Myo-Inositol Boosts Doxorubicin-Induced Apoptosis in Breast Cancer Cells in Vitro

The combination treatment induced apoptotic cell death in 25.12 percent of MCF7 breast cancer cells, and the effect was accompanied by an unexpected drop in the cellular levels of total p38 MAPK, a stress-responsive signaling protein that is often described as pro-apoptotic. The work, led by researchers including Berna Özdem and Cemil Demir, was published on 5 October 2026 as an open access article.

The researchers set out to test whether myo-inositol could act as a chemosensitizer, a compound that makes cancer cells more vulnerable to chemotherapy. Myo-inositol is the most abundant natural polyol among the nine possible structural isomers found in living organisms, an isomer of glucose that serves as a precursor in the phosphatidylinositol cycle and plays an important role in cellular signaling. It accumulates at high levels in the brain, contributes to neurotransmitter biosynthesis, mediates signal transduction pathways activated by neurotransmitters, hormones and growth factors, and participates in cellular osmoregulation, intracellular calcium control, insulin signaling and gene expression modulation. Because it exhibits low toxicity, the team considered it an attractive candidate for combination therapy.

Doxorubicin, by contrast, is a cornerstone chemotherapeutic agent for breast cancer. It exerts its cytotoxic effects by intercalating into DNA and inhibiting topoisomerase II, which leads to DNA damage and induces apoptosis in breast cancer cells through multiple pathways. The p38 MAPK pathway, which the study focused on, is activated by stress signals such as DNA damage and reactive oxygen species, and is generally understood to promote apoptosis through mitochondrial disruption and caspase activation. Whether myo-inositol modulates p38 MAPK in conjunction with doxorubicin had not been previously explored, according to the authors.

The experimental system was deliberately simple. The team used MCF7 cells, a breast cancer cell line representing the estrogen receptor-positive subtype, and MCF 10A cells, a normal breast epithelial line, as a comparison. Cell viability was measured with the MTS assay, a colorimetric test in which metabolically active cells bio-reduce a tetrazolium compound into a colored formazan product, with absorbance read at 492 nanometers. Apoptosis, p38 MAPK levels and reactive oxygen species were all assessed by flow cytometry on a Beckman Coulter CytoFLEX instrument, with at least 10,000 cells analyzed per sample in the apoptosis assay.

The dose-response data showed that myo-inositol alone inhibited MCF7 cell viability in a concentration-dependent manner, with an estimated half-maximal inhibitory concentration of approximately 713 micromolar. Cell viability declined from 100 percent in the untreated control group to 64.13, 60.43, 47.11, 43.39 and 31.80 percent following treatment with 200, 400, 800, 1600 and 3200 micromolar myo-inositol respectively, corresponding to growth inhibition rising progressively from 35.87 percent at the lowest dose to 68.20 percent at the highest. In the normal MCF 10A breast epithelial cells, the researchers observed only a relatively limited cytotoxic response, even at the highest 3200 micromolar concentration, which they interpreted as suggesting selective cytotoxicity toward the cancer cells.

For the combination experiments, the team used a fixed doxorubicin concentration of 2 micromolar alongside the myo-inositol doses. The combined treatment significantly reduced MCF7 cell proliferation in a dose- and time-dependent manner, and the Annexin V-FITC and propidium iodide staining assay, which distinguishes viable, early apoptotic, late apoptotic and necrotic cells, confirmed that the combination induced 25.12 percent total apoptotic cell death, calculated as the sum of early and late apoptotic populations. The authors also reported a marked increase in intracellular reactive oxygen species in the combination group, with the ROS-positive fraction reaching 54.23 percent compared with 6.04 percent in the control.

The p38 MAPK result was the most counterintuitive finding. Flow cytometry using an antibody that recognizes endogenous total p38 alpha, beta and gamma proteins showed that the fraction of p38-positive cells fell to 58.82 percent in the combination group, compared with 99.77 percent in untreated controls, 90.17 percent with doxorubicin alone and 72.51 percent with myo-inositol alone. The authors described this as a 1.53-fold reduction relative to doxorubicin and a 1.23-fold reduction relative to myo-inositol alone, with the former difference statistically significant at p < 0.01 and the latter at p < 0.05. Because p38 MAPK is commonly associated with pro-apoptotic signaling, the team proposed that in this context its reduced levels coincided with enhanced apoptosis, possibly because excessive reactive oxygen species accumulation can trigger mitochondrial-dependent apoptosis independently of p38 MAPK, and that p38 MAPK may instead play a protective or adaptive role in MCF7 cells under stress.

An important caveat concerns what the flow cytometry assay actually measured. The antibody used detects total p38 MAPK protein and, at the dilution employed, does not detect the phosphorylated form of the protein, which is the activated state. The authors acknowledged this directly in their discussion, noting that because only total intracellular p38 MAPK levels were evaluated, without assessment of phosphorylation status or kinase activity, the findings do not demonstrate direct pathway activation or inhibition. Instead, they indicate an association between altered total p38 MAPK levels and the enhanced apoptotic response induced by the combination treatment. The abstract’s reference to lower phospho-p38 levels therefore sits uneasily with the methods as described, and readers should treat the mechanistic interpretation as tentative.

To explore possible mechanistic links, the researchers also ran computational analyses. SwissADME predictions characterized myo-inositol as highly hydrophilic, with low lipophilicity and high topological polar surface area, making it poorly permeable across lipid membranes and suggesting low gastrointestinal absorption and suboptimal oral delivery despite a moderate predicted bioavailability of 0.55. The molecule was predicted not to inhibit major CYP450 enzymes, indicating minimal potential for drug-drug interactions, and to lack PAINS or reactive groups. KEGG pathway and subcellular compartment enrichment analyses of predicted targets highlighted MAPK signaling and apoptosis-related pathways with very low false discovery rates. These are in silico predictions and target-gene enrichments, however, not direct measurements of pathway activity in the treated cells.

The authors themselves list substantial limitations. The cellular and molecular evaluations relied mainly on a single breast cancer cell line, and although myo-inositol alone showed limited cytotoxicity in MCF 10A cells, breast cancer is molecularly heterogeneous, so further studies with additional models are required. Because the interaction was tested at a single fixed doxorubicin concentration, combination index, isobologram or Chou-Talalay analyses could not be performed, meaning the study demonstrates an enhanced combination effect rather than a formally confirmed synergistic interaction. The team also notes that mechanistic studies using specific pathway inhibitors or genetic approaches are needed to confirm any causal role for p38 MAPK signaling, and that in vivo validation is warranted. Within those boundaries, the study adds a testable lead: in this one estrogen receptor-positive cell line, pairing a widely available, well-tolerated inositol isomer with doxorubicin increased apoptotic death while total p38 MAPK levels fell, a pattern the authors suggest could, if confirmed, eventually support dose-sparing strategies, though no clinical implication follows from cell culture data alone.

Subject of Research: A laboratory study of doxorubicin combined with myo-inositol inducing apoptosis in estrogen receptor-positive breast cancer cells in association with altered total p38 MAPK levels

Article Title: Combination of doxorubicin and myo-inositol induces apoptosis in MCF7 cells associated with altered total p38 MAPK levels

Article References: Özdem, B., Kaymaz, T., Yıldırım, I., & Demir, C. (2026). Combination of doxorubicin and myo-inositol induces apoptosis in MCF7 cells associated with altered total p38 MAPK levels. Applied Biological Chemistry, 69(1), Article 72. https://doi.org/10.1186/s13765-026-01124-y

Image Credits: AI Generated

DOI: 10.1186/s13765-026-01124-y

Keywords: doxorubicin, myo-inositol, MCF7 cells, apoptosis, p38 MAPK, breast cancer, flow cytometry, reactive oxygen species, chemosensitizer, cell culture study, MTS assay, estrogen receptor-positive