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Though Isoimperatorin from Angelicae dahuricae is known to have antiviral, antidiabetic, anti-inflammatory and antitumor effects, its underlying antitumor mechanism remains elusive so far. Hence, the apoptotic mechanism of Isoimperatorin was explored in hepatocellular carcinomas (HCCs). In this study, Isoimperatorin inhibited the viability of Huh7 and Hep3B HCCs and increased the subG1 apoptotic portion and also abrogated the expression of pro-poly-ADP ribose polymerase (pro-PARP) and pro-caspase 3 in Huh7 and Hep3B cells. Also, Isoimperatorin abrogated the expression of cyclin D1, cyclin E1, CDK2, CDK4, CDK6 and increased p21 as G1 phase arrest-related proteins in Huh7 and Hep3B cells. Interestingly, Isoimperatorin reduced the expression and binding of c-Myc and Sirtuin 1 (SIRT1) by Immunoprecipitation (IP), with a binding score of 0.884 in Huh7 cells. Furthermore, Isoimperatorin suppressed the overexpression of c-Myc by the proteasome inhibitor MG132 and also disturbed cycloheximide-treated c-Myc stability in Huh7 cells. Overall, these findings support the novel evidence that the pivotal role of c-Myc and SIRT1 is critically involved in Isoimperatorin-induced apoptosis in HCCs as potent molecular targets in liver cancer therapy. [ABSTRACT FROM AUTHOR]

Dysregulation of cyclin-dependent kinase 8 (CDK8) activity has been associated with many diseases, including colorectal and breast cancer. As usual in the CDK family, the activity of CDK8 is controlled by a regulatory protein called cyclin C (CycC). But, while human CDK family members are generally activated in two steps, that is, the binding of the cyclin to CDK and the phosphorylation of a residue in the CDK activation loop, CDK8 does not require the phosphorylation step to be active. Another peculiarity of CDK8 is its ability to be associated with CycC while adopting an inactive form. These specificities raise the question of the role of CycC in the complex CDK8–CycC, which appears to be more complex than the other members of the CDK family. Through molecular dynamics (MD) simulations and binding free energy calculations, we investigated the effect of CycC on the structure and dynamics of CDK8. In a second step, we particularly focused our investigation on the structural and molecular basis of the protein–protein interaction between the two partners by finely analyzing the energetic contribution of residues and simulating the transition between the active and the inactive form. We found that CycC has a stabilizing effect on CDK8, and we identified specific interaction hotspots within its interaction surface compared to other human CDK/Cyc pairs. Targeting these specific interaction hotspots could be a promising approach in terms of specificity to effectively disrupt the interaction between CDK8. The simulation of the conformational transition from the inactive to the active form of CDK8 suggests that the residue Glu99 of CycC is involved in the orientation of three conserved arginines of CDK8. Thus, this residue may assume the role of the missing phosphorylation step in the activation mechanism of CDK8. In a more general view, these results point to the importance of keeping the CycC in computational studies when studying the human CDK8 protein in both the active and the inactive form. [ABSTRACT FROM AUTHOR]

Alpha-1 antitrypsin-overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AAT's effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3β and β-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate. [ABSTRACT FROM AUTHOR]