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  • br Figure Ceramide analogues decrease Bcl xL


    Figure 3. Ceramide analogues decrease Bcl-xL and xIAP in human colon carcinoma cells. The metastatic human colon carcinoma SW620 Talaporfin were treated with the indicated ceramide analogues at the indicated concentrations for 48 h, and analyzed for Bcl-xL and xIAP protein levels by Western blotting analysis.
    4. Discussion
    For the synthesis of ceramide analogues, L-serine has been widely used as a starting material.10,11 A series of novel ceramide analogues have been synthesized by changing sphingosine backbone to a long-chain L-serinamide and introducing amide or imine substituents on the original nitrogen.14 These analogues exhibit high efficacy as anti-pro-liferative agents against human cancer cell lines.15,16 L-threonine, one of essential amino acids, has one more methyl and one more chiral carbon as compared to L-serine. Based on the medicinal chemistry phenomenon of “magic methyl” effect that links the dramatic change in affinity to the addition of a single methyl group in just the right place, we hypothesized that L-threonine is a better substrate for synthesis of ceramide analogues with higher solubility and greater bioactivity and made use of L-threonine as a starting material for the synthesis of novel ceramide analogues. We successfully synthesized 47 novel ceramide analogues with great solubility and high yield. Therefore, we have es-tablished a simple and yet efficient synthesis procedure to chemically synthesize new ceramide analogues starting from L-threonine.
    In vitro cytotoxicity analysis revealed that the majority of these 47 new ceramide analogues are bioactive as they can effectively suppress human cancer cell growth. Several analogues (i.e. 5ch and 5cm) ex-hibited super ant-proliferative activity with IC50 of 4.8–11 μM. Initial testing of four of these new ceramide analogues indicate that they are also effective in suppression of established human cancer xenograft growth in vivo. However, it is interesting to notice that the analogue with the most potent in vitro cytotoxicity (i.e.5ch) does not exhibit the greatest suppressive efficacy against human xenograft growth in vivo, suggesting that in addition to its cytotoxicity potential, other factors, such as pharmacokinetics and pharmacodynamics of the analogues may also be important determinants of in vivo anti-cancer activity. More studies are apparently needed to expand the in vivo studies to more analogues to identify the most effective and low toxic ceramide ana-logues among these 47 compounds for cancer therapy.
    TRAIL is a cancer-selective agent that has been extensively tested in 
    human cancer patients for Talaporfin the last decade. However, so far, the test results in human cancer patients are not encouraging and the hope for developing TRAIL or TRAIL receptor agonist for human cancer therapy
    is fading. This outcome is not totally surprising since it is known that most human cancer cells are resistant to TRAIL.33,34 We screened some
    of these new ceramide analogues with IC50 greater than 50 µM in five types of human cancer cells, and observed that seven analogues are effective in overcoming human cancer cell resistant to TRAIL. Our data thus indicate that these new ceramide analogues have the potential to be developed as an adjunct agent for TRAIL-based cancer therapy to improve the efficacy of TRAIL therapy.
    Numerous studies have demonstrated that ceramide targets the Bcl-2 family proteins and IAPs to induce cellular apoptosis.24–29 We ob-served that indeed the new ceramide analogues can significantly de-crease the anti-apoptotic Bcl-xL and xIAP protein level in human cancer cells.31 However, the although degree of decrease in xIAP and Bcl-xL protein level induced by a particular analogue is generally associated with the in vitro cytotoxicity, the decrease of Bcl-xL and xIAP is only partially associated with the in vivo tumor suppression efficacy of a particular ceramide analogue. These observations suggest that these ceramide analogues suppress tumor growth only partially through in-hibiting these anti-apoptotic mediators. Therefore, further studies are needed to further elucidate the molecular mechanisms underlying ceramide-mediated apoptosis and tumor suppression.
    In summary, we have developed a simple and efficient new proce-dure to chemically synthesize ceramide analogues using L-threonine as substrate. A total of 47 novel ceramide analogues have been synthe-sized. Initial analysis indicates that many of these ceramide analogues are bioactive as anti-cancer agents with IC50 as low as 4.8 μM. Furthermore, these ceramide analogues also exhibited potent sensiti-zation activity to overcome human cancer cell resistant to TRAIL. More significantly, several of these new ceramide analogues suppressed es-tablished human cancer xenograft growth in vivo without significant toxicity. These ceramide analogues thus hold great potential to be de-veloped as a class of new anti-cancer agents.