Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism

Abstract

To investigate the effects of a novel polyamine metabolism enzyme inhibitor SI-4650 on autophagy and apoptosis of colon cancer CT-26 cells as well as their correlation. CT-26 cells treated with 40, 80 μmol·L-1 SI-4650 alone or in combination with 3-MA were used as experimental group. CT-26 cells treated with 0 μmol·L-1 SI-4650 alone or in combination with 3-MA were used as control group. Chemiluminescence was used to analyze the effect of SI-4650 on spermine oxidase (SMO) and acetylpolyamine oxidase(APAO) activity. High performance liquid chromatography (HPLC) was performed to detect cellular polyamine content.The CCK8 method was used to detect the inhibitory effect of SI-4650 on proliferation of CT-26 cells. PI single-staining/flow cytometry (FCM) were used to analyze cell cycle. Western blot were used to analyze autophagy. Apoptosis was analyzed by PI/FITC-Annexin V double staining, JC-1 fluorescent probe and Fluo-3 AM calcium ion fluorescent probe combined with flow cytometry and Western blot. CCK8 assay showed that 24-,48-,72-hours treated with SI-4650 all could inhibit the proliferative activity of CT-26 cells in a dose- and time-dependent manner (P＜0.01) . The inhibition rate was 36.98% and 46.91% in 40 μmol·L-1 SI-4650 group and 80 μmol·L-1 SI-4650 group respectively. SI-4650 could significantly inhibit the activities of SMO and APAO interfere with polyamine metabolism and reduce the content of total polyamine in CT-26 cells (P＜0.01). SI-4650 could block CT-26 cells in G0/G1 phase, significantly reduce the number of cells in S phase(P＜0.01), and lead to a significant increase in the contents of autophagy-related Beclin-1, LC3-II in CT-26 cells(P＜0.01); At the same time, the concentration of calcium in CT-26 cells was increased, the mitochondrial membrane potential was decreased, the expressions of c-PARP and Bax were increased, the content of Bcl-2 was decreased, and the number of apoptotic cells was increased. After SI-4650 combined with autophagy inhibitor 3-MA treatment of CT-26 cells, the level of autophagy, the apoptosis-related protein, mitochondrial membrane potential and calcium ion concentration were decreased, and the number of apoptotic cells was decreased. SI-4650 has the pharmacological activity of killing colon cancer CT-26 cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy. In this process, autophagy is inhibited to block apoptosis, autophagy and apoptosis combined to kill tumor cells.