Exogenous Sphingolipids To Prevent Cancer In Vivo

The amphiphilic nature of sphingolipid metabolites and their inherent toxicity makes their site-directed administration in vivo a challenge. However, topical, intraperitoneal, and intravenous injections and oral administration of complex sphingolipids and sphingolipid metabolites have been reported.

25.3.1 Topical Administration

Sencar mice were treated with dimethylbenz-[a]-anthracene to induce skin cancer. Topical application of sphingosine, methylsphingosine, and N-acetylsphingosine did not inhibit the development of papillomas, and at high doses, the formation of papillomas was even enhanced.48,49 This was also seen in a follow-up study measuring the efficacy of sphingosine, N-methylsphingosine, and N-acetylsphin-gosine; again, the sphingolipids did not change papilloma incidence,50 but both N-methylsphingosine and N-acetylsphingosine increased cancer-free survival. Furthermore, weekly application of sphingosine and N-acetylsphingosine for 10 weeks after treatment with phorbol esters suppressed tumor progression.50

Another sphingolipid derivative, safingol, the L-threo isomer of sphinganine, a potent inhibitor of protein kinase C, was developed to treat dermatoses and cancer. Topical application of safingol, however, caused liver damage that was more pronounced in female than in male rats possibly due to insufficient clearance of safingol by cytochrome P450 isozymes.51

Topical application of a 1% mixture of short-chain ceramides twice per day to patients with cutaneous breast cancer resulted in a partial response in only 1 of 26 patients, which was found to be not promising enough to conduct further studies using this approach.52

25.3.2 Administration of Sphingolipids via Injection Intraperitoneal Injection

To the best of our knowledge, chemopreventive studies with sphingolipids administered via intraperitoneal (i.p.) injection have not been reported. Some chemo-therapeutic studies used ceramide analogs that induced apoptosis in vitro also induced apoptosis in vivo in human colon cancer xenografts, and significantly reduced their tumor size.53 However, this study did not mention the toxic side effects of the successful dose. Intravenous Injection

The systemic delivery of sphingolipids is hindered by their hydrophobicity, their possible degradation by enzymes, and their toxicity. Single doses of safingol (l-threo-sphinganine, see above) up to 5 mg/kg body weight did not cause adverse effects in rats or dogs,54 but repeated injections increased its plasma concentration, and caused hemolysis and marked renal and hepatoxicity at higher dosages. Furthermore, safingol caused degeneration and necrosis of the intima of the venes at the injection sites. In contrast, injection of sphingosine, dimethylsphingosine, or trimethylsphingosine in mice that have been inoculated with MKN74 human gastric cancer cells showed a pronounced and sustained inhibition of tumor growth by the methylated sphingosine derivatives that did not cause side effects.55 The delivery of sphingolipids via liposomes has been shown to be very effective in cell culture56 and was intended to protect ceramide from degradation or precipitation, and generate a maximum ceramide increase in cancer cells. Repeated injections of ceramide-containing liposomes into mice carrying breast tumors resulted in a significant decrease of tumor size without causing severe toxic side effects.57 Ceramide liposomes also suppressed ovarian cancer cell dissemination.58 Ceramides dissolved in soybean oil reduced pulmonary metastases derived from Meth A-T tumor cells.59 However, these are again chemotherapeutic approaches; whether there are chemopreventive applications of these methods is not known.

25.3.3 Oral Administration

Topical administration appears to be effective in inhibiting skin cancer progression, and either route of injection is not ideal for long-term cancer prevention strategies and, thus, likely is more important in treatment strategies. In contrast, oral administration is a convenient way to deliver sphingolipids and, therefore, appears to be a more relevant route in the prevention of colon cancer and possibly other sites. Natural Sphingolipids

Sphingolipids are minor components of food, but milk and meat products, eggs, and soybeans are rich in sphingolipids.60 In animal products, the major sphin-golipid is sphingomyelin, containing a sphingosine backbone and amid-bound fatty acids that are mostly saturated and contain 16, or 22 to 24 carbons. In plants, the prominent sphingolipids are cerebrosides, containing a variety of different sugar headgroups (glucose, galactose, mannose), sphingoid bases, and fatty acids (see Figure 25.1). Intestinal cells are constantly exposed to bioactive sphingolipid metabolites when dietary complex sphingolipids are hydrolyzed to ceramide and sphingosine by intestinal enzymes. Ceramides and sphingoid bases are efficiently absorbed by small intestinal cells,61 and it seems unlikely that orally administered sphingosine could reach the colon in appreciable amounts. However, about 10% of complex sphingolipids reach the colon intact and can be hydrolyzed by the colonic microflora to ceramides and sphingoid bases.61 Due to a limited digestion of complex sphingolipids to ceramide and sphingoid bases,62 even high amounts of sphingolipids (1% of the diet by weight or 50 to 100 times more than the estimated average consumption) did not cause side effects in mature rats or their offspring.63 This indicates that oral administration of complex sphingolipids is a safe route of delivering bioactive molecules to colonic cells. To test if this is sufficient to prevent colon tumor formation, complex sphingolipids isolated from buttermilk (sphingomyelin, glucosylceramide, lactosylceramide, and ganglioside GD3) were mixed into an essentially sphingolipid-free AIN 76A diet at 0.1% and fed to carcinogen-treated CF1 mice (Figure 25.2A). The end point of these studies was the appearance of early morphological changes in colon tumorigenesis, the aberrant crypt foci (ACF), precursors of adenomas, and adenocarcinomas. All the tested complex sphingolipids reduced ACF formation by 50 to 70%64-66 (Figure 25.3). This was not unexpected because the same metabolites are released in the intestinal tract from these complex sphingolipids.66 By using the same study design, ganglioside GM1 extracted from brain64 or glucosylceramide derived from soy that contains a sphingoid base with an 8.9-trans double bond also prevented ACF formation.67 These studies demonstrate that the beneficial effects of orally administered natural sphingolipids are not specific for those containing certain headgroups or sphingoid bases, and also not specific for the mouse model since sphingolipids (sphingomyelin) also reduced chemically induced ACF in rats.68

To confirm that the suppression of early stages of colon cancer indeed translates into a suppression of colon tumors, CF1 mice were fed sphingomyelin for 52 weeks, beginning before tumor initiation. This reduced the tumor formation by more than 70%69 (Figure 25.4A). However, administration of sphingolipids only after tumor initiation, e.g., when the damage to the colonic cells already has occurred, yielded in the same suppression of tumor formation, suggesting that there is a "window of opportunity" for the prevention of colon cancer with orally administered sphingolipids that includes predisposed and premalignant cells. How long carcinogen-treated cells respond to these doses of sphingolipids, i.e., if this effect is limited to pre-neoplastic cells (chemopreventive approach) or if the regulation of already more progressed cells is also possible with these doses (chemotherapeutic approach) remains to be determined.

Rodent models are available that closely resemble the human disease to assess the efficacy of orally administered sphingolipids on human colon cancer. C57/B6JMin/+ mice (multiple intestinal neoplasia, Min mice) carry mutations in the APC (adenomatous polyposis coli) gene that is found in almost all patients with familial adenomatous polyposis and 40 to 80% of sporadic colon cancer.70 71 Feeding Min mice with a mixture of complex sphingolipids (70% sphingomyelin, 5% lactosylceramide, 7.5% glucosylceramide, and 7.5% ganglioside GD3) at 0.1% of the diet for 65 days reduced tumor formation throughout the intestinal tract by 40%. Adding ceramide to this mixture but maintaining sphingolipids at 0.1% of the diet increased tumor suppression to 50%.72 Similar results were observed


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