High-effective biosynthesis of baccatin â ¢ by using the alternative acetyl substrate, N-acetyl-d-glucosamine.

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.

Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression.

BACKGROUND: Fatty acid synthase (FAS) performs the anabolic conversion of dietary carbohydrate or protein to fat. FAS expression is low in most normal tissues, but is elevated in many human cancers, including androgen-sensitive and androgen-independent prostate cancer. METHODS: Immunohistochemical evaluation of FAS expression was performed in human prostate cancer specimens under various states of androgen ablation. In vitro and in vivo prostate cancer models were evaluated for FAS expression and activity under androgenic and androgen-depleted conditions, and were tested for sensitivity to antimetabolite drugs that target fatty acid synthesis. RESULTS: While FAS expression in the prostate was androgen responsive, it persisted or was reactivated in human prostate carcinoma after androgen ablation, and was high in 82% of lethal tumors examined at autopsy. Similar patterns of FAS expression and fatty acid synthesis were seen in cell culture and xenograft models of human prostate cancer. Pharmacologic inhibition of FAS resulted in a dose-dependent reduction of tumor growth in these models, including fourfold inhibition of an androgen-independent human prostate cancer xenograft with little associated toxicity. CONCLUSIONS: The data suggest that FAS expression/FA synthesis provides an important functional aspect of the malignant phenotype in prostate cancer, perhaps supporting cell growth or survival. FAS expression may be upregulated by alternate signaling pathways important for prostate cancer growth under androgen withdrawal. The re-emergence of FAS expression and activity during the development of androgen independence demonstrate that FAS may serve as a novel target for antimetabolite therapy in prostate cancer.

Fatty acid synthase (FAS) expression in human breast cancer cell culture supernatants and in breast cancer patients.

Fatty acid synthase (FAS) is selectively expressed in certain human cancers, including carcinoma of the breast, prostate, colon, ovary, and endometrium, compared to normal human tissues and therefore is a putative tumor marker. In this study, we found FAS concentrations were elevated in cell culture supernatants during cell growth in two human breast cancer cell lines but not other cancer cell lines. A quantitative enzyme-linked immunosorbent assay and Western blot analysis were employed in this study. In addition, serum FAS levels were significantly higher in breast cancer patients with different clinical stages (Stage II: 0.59+/-0.09 units/l, Stage III: 0.79+/-0.13 units/l, and Stage IV: 1.39+/-0.35 units/l) compared with healthy subjects (0.27+/-0.02 units/l, P<0.05). Taken together, our data suggest that FAS expression may be a useful tumor marker for breast cancer and play a role in assessing cancer virulence.

Pharmacological inhibition of fatty acid synthase activity produces both cytostatic and cytotoxic effects modulated by p53.

Fatty acid synthetic metabolism is abnormally elevated in tumor cells, and pharmacological inhibitors of the anabolic enzyme fatty acid synthase (FAS), including the natural product cerulenin and the novel synthetic compound c75, are selective inhibitors of tumor cell growth. We have recently reported that these two FAS inhibitors both produce rapid, potent inhibition of DNA replication and S-phase progression in human cancer cells, as well as apoptotic death. Here we report an additional characterization of the cellular response to FAS inhibition. RKO colon carcinoma cells were selected for study because they undergo little apoptosis within the first 24 h after FAS inhibition. Instead, RKO cells exhibited a biphasic stress response with a transient accumulation in S and G2 at 4 and 8 h that corresponds to a marked reduction in cyclin A- and B1-associated kinase activities, and then by accumulation of p53 and p21 proteins at 16 and 24 h and growth arrest in G1 and G2. The response of RKO cells to FAS inhibition resembled a genotoxic stress response, but DNA damage did not appear to be an important downstream effect of FAS inhibition, because none was detected using the single cell gel electrophoresis assay (comet assay) to assess DNA damage. p53 function is probably important in protecting RKO cells from FAS inhibition because, similar to many other tumor lines, RKO cells expressing a dominant negative mutant p53 gene underwent extensive apoptosis within 24 h after FAS inhibition. Sensitization of cells to FAS inhibitors by the loss of p53 raises the possibility that these agents may be clinically useful against malignancies carrying p53 mutations. Whereas induction of apoptosis appeared related to accumulation of the substrate, malonyl-CoA, after FAS inhibition, the cytostatic effects were independent of malonyl-CoA accumulation and may have resulted from product depletion.

Sterol regulatory element-binding protein-1 participates in the regulation of fatty acid synthase expression in colorectal neoplasia.

Endogenous fatty acid synthesis has been observed in certain rapidly proliferating normal and neoplastic tissues. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of lipogenic genes including fatty acid synthase (FAS), the major biosynthetic enzyme for fatty acid synthesis. We have previously shown that SREBP-1, FAS, and Ki-67, a proliferation marker, colocalized in the crypts of the fetal gastrointestinal tract epithelium. This study sought to determine whether SREBP-1 participates in the regulation of proliferation-associated fatty acid synthesis in colorectal neoplasia. An immunohistochemical analysis of SREBP-1, FAS, and Ki-67 expression in 25 primary human colorectal carcinoma specimens showed colocalization in 22 of these. To elucidate a functional linkage between SREBP-1 activation and proliferation-associated FA synthesis, SREBP-1 and FAS content were assayed during the adaptive response of cultured HCT116 colon carcinoma cells to pharmacological inhibition of FA synthesis. Cerulenin and TOFA each inhibited the endogenous synthesis of fatty acids in a dose-dependent manner and each induced increases in both precursor and mature forms of SREBP-1. Subsequently, both the transcriptional activity of the FAS promoter in a luciferase reporter gene construct and the FAS expression increased. These results demonstrate that tumor cells recognize and respond to a deficiency in endogenous fatty acid synthesis by upregulating both SREBP-1 and FAS expression and support the model that SREBP-1 participates in the transcriptional regulation of lipogenic genes in colorectal neoplasia.

Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts.

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines.

Pharmacological inhibitors of the anabolic enzyme, fatty acid synthase (FAS), including the natural product cerulenin and the novel compound c75, are selectively cytotoxic to cancer cells via induction of apoptosis, apparently related to the tumor cell phenotype of abnormally elevated fatty acid synthetic metabolism. As part of a larger effort to understand the immediate downstream effect of FAS inhibition that leads to apoptosis, the effects of these inhibitors on cell cycle progression were examined. Both FAS inhibitors produce rapid, profound inhibition of DNA replication and S phase progression in human cancer cells. The dose responses for fatty acid synthesis inhibition and DNA synthesis inhibition are similar. The kinetics of both effects are rapid, with fatty acid synthesis inhibition occurring within 30 min and DNA synthesis inhibition occurring within 90 min of drug exposure. Meanwhile, apoptotic changes are not detected until 6 h or later after inhibitor exposure. Fatty acid synthetic pathway activity and the magnitude of DNA synthesis inhibition by FAS inhibitors are increased in parallel by withdrawal of lipid-containing serum from the cultures. The mechanism of DNA synthesis inhibition by cerulenin is indirect, because expression of certain viral oncogenes rescues DNA synthesis/S phase progression in cerulenin-exposed cells. The data suggest a direct linkage at a regulatory level, between fatty acid synthesis and DNA synthesis in proliferating tumor cells.

Clonal diversity of IL-4 and IL-13 expression in human allergen-specific T lymphocytes.

The expression of IL-4 and IL-13 was analyzed in a panel of short ragweed allergen (Amb a 1)-specific T-cell clones from an allergic subject and a nonallergic individual. The T cells from the allergic subject showed a predominantly TH0 phenotype. The T cells from the nonallergic individual produced undetectable levels of IL-4 and high level of interferon-gamma, suggesting a TH1 cytokine profile. However, all T-cell clones showed significantly higher levels of IL-13 secretion than IL-4 secretion, and no quantitative correlation could be found between the levels of IL-4 and IL-13 in the clones tested. Furthermore, both cytokines showed similar kinetics of expression in antigen-induced steady-state messenger RNA. Finally, both cytokines were induced by stimulation of the cells with either ionomycin alone or with a combination of ionomycin and phorbol myristate acetate. These results demonstrate that there is a significant clonal diversity and quantitative difference in the levels of IL-4 and IL-13 expression in allergen-specific human T cells.