Narrow Results

Cinobufagin is a Na⁺/K⁺-ATPase (NKA) inhibitor with excellent anticancer effects to prolong the survival of patients. The purpose of the present study was to clarify the underlying mechanism of the anticancer effects of cinobufagin using overexpression or inhibition of aurora kinase A (AURKA) signaling. First, high expression of Na⁺/K⁺-ATPase alpha 1 subunit (ATP1A1) and AURAK resulted in increased malignant transformation in hepatocellular carcinoma (HCC) patients using the cancer genome atlas (TCGA) data and tissue samples. After treatment with cinobufagin, we successfully screened 202, 249, and 335 changing expression proteins in Huh-7 cells under normal, overexpression, and inhibition of AURKA using tandem mass tags (TMT)-labeled quantitative proteomics coupled to 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these molecules were closely associated with chromosome segregation, DNA damage, and regulation of translation processes. We further confirmed that cinobufagin induced DNA damage and chromosome segregation disorders and suppresses translational processing in oncogenes by decreasing the expression of AURKA, mechanistic target of rapamycin kinase (mTOR), p-mTOR, p-extracellular regulated protein kinases (ERK), eukaryotic translation initiation factor 4E (eIF4E), and p-eIF4E, while increasing the expression of p-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (S65, T37, T46, T45) and increasing the interaction between eIF4 and 4E-BP1. Our results suggested that cinobufagin performed an antitumor effects in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein with dual phosphatase activity, is found to be frequently mutated in various cancers. PTEN is post-translationally modulated at various amino acid residues which are crucial for sub-cellular localization as well as its catalytic functions rendering genomic stability. Recent reports suggest that PTEN also acts as a DNA repair protein. But how post-translational modulation of PTEN affects cytological damage and aneuploidy is not studied in detail. Here, we focus on the role of sumoylation of PTEN in context with DNA damage induced cytological damage like micronucleus (MNi), nuclear bud (NB), and nuclear bridge formation. Our data suggest that wild type PTEN but not sumo-dead PTEN significantly reduces cytological damage in PTEN mutant PC3 cells. In case of sumo-dead PTEN, the cytological parameters are increased during 24h recovery time point after DNA damage. Next, we measured the effectiveness of the sumo-dead (PTEN-K254R) mutant on aneuploidy, where we found that sumoylation is essential for maintaining chromosome number. As chromosome number variation in daughter cell is due to multiple spindle pole formation, we qualitatively and quantitatively evaluate the $\gamma$ tubulin pole formation in PTEN-K254R clone transfected cells. We found aberrant pole formation is significantly increased in PTEN-K254R transfected cells compared to wild-type PTEN. Further depletion of sumoylation activity of PTEN increases the expression of phosphorylated form of Aurora kinase A (AURKA) (T288) and PLK1 (T210) proteins with or without nocodazole, a microtubule depolymerizing agent compared to cells expressing wild-type PTEN. Thus, sumoylation of PTEN is essential for maintaining genomic stability.