Effectiveness of tumor­treating fields to reduce the proliferation and migration of liposarcoma cell lines.

Liposarcoma (LPS) is a rare type of soft tissue sarcoma that constitutes 20% of all sarcoma cases in adults. Effective therapeutic protocols for human LPS are not well-defined. Tumor-treating fields (TTFields) are a novel and upcoming field for antitumor therapy. TTFields combined with chemoradiotherapy have proven to be more effective than TTFields combined with radiotherapy or chemotherapy alone. The present study aimed to assess the effectiveness of TTFields in inhibiting cell proliferation and viability for the anticancer treatment of LPS. The present study used TTFields (frequency, 150 kHz; intensity, 1.0 V/cm) to treat two LPS cell lines (94T778 and SW872) and analyzed the antitumor effects. According to trypan blue and MTT assay results, TTFields markedly reduced the viability and proliferation of LPS cell lines along with the formation of colonies in three-dimensional culture. Based on the Transwell chamber assay, TTFields treatment also markedly reduced the migration of LPS cells. Furthermore, as shown by the higher activation of caspase-3 in the Caspase-3 activity assay and the results of the reactive oxygen species (ROS) assay, TTFields increased the formation of ROS in the cells and enhanced the proportion of apoptotic cells. The present study also investigated the inhibitory effect of TTFields in combination with doxorubicin (DOX) on the migratory capacity of tumor cells. The results demonstrated that TTFields treatment synergistically induced the ROS-induced apoptosis of LPS cancer cell lines and inhibited their migratory behavior. In conclusion, the present study demonstrated the potential of TTFields in improving the sensitivity of LPS cancer cells, which may lay the foundation for future clinical trials of this combination treatment strategy.

Bacterial synthesis of N-hydroxycinnamoyl phenethylamines and tyramines.

BACKGROUND: Hydroxycinnamic acids (HCAs) including cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, are C6-C3 phenolic compounds that are synthesized via the phenylpropanoid pathway. HCAs serve as precursors for the synthesis of lignins, flavonoids, anthocyanins, stilbenes and other phenolic compounds. HCAs can also be conjugated with diverse compounds including quinic acid, hydroxyl acids, and amines. Hydroxycinnamoyl (HC) amine conjugates such as N-HC tyramines and N-HC phenethylamines have been considered as potential starting materials to develop antiviral and anticancer drugs. RESULTS: We synthesized N-HC tyramines and N-HC phenethylamines using three different approaches in Escherichia coli. Five N-HC phenethylamines and eight N-HC tyramines were synthesized by feeding HCAs and phenethylamine or tyramine to E. coli harboring 4CL (encoding 4-coumarate CoA:ligase) and either SHT (encoding phenethylamine N-HC transferase) or THT (encoding tyramine N-HC transferase). Also, N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid using E. coli harboring an additional gene, PDC (encoding phenylalanine decarboxylase) or TDC (encoding tyrosine decarboxylase). Finally, we synthesized N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine from glucose by reconstructing the metabolic pathways for their synthesis in E. coli. Productivity was maximized by optimizing the cell concentration and incubation temperature. CONCLUSIONS: We reconstructed the metabolic pathways for synthesis of N-HC tyramines and N-HC phenethylamines by expressing several genes including 4CL, TST or SHT, PDC or TDC, and TAL (encoding tyrosine ammonia lyase) and engineering the shikimate metabolic pathway to increase endogenous tyrosine concentration in E. coli. Approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine and 495.4 mg/L N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid. Furthermore, 152.5 mg/L N-(p-coumaroyl) phenethylamine and 94.7 mg/L N-(p-coumaroyl) tyramine were synthesized from glucose.

Biological synthesis of coumarins in Escherichia coli.

BACKGROUND: Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives. RESULTS: Constructs harboring two genes, F6'H (encoding feruloyl CoA 6' hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway. CONCLUSIONS: To produce coumarin derivatives (umbelliferone, scopoletin, and esculetin) in E. coli, several constructs containing F6'H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6'H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.

Production of kaempferol 3-O-rhamnoside from glucose using engineered Escherichia coli.

Flavonoids are ubiquitous phenolic compounds and at least 9,000 have been isolated from plants. Most flavonoids have been isolated and assessed in terms of their biological activities. Microorganisms such as Escherichia coli and Saccharomyces cerevisiae are efficient systems for the synthesis of flavonoids. Kaempferol 3-O-rhamnoside has notable biological activities such as the inhibition of the proliferation of breast cancer cells, the absorption of glucose in the intestines, and the inhibition of the self-assembly of beta amyloids. We attempted to synthesize kaempferol 3-O-rhamnoside from glucose in E. coli. Five flavonoid biosynthetic genes [tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), and flavonol 3-O-rhamnosyltransferase (UGT78D1)] from tyrosine were introduced into E. coli that was engineered to increase tyrosine production. By using this approach, the production of kaempferol 3-O-rhamnoside increased to 57 mg/L.