Affiliations of authors: Glycobiology Program, Center for Cancer and Transplantation Biology, Children's Research Institute, Washington, DC, and Departments of Pediatrics and Biochemistry/Molecular Biology, The George Washington University School of Medicine, Washington, DC.
Correspondence to: Stephan Ladisch, M.D., Center for Cancer and Transplantation Biology, Children's Research Institute, 111 Michigan Ave., N.W., Washington, DC 20010 (e-mail: SLadisch{at}cnmc.org).
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ABSTRACT |
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INTRODUCTION |
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One of these factors may be the sialic acid-containing glycosphingolipids, termed "gangliosides." They are component molecules of the outer leaflet of the plasma membrane and are believed to play a role in tumor formation and progression (1). Gangliosides contain a hydrophobic ceramide anchored in the membrane and a hydrophilic oligosaccharide chain with one or more sialic acids facing the extracellular environment and, along with neutral glycosphingolipids (2), are shed by tumor cells (35). Gangliosides are involved in various cellular functions, including signal transduction, regulation of cell proliferation and differentiation, cellcell recognition and adhesion, and cell death (610).
Properties of shed gangliosides include antagonism of host immune function by multiple immunosuppressive effects [such as inhibition of normal lymphoproliferative responses to mitogens and antigens (3,11), interleukin 2-dependent cell proliferation (12), helper T-cell proliferation (13), and natural killer cell activity (14)] and enhancement of apoptotic cell death of thymocytes (15). Therefore, shedding of immunosuppressive gangliosides may be one mechanism used by tumor cells to inhibit host immune responses and thus escape destruction by the immune system.
In line with these observations, past studies of the tumor-forming characteristics of various types of tumor cells containing different concentrations of gangliosides (1619) have suggested that tumor cells with higher concentrations of gangliosides are more tumorigenic than comparable cells with lower concentrations of gangliosides. In one of these studies (17), tumor formation by a poorly tumorigenic, ganglioside-deficient murine lymphoma cell line was greatly enhanced by increasing the ganglioside concentration of the cells with purified gangliosides from a related, but highly tumorigenic, cell line.
In this study, we have also observed such a relationship, comparing tumor formation by MEB4 melanoma cells and a related ganglioside-deficient subline, GM95 (20). However, whether tumor formation would be affected by pharmacologically modifying the intrinsic ganglioside content of tumor cells has not been determined. We hypothesized that such a modification (i.e., depletion of the ganglioside content of tumor cells) would reduce their ability to form tumors in vivo. To test this hypothesis, we studied tumor formation and metastasis by a ganglioside-rich B16 murine melanoma subline, MEB4. To deplete the MEB4 cells of gangliosides, we used a new selective inhibitor of glycosphingolipid synthesis, 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) (20), which blocks cellular ganglioside synthesis without reducing the cell proliferation rate. We report that pharmacologically induced cellular ganglioside deficiency results in a striking reduction of tumor formation and metastasis in mice.
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MATERIALS AND METHODS |
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MEB4 and GM95 cells (20), two sublines of the B16 murine melanoma, were obtained from the RIKEN Cell Bank, Saitama, Japan. The cells were maintained in Dulbecco's modified Eagle medium (product 12430-054; Life Technologies, Inc. [GIBCO], Gaithersburg, MD) supplemented with 10% fetal bovine serum. Cultures were maintained in an atmosphere of 5% CO2/95% air in a humidified incubator at 37°C.
Inhibition of Glucosylceramide Synthase by PPPP
PPPP (>98% pure by high-performance thin-layer chromatographic [HPTLC] analysis) was purchased from Matreya, Inc., Pleasant Gap, PA. A stock solution of 1 mM PPPP was prepared by dissolving PPPP in 1% ethanol in phosphate-buffered saline (PBS) with sonication and was stored at 4°C. Before addition to the cell cultures, the stock solution was warmed to 37°C and diluted 1:10 in culture medium. This solution in turn was added to the cell cultures to give the desired final concentration. After exposure of the cells to PPPP for up to 5 days, cells were harvested and gangliosides were analyzed.
To assess the effect of PPPP on the proliferative characteristics of MEB4 cells, we cultured the cells in 96-well plates with or without PPPP for up to 4 days. Before harvest, cells were incubated for 4 hours with [3H]thymidine at 3.3 µCi/mL and then rinsed twice with ice-cold PBS. Cells were detached in 1% trypsinEDTA, and incorporated [3H]thymidine was quantified by ß-scintillation counting (21).
Ganglioside Analysis
Total ganglioside fractions were isolated from trypsinized MEB4 and GM95 cells that had been pelleted by centrifugation at 4°C at 300g for 10 minutes. Cell pellets were lyophilized and extracted twice with chloroform/methanol, 1:1 (vol/vol). The combined extracts were reduced to 25% of the original volume and cooled to 4°C overnight, and then insoluble material was removed by centrifugation at 4°C at 1000g for 10 minutes. The supernatant containing the total lipid was dried under nitrogen and lyophilized, and the total lipid extract from 108 MEB4 or GM95 cells was partitioned twice in 6 mL of diisopropyl ether/1-butanol, 60:40 (vol/vol), and 3 mL of 0.1% aqueous NaCl. The final aqueous phase was lyophilized and redissolved in a small volume of distilled water. Salts and low-molecular-weight impurities were removed from the total ganglioside fraction by Sephadex G-50 gel filtration. Gangliosides, recovered in the void volume, were lyophilized, and the colorimetric resorcinol assay (22) was used to quantify lipid-bound sialic acid. For HPTLC analysis, recovered gangliosides were spotted on precoated Silica Gel-60 HPTLC plates (10 x 20 cm), which were then developed in chloroformmethanol0.25% CaCl2 2H2O, 60:40:9 (vol/vol). Gangliosides were visualized as purple bands with the resorcinolHCl reagent (22). Densitometric scanning analysis of the samples was performed with a Shimadzu CS-930 dual-wavelength HPTLC scanner. The molecular structure of the gangliosides of MEB4 cells was determined by negative-ion, fast-atom bombardment mass spectrometry (23).
Tumor Formation
Female syngeneic C57BL/6 mice, 68 weeks old, cared for in accordance with institutional guidelines, were used. MEB4 cells were cultured with or without 0.5 µM PPPP for 4 days, and then these cells and GM95 cells were washed in PBS and resuspended in 0.9% NaCl. Cell viability was assessed by trypan blue dye exclusion. The resuspended cells (20 µL containing 105 or 104 cells) were injected intradermally on the back of each mouse. Mice were examined for tumor formation twice weekly for 10 weeks.
Experimental Metastasis
The effect of ganglioside depletion on the ability of MEB4 cells to form metastases was assessed, as described (24). Control MEB4 cells, ganglioside-depleted MEB4 cells, and GM95 cells were prepared as above, and 12 x 105 cells were suspended in 100 µL of 0.9% NaCl and injected into the tail vein of C57BL/6 mice. Four weeks after injection, the mice were killed and visually inspected under a dissecting microscope for pulmonary and extrapulmonary metastases, which were readily identified by their melanotic appearance.
Statistical Analysis
The statistical significance of differences in tumor formation between control groups and treatment groups was determined with the 2 and Fisher's exact tests. The statistical significance of differences in metastasis between control groups and treatment groups was assessed by determination of the 97% confidence intervals. All P values are from two-sided tests.
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RESULTS |
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Characterization of the purified gangliosides from MEB4 cells by HPTLC revealed a prominent doublet with the mobility of GM3 ganglioside, as previously reported (20). MEB4 cells have 37 nmol of gangliosides per 108 cells, and ganglioside-deficient GM95 cells have 2.5 nmol of gangliosides per 108 cells. With this relatively high ganglioside content, MEB4 cells actively shed GM3 ganglioside into culture medium at a rate of 0.35% of their total content of gangliosides per hour or 130 pmol per 108 cells per hour.
The structure of GM3, the major ganglioside, was confirmed by mass spectrometry (22). There were three major molecular ion peaks at m/z 1152, 1262, and 1264 and three less prominent peaks at m/z 1208, 1180, and 1236 (Table 1). These values correspond to ganglioside GM3 with proposed ceramide structures of d18:1-C16:0, d18:1-C24:1, d18:1-C24:0, d18:1-C20:0, d18:1-C18:0, and d18:1-C22:0, respectively, reflecting a heterogeneity of ceramide structure that is characteristic of tumor cells.
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In initial experiments, we assessed the effects of ganglioside content on the tumorigenicity of ganglioside-rich MEB4 cells and ganglioside-poor GM95 cells (both of which were derived from B16 murine melanoma cells). We expected that, if the cellular ganglioside content impacted tumorigenicity, then we might detect a difference in the ability of these melanoma cell lines to form tumors. Syngeneic C57BL/6 mice were given an intradermal injection of either 105 or 104 cells. We used the intradermal route to optimize the intercellular and cellularextracellular environmental interactions that may, in turn, modulate the process of tumor formation (17). As shown in Table 2, ganglioside-rich MEB4 cells were much more tumorigenic; 105 MEB4 cells caused tumors in 100% of the mice, but 105 GM95 cells caused only one tumor among 15 mice (P<.001, Fisher's exact test). A similar difference was observed when 104 cells were injected (Table 2
). These results suggest that the differences in ganglioside content and shedding in these two cell lines may account, at least in part, for the differences in tumorigenicity.
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Having shown that there is a difference in tumor formation between the ganglioside-rich and ganglioside-poor B16 melanoma sublines, we tested the influence of endogenous tumor cell gangliosides on tumor formation and metastasis by pharmacologically modifying the ganglioside content of tumor cells. We inhibited ganglioside synthesis of MEB4 cells with PPPP under conditions that effectively depleted gangliosides of the MEB4 cells without reducing the rate of cell proliferation. Exposure of MEB4 cells to up to 0.5 µM PPPP for 4 days resulted in a concentration-dependent decrease in the content of gangliosides (Fig. 1, A); 0.5 µM PPPP resulted in a 90% reduction in the content of total cellular gangliosides, as shown by HPTLC densitometry. The PPPP-mediated reduction of gangliosides was also time dependent; 0.5 µM PPPP reduced ganglioside content by 50% within 24 hours and by 90% after 45 days (Fig. 1, B
).
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We next evaluated PPPP for potential cytotoxicity and an effect on cell proliferation, which could influence tumor formation. Specifically, if PPPP reduced the rate of cell proliferation, as has been observed with other inhibitors such as D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (25), this, in itself, could adversely affect (inhibit) tumor formation. Under the experimental conditions described above, 0.5 µM PPPP was not cytotoxic as assessed by trypan blue dye exclusion: 98% or more of the MEB4 cells remained viable after incubation with 0.5 µM PPPP for up to 4 days. Furthermore, 0.5 µM PPPP did not reduce the proliferation rate of MEB4 cells, as quantified by [3H]thymidine uptake, at any time tested (Fig. 2), even when cellular gangliosides were almost completely depleted (day 4). Overall, these studies demonstrate that survival and proliferation of MEB4 cells are not reduced by PPPP, even under conditions where ganglioside synthesis was almost completely inhibited. This excludes inhibition of tumor cell proliferation as a cause of decreased tumor formation in vivo.
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To assess the effects of endogenous ganglioside depletion on tumor formation, we compared the tumorigenicity of control MEB4 cells with that of ganglioside-depleted MEB4 cells. MEB4 cells were incubated with 0.5 µM PPPP for 4 days to deplete endogenous gangliosides. The cells were then harvested, washed, resuspended in 0.9% NaCl, and injected intradermally into syngeneic C57BL/6 mice. The combined results of three experiments are shown in Fig. 3. When 105 cells were injected per mouse, untreated MEB4 (control) cells caused tumors in 100% of the mice at 8 weeks, whereas PPPP-treated cells caused tumors in 40% of the mice (in seven of 15 mice, in seven of 14, and in three of 15) (P>.001, Fisher's exact test, two-sided; Fig. 3, A
). Even when only 104 cells were injected per mouse (Fig. 3, B
), MEB4 control cells caused tumors in 45% of the mice (in seven of 14 mice, in seven of 15, and in six of 15), whereas PPPP-treated MEB4 cells caused tumors in 9% of the mice (in one of 15 mice, in three of 15, and in none of 15) (P = .005, Fisher's exact test). These results clearly show that inhibition of glucosylceramide synthase, with its consequent inhibition of ganglioside synthesis and reduction of cellular ganglioside content, results in markedly reduced tumorigenicity of MEB4 melanoma cells.
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To complement the studies of tumor formation, we tested the metastasizing capability of tumor cells in an established model of pulmonary metastasis (26), in which 1 or 2 x 105 control or ganglioside-depleted MEB4 cells or ganglioside-deficient GM95 cells were injected via tail vein into 8-week-old syngeneic C57BL/6 mice. As above, MEB4 cells were incubated in 0.5 µM PPPP for 4 days to deplete gangliosides. A statistically significant difference in metastatic potential was observed (Table 3); ganglioside-depleted MEB4 cells caused statistically significantly fewer pulmonary metastases than did the control cells. When 2 x 105 ganglioside-depleted versus control cells were injected, a mean of five versus 25 pulmonary metastases per mouse, respectively, was observed; when 1 x 105 cells were injected, a mean of one versus three pulmonary metastases per mouse was observed. We also examined mice for the development of metastases in other tissues, such as liver, kidney, and intestines. Extrapulmonary metastases were reduced after ganglioside depletion of MEB4 cells; a total of 16 extrapulmonary metastases were identified in 10 mice receiving 2 x 105 control MEB4 cells compared with two extrapulmonary metastases in mice receiving PPPP-treated MEB4 cells. Finally, as shown in Table 3
, ganglioside-deficient GM95 cells and PPPP-treated, ganglioside-depleted MEB4 cells gave similar results, confirming the importance of cellular gangliosides in enhancing the metastatic potential of tumor cells.
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DISCUSSION |
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Earlier studies of this issue generally compared the biologic properties of cell lines or sublines with different cellular ganglioside contents. Ladisch et al. (17) found that, among several murine AKR lymphoma cell sublines, cells with high ganglioside content were highly tumorigenic, cells with low ganglioside content were poorly tumorigenic, and the addition of gangliosides isolated from highly tumorigenic cells greatly enhanced tumor formation by ganglioside-deficient, poorly tumorigenic cells. Other studies have also demonstrated that a high ganglioside content was associated with high tumorigenicity (16,18,19) and a high metastatic potential (2729). The importance of tumor cell gangliosides to tumorigenicity and metastasis is underscored, for example, by the following observation: Compared with nontransfected melanoma cells, murine melanoma cells transfected with a complementary DNA for sialidase, an enzyme that destroys intact gangliosides by hydrolyzing their sialic acids, resulted in fewer metastases when injected into mice (26). Thus, the prior studies clearly suggest that cellular gangliosides have an important influence on tumor formation and/or progression. Similarly, in the present study, ganglioside-poor GM95 cells were poorly tumorigenic compared with ganglioside-rich MEB4 cells. However, these two cell lines, as in the prior comparative studies, differ in other characteristics [e.g., proliferative properties and morphologic characteristics (20)], which may also contribute to differences in tumorigenicity. Thus, critical evidence essential to proving the hypothesis that tumor cell gangliosides have an enhancing role in the initial formation of tumors by a transformed cell has been lacking. This evidence is that reduction of the ganglioside content of ganglioside-rich, highly tumorigenic cells (such as MEB4), by a specific pharmacologic approach, would reduce their ability to form tumors.
The pharmacologic approach that we used to assess the role of gangliosides in tumor formation overcomes a substantial drawback of many previous studies. This drawback is that the studies were primarily based on the addition of exogenous gangliosides, on an enzyme treatment of the tumor cells, or on a comparison of pairs of tumor cell lines with most likely multiple phenotypic and or genotypic differences. These problems can be circumvented by using inhibitors of ganglioside synthetic enzymes to block endogenous cellular ganglioside production. One such molecule is PDMP, an inhibitor of glucosylceramide synthase (24,30,31). PDMP treatment of mice carrying Ehrlich ascites carcinoma cells or rats carrying C6 glioma cells reduced the rate of tumor growth (30), and preincubation of murine Lewis lung carcinoma cells with PDMP reduced their ability to metastasize (24). However, PDMP also substantially inhibits cell proliferation (25,31,32) and causes intracellular accumulation of ceramide (25,33), which, in turn, triggers apoptotic cell death (8), both potential causes of reduced tumor growth and metastasis that are independent of changes in cellular ganglioside content. We, therefore, turned to PPPP, a derivative of PDMP, in which the acyl chain length is increased from 10 to 16 carbons and a morpholine ring is replaced with a pyrrolidine (34). PPPP is a more specific inhibitor of glucosylceramide synthase that does not alter the cell growth rate in vitro or cause the accumulation of ceramide at concentrations that efficiently block glucosylceramide synthesis and consequently inhibit ganglioside synthesis (34,36).
The reduction in the incidence of tumor formation and metastasis that was associated with the abrogation of cellular ganglioside synthesis and effective ganglioside depletion by PPPP was striking and shows, to our knowledge, for the first time that a reduction in tumor cell ganglioside content substantially reduces tumor incidence. It will be of great interest to confirm these findings in other tumor systems, especially since PPPP has a similar effect on glycosphingolipid metabolism in other tumor cell types (36,37). How tumor formation and metastasis are reduced remains to be elucidated, but several possibilities exist and knowledge of certain biologic properties of gangliosides gives some clues. First, ganglioside shedding, including the shedding of immunosuppressive gangliosides, is inhibited by the blockade of ganglioside synthesis (31). These immunosuppressive gangliosides inhibit host immune responses to tumors by suppressing immune cell function at the tumor site (3,17,3840). In fact, we recently have obtained direct evidence that the GM1b ganglioside shed by a murine lymphoma inhibits the syngeneic, specific immune response against this tumor in vivo (41). This includes the primary antitumor immune response, the secondary response, and the generation of tumor-specific cytotoxic lymphocytes. Second, ganglioside expression on the tumor cell surface may promote (7,29) [and decreased ganglioside expression caused by PPPP may hamper (26)] the processes of cell migration and adhesion, which facilitate tumor cell metastasis. Therefore, although the cellular mechanisms are not fully elucidated, the substantial reduction of tumor formation and metastasis by depletion of tumor cell gangliosides with PPPP suggests that this highly specific pharmacologic interference with glycosphingolipid metabolism warrants further study as a potential experimental therapeutic approach to cancer.
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NOTES |
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We thank Dr. Douglas Gage, Department of Biochemistry, and the National Institutes of Health-Michigan State University Mass Spectrometry Facility, Michigan State University, for performing the mass spectrometric analysis and Dr. Bonnie LaFleur for her statistical analyses.
Presented in part at the 89th annual meeting of the American Association for Cancer Research, New Orleans (LA), 1998.
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Manuscript received August 4, 1999; revised March 22, 2000; accepted March 27, 2000.
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