Effect of rice cell-derived human granulocyte-macrophage colony-stimulating factor on 5-fluorouracil-induced mucositis in hamsters.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.

Age-related effects of sodium arsenite on splenocyte proliferation and Th1/Th2 cytokine production.

Aging is associated with immune dysfunction and conditions such as inflamm-aging and immunosuppression. Arsenic, an environmental contaminant distributed worldwide, affects the immune system. This study tested the hypothesis that arsenic has distinct effects on T cell proliferation and the production of cytokines by activated T cells. Murine splenocytes from young (2 months) and aged (24-26 months) C57BL/6 mice were exposed to arsenite (As(3+)), the most toxic form of inorganic arsenic, and stimulated with concanavalin A (Con A) or anti-CD3 antibody. T cell proliferation decreased significantly in response to Con A and anti-CD3 at subtoxic doses of arsenite in splenocytes from both young and aged mice. Arsenite, added concurrently with Con A or anti-CD3, significantly inhibited the production of interleukin-2 (IL-2), interferon-γ (IFN-γ), and interleukin-4 (IL-4) by splenocytes from young mice and significantly reduced the production of IL-10 by splenocytes from aged mice. In contrast, the production of IL-2 and IL-4 by splenocytes from aged mice was only slightly affected by arsenite. The results show that arsenic exposure reduces the immune response in splenocytes. Moreover, this effect may be influenced by aging.

C(6)-ceramide enhances phagocytic activity of Kupffer cells through the production of endogenous ceramides.

Ceramide has been suggested to be not only a tumor-suppressive lipid but also a regulator of phagocytosis. We examined whether exogenous cell-permeable C(6)-ceramide enhances the phagocytic activity of Kupffer cells (KCs) and affects the level of cellular ceramides. Rat KCs were isolated by collagenase digestion and differential centrifugation, using Percoll system. Phagocytic activity was measured by FACS analysis after incubating KCs with fluorescence-conjugated latex beads, and the level of cellular ceramide was analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). In this study we found that permeable C(6)-ceramide increases the cellular levels of endogenous ceramides via a sphingosine-recycling pathway leading to enhanced phagocytosis by KCs.

Identification and evaluation of neutral sphingomyelinase 2 inhibitors.

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg(2+)-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg(2+)-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2.

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium.

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C(6)-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C(6)-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.

Hypoxia-induced neuronal apoptosis is mediated by de novo synthesis of ceramide through activation of serine palmitoyltransferase.

Cellular hypoxia can lead to cell death or adaptation and has important effects on development, physiology, and pathology. Here, we investigated the role and regulation of ceramide in hypoxia-induced apoptosis of SH-SY5Y neuroblastoma cells. Hypoxia increased the ceramide concentration; subsequently, we observed biochemical changes indicative of apoptosis, such as DNA fragmentation, nuclear staining, and poly ADP-ribose polymerase (PARP) cleavage. The hypoxic cell death was potently inhibited by a caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone). l-Cycloserine, a serine palmitoyltransferase (SPT) inhibitor, and fumonisin B(1) (FB(1)), a ceramide synthase inhibitor, inhibited the hypoxia-induced increase in ceramide, indicating that the increase occurred via the de novo pathway. Hypoxia increased the activity and protein levels of SPT2, suggesting that the hypoxia-induced increase in ceramide is due to the transcriptional up-regulation of SPT2. Specific siRNA of SPT2 prevented hypoxia-induced cell death and ceramide production. However, hypoxia also increased the cellular level of glucosylceramide, which was inhibited by a glucosylceramide synthase (GCS) inhibitor and specific siRNA, but not a ceramidase inhibitor. The increase in glucosylceramide was accompanied by increases in both PARP cleavage and DNA fragmentation. Together, the current results suggest that both SPT and GCS may regulate the cellular level of ceramide, and thus may be critical enzymes for deciding the fate of the cells exposed to hypoxia.

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

Ganciclovir augments the lytic induction and apoptosis induced by chemotherapeutic agents in an Epstein-Barr virus-infected gastric carcinoma cell line.

Epstein-Barr virus is an oncogenic herpesvirus and has been associated with several human malignancies, including gastric cancer. In Epstein-Barr virus-associated gastric cancer, Epstein-Barr virus is found in virtually all tumor cells, but rarely in normal epithelial cells, thus implying that Epstein-Barr virus-targeting therapies are likely to be an effective treatment strategy. Using the SNU-719 gastric cancer cell line, which is naturally infected with Epstein-Barr virus, we found that the chemotherapeutic agents, such as 5-fluorouracil, cis-platinum and taxol, induced the expressions of BMRF1, BZLF1 and BRLF1 proteins that are usually found in the lytic form of the virus. This effect was found to require various signal transduction pathways involving phosphatidylinositol 3' kinase, mitogen-activated protein/extracellular signal-regulated kinase, protein kinase C delta and p38 mitogen-activated protein kinase. Moreover, the combination of ganciclovir with these agents increased the lytic transformation and induced apoptosis in Epstein-Barr virus-associated gastric carcinoma. We conclude that ganciclovir enhances the therapeutic efficacy of 5-fluorouracil, cis-platinum and taxol in Epstein-Barr virus-positive gastric cancer cells. It is hoped that this information will be found useful during the establishment of treatment strategies for Epstein-Barr virus-associated gastric cancer.