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CHAPTER 2 Literature Review
2.18 APOPTOSIS BY SPHINGOID BASES
in cell number were also similar with respect to elevation of Sa, which is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with myriocin (ISP-l) prevented the elevation in Sa, DNA fragmentation, and apoptosis induced by FBI.
Therefore, these effects of FBI on HT29 cells can be attributed to the accumulation of Sa (Schmelz et aI., 1998).
Wang et al. (1996) established that FBI-induced apoptosis, or cell cycle arrest, in CVl Mrican green monkey kidney fibroblasts, by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. The addition of FBI to CVl cells induced formation of DNA ladders, compaction of nuclear DNA, and appearance of apoptotic bodies. Fumonisin BI also induced cell cycle arrest in the G 1 phase in CVl cells (Wang et aI., 1996).
In a subsequent study, Jones et al. (2001) identified genes that inhibit FBI-induced apoptosis in CVl cells and two mouse embryo fibroblast (MEF) lines. A baculovirus gene p53, and inhibitor of apoptosis, CpIAP, protected these cells from apoptosis. CpIAP blocks apoptosis induced by the tumour necrosis factor (TNF) pathway, as well as via other mechanisms. Further support for the involvement of the TNF signal transduction pathway in FBI induced apoptosis, was the cleavage of caspase 8. Inhibition of caspases by the baculovirus gene p35 also inhibited FBI-induced apoptosis. The tumour suppressor gene p53 was not required for FBI induced apoptosis because p53-/- MEF undergoes apoptosis following FBI treatment. Furthermore, BcI-2 was not an effective inhibitor of FBI-induced apoptosis in CVl cells or p53+/+ MEF. These results provide information to understanding the mechanism by which FBI induces apoptosis (Jones et al., 2001).
effective levels of CER. Thus, So and Sa increased both the potency and efficacy of CER (Jarvis et al., 1996).
Sweeney et al. (1996) reported that So and its methylated derivative N,N,-dimethyl So (DMS) induced apoptosis in cancer cells of both haematopoietic and carcinoma origin. In human leukaemic cell lines CMK-7, HL60 and U937, treatment with 20~M So for six hours caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRTI8, MKN74 and COL0205 were shown to be more susceptible to apoptosis upon addition ofDMS (>50%) than of So «50%), yet were weakly or not sensitive to N, N, N- trimethyl So (TMS). Under the same conditions in the presence of serum, neither So-I- phosphate nor CER analogues C2-, C6- or C8-CER were able to induce apoptosis in any cell lines. However, in the absence of serum, CER analogues induced apoptosis in leukaemic cell lines after 18 hours, yet much less so than So or DMS. Furthermore, apoptosis induced by So or DMS could not be inhibited by FBI. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by So, DMS or C2-CER were inhibited by protease inhibitors. These data support evidence that the catabolic pathway of SM involving So and other metabolites is an integral part of the apoptosis pathway (Sweeney et al., 1996).
Gennero et at. (2002) studied the apoptotic effect of So-I-phosphate and increased So-I-phosphate hydrolysis on mesangial cells, and found that So-I-phosphate stimulated proliferation of rat mesangial cells and phosphorylation of MAPKs at sub-confluent cell density. Mesangial cells expressed several So-I-phosphate receptors of the endothelial differentiation gene (EDG) family: EDG-I, -3, -5, and -8. Conversely, So-I-phosphate induced apoptosis at low cell density (2x104cells/cm2), which was demonstrated by flow cytometry and Hoechst staining. Apoptosis was observed also in quiescent or growing cells. Incubation with e3p] So-I-phosphate, eH] So-I-phosphate and CH] SO demonstrated increased So-I-phosphate hydrolysis, resulting in enhanced intracellular So levels and decreased So-I-phosphate levels. Therefore, Gennero et at. (2002) suggested that So and decreased So-I-phosphate are primarily responsible for So-I-phosphate- induced apoptosis, and that incubation of low density mesangial cells with So-I-phosphate results in apoptosis, presumably due to increased So-I-phosphate hydrolysis.
2.19 INHmITION OF DE NOVO SPHINGOLIPID BIOSYNTHESIS IN CULTURED CELLS
Theoretically, the balance between the intracellular concentration of effectors that protect cells from apoptosis, and those that induce apoptosis, determine cellular responses. As the balance between the rates of apoptosis and proliferation are important in tumorigenesis, cells sensitive to the proliferative effect of decreased CER and increased So-I-phosphate may be selected to survive and proliferate when free sphingoid base concentration is not growth inhibitory. Conversely, when the increase in free sphingoid bases exceeds a cell's ability to convert Sa:So to dihydroceramide/CER, or their sphingoid base I-phosphate, then free sphingoid bases will accumulate. In this case, cells that are sensitive to sphingoid base-induced growth arrest will die, and non-sensitive cells will survive. If the cells selected to die are of normal phenotypes, and the cells selected to survive are abnormal, then cancer risk will increase (Riley et al., 2001).
The potential for fumonisins to inhibit de novo sphingolipid biosynthesis was examined in cultures of hepatocytes obtained from male Sprague-Dawley rats and in rat liver microsomes. Fumonisin Bl caused almost complete inhibition of [14C] So formation by hepatocytes. Similar inhibition occurred when [14C] serine and FBI were added together for 2 or 16 hours, and when the cells were incubated for 16 hours with FBI before addition of
e
4C] serine. Fumonisin Bz produced a comparable degree of inhibition. Wang et al. (1991) speculated that similarities between the fumonisins and sphingoid bases allowed their recognition as substrate (or transition state or product) analogues of CER synthase. It was further speculated that the absence of a hydroxyl group at carbon 1 may have altered their orientation in the active site of the enzyme and precluded acylation or, if acy lated, resulted in an inhibitory CER that could not be removed by addition of a sphingolipid headgroup at that position.Wang et at. (1991) conducted further in vitro assays of So N-acyltransferase using rat liver microsomes, and followed the conversion of eH] So to eH]CER by rat hepatocytes.
Results showed that O.I!lM of FBI caused 50% inhibition in So N-acyltransferase activity.
When rat hepatocytes were incubated with I!lM FB I and 1 !lCi of eH] So for one hour, the conversion of eH] So to CER was significantly inhibited (p<0.05) compared to that of untreated control cultures, with an approximate concentration giving 50% inhibition of cell proliferation (IC50 ) of 0.1 !lM. These results provide identification of a biochemical target
for the action of fumonisins and imply that inhibition of de novo sphingolipid biosynthesis in vitro may underlie the hepatotoxicity and hepatocarcinogenicity of this mycotoxin in vivo (Wang et at., 1991).
Cytotoxicity studies in primary rat hepatocytes and binding studies using subcellular fractions indicate that
e
4C] FBI binds tightly to hepatocytes and microsomal and plasma membrane fractions (Cawood et at., 1994).Investigations on pig kidney epithelial cells showed that the fumonisins were potent inhibitors of sphingolipid biosynthesis in the cells, killing the renal cells after three days at
70~M of FBI (Yoo et al., 1992). Fumonisin BI and FB2 between 10 and 35~M inhibited cell proliferation in the LLC-PKI cells, whereas higher concentrations (>35~M) killed cells. Inhibition of cell proliferation and cell death were preceded by a lag period of at least 24 hours during which cells appeared to be functioning normally. Inhibition of de novo sphingolipid biosynthesis occurred before inhibition of cell proliferation or cytotoxicity, and the dose response for decrease in eH] So:eH] Sa ratio at seven hours closely paralleled the dose response for effects on proliferation and cytotoxicity at three-five days.
In addition, the level of free Sa and to a lesser extent So, increased in fumonisin treated- cells in a dose dependent manner. The fact that Sa accumulated to a much greater extent than So, suggested that the de novo pathway was the primary target for inhibition, and as a result of this differential inhibition, the Sa:So ratio increased after exposure to fumonisin.
These increases occurred long before any indication of cytotoxicity. These results support the hypothesis that inhibition of de novo sphingolipid biosynthesis is an early event in the toxicity of fumonisins to LLC-PK 1 cells (Yoo et al., 1992).
Other consequences of fumonisin inhibition of CER synthase include increase in degradation products from catabolism of free sphingoid bases (Merrill et at., 1993a;1993b), increase in lipid products derived from increases in sphingoid base degradation products (Smith and Merrill, 1993), and increase in free So presumably from inhibition of reacylation of So derived from either the diet or catabolism of complex sphingolipids (Yoo et al., 1992; Merrill et al., 1993b).
Fumonisin B I stimulates Sa-dependent DNA synthesis in Swiss 3T3 cells, but is mitoinhibitory and growth inhibitory 10 many cell systems (Merrill, 1983;
Stevens et aI., 1990; Zhang et aI., 1990) Schroeder et at. (1994) found that free sphingoid bases can act as mitogens, however that the critical concentration of cellular sphingoid bases necessary to stimulate mitogenesis is within a range and the effects were cell-type specific. Fumonisins that cause sphingoid base accumulation can therefore be regarded as tumour promoters, possibly by stimulating cell proliferation. The exogenous addition of So and So-I-phosphate to Swiss 3T3 cells was also shown to stimulate DNA synthesis (Zhang et al., 1990; Spiegel et aI., 1993). In contrast, other studies indicate that accumulation of sphingoid bases and depletion of complex sphingolipids were contributing factors to growth inhibition and increased cell death (Spiegel and Merrill, 1996).
While So has little difficulty in crossing cell membranes (Hannun et al., 1991), the half-life of Sa inside LLC-PKI cells is much longer than that of FBI in LLC-PKI cells (Riley et al., 1998), which suggests either that the inhibition of Sa N-acyltransferase is persistent, that Sa does not easily diffuse out of cells, or that Sa degradation is slow relative to its biosynthesis. Studies with LLC-PKI cells also indicate that a low, but persistent pool of [14C] labelled material is retained inside cells long after the rapidly diffusible pool of [14C] fumonisin has exited the cells (Riley et aI., 1998). This retained pool appears to be capable of maintaining the elevation of cellular free sphingoid base concentration, a biomarker of fumonisin exposure (Solfrizzo et al., 1997; Riley et aI., 1998; Wang et aI., 1999). Riley et al. (1999) using the same cell system showed that fumonisin inhibition of cell proliferation and increased cell death (apoptosis) are prevented by >90%
using the SPT specific inhibitor, ISP 1 (Riley et aI., 1999). Similar results were obtained with the HT29 human colonic cell line (Schmelz et aI., 1998).
Fumonisin BI elevated intracellular free Sa levels in both LLC-PK1 and Chinese hamster ovary fibroblast (CHO) cells (Yu et aI., 2001). However, CHO cells were resistant to fumonisin cytotoxicity at 50~M, while LLC-PK1 cells were sensitive at concentrations
>35~M Adding exogenous Sa to the CHO cells along with 50~M FBI treatment for 72 hours caused both necrosis and apoptosis. It was concluded that elevated endogenous Sa acts as a contributing factor to fumonisin-induced cell death (Yu et aI., 2001).
2. 20 THE EFFECT OF FUMONISIN Bl ON OXYGEN TRANSPORT
Yin et at. (1996a) used electron spin resonance (ESR) spin label oximetry to study the effect of FBI on oxygen transport in phosphotidylcholine bilayers. Moreover, the use of
spin label attached to different carbons of fatty acids facilitated structural and oximetric determinations with the same test sample. The incorporation of 10 mol %FB I increased the oxygen transport properties of both saturated and unsaturated membranes at 37°C by approximately 30% and decreased the ordering of the hydrocarbon chains near the surface of the membranes. Concomitantly, oxygen transport near the centre of bilayers was diminished slightly, and the relative oxygen diffusion-concentration product profile curves were markedly flattened.
Yin et at. (1996b) used ESR spectroscopy and spin label techniques further to study the effects of FBI and APJ on the structural and dynamic properties of phosphatidylcholine membranes at the molecular level. Multilamellar liposomes consisting of dimyristoylphosphatidyl-choline (DMPC) and egg yolk phosphatidylcholine (EYPC) were used. In the fluid phase membrane, FBI significantly increased the fluidities of n-doxylstearic acid spin labels attached to carbons 5 and 7, which disordered the alkyl chains and perturbed the surface region of the bilayer. By comparison, minimal effects were detected near the middle of the bilayer. In the gel phase, FBI and API decreased membrane fluidity, which further enlarged the change in ordering on the phase transition.
Fumonisin BI also restricted the mobility of the rigid cholestane spin ]abel. A reduction in mobility of the temp-stearate spin label suggests that the TCA moieties of FBI might mimic the structure of polar head groups in phospholipids. These results that show fumonisins disturbed the ordering of membranes, provide additional mechanisms to elucidate toxicological activities of fumonisins.
Subsequently Yin et at. (1998) studied the effects of FBI on lipid peroxidation in membranes. In this study of the interaction between FBI and lipid bilayers, fumonisins disturbed the ordering of membranes, enhanced oxygen transport in membranes, and also increased membrane permeability. The results provided the first evidence that fumonisin appears to increase the rate of oxidation, promote free radical intermediate production and accelerate the chain reactions associated with lipid peroxidation. The disruption of membrane structure, enlargement of relative oxygen diffusion-concentration products, and enhancement effects on membrane permeability, provide additional insights into potential mechanisms by which fumonisins could enhance oxidative stress and cell damage.
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2.21 FUMONISIN-INDUCED DISRUPTION OF SPHINGOLIPID