immune response in such a way that tumor growth is suppressed. Unlike immunosuppressive conventional chemotherapy, IYIY-I2-BODIPY can act as an immune-stimulatory chemotherapeutic agent with potential applications in clinical cancer treatment.

Chitosan-Coated Poly(lactic-co-glycolic acid)-Diiodinated boron-Dipyrromethene Nanoparticles Improve Tumor Selectivity and Stealth Properties in Photodynamic Cancer Therapy.

Their limited solubility and lack of tumor selectivity limit the clinical usefulness of photosensitizers. Various nanostructures have been evaluated as delivery agents for photosensitizers in an attempt to overcome these obstacles, but these have typically been limited by premature clearance by the reticuloendothelial system (RES) and non-specific interactions with normal cells that result from their hydrophobic surfaces. In this study, we report our attempt to circumvent these problems by applying a low molecular weight chitosan (25 kDa) coating to a poly(lactic-co-glycolic acid)-diiodinated boron dipyrromethene (PLGA-I2BODIPY) nanoparticle-photosensitizer construct. This chitosan coating increased the hydrophilicity and decreased the charge of PLGA-I2 BODIPY nanoparticle surfaces without changing their size (average diameter 147 nm) or morphology. In comparison to the uncoated controls, the coated nanoparticles reduced the burst release of I2BODIPY, increased its predominantly lysosomal cellular uptake, and enhanced its photocytotoxicity in 4T1 murine and MDA-MB-231 human breast cancer cells. PLGA-Chitosan-I2BODIPY nanoparticles also showed reduced serum protein adsorption and macrophage uptake compared to the uncoated controls. In 4T1 tumor-bearing mice, the PLGA-Chitosan-I2BODIPY nanoparticles exhibited better tumor-targeting selectivity and significantly reduced accumulation in RES tissues, including the lymph nodes, spleen and liver (by 10.2-, 2.1- and 1.3-fold, respectively), and in non-tumorous organs, such as the skin and eyes (by 22.7- and 4-fold, respectively). The PLGA-Chitosan-I2BODIPY and PLGA-I2BODIPY nanoparticles also showed increased anticancer efficacy compared to free I2BODIPY. These results suggest that the low molecular weight chitosan (25 kDa) is a promising nanoparticle "stealth coating" that improves tumor selectivity.

Chick embryo chorioallantoic membrane (CAM): an alternative predictive model in acute toxicological studies for anti-cancer drugs.

The chick embryo chorioallantoic membrane (CAM) is a preclinical model widely used for vascular and anti-vascular effects of therapeutic agents in vivo. In this study, we examine the suitability of CAM as a predictive model for acute toxicology studies of drugs by comparing it to conventional mouse and rat models for 10 FDA-approved anticancer drugs (paclitaxel, carmustine, camptothecin, cyclophosphamide, vincristine, cisplatin, aloin, mitomycin C, actinomycin-D, melphalan). Suitable formulations for intravenous administration were determined before the average of median lethal dose (LD50) and median survival dose (SD(50)) in the CAM were measured and calculated for these drugs. The resultant ideal LD(50) values were correlated to those reported in the literature using Pearson's correlation test for both intravenous and intraperitoneal routes of injection in rodents. Our results showed moderate correlations (r(2)=0.42 - 0.68, P<0.005-0.05) between the ideal LD(50) values obtained using the CAM model with LD(50) values from mice and rats models for both intravenous and intraperitoneal administrations, suggesting that the chick embryo may be a suitable alternative model for acute drug toxicity screening before embarking on full toxicological investigations in rodents in development of anticancer drugs.

Targeted PDT agent eradicates TrkC expressing tumors via photodynamic therapy (PDT).

This contribution features a small molecule that binds TrkC (tropomyosin receptor kinase C) receptor that tends to be overexpressed in metastatic breast cancer cells but not in other breast cancer cells. A sensitizer for (1)O2 production conjugated to this structure gives 1-PDT for photodynamic therapy. Isomeric 2-PDT does not bind TrkC and was used as a control throughout; similarly, TrkC- cancer cells were used to calibrate enhanced killing of TrkC+ cells. Ex vivo, 1- and 2-PDT where only cytotoxic when illuminated, and 1-PDT, gave higher cell death for TrkC+ breast cancer cells. A 1 h administration-to-illumination delay gave optimal TrkC+/TrkC--photocytotoxicity, and distribution studies showed the same delay was appropriate in vivo. In Balb/c mice, a maximum tolerated dose of 20 mg/kg was determined for 1-PDT. 1- and 2-PDT (single, 2 or 10 mg/kg doses and one illumination, throughout) had similar effects on implanted TrkC- tumors, and like those of 2-PDT on TrkC+ tumors. In contrast, 1-PDT caused dramatic TrkC+ tumor volume reduction (96% from initial) relative to the TrkC- tumors or 2-PDT in TrkC+ models. Moreover, 71% of the mice treated with 10 mg/kg 1-PDT (n = 7) showed full tumor remission and survived until 90 days with no metastasis to key organs.

C6-ceramide in combination with transforming growth factor-ß enhances Treg cell differentiation and stable FoxP3 expression in vitro and in vivo.

Ceramides, sphingosine-based lipid molecules, are generated mainly from the hydrolysis of sphingomyelin and play pivotal roles in biological processes including cell growth, differentiation, and inflammation. In this study, we investigated the effect of exogenous ceramides on the differentiation of regulatory T (Treg) cells and expression of FoxP3 gene in Treg cells. A cell-permeable C6-ceramide (C6) was capable of upregulating Treg cell differentiation when acting together with transforming growth factor-beta (TGF-ß), and this induction was independent of T-cell receptor (TCR) and CD28 strength. Additionally, TGF-ß/C6 treatment sustained the expression of FoxP3 gene in Treg cells, as the percentages of FoxP3(+) Treg cells in the TGF-ß/C6-treated group remained high for prolonged periods compared to those in the group treated with TGF-ß alone. Furthermore, C8-ceramide was also capable of sustaining Treg cell populations and FoxP3 expression, whereas C2-, C16-, and C24-ceramides did not. Importantly, adoptive transfer of the TGF-ß/C6-induced Treg cells into syngenic mice showed that TGF-ß/C6-induced Treg cells maintained their FoxP3 expression in vivo significantly longer periods than the TGF-ß-induced Treg cells. Taken together, our findings indicate that C6 can be utilized to increase Treg cell populations and also to sustain their FoxP3 expression in the treatment of autoimmune diseases or graft rejection.

C6-ceramide enhances Interleukin-12-mediated T helper type 1 cell responses through a cyclooxygenase-2-dependent pathway.

Ceramides, lipid molecules located predominantly within the plasma membrane of a cell, can function as second messengers, and have been known to carry out a number of cellular functions. T helper type 1 (Th1) immune responses are known to be involved in the cellular immunity, which is crucial in the cancer and allergy immunotherapy. This study was designed to evaluate the effects of ceramides on T helper cell responses and their underlying mechanisms. We demonstrated that a cell-permeable C6-ceramide (C6) together with IL-12 enhanced Th1 cell differentiation, whereas C6 alone had no effects, as demonstrated by the increased populations of IFN-γ expressing CD4(+) T cells and the up-regulation of IFN-γ production from CD4(+) T cells. In contrast, C2-ceramide and long chain ceramides (C16 and C24) did not affect the Th1 responses. C6 treatment was shown to increase the expression of T-bet, a master transcription factor of Th1 responses, in a dose-dependent fashion. Furthermore, C6 increased the expression of cyclooxygenase-2 (COX-2) in CD4(+) T cells. The C6-mediated increase of IFN-γ production and IFN-γ expressing CD4(+) T cell populations were significantly suppressed by a COX-2 specific inhibitor (NS-398) in a dose-dependent manner. T-bet expression was also decreased by NS-398 treatment, thereby indicating that C6 ceramide enhances Th1 responses via a COX-2 dependent pathway. This result demonstrates that C6 may be utilized in therapies for the treatment of immune diseases such cancer and allergy by enhancing the Th1 activity.