1. Apoptosis of NG108-15 cells induced by buprenorphine hydrochloride occurs via the caspase-3 pathway
F Kugawa, A Ueno, M Aoki Biol Pharm Bull. 2000 Aug;23(8):930-5. doi: 10.1248/bpb.23.930.
Apoptosis of NG108-15 neuroblastoma x glioma hybrid cells (NG108-15 cells) is induced by a morphine alkaloid derivative, buprenorphine hydrochloride (Bph). In a previous report, we used various apoptosis inhibitors to identify the "death pathway," and found that caspase inhibitors Ac-YVAD-CHO (Ac-Tyr-Val-Ala-Asp-CHO) and Ac-DEVD-CHO (Ac-Asp-Glu-Val-Asp-CHO) did not inhibit this particular apoptosis. Here, we tested Z-VAD-FMK (Z-Val-Ala-Asp[OMe]-CH2F) and Z-DEVD-FMK (Z-Asp[OMe]-Glu-[OMe]Val-Asp[OMe]-CH2F) for their ability to inhibit Bph-induced NG108-15 apoptosis. These tri- or tetra-peptide caspase inhibitors have a fluoromethyl ketone in their C-terminus instead of an aldehyde, and thus are more permeable than Ac-YVAD-CHO and AC-DEVD-CHO. Our observations of DNA ladder formation, cell morphology changes, and caspase-3 activities all indicated that these cell membrane-permeable caspase inhibitors completely inhibited the apoptosis, providing strong evidence that this apoptosis occurs through the caspase cascade "death pathway." Our previous report also showed that pretreatment of NG108-15 cells with TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) prevented DNA fragmentation and decreased the cell viability in Bph-induced apoptosis. The comparison of caspase-3 activities in Bph-induced samples with or without TPCK pretreatment revealed that caspase-3 was activated in both samples. Taken together, these results indicate that the Bph-induced apoptosis of NG108-15 cells occurs via the conventional caspase-dependent death pathway and that TPCK pretreatment results in a DNA ladder-deficient apoptosis.
2. Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway
Shanshan Qi, Lingyuan Guo, Shuzhen Yan, Robert J Lee, Shuqin Yu, Shuanglin Chen Acta Pharm Sin B. 2019 Mar;9(2):279-293. doi: 10.1016/j.apsb.2018.12.004. Epub 2018 Dec 15.
Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.
3. Enhancement of Z-aspartame synthesis by rational engineering of metalloprotease
Fucheng Zhu, Tianyue Jiang, Bin Wu, Bingfang He Food Chem. 2018 Jul 1;253:30-36. doi: 10.1016/j.foodchem.2018.01.108. Epub 2018 Jan 31.
Metalloprotease PT121Y114S, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S. One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121Y114S and its mutants revealed that the affinity of mutants toward the l-Phe-OMe was significantly higher than that of PT121Y114S. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-l-Phe-OMe complex.
