ß-Sitosterol modulates TLR4 receptor expression and intracellular MyD88-dependent pathway activation in J774A.1 murine macrophages.

Recent evidence has shown that dietary phytosterols (PS) possess anti-inflammatory properties both in vivo and in vitro. Our previous work shows that PS ß-Sitosterol (SIT), may function by down-regulating pro-inflammatory transcription factors NF-kB and STAT1 in response to LPS stimulation, possibly through modulation of the TLR4 pathway. The objective in this study was to determine the effects of SIT on TLR4 surface expression and localization into lipid rafts, as well as to investigate its effects on intracellular MyD88 dependent pathway activation. J774A.1 macrophages were pre-treated with cyclodextrin vehicle loaded with cholesterol or SIT, then stimulated with LPS (100 ng/ml) for 30 min. ImageStream cytometry demonstrated that SIT down-regulates TLR4 expression without affecting lipid raft distribution. Western blot demonstrated that SIT down-regulated the adaptor protein MyD88 and the activity of IRAK1 but increased SOCS3 expression. Together, these results provide evidence that SIT may indeed elicit anti-inflammatory properties by down-regulating some components of the TLR4 pathway.

beta-Sitosterol activates Fas signaling in human breast cancer cells.

beta-Sitosterol is the most abundant phytosterol. Phytosterols are enriched in legumes, oil seeds and unrefined plant oils as found in foods such as peanut butter, pistachios and sunflower seeds. beta-Sitosterol inhibits the growth of several specific types of tumor cells in vitro and decreases the size and the extent of tumor metastases in vivo. The effects of beta-sitosterol on the extrinsic apoptotic programmed cell death pathway in human breast MCF-7 and MDA-MB-231 adenocarcinoma cells were examined, along with the extent of its incorporation into cellular membranes and its effects on cell growth, expression of Fas receptor pathway proteins, and caspase-8 activity. The results show that beta-sitosterol exposure promotes its enrichment in transformed cell membranes and significantly inhibits tumor cell growth. Concurrently, Fas levels and caspase-8 activity are significantly increased. These actions are specific, as expression of other proteins of the Fas receptor pathway, including Fas ligand, FADD, p-FADD and caspase-8, remain unchanged. These findings support the hypothesis that beta-sitosterol is an effective apoptosis-promoting agent and that incorporation of more phytosterols in the diet may serve a preventive measure for breast cancer.

Measurement variability of plasma beta-sitosterol and campesterol, two new biomarkers for cancer prevention.

Phytosterols are plant sterols that are structurally similar to cholesterol and are characterized by anti-carcinogenic and anti-atherogenic properties. Beta-sitosterol and campesterol are the predominant phytosterols in blood. The present study aimed to analyse the reproducibility and overtime reliability of plasma beta-sitosterol and campesterol measurements. In order to study the reproducibility of the measurement (technical variability), three healthy premenopausal women donated a sample of their blood. Each blood sample was subdivided into six aliquots and analysed within the same run by the same laboratory technician. The intraclass correlation coefficients (ICCs) of the assay for plasma beta-sitosterol and campesterol were 0.88 and 0.94 (95% confidence intervals low bounds (95% CI(low)) were 0.66 and 0.82), respectively. To study the reliability of beta-sitosterol and campesterol measurement over time, seven premenopausal women were recruited. Over a 6-month period, each woman provided a fasting blood sample once a month at the same time of day, and the same numerical day of the luteal phase of her menstrual cycle (between the 20th and 24th day of her menstrual cycle). All plasma samples from the same individual were processed together at the same time by the same technician at the end of the 6-month period. The overtime ICCs of plasma beta-sitosterol and campesterol were 0.91 (95% CI(low) 0.49) and 0.58 (95% CI(low) 0.31), respectively. The high reproducibility and good overtime reliability of plasma beta-sitosterol and campesterol measurements indicate that they may be suitable for potential clinical and population-based studies on cancer prevention.

Plant sterols regulate rat vascular smooth muscle cell growth and prostacyclin release in culture.

Cardiovascular disease (CVD) is the leading cause of death in the USA and other industrialized countries. A large number of epidemiological studies have established a direct correlation between diet and the development and progression of atherosclerosis. Several studies have shown the incidence of CVD to be lower in populations consuming a predominantly plant-based diet, as compared to meat-based diets. Besides being low in fat and cholesterol, vegetarian and Asian diets contain a large variety of phytochemicals, which may function in the body. For example, phytosterols (PS) are plant sterols that interfere with the absorption of cholesterol from the intestine when present in adequate amounts. Although PS may also function at a cellular level in the body, there are few studies examining the action of PS on cells involved in atherosclerosis. The purpose of this study was to examine the effect of dietary PS on vascular smooth muscle cell (VSMC) growth and function, since VSMC play a central role in the development of atherosclerosis. VSMC were treated with 16 microM cholesterol, 25-hydroxycholesterol, campesterol and beta-sitosterol (SIT) using an ethanol as a vehicle. Cell growth was determined by cell counting and cell proliferation by DNA synthesis, which was measured by [(3)H]-thymidine incorporation. Cholesterol supplementation had no effect on cell growth and proliferation. 25-Hydroxycholesterol decreased cell growth by 68% and DNA synthesis by 99%. SIT was found to inhibit VSMC growth more effectively than campesterol. Of the two PS, campesterol decreased cell growth by 16% and SIT decreased cell growth by 30%. DNA synthesis was decreased 25% by SIT supplementation but was not influenced by campesterol or cholesterol supplementation. Cholesterol, campesterol and SIT were not cytotoxic to VSMC and did not significantly alter cell viability. 25-Hydroxycholesterol, however, was cytotoxic and decreased cell viability by 45% as determined by lactate dehydrogenase release and a trypan blue dye exclusion test. De novo cholesterol synthesis was decreased 28% by campesterol, 49% by SIT and 23% by cholesterol. Beta-sitosterol exhibited a greater effect on cholesterol synthesis than campesterol or cholesterol supplementation. Measurement of cell sterol content demonstrated incorporation of PS into VSMC at the expense of cholesterol. Campesterol decreased VSMC cholesterol content by 36%, representing 40% of the total sterol content following treatment. Beta-sitosterol decreased VSMC cholesterol by 41% following supplementation and represented 49% of the total sterol amount. Cholesterol treatment did not alter the cholesterol content of the cells. Prostacyclin production was significantly altered by PS treatment. Basal prostacyclin release was increased 43% by campesterol and 81% by SIT. A23187 stimulated prostacyclin release was increased 25% by campesterol and 54% by SIT. SIT supplementation induced a greater effect on prostacyclin release from VSMC than cholesterol or campesterol supplementation. The in vitro results presented here suggest that dietary PS, especially SIT, may offer protection from the VSMC hyperproliferation found in atherosclerosis. Further in vivo research is needed to support these observations.

In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells.

The dietary effect of phytosterols (PS) versus cholesterol on the growth and metastasis of the PC-3 human prostate cancer cells in SCID mice was studied. Also, their direct effect on the growth and migration of these cells in vitro was analysed. In the in vivo experiment, SCID mice were fed a diet containing 2% of either PS mixture or cholesterol plus 0.2% cholic acid and implanted with 2 x 10(6) tumour cells per mouse. Tumour growth was monitored for 8 weeks post inoculation. Animals fed the PS diet had tumours 40-43% smaller than those fed the cholesterol diet. Furthermore, the number of mice with lymph node and lung metastasis was almost one-half that of the cholesterol-fed group. In the in vitro studies, both beta-sitosterol and campesterol inhibited the growth of PC-3 cells by 70% and 14%, respectively, while cholesterol supplementation increased the growth by 18% when compared with controls. PS inhibited the invasion of PC-3 cells into Matrigel-coated membranes by 78% while cholesterol increased it by 43% as compared with the cells in the control media. Migration of tumour cells through 8 microm pore membranes was reduced by 60-93% when the PC-3 cells were in PS media, as compared with a 67% increase after cholesterol supplementation. PS supplementation reduced the binding of PC-3 cells to laminin by 15-38% and fibronectin by 23% while cholesterol increased binding to type IV collagen by 36%. It was concluded that PS indirectly (in vivo as a dietary supplement) and directly (in tissue culture media) inhibited the growth and metastasis of PC-3 cells. beta-Sitosterol was more effective than campesterol in offering this protection in most of the parameters studied.

Phytosterols reduce in vitro metastatic ability of MDA-MB-231 human breast cancer cells.

Metastasis plays a major role in morbidity and mortality from breast cancer. Differences in the incidence and mortality of breast cancer between societies suggest that environmental factors such as diet may play a role in the disease. Previous work from this laboratory suggests that dietary phytosterols (PS) may offer protection from breast cancer by inhibiting growth of the tumor and its metastasis in severe combined immunodeficient mice. Because metastasis is a multistep process, the aim of the present study was to investigate the effect of PS on some steps of the metastatic process: tumor cell invasion, adhesion, and migration. In addition, cell growth and cell cycle progression were evaluated. MDA-MB-231 cells were supplemented with cholesterol, beta-sitosterol, and campesterol. Cells were treated for 3 days with 16 microM sterol that was loaded on 5 mM cyclodextrin. beta-Sitosterol inhibited tumor cell invasion through Matrigel and adhesion of cells to plates coated with collagen I, collagen IV, fibronectin, and laminin compared with cholesterol treatments and controls. Cholesterol treatment resulted in increased adhesion to laminin and collagen IV, two basement membrane (BM) components that are implicated in signaling tumor cell invasion in this cell line. Only cholesterol treatment increased cellular migration. beta-Sitosterol inhibited cell growth by 70% compared with controls and induced cell cycle arrest at the G2/M phase. It is concluded that, among PS, beta-sitosterol may offer protection from breast cancer metastasis by inhibiting cell invasion of the BM, which may be mediated by its ability to limit the adhesive interaction of the tumor cell and the BM.

Phytosterols as anticancer dietary components: evidence and mechanism of action.

Phytosterols (PS) or plant sterols are structurally similar to cholesterol. The most common PS are beta-sitosterol, campesterol and stigmasterol. Epidemiologic and experimental studies suggest that dietary PS may offer protection from the most common cancers in Western societies, such as colon, breast and prostate cancer. This review summarizes the findings of these studies and the possible mechanisms by which PS offer this protection. These include the effect of PS on membrane structure and function of tumor and host tissue, signal transduction pathways that regulate tumor growth and apoptosis, immune function of the host and cholesterol metabolism by the host. In addition, suggestions for future studies to fill the gaps in our knowledge have been given.

Peanuts as a source of beta-sitosterol, a sterol with anticancer properties.

Work from our laboratory, as well as others, suggests a protective role of phytosterols (PS), especially beta-sitosterol, from colon, prostate, and breast cancer. Asians and vegetarians consume higher amounts of PS than Western societies. The latter societies have a higher incidence of these cancers than Asians and vegetarians. The aim of this study was to evaluate peanuts and its products as sources of PS in the American diet. Roasted peanuts contain 61-114 mg PS/100 g depending on the peanut variety, 78-83% of which is in the form of beta-sitosterol. Unrefined peanut oil contains 207 mg PS/100 g, which is similar to that of the US Department of Agriculture Nutrient Database. This value is higher than that of unrefined olive oil. Refining these oils results in reduction in PS concentration in the oil. This loss is greater in the case of olive oil than peanut oil. Further refining, such as deodorization, results in significant loss in PS, but hydrogenation after refining has a minimal effect on PS loss. Peanut butter, which represents 50% of the peanuts consumed in the United States, contains 144-157 mg PS/100 g. Peanut flour, which results from partial removal of oil from peanuts, contains 55-60 mg PS/100 g. The data suggest that peanuts and its products, such as peanut oil, peanut butter, and peanut flour, are good sources of PS.

Dietary phytosterol inhibits the growth and metastasis of MDA-MB-231 human breast cancer cells grown in SCID mice.

The objective of the present study was to investigate the effect of dietary phytosterols on the growth and metastasis of the human breast cancer MDA-MB-231 cell line xenografted in SCID mice. Two groups of animals were fed AIN-93G diet supplemented with 0.2% cholic acid and 2% sterol (cholesterol or phytosterol mixture) for 15 days before inoculation of the tumor into the right inguinal mammary fat pad. Tumor growth and food consumption were recorded weekly throughout the 8 weeks of the experiment. At the end of the experiment, the animals fed phytosterol had a 40% lower serum cholesterol and 20 and 30 fold higher serum beta-sitosterol and campesterol, respectively as compared to those fed cholesterol. There was no difference between the two groups in body weight and food consumption. However, the tumor size in animals fed phytosterols was 33% smaller (P < 0.03) and had 20% fewer metastases to lymph nodes and lungs than the cholesterol group. At termination, the tumor weight of the animals fed the phytosterol diet was also less (P < 0.07) than that of the cholesterol group. It is concluded that dietary phytosterols retard the growth and spread of breast cancer cells.

Effect of beta-sitosterol, a plant sterol, on growth, protein phosphatase 2A, and phospholipase D in LNCaP cells.

Previous work from this laboratory suggests an activation of the sphingomyelin cycle as a mechanism for growth inhibition with the incorporation of beta-sitosterol (SIT) into human prostate cancer LNCaP cells. In the present study we examined two key enzymes that have been shown to play a role in the sphingomyelin cycle. Dietary sterols (SIT and cholesterol) were compared for their effect on LNCaP cell growth, phospholipase D (PLD) activity, and protein phosphatase 2A (PP 2A) activity and expression. PP 2A has been suggested as a direct in vitro target of ceramide action on cell growth and apoptosis. Ceramide also inhibits phorbol myristate acetate-stimulated PLD. SIT (16 microM) increased PP 2A activity by 50% compared with cholesterol treatment in LNCaP prostate cells; however, SIT did not alter protein levels of PP 2A. There was an increase in PLD activity in the presence of phorbol myristate acetate in cells supplemented with 16 microM SIT compared with those supplemented with cholesterol after five days of treatment. The present study suggests that the activation of PP 2A added support to the role of the activation of the sphingomyelin cycle by SIT treatment. However, the increase in PLD activity, which was modest but significant, with SIT supplementation suggests that this pathway may be modulated by other mechanisms. This includes the incorporation of SIT into cell membranes that may alter fluidity and, thus, influence the activation of membrane-bound enzymes such as PLD.

Inhibition of growth and stimulation of apoptosis by beta-sitosterol treatment of MDA-MB-231 human breast cancer cells in culture.

Epidemiological and experimental studies have suggested a protective role of phytosterols (PS) in the development of some types of cancer such as colon and prostate cancer. No work has been reported on the role of PS in the development of breast cancer, the second leading cancer in woman. The present study was designed to examine the effect of the two most common dietary PS, beta-sitosterol (SIT) and campesterol, as compared to cholesterol, the main sterol in the Western diet, on growth, apoptosis and cytotoxicity of MDA-MB-231 human breast cancer cells in culture. In addition, we investigated the possible role of protein phosphatase 2A (PP2A), an enzyme that has been shown to regulate growth and apoptosis in tumor parameters studied. Breast cancer cell growth was found to be inhibited by 66% after 3 days and 80% after 5 days with 16 microM SIT. Both campesterol and cholesterol sustained tumor growth at levels comparable to that of the vehicle control. None of the sterols tested at this level (16 microM) induced cytotoxicity as measured by lactic dehydrogenase release. SIT supplementation for 3 days at 16 microM resulted in a 6-fold increase in apoptosis in cells when compared to cholesterol treated cells. SIT treatment was found to have no effect on the level and content of tumor cell PP2A. It is concluded that SIT, by a still unknown mechanism, may offer protection from breast cancer by inhibiting growth and stimulating apoptosis.

Effect of sterols and fatty acids on growth and triglyceride accumulation in 3T3-L1 cells.

Epidemiologic studies suggest a role of dietary fat in the development of obesity. Populations that consume Western diets have a higher incidence of obesity than do those that consume a vegetarian type diet such as Asians. Because dietary fats are made up mostly of triglyceride with minor lipids such as sterols, the objective of this study was to examine the effect of different fatty acids, the main component of triglycerides, and sterols on cell growth and triglyceride accumulation in 3T3-L1 cells. These cells are being used as an in vitro model for studying obesity because upon differentiation in culture they accumulate triglycerides. Cells were seeded at 5,000 cells/cm(2) and supplemented with 0, 3, 10, or 30 microM of oleic acid, elaidic acid, or docosahexaenoic acid (DHA). Similarly, cells were supplemented with 0, 2, 8, or 16 microM of cholesterol, beta-sitosterol (SIT), or campesterol. Cell growth was measured by cell counting. Cellular triglycerides were measured by the Oil Red O method. In some experiments, fatty acids were combined with sterols and growth and triglyceride content were assessed as described. Both DHA and SIT had inhibitory effects on 3T3-L1 cell growth. However, SIT was more potent than DHA in this regard. The combination of SIT and oleic acid was the most potent in inhibiting cell growth and increasing cellular triglyceride content. It is concluded that cell growth and triglyceride accumulation in 3T3-L1 cells is influenced by fatty acid and sterols. When used alone, DHA and SIT inhibit cell growth. SIT was more effective in this process than was DHA. There was an interaction between fatty acids and sterols. The most effective combination inhibiting cell growth and triglyceride concentration was the combination of SIT and oleic acid. This combination reduced cell growth and increased triglyceride accumulation. These data suggest that diets rich in both monounsaturated fatty acids and phytosterols may play a role in controlling obesity.

beta-Sitosterol activates the sphingomyelin cycle and induces apoptosis in LNCaP human prostate cancer cells.

Epidemiological evidence has shown that men consuming a low-fat, high-fiber diet containing high amounts of plant products have a lower risk of prostate cancer than men consuming a Western diet. One of the main differences between these two diets is the type of dietary fat, including dietary sterols. This study was undertaken to compare the effect of two dietary sterols on prostate cancer cells in vitro. beta-Sitosterol (SIT), the most common plant sterol, and cholesterol, an animal sterol, were compared for effect on LNCaP cell growth, differentiation, apoptosis, and sphingomyelin cycle intermediates. Cells were treated for up to seven days with sterols delivered by a cyclodextrin vehicle. Compared with cholesterol, SIT (16 microM) decreased growth by 24% and induced apoptosis fourfold, which was accompanied by cell rounding and a 50% increase in ceramide production. No effect was observed on differentiation as measured by prostate-specific antigen and prostatic acid phosphatase, although total acid phosphatase increased with SIT treatment for up to seven days. The results suggest that the decrease in cell number and increase in apoptosis associated with SIT treatment are mediated by activating the sphingomyelin cycle.

beta-Sitosterol inhibits growth of HT-29 human colon cancer cells by activating the sphingomyelin cycle.

The present study examined the SM cycle as a mechanism to explain the inhibitory effect of SIT on HT-29 cell growth. SIT was the main phytosterol in the diet. Supplementation of SIT at 16 microM for 5 days in the media inhibited growth by 55% as compared to cholesterol. SIT supplementation had no effect on sphingosine production. Ceramide production increased 45% with SIT supplementation as compared to cholesterol. Sterol supplementation had no effect on phospholipase C, a key enzyme in the PKC pathway. We concluded that the activation of the SM cycle may play a role in growth inhibition of HT-29 cells by SIT.

Effect of dietary phytosterols on rat tissue lipids.

The present study was designed to examine the incorporation of phytosterols (PS) in membranes and tissues of rats fed a diet containing 2% PS in the presence of 0.2% cholic acid for 22 days. The control diet contained 12 mg PS/100 g compared with 2,012 mg/100 g. Liver, kidney, testis, and prostate microsomes, plasma, and epididymal fat pads were examined for sterols. Fatty acid composition and phospholipid pattern were also examined in some tissues. The PS diet resulted in a fivefold increase in plasma PS compared with controls. PS was found to accumulate in adipose tissue and liver microsomes in rats fed the PS-supplemented diet. There was no effect of PS incorporation on microsomal cholesterol content, except in the testes, in which dietary PS reduced cholesterol content by 25%. Dietary PS increased 20:4n-6 and 22:5n-3 fatty acids in membranes of the liver, testis, and prostate but decreased 16:1 in liver microsomes. PS incorporation had no effect on the phospholipid pattern of the liver and testis.

Type of dietary fiber, not fat, alters phospholipase D and ornithine decarboxylase activities in the rat large intestine.

We investigated the effect of dietary fatty acid composition (n-6 vs. n-3) and fiber (highly fermentable vs. less fermentable) on the activities of phospholipase D (PLD) and ornithine decarboxylase (ODC) in the rat large intestine (cecum and proximal and distal colon). Twenty-four Sprague-Dawley rats (215-270 g) ate synthetic diets with 2% safflower oil plus 21.5% safflower or fish oil and 10% cellulose or guar gum for four weeks. Cecal bile acids and free fatty acids were higher in rats fed guar gum than in rats fed cellulose. Rats fed fish oil had more proximal colonic mucosal and cecal bile acids than those fed safflower oil. PLD activity was 23% lower in the proximal colon of rats fed guar gum than in those fed cellulose, but the mucosal weight was not different. ODC activity was lower but cecal mucosal wet weight was higher in the cecum of the rats fed guar gum than in the cecum of the rats fed cellulose. The activities of PLD and ODC are affected by dietary fiber and may not be accurate markers for tissue growth in the colonic mucosa.

Effect of dietary phytosterols on cell proliferation and protein kinase C activity in rat colonic mucosa.

The present study investigated the role of phytosterols in colonic cell proliferation and examined the possible role of protein kinase C (PKC) in this process. A total of 18 male Sprague-Dawley rats weighing 240-270 g were fed, for a period of 22 days, one of three experimental diets: a control diet, a diet supplemented with 0.2% cholic acid, or a diet supplemented with 0.2% cholic acid + 2% dietary phytosterols. Two hours before decapitation, animals were injected with 5'-bromo-2'-deoxyuridine (BrdU, 50 mg/kg body wt ip). Cell proliferation in the proximal colon was measured using a monoclonal antibody to BrdU. PKC activity in the proximal colonic mucosa was assayed using a myelin basic protein as a substrate. Cell proliferation was significantly increased by 276% with 0.2% cholic acid feeding compared with controls. The presence of 2% phytosterols in the diet abolished the cholic acid-induced hyperplasia. Cholic acid induced a 31% expansion of the proliferative zone. Only the cytosolic PKC was significantly lower in the phytosterol-fed group. Neither the total PKC nor the particulate PKC demonstrated an effect of phytosterols on enzyme activity. In conclusion, we found that dietary supplementation with 2% phytosterol has a significant protective effect on enhanced cell proliferation and that this effect is not mediated through the PKC system.

Effect of membrane lipid alteration on the growth, phospholipase C activity and G protein of HT-29 tumor cells.

The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12 x 10(3) cells/cm2 and supplemented with 30 microM of either 18:0, 18:2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18:0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18:2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18:0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18:2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 micrograms alamethicin and 42 microM free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation. PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.

The effect of unsaturated fatty acids on membrane composition and signal transduction in HT-29 human colon cancer cells.

The objective of the present study was to investigate the effect of membrane fatty acid (FA) composition on the activity of phospholipase C (PLC) in HT-29 human colon cancer cells. The membrane FA composition was altered by supplementing cultured cells with FAs of different composition. The FAs were stearic acid (18:0; SA), gamma linolenic acid (18:3 omega 6; gamma LnA); alpha linolenic acid (18:3 omega 3; alpha LnA;); eicosapentaenoic acid (20:5 omega 3; EPA) and docosahexaenoic acid (22:6 omega 3; DHA). The fatty acids were supplemented as a FA/BSA complex. Cells supplemented with SA served as the control. Tumor growth was followed by counting the number of cells in culture. The results indicate that polyunsaturated fatty acid (PUFA) supplementation had no consistent effect on tumor growth from 1 day to another throughout the 15 days of growth. The fatty acid composition of membranes indicates that cells incorporated and modified the supplemented fatty acids by desaturation, elongation and retroconversion. The unsaturation index (UI) of membranes of cells supplemented with EPA and DHA was higher than other groups. PLC activity; measured in the absence of GTP gamma(S) in the assay mixture; was not influenced by membrane FA modification. However, in the presence of GTP gamma(S) PLC of cells supplemented with 18:3(omega 6) was the lowest among the groups. It has been shown that 18:3(omega 6) accumulated the most in the phosphatidylethanolamine (PE) fraction. There was a negative correlation between the activity of PLC in the presence of G protein activation and PE 18:3 (omega 6) content without affecting UI. It was concluded that G protein may be sensitive to the level of 18:3(omega 6) content and not to the general fluidity of the membranes.

beta-Sitosterol inhibits HT-29 human colon cancer cell growth and alters membrane lipids.

The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.