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The present study investigated the effects of the combined treatment of two peptide nucleic acids (PNAs), directed against microRNAs involved in caspase‑3 mRNA regulation (miR‑155‑5p and miR‑221‑3p) in the temozolomide (TMZ)‑resistant T98G glioma cell line. These PNAs were conjugated with an octaarginine tail in order to obtain an efficient delivery to treated cells. The effects of singularly administered PNAs or a combined treatment with both PNAs were examined on apoptosis, with the aim to determine whether reversion of the drug‑resistance phenotype was obtained. Specificity of the PNA‑mediated effects was analyzed by reverse transcription‑quantitative polymerase‑chain reaction, which demonstrated that the effects of R8‑PNA‑a155 and R8-PNA-a221 anti‑miR PNAs were specific. Furthermore, the results obtained confirmed that both PNAs induced apoptosis when used on the temozolomide‑resistant T98G glioma cell line. Notably, co‑administration of both anti‑miR‑155 and anti‑miR‑221 PNAs was associated with an increased proapoptotic activity. In addition, TMZ further increased the induction of apoptosis in T98G cells co‑treated with anti‑miR‑155 and anti‑miR‑221 PNAs.

The biological activity of a combined treatment of U251, U373 and T98G glioma cell lines with two anti-miR PNAs, directed against miR‑221 and miR‑222 and conjugated with an ocataarginine tail (R8-PNA-a221 and R8-PNA-a222) for efficient cellular delivery, was determined. Apoptosis was analyzed, and the effect of the combined treatment of glioma cells with either or both PNAs on the reversion of drug-resistance phenotype was assessed in the temozolomide-resistant T98G glioma cell line. Selectivity of PNA/miRNA interactions was studied by surface plasmon resonance (SPR)-based Biacore analysis. Specificity of the PNA effects at the cellular level was analyzed by RT-qPCR. These experiments support the concept that the effects of R8-PNA-a221 and R8-PNA-a222 are specific. The studies on apoptosis confirmed that the R8-PNA-a221 induces apoptosis and demonstrated the pro-apoptotic effects of R8-PNA-a222. Remarkably, increased pro-apoptotic effects were obtained with the co-administration of both anti-miR‑221 and anti-miR‑222 PNAs. In addition, co-administration of R8-PNA-a221 and R8-PNA-a222 induced apoptosis of TMZ-treated T98G cells at a level higher than that obtained following singular administration of R8-PNA-a221 or R8-PNA-a222.

O-6-methylguanine-DNA methyltransferase (MGMT) is an abundantly expressed nuclear protein dealkylating O6-methylguanine (O6-MG) DNA residue, thus correcting the mismatches of O6-MG with a thymine residue during DNA replication. The dealkylating effect of MGMT is relevant not only in repairing DNA mismatches produced by environmental alkylating agents promoting tumor pathogenesis, but also when alkylating molecules are applied in the chemotherapy of different cancers, including glioma, the most common primary tumor of the central nervous system. Elevated MGMT gene expression is known to confer resistance to the treatment with the alkylating drug temozolomide in patients affected by gliomas and, on the contrary, methylation of MGMT gene promoter, which causes reduction of MGMT protein expression, is known to predict a favourable response to temozolomide. Thus, detecting expression levels of MGMT gene is crucial to indicate the option of alkylating agents or to select patients directly for a second line targeted therapy. Further study is required to gain insights into MGMT expression regulation, that has attracted growing interest recently in MGMT promoter methylation, histone acetylation and microRNAs expression. The review will focus on the epigenetic regulation of MGMT gene, with translational applications to the identification of biomarkers predicting response to therapy and prognosis.