Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones

Abstract

Purpose Previously, compounds containing a piperidone structure have been shown to be highly cytotoxic to cancer cells. Recently, we found that the piperidone compound P2 exhibits a potent anti-neoplastic activity against human breast cancer-derived cells. Here, we aimed to evaluate two piperidone compounds, P1 and P2, for their potential anti-neoplastic activity against human leukemia/lymphoma-derived cells. Methods Cytotoxicity and apoptosis induction were evaluated using MTS, annexin V-FITC/PI and mitochondrial membrane potential polychromatic assays to confirm the mode of action of the piperidone compounds. The effects of compound P1 and P2 treatment on gene expression were assessed using AmpliSeq analysis and, subsequently, confirmed by RT-qPCR and Western blotting. Results We found that the two related piperidone compounds P1 and P2 selectively killed the leukemia/lymphoma cells tested at nanomolar concentrations through induction of the intrinsic apoptotic pathway, as demonstrated by mitochondrial depolarization and caspase-3 activation. AmpliSeq-based transcriptome analyses of the effects of compounds P1 and P2 on HL-60 acute leukemia cells revealed a differential expression of hundreds of genes, 358 of which were found to be affected by both. Additional pathway analyses revealed that a significant number of the common genes were related to the unfolded protein response, implying a possible role of the two compounds in the induction of proteotoxic stress. Subsequent analyses of the transcriptome data revealed that P1 and P2 induced similar gene expression alterations as other well-known proteasome inhibitors. Finally, we found that Noxa, an important mediator of the activity of proteasome inhibitors, was significantly upregulated at both the mRNA and protein levels, indicating a possible role in the cytotoxic mechanism induced by P1 and P2. Conclusions Our data indicate that the cytotoxic activity of P1 and P2 on leukemia/lymphoma cells is mediated by proteasome inhibition, leading to activation of pro-apoptotic pathways.

Pyronaridine exerts potent cytotoxicity on human breast and hematological cancer cells through induction of apoptosis

“…An early biochemical event triggering the intrinsic apoptosis pathway is mitochondrial depolarization, which can be quantified by using a polychromatic JC-1 reagent and flow cytometry [ 18 , 19 , 20 ]. JC-1 emits a red or green fluorescence signal when the mitochondria are polarized or depolarized, respectively.…”

A new pyridazinone exhibits potent cytotoxicity on human cancer cells via apoptosis and poly-ubiquitinated protein accumulation

“…The HL-60 cell line was selected for further experimentation for two reasons. We first investigated whether Pyr-1 acts as a proteasome inhibitor by comparing its activity to two previously reported proteasome inhibitors that were primarily tested in the HL-60 cell line (Contreras et al 2018). In addition, apoptosis assays on the HL-60 non-adherent cancer cells are easier to process than the adherent MDA-MB-231 cells by flow cytometry as the adherent cells require additional cellular manipulations to process (use of cell scrappers or trypsin).…”

“…To corrobotate whether Pyr-1 utilizes apoptosis to induce cell death, we measured phosphatidylserine (PS) externalization in Pyr-1-treated HL-60 cells by using the annexin V-FITC/PI assay (Contreras et al 2018; Robles-Escajeda et al 2016). For these experiments, two concentrations of Pyr-1 were used, 0.39 μM (CC50) and 0.78 μM (2× CC50).…”

“…To further investigate the mechanism by which Pyr-1 induces apoptosis, we examined its ability to inhibit proteasome activity. In the past, it has been observed that accumulation of poly-ubiquitinated proteins is an indication of proteasome impairment (Brnjic et al 2014; Contreras et al 2018; D’Arcy et al 2011; Wada et al 2015). Therefore, we measured poly-ubiquitinated proteins on HL-60 cells exposed to Pyr-1 at different time points by Western blotting.…”

“…However, not all cancer patients responded to bortezomib therapy, especially those with solid tumors, which are often resistant to this drug (Dou and Zonder 2014). To overcome the limitations of bortezomib therapy, second-generation proteasome inhibitors have been developed, mainly to increase anti-tumor efficacy and to reduce toxicity (Dou and Zonder 2014; Contreras et al 2018).…”