Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis

Abstract

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a “combo-therapy” associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

Synergistic Effects of A Combined Treatment of Glioblastoma U251 Cells with An Anti-miR-10b-5p Molecule and An AntiCancer Agent Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole Scaffold

“…We have already published two studies on synergistic effects of miRNA inhibitors on GBM cell lines when they are co-administered with anticancer agents [ 13 , 19 ]. In the first study, a peptide-nucleic acid (PNA) targeting miR-221-3p was co-administered with a tubulin inhibitor different from that employed in the present study [ 19 ].…”

“…In the first study, a peptide-nucleic acid (PNA) targeting miR-221-3p was co-administered with a tubulin inhibitor different from that employed in the present study [ 19 ]. In the second, a PNA targeting miR-15b-5p was co-administered with sulforaphane [ 13 ]. The reason behind studying different miRNA inhibitors combined with different anti-GBM agents is due to the person-to-person variability of response to chemotherapy on the one hand and of onco-miRNAs upregulation on the other.…”

“…The isolated RNA was washed once with cold 75% ethanol and stored at −80 °C until use. Obtained RNA was dried and dissolved in nuclease-free water before use [ 13 , 18 ] and quantified using a SmartSpec TM Plus Spectrophotometer (Bio-Rad, Hercules, CA, USA) before proceeding with reverse transcription reaction.…”

“…The expression of the Caspase-3 gene was verified by RT-qPCR, as described elsewhere [ 13 ]. Total RNA was reverse transcribed using random hexamers and TaqMan Reverse Transcription PCR Kit (Thermo Fischer Scientific).…”

“…In this respect, we have recently reported a possible combined therapeutic approach for GBM based on the use of microRNA inhibitors and anti-GBM molecules. For example, we have found that sulforaphane (SFN) and a peptide-nucleic acid (PNA) targeting microRNA miR-15b-5p synergistically act by inducing apoptosis of the glioblastoma U251 cells [ 13 ]. Therefore, the PNA-a15b might be proposed in “combo-therapy” associated with SFN.…”