Ohe, K. | bioRxiv Science

bioRxiv2026

Sodium butyrate inhibits the proliferation of temozolomide-resistant glioblastoma (A172) and lung adenocarcinoma (A549) cells and biphasically regulates EGFR expression

Glioblastoma carries one of the poorest prognoses of any solid tumour, and once resistance to temozolomide (TMZ) emerges there is little left to offer. Because the TMZ response is governed largely by epigenetic silencing of MGMT, histone deacetylase (HDAC) inhibitors are a rational partner for TMZ. We asked whether sodium butyrate (NaB), a short-chain fatty acid produced by the gut microbiota and a class I/IIa HDAC inhibitor, retains activity against TMZ-resistant cells. In A172 glioblastoma and A549 lung adenocarcinoma cells, NaB changed EGFR expression biphasically: EGFR exon 2-5 mRNA was induced at 0.15 mM in A172 and at 0.30 mM in A549, and was suppressed at higher concentrations, with EGFR protein following the same pattern in A172. NaB reduced proliferation of both lines from 0.31 mM in a concentration-dependent manner, more markedly in A172 than in A549. At concentrations that were inactive alone (NaB 0.30 mM, TMZ 3 {micro}M), the combination inhibited proliferation of A172 on day 3 and of A549 on days 2 and 3. TMZ-resistant sublines (A172 rTMZ, A549 rTMZ) established by stepwise exposure showed no response to TMZ 10 {micro}M alone, but NaB at 0.5 and 2.5 mM inhibited their proliferation concentration-dependently. The ratio of combination to TMZ alone was not lower in the resistant sublines than in the parental lines (A172 0.70 vs 0.86; A549 0.49 vs 0.58), indicating that the effect in resistant cells is attributable to NaB itself rather than to restored TMZ sensitivity. Millimolar exposure is the main barrier to translation.

molecular biology↗

bioRxiv2026

Decoy and antisense RNA oligonucleotides targeting the EGFR exon 3 splice site suppress proliferation of A172 glioblastoma cells

Short RNA oligonucleotides directed at the 3' splice site region of EGFR exon 3 reduced the proliferation of A172 glioblastoma cells, while the same oligonucleotides had no detectable effect on A549 lung adenocarcinoma cells. Glioblastoma carries EGFR amplification in roughly a third of IDH-wildtype cases, and the A172 line harbours a tandem duplication in the EGFR tyrosine kinase domain. Because exon definition in human genes depends on RNA-binding proteins that engage exonic sequence near splice sites, we used the SpliceAid database to identify candidate RNA-binding proteins at EGFR exons 2 and 3. Y-box binding protein 1 (YB-1, encoded by YBX1), reported to drive glioblastoma growth through its RNA-binding function, scored highly immediately adjacent to the exon 3 3' splice site. We designed four 2'-modified RNA oligonucleotides carrying a nuclear localisation signal: two antisense oligonucleotides against the exon 3 region (asRNA1, spanning the splice site; asRNA2, downstream of it), a decoy complementary to the asRNA2 target (dRNA2), and a decoy carrying the YB-1 consensus binding sequence (dYB-1). In A172 cells, all four reduced EGFR protein in a single Western blot experiment, and asRNA1, asRNA2 and dRNA2 reduced proliferation relative to scrambled RNA at days 1 to 3; dYB-1 produced a comparable reduction at day 3. None of these effects was seen in A549 cells. YB-1 protein level and subcellular distribution were unchanged by the oligonucleotides, arguing against an indirect route through altered YB-1 abundance. These observations are preliminary. Every experiment reported here was performed once: the proliferation data come from single plates with three wells per group, analysed by Dunnett's test against the scrambled-RNA control, and the Western blot, RT-PCR and immunocytochemistry panels are single runs presented as representative observations. Direct binding of YB-1 to the targeted EGFR sequence has not been demonstrated. We post this work as a preprint so that the design strategy and the initial observations are available while these questions remain open.

molecular biology↗