# Sensitising PDAC to Gemcitabine by Suppressing NF-κB Pathway and Enhancing Apoptosis

## TL;DR

This study shows that a molecule called B12 can make pancreatic cancer cells more responsive to the drug gemcitabine, potentially improving treatment outcomes.

## Contribution

The novel contribution is the identification of B12 as a sensitiser that enhances gemcitabine efficacy in PDAC cells by suppressing NF-κB and promoting apoptosis.

## Key findings

- B12 reduced gemcitabine IC50 in PANC-1 cells without significant toxicity to normal pancreatic cells.
- B12 co-treatment increased apoptosis and mitochondrial depolarisation in PDAC cells.
- Transcriptomic analysis revealed downregulation of NF-κB-related genes and reduced p65 nuclear accumulation with B12.

## Abstract

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) exhibits poor clinical response to gemcitabine, largely due to intrinsic and acquired mechanisms of chemoresistance. Identifying agents capable of enhancing gemcitabine efficacy without increasing cytotoxicity remains an unmet therapeutic need. Here, we characterise a small drug sensitiser molecule, B12, and evaluate its potential to sensitise PDAC cells to gemcitabine. Methods: Gemcitabine’s dose–response was assessed by MTT assay to determine IC50 values and dose-modifying factor (DMF). Phenotypic consequences of co-treatment were examined using colony formation and wound scratch assays. Mitochondrial membrane potential (JC-1) and apoptosis (Annexin V/PI) were measured using flow cytometry. Transcriptomic profiling was performed using mRNA-seq with differential expression analysis and pathway enrichment (KEGG/GSEA). NF-κB activity was assessed by nuclear and cytoplasmic fractionation of p65, and RT-qPCR validation of NF-κB associated target genes. Results: B12 alone displayed minimal cytotoxicity in the PANC-1 cell line and normal pancreatic ductal HPDE cells, yet shifted the gemcitabine dose–response curve in PANC-1 cells, reducing the IC50 and yielding a dose-modifying factor of 1.39. Functionally, B12 enhanced gemcitabine-induced suppression of colony formation and reduced wound closure relative to gemcitabine alone. The co-treatment also increased both mitochondrial depolarisation and apoptotic cell populations, with increased cell proliferation inhibition over time. Transcriptomic profiling identified a set of B12-associated genes downregulated both in B12-treated and B12 + gemcitabine conditions, including factors linked to growth, survival, inflammation, metabolism, and drug inactivation. Gene set enrichment analysis revealed negative enrichment of NF-κB associated pathways during B12 co-treatment. Consistently, nuclear-cytoplasmic fractionation showed that B12 reduced gemcitabine-induced nuclear accumulation of p65, accompanied by decreased expression of NF-κB associated targets such as BCL2L1, CCL20, SLC2A1, and MAP3K14. Conclusions: In PDAC cell models, B12 enhances gemcitabine cytotoxic response while displaying minimal intrinsic toxicity under the conditions tested. The sensitising phenotype is accompanied by increased apoptotic susceptibility and is associated with reduced NF-κB signalling at the pathway, transcript, and p65 nuclear localisation levels. However, to establish causality, the lack of sensitisation in HPDE cells will require further validation.