The Cytotoxicity of Analogues Carnosine Dipeptide on Human Glioblastoma Multiforme

Abstract

Background & Aims: The present study evaluated the effect of some synthesized linear and cyclic Carnosine analogues on the cells of human glioblastoma multiforme (GBM). The purpose of this research was to study the exposure of Carnosin analogues on astrocytoma using several experiments. Methods: The mass spectral measurements were performed on a 6410 Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface. All tests were carried out six times. The concentration used for peptides (10 µg/mL) was selected based on MTT assay. The effect of peptides on the activity of SDH was assayed by MTT test. 100 μL mitochondrial suspension from GBM and normal groups were incubated with applied concentration of peptides (10μg/mL) at 37°C for 30 min. DCFH (final concentration, 10µM) was added to cells of both groups and incubated at 37°C for 15 min. The fluorescence intensity of DCFH which is an indicator of ROS concentration was then assayed by a Shimadzu RF-5000U fluorescence spectrophotometer. The cationic fluorescent dye, rhodamine 123 (concentration10 µM), from mitochondria into the cytosol have been used for the determination of MMP collapse. Mitochondria from astrocytoma and normal astocyte groups were suspended in corresponding assay buffer and incubated at 37°C with 10µg/mL peptides. The release was assayed by the Quantikine Cytochrome c. Carnosine peptide analogues on the activation of caspase-3 in the mitochondria isolated from GBM and normal groups using Sigma’s caspase-3 colorimetric assay kit. Results: Our study showed that a various range of toxicity on GBM cells was resulted by the linear and cyclic Carnosine analogues using MTT assay. Also, applying peptides on the mitochondria of GBM cells, a raise of mitochondrial reactive oxygen species (ROS) level, mitochondrial swelling, mitochondrial membrane potential (∆ψm) collapse, release of cytochrome c and caspase-3 activation of the affected mitochondria were detected. Conclusion: Based on the overall results, cyclic Carnosine analogues, especially compound 1c [Cyclo-(β-alanine-¬His-Pro-¬β-alanine-¬His)] showed more toxic activity than linear Carnosine analogues, which would be supporting to develop Carnosine cyclic peptide analogues as new anticancer and complementary therapeutic agents for the treatment of glioblastoma.