D(−)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent

Abstract

We have recently found that 𝒟(−)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by 𝒟−)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that 𝒟−)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5–3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3–2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 μM 𝒟−)lentiginosine, whereas apoptosis by 𝒟−)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.

Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB

“…For studying mitochondrial membrane potential (MMP) in U937 cells subjected to different treatments the MitoProbe™ JC-1 Assay Kit for Flow Cytometry (Molecular Probes Europe BV, Leiden, Netherlands) was utilized, as previously described [31].…”

“…For reverse-transcription, 0.25 μg of total RNA extracted from each experimental sample were processed for cDNA generation using a highcapacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer's instructions. RQ-PCR was performed and relative mRNA levels were calculated as previously described in detail [31]. As a housekeeping gene, glucuronidase beta (GUSB) was used.…”

Molecular dynamics of the full-length p53 monomer

“…56,57 The regulation of cell death by the p53 protein is quite complex, acting both at the level of autophagy, 58 lysosomes, 59 or at the core machinery of programmed cell death. [60][61][62][63][64][65] In addition to DNA damage response and cell death, p53 plays a crucial role in regulating cellular senescence [66][67][68] by interacting, for example, with MageA2, 69 PATZ1, 70 4E-BP1, 71 mTOR, 72,73 highlighting the vast complexity of this crucial regulation.…”

Synthesis of biotin and fluorescein labeled (–)-lentiginosine

“…To this end, the inexpensive 11-aminoundecanoic acid (12) was protected with the Cbz group, 19 and then the free carboxylic group was converted into isocyanate through Curtius rearrangement of the corresponding acyl azide.…”

“…Accordingly, the development of new pro-apoptotic molecules and the knowledge of their mechanism of action are major challenges in organic chemistry and biomedical sciences. Initial studies have disclosed that the activity of 2 is caspase dependent and involves activation of the intrinsic pathway of apoptosis, 12 but still many aspects of the mechanism of action of 2 need to be investigated. In particular, the specific receptor of the molecule that is able to start the cascade of events leading to apoptosis has yet to be identified.…”