Cyclosporin A and sanglifehrin A enhance chemotherapeutic effect of cisplatin in C6 glioma cells.

Glioma is the most common type of brain tumors in adults, and treatment of high-grade gliomas is still palliative. Studies to date have revealed only modest effect in attenuating growth of these tumors with single agent therapy, but combination treatment appears to be more effective. Cyclophilin A (CypA), a target of immunosuppressive drugs cyclosporin A (CsA) and sanglifehrin A (SFA), is an intracellular protein that has peptidyl-prolyl cis-trans isomerase (PPIase) enzymatic activity. Previously, we showed that overexpressed CypA induced chemoresistance in cancer cells. Here we provide evidence that combination of cisplatin with either CsA or SFA synergistically enhances apoptotic cell death in C6 glioma cells, compared with single agent treatment. Enhanced apoptotic cell death is a result of an increase in ROS generation and a decrease in intracellular glutathione levels. Consistently, CypA knockdown by siRNA also enhances cisplatin-induced apoptosis. Immunohistochemical analysis showed increased expression of CypA in human glioblastoma multiforme, but not in normal human astrocytes. CypA was also shown to be up-regulated in C6 glioma cells during hypoxia. In conclusion, CsA or SFA in combination with cisplatin synergistically enhances cisplatin-induced apoptosis in C6 glioma cells via inhibition of PPIase activity of CypA, indicating that development of new drugs that selectively inhibit the CypA PPIase activity without immune suppression may facilitate alleviation of chemoresistance in treatment of high-grade glioma.

Optimal production of poly-gamma-glutamic acid by metabolically engineered Escherichia coli.

Metabolically-engineered Escherichia coli strains were developed by cloning poly-gamma-glutamic acid (gamma-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of gamma-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P(HCE)) derived from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of gamma-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH(4))(2)SO(4 )was added at 40 g/l into the feeding solution, the final gamma-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.