METHODS: α-Mangostin (AM) was isolated from C. arborescens and its cell death mechanism was investigated. AM-induced cytotoxicity was observed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Acridine orange/propidium iodide staining and annexin V were used to detect cells in early phases of apoptosis. High-content screening was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release. The role of caspases-3/7, -8, and -9, reactive oxygen species, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. To determine the role of the central apoptosis-related proteins, a protein array followed by immunoblot analysis was conducted. Moreover, the involvement of nuclear factor-kappa B (NF-κB) was also analyzed.
RESULTS: Apoptosis was confirmed by the apoptotic cells stained with annexin V and increase in chromatin condensation in nucleus. Treatment of cells with AM promoted cell death-transducing signals that reduced MMP by downregulation of Bcl-2 and upregulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase-9 followed by the executioner caspase-3/7 and then cleaved the PARP protein. Increase of caspase-8 showed the involvement of extrinsic pathway. AM treatment significantly arrested the cells at the S phase (P<0.05) concomitant with an increase in reactive oxygen species. The protein array and Western blotting demonstrated the expression of HSP70. Moreover, AM significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus.
CONCLUSION: Together, the results demonstrate that the AM isolated from C. arborescens inhibited the proliferation of MDA-MB-231 cells, leading to cell cycle arrest and programmed cell death, which was suggested to occur through both the extrinsic and intrinsic apoptosis pathways with involvement of the NF-κB and HSP70 signaling pathways.
METHODOLOGY: A 12-mer random peptide library expressed on the surface of the filamentous phage, M13, was used to select the mimotopes of two S. Typhi heat shock proteins by biopanning with monoclonal antibodies (mAbs), DnaK and HSP90. The immunogenicity of the selected peptides was determined through binding affinity with polyclonal antibodies from pooled typhoid-confirmed patients' sera and purified HSPs mAb using Western blotting and ELISA.
RESULTS: Five rounds of biopanning resulted in enrichment of phage clones expressing the binding motifs TDxSTRP and FPSHYWLYPPPT, respectively. The selected peptides showed strong immunoreactivity with patients' sera. Thus, monoclonal antibodies against HSP and patient sera can select common mimotopes from the random peptide library.
CONCLUSION: These findings may provide fundamental information for further studies on diagnostic application or vaccine design against this aetiologic agent of typhoid fever.